# Effects of Crude Extracts from Medicinal HerbsRhazya stricta and Zingiber officinale on Growth and Proliferation of Human Brain Cancer Cell Line In Vitro

Authors: Ayman I. Elkady; Rania Abd El Hamid Hussein; Osama A. Abu-Zinadah
Journal: BioMed Research International
 (2014)

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## Abstract

Hitherto, limited clinical impact has been achieved in the treatment of glioblastoma (GBMs). Although phytochemicals found in medicinal herbs can provide mankind with new therapeutic remedies, single agent intervention has failed to bring the expected outcome in clinical trials. Therefore, combinations of several agents at once are gaining increasing attractiveness. In the present study, we investigated the effects of crude alkaloid (CAERS) and flavonoid (CFEZO) extracts prepared from medicinal herbs,Rhazya stricta and Zingiber officinale, respectively, on the growth of human GBM cell line, U251. R. stricta and Z. officinale are traditionally used in folkloric medicine and have antioxidant, anticarcinogenic, and free radical scavenging properties. Combination of CAERS and CFEZO treatments synergistically suppressed proliferation and colony formation and effectively induced morphological and biochemical features of apoptosis in U251 cells. Apoptosis induction was mediated by release of mitochondrial cytochrome c, increased Bax : Bcl-2 ratio, enhanced activities of caspase-3 and -9, and PARP-1 cleavage. CAERS and CFEZO treatments decreased expression levels of nuclear NF-κBp65, survivin, XIAP, and cyclin D1 and increased expression level of p53, p21, and Noxa. These results suggest that combination of CAERS and CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of GBM.


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## Body

## 1. Introduction

Glioblastoma multiforme (GBM) is the most common primary central nervous system neoplasm, accounting for more than half of all brain tumors, and is among the most lethal phenotypes of all cancers. Despite significant advances in molecular analysis and several promising therapies for GBM, the prognosis for patients with GBM remains poor [1]. The poor prognosis of those patients is owing to the intrinsic resistance of the GBM cells to apoptosis [2]. Therefore, induction of apoptosis in glioblastoma cells has come to be appreciated as targets for the management of GBM [3].Apoptosis is a highly sophisticated and elaborate mode of cell death that requires precise regulation of different intracellular signaling pathways to ensure the continuation of the transmission of the death signal. Apoptosis is tightly regulated by two opposing groups of hub proteins: death antagonists (Bcl-2, Bcl-XL, and Mcl-1) and death agonists (Bax and Bak and other proteins) [4]. The typical executioners of apoptosis are intracellular cysteine proteases called caspases, stored in most cells as zymogens or procaspases [5]. These caspases are activated by two pathways: the death receptor (extrinsic) and mitochondrial (intrinsic) pathways. A third less well-known initiation pathway is the intrinsic endoplasmic reticulum pathway [4]. The mitochondrial pathway initiates apoptosis in most physiological and pathological situations and is triggered by a variety of apoptotic stimuli, which converge at the mitochondria, leading to the release of cytochromec from the mitochondria to the cytoplasm. Both extrinsic and intrinsic pathways eventually converge on a common pathway, or the execution phase of apoptosis, activation of caspase-3 that provokes engagement of the effector caspases [4]. These latter caspases mediate cleavage of proteins that are essential for cell viability, resulting in morphological hallmarks of apoptosis. These include cytoplasm and chromatin condensation, nuclear breakdown, and shrinkage of the cell and fragmentation into membrane-bound apoptotic bodies, eventually subjected to rapid phagocytosis by surrounding cells [6].Recently, considerable attention has been focused on dietary and medicinal phytochemicals derived from natural sources, as a rich reservoir for discovery of novel anticancer drugs [7]. Nonetheless, dietary agents have relatively low potency compared with pharmacological compounds [8]. Furthermore, cancer is a complex disease, in which there is genetic variability among not only different types of cancer but also among different patients with the same type of cancer, and even among different cells within the same tumor [9]. Therefore, relying on a single dietary agent to target a distinct molecular target, for therapeutic purposes, might not be sufficient to elicit the desired outcome. In this regard, it might be possible to achieve additive or synergistic preventive effects and improve therapeutic index by combining dietary agents [10]. The underlying theory is that interactions among the chemical entities, present in different herbs in a formula, exert synergistic pharmacodynamic actions and neutralize the adverse effects and toxicities of specific individual chemicals. Indeed, considerable data indicate that combinations of dietary agents are more effective than a single agent [8]. Thus, optimization of combination chemotherapy based on molecular mechanism may improve therapeutic index, for the treatment of GBM patients.Rhazya stricta Decne (Harmal), a member of the Apocynaceae family, is an important medicinal species used in folkloric medicine to cure various diseases in South Asia and the Middle East [11, 12]. Extracts ofR. strictaleaves have been prescribed for the treatment of various disorders [11, 12]. It has been reported thatR. stricta is a good source of antioxidants [13]. We previously have reported that an aqueous extract ofR. stricta inhibited cell proliferation and induced apoptotic cell death in the breast cancer cell lines MCF-7 and MDA MB-231 [14]. Although some compounds have been identified fromR. stricta and their anticancer activities have been demonstrated [11, 12], new compounds and action mechanisms underlying their anticancer effects have been not fully studied. The herb is particularly rich in alkaloid, where over 100 alkaloids have been isolated, characterized, and identified from leaves, stems, roots, and legumes of the herb [11]. The fact thatR. stricta is an alkaloid-rich herb deserves attention for many reasons. First, alkaloids are among the most important active components in natural herbs, where several alkaloids, isolated from natural herbs, have been shown to exhibit antiproliferation and antimetastasis effects on various types of cancers bothin vitro andin vivo [15]. Seconds, other alkaloids, such as camptothecin [14] and vinca alkaloids (vincristine and vinblastine) isolated fromCatharanthus roseus (which, likeR. stricta, belongs to the Apocynaceae family) [16], have already been successfully developed into anticancer drugs. Likewise, indole alkaloids inR. stricta have been found to exhibit numerous biological activities such as antimicrobial and antihypertensive activities [17] and anticancer potentiality [11, 18]. Recently, we found that a crude alkaloid extract fromR. stricta inhibited cell growth and sensitizedhuman lung cancer cells, A549, to cisplatin through induction of apoptosis [19]. Finally, a recent study demonstrated that the active strongly basic alkaloid fraction inR. stricta induced the chemopreventative enzyme, Nqo1, which could be, at least in part, a novel mechanism for the traditional use ofR. stricta’s alkaloid as an antitumor agent [20]. Therefore, continued research into the action mechanisms of suchRhazya’s alkaloids will be necessary for credible assessments of the cancer chemopreventive qualities of the herb.Zingiber officinale Rosc. (Ginger), a member of the Zingiberaceae family, has been used in traditional oriental medicine for centuries to treat various gastrointestinal illnesses, arthritis, rheumatism, pain, muscle discomfort, various cardiovascular diseases, and metabolic diseases [21]. It is generally accepted that the bioactive molecules of the herb are 6-gingerol, flavonoids, and phenolic acids [22]. However, most researchers dealt with phenolic ingredients, such as gingerols and 6-shogaols, as anticancer bioactive compounds inZ. officinale and have paid little attention, if any, to flavonoids. Flavonoids display a wide range of pharmacological properties, such as antimicrobial, antiviral, anti-inflammatory, antiallergic, analgesic, antioxidant, and hepatoprotective activities [23]. Emerging evidence has also shown that many flavonoids have various biological activities, such as apoptosis induction, cell cycle arrest, antiangiogenesis, antioxidation, andin vitroandin vivo cancer chemopreventive and chemotherapeutic potentialities [23–25]. Flavonoids have been found to affect the overall process of carcinogenesis by several mechanisms. For example, they modulate activities of cyclin-Cdk4 regulators and ERK-MAP kinase to release cytochrome c with subsequent activation of caspase-9 and caspase-3, to increase levels of caspase-8, to downregulate expression of Bcl-2 and Bcl-xL, to enhance expression of Bax and Bak, and to modulate activity of nuclear factor-κB (reviewed in [23–25]). Despite these studies, the anticancer potentialities of flavonoids present inZ. officinale on GBM have never been reported yet.To our knowledge, previous studies have shown the chemopreventive effect ofZ. officinale orR. stricta in other cancer cells but no reported evidence on GBM cells. In the present study we have evaluated the antiproliferative potentialities and mechanisms of crude flavonoids and alkaloids isolated fromZ. officinale orR. stricta, respectively, and found to be highly effective in GBM cell line.


## 2. Materials and Methods

### 2.1. Preparation and Phytochemical Examination of Crude Flavonoid and Alkaloid Extracts

A crude alkaloid extract ofR. stricta leaves was prepared essentially as described elsewhere [19]. Briefly, air-dried leaves ofR. stricta (350 g) were soaked in 80% methanol (1 L) at ambient temperature for seven days after which the methanolic extract was evaporated in a rotatory evaporator and the remaining residue was suspended in water and filtered. The aqueous extract was then acidified with 10% glacial acetic acid and extracted with chloroform. This chloroform fraction contained weakly basic alkaloids and neutral compounds. The remaining aqueous solution was alkalinized using NaOH and the pH was adjusted to 11. The alkaline aqueous layer was extracted with chloroform to yield a chloroform fraction enriched in strongly basic alkaloids. The chloroform layer was evaporated to dryness to obtain a crude extract of alkaloids. Before use, the stock was further diluted in DMSO to give the final indicated concentrations and termed as crude alkaloid extract ofR. stricta (CAERS).For preparation of crude flavonoid extract fromZ. officinale, a rhizome of the herb was purchased from local market and powdered. The dried powder was extracted by cold percolation with 70% (2 L) ethanol for 72 h at room temperature and then filtered. The extraction was repeated twice. The combined filtrates were concentrated in a vacuum evaporator to afford a syrupy brown residue. This residue was suspended in 250 mL hot water (60°C), for h, filtered, and defatted by using petroleum ether (250 mL × 3). The aqueous portion was then separated, collected, and fractionated with N-butanol saturated water (250 mL × 3). The aqueous portion was discarded and the N-butanol portion was then separated, collected before being fractionated with 1% KOH. The KOH portion was then fractionated with dilute HCl (2%) and N-butanol saturated water. The dilute HCl portion was discarded. The N-butanol portion was then separated, collected, and dried to obtain a crude extract of flavonoids. Before use, the stock was further diluted in DMSO to give the final indicated concentrations and termed as crude flavonoid extract ofZ. officinale (CFEZO).Phytochemical examination for testing the presence of alkaloids in CAERS was carried out using Dragendorff’s test and Mayer’s test. Few quantity of each portion was stirred with 5 mL of 1% aqueous HCl on water bath and then filtered. Of the filtrate, 1 mL was taken individually into 2 test tubes. To the first portion, few drops of Dragendorff’s reagent (solution of potassium bismuth iodide) were added; occurrence of orange-red precipitate was taken as positive. To the second 1 mL, Mayer’s reagent (potassium mercuric iodide) was forming a buff-colored precipitate indicating the presence of alkaloids [26].Phytochemical examination for testing the presence of flavonoids in CFEZO was carried out using sodium hydroxide test. Few quantity of the each portion was dissolved in water and filtered; to this 2 mL of the 10% aqueous sodium hydroxide was later added to produce a yellow colouration. A change in colour from yellow to colourless on addition of dilute hydrochloric acid was an indication of the presence of flavonoids [26].


### 2.2. Cell Culture

The human GBM, cancer breast (MCF-7), cervical carcinoma (HeLa), and nonmalignant human foreskin fibroblast (HF-5) cell lines were obtained from King Fahd Center for Medical Research, King Abdulaziz University, Saudi Arabia. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Promega) containing 10% fetal bovine serum (FBS, Promega) and 1% penicillin-streptomycin antibiotics (Promega) in tissue culture flasks under a humidifying atmosphere containing 5% CO2 and 95% air at 37°C. The cells were subcultured at 3-4-day interval.


### 2.3. Cell Viability and Clonogenic and Soft Agar Colony-Forming Assays

Cell viability and the effects of CAERS and/or CFEZO on the growth of U251 cells were assessed by tetrazolium salt WST-1 kit (Cayman Chemicals, USA) following the manufacturer’s instructions. Briefly, U251 cells were seeded, at a density of 104/well in 96 wells plate for 24 h; then cells were treated with the indicated concentrations of the extracts and incubated for 24, 48, or 72 h. After the specified time of treatment, 10 μL of freshly prepared WST-1 solution was added to each well. Culture medium and WST-1/solution were added in an empty well as a blank for the microtiter plate reader. The absorbance of the treated and untreated samples was measured after 2 hours by (BioTek Synergy HT, USA) micro plate reader, at 450 nm with a reference wavelength 630 nm to avoid the interference of cell layer absorbance that blocks light passing through. In all experiments, the effects of CAERS and/or CFEZO on growth inhibition were assessed as percent cell viability, where nontreated cells were taken as 100% viable. For these studies, all experiments were repeated three or more times in triplicate.For clonogenic assay, approximately 500 cells were seeded into six-well plates in triplicate and allowed to adhere overnight. Thereafter, cell culture media were changed and cells were treated with indicated concentrations of CAERS and/or CFEZO extract(s). The cells were allowed to incubate at 37°C in the incubator undisturbed for 15 days. During this period each individual surviving cell would proliferate and form colonies. On day 15, the colonies were washed with cold phosphate buffer saline and fixed with −20°C cold methanol before being stained with 0.1% Coomassie Brilliant Blue. The colonies that had >50 cells/colony were counted and expressed as percent control.For soft agar colony-forming assay, U251 cells were seeded at 5,000 cells per well in 0.35% top agarose (Promega) with a base agarose of 0.7% agarose supplemented with complete medium. Cultures were treated with indicated concentrations of CAERS and/or CFEZO extract(s) and incubated in a humidified incubator at 37°C for 3 weeks. Cells were then stained with 0.5 mL of 0.0005% crystal violet, and colonies were counted visually. All experiments were done in triplicate with two independent experiments.


### 2.4. Assessment of Cell Morphological Changes

The nuclear morphological changes associated with apoptosis were analyzed using DAPI staining. Briefly, cells (2
×
10
4) were plated on coverslips, allowed to attach overnight, and exposed to indicated concentrations of the CAERS and/or CFEZO extract(s) for 48 h. The cells were washed with PBS and fixed with 3.7% paraformaldehyde (Sigma-Aldrich, USA) in PBS for 10 min at room temperature. Fixed cells were washed with PBS and stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) solution for 10 min at room temperature. The cells were washed two more times with PBS and analyzed. The cells with condensed and fragmented DNA (apoptotic cells) were scored under a fluorescence microscope (Carl Zeiss, Germany) using the magnification indicated in the figures.


### 2.5. Photomicrograph Images

Cells (2
×
10
4) were plated on coverslips, allowed to attach overnight, and exposed to indicated concentrations of the CAERS and/or CFEZO extract(s) for 48 h. After incubation, cells were trypsinized and centrifuged for 5 min at room temperature. Then, the supernatant was decanted and pellets were dried and fixed with 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Then, fixed samples were washed in 0.1 M sodium cacodylate buffer (pH 7.2) and postfixed in 1% osmium tetroxide in the same buffer. The cells were dehydrated in a graded ethanol series and propylene oxide. The resin infiltration was performed with a 1 : 1 mixture of propylene oxide and epon for 5 h, followed by 100% epon for another 5 h. Next, the material was embedded, followed by 48 h of polymerization. Thin sections were produced using an ultramicrotome (LEICA EM UC6) and these were stained with toluidine blue.


### 2.6. Flow Cytometer

The ability of CAERS and/or CFEZO treatments to induce apoptosis in U251 cells was determined using an Annexin V-FITC Apoptosis Detection Kit (Sigma), according to manufacturer’s instructions. Briefly, U251 cells were seeded in 6-well plate (20
×
10
4 cells/well) treated with the indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h, harvested, washed in PBS, centrifugated at 12,500 ×g for 5 min, and resuspended in binding buffer. Then, 5 μL of Annexin V-FITC conjugate and 10 μL of propidium iodide (PI) solutions were added to the cell suspension and the cells were incubated at room temperature for 10 minutes protected from light. The FITC/PI fluorescence intensity was measured by flow cytometry to differentiate between viable (annexin V-negative and PI-negative), early apoptotic (annexin V-positive, PI-negative), and late apoptotic (annexin V-positive and PI-positive) cells. The extent of apoptosis was quantified as percentage of annexin V-positive cells.


### 2.7. DNA Fragmentation Assay

DNA gel electrophoresis was used to determine the presence of internucleosomal DNA cleavage as described previously [23]. Briefly, U251 cells (3
×
10
6 cells/100 mm dish) treated with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h were collected, washed in PBS, and centrifugated at 12,500 ×g for 5 min. Cell pellets were then lysed in 600 μL lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100), kept on ice for 30 min, and centrifuged at 12,500 ×g for 20 min. The supernatant from the lysate was treated with 2 μL RNase A (20 mg/mL) at 37°C for 1 h, followed by proteinase K digestion, 2 μL proteinase K (20 mg/mL) at 56°C for 15 min and then phenol chloroform extraction and isopropanol precipitation. After centrifugation at 12,500 ×g for 20 min, the DNA pellets were dissolved in 20 μL TE buffer (10 mM Tris (pH 7.4) and 1 mM EDTA (pH 8.0)) and the concentration of DNA was determined spectroscopically. Then, DNA was resolved by electrophoresis on 1.5% agarose gel at 80 100 V, stained with ethidium bromide, and visualized by a UV transilluminator (BIO-RAD).


### 2.8. Single-Cell Gel Electrophoresis (Comet Assay)

CAERS and/or CFEZO-induced DNA damage was determined using the comet assay. Cells were treated with 25 and 50μg/mL EENS for 24 h in complete medium, and the comet assay was done as described earlier [27]. Briefly, after treatment with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h, the cells were harvested and resuspended in ice-cold PBS. Approximately, 10 × 103 cells in a volume of 75 μL of 0.5% (w/v) low-melting-point agarose were pipetted onto a frosted glass slide coated with a thin layer of 1.0% (w/v) agarose, covered with a coverslip, and allowed to set on ice for 10 min. Following removal of the coverslip, the slides were immersed in ice cold lysis buffer containing 2.5 mol/L NaCl, 10 mmol/L Tris, 100 mmol/L Na2-EDTA, and 1% (w/v) N-lauroylsarcosine, adjusted to pH 10.0, and 1.0% Triton X-100 was added immediately before use. After 2 h at 4°C, the slides were placed into a horizontal electrophoresis tank filled with buffer (0.3 mol/L NaOH, 1 mmol/L EDTA (pH 13)) and subjected to electrophoresis for 30 min at 300 mA. Slides were transferred to neutralization buffer (0.4 mol/L Tris-HCl) for 3 to 5 min washes and stained with ethidium bromide for 5 min. After a final wash in double-distilled water, the gels were covered with glass coverslips. To prevent additional DNA damage from visible light, all the steps described above were conducted under a dimmed light. Slides were viewed and nuclei images were visualized and captured at 400x magnifications with an Axioplan 2 fluorescence microscope (Zeiss) equipped with a CCD camera (Optronics). Hundreds of cells were scored to calculate the overall percentage of comet tail-positive cells.


### 2.9. RNA Extraction and qRT-PCR

Cells were seeded (250 × 103/well) onto 6-well plates and treated with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h. After this period, floating and adherent cells were collected (with care being taken that none of the floating cells were lost during washes) and pelleted by centrifugation (700 g, 5 min). RNA extraction and reverse transcriptase-PCR were done as previously described [19]. Briefly, total RNA was extracted using SV Total RNA Isolation System (Promega) before being reverse-transcribed and amplified by PCR using GoTaqR 1-Step RT-qPCR System (Promega) according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was done using gene-specific primers. The primer sequences for Bcl-2, survivin (BIRC5), Noxa, XIAP, and GAPDH were described earlier [27]. Amplification products obtained by PCR were separated electrophoretically on 1% agarose gels and visualized by ethidium bromide (0.5 μg/mL) staining.


### 2.10. Preparation of Mitochondrial Cytosolic Extracts and Nuclear Extracts

To detect cytc release by western immunoblotting, mitochondrial and cytosolic extracts were obtained as described previously [19]. Briefly, cells were seeded (250
×
10
3/well) onto 6-well plates, treated with the indicated concentrations of CAERS and/or CFEZO extract(s), and incubated for 24 h. After this incubation, the cells were collected by centrifugation, washed twice with cold PBS, and resuspended in 500 μL of ice-cold cytosol extraction buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) containing a protease inhibitor cocktail (1 mM PMSF, 1% aprotinin, 1 mM leupeptin, and 1 μg of pepstatin A/mL). After 30 min incubation on ice, the cells were homogenized in the same buffer using a dounce homogenizer (30 strokes) and centrifuged (1000 ×g, 10 min, 4°C). The supernatant was collected and centrifuged again (14,000 ×g, 30 min) to collect the mitochondria-rich (pellet) and cytosolic (supernatant) fractions. The supernatant was used as cytosolic lysate while the pellet was suspended in lysis buffer (137 mM NaCl, 20 mM Tris, pH 7.9, 10 mM NaF, 5 mM EDTA, 1 mM EGTA, 10% (v/v) glycerol, and 1% Triton X-100) supplemented with a protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem) before being centrifuged to obtain the mitochondrial lysate. Proteins concentrations were determined with a BCA protein assay kit (Pierce) and equal amounts of protein fractions were subjected for further analyses as described below. For preparation of nuclear extracts, treated cells were collected by centrifugation and washed twice with cold PBS, and nuclear extract was prepared using Pierce BCA Protein Assay Kit (Thermo Scientific) following the manufacture’s procedure.


### 2.11. Western Blot Analysis

The western blot analyses were carried out as detailed previously [19]. Briefly, cells were seeded (250 × 103/well) onto 6-well plates, treated with the indicated concentrations of CAERS and/or CFEZO extract(s), and incubated for 24 h. The cells were washed three times with PBS and lysed in cold lysis buffer containing 0.05 mmol/L Tris-HCl, 0.15 mmol/L NaCl, 1 mol/L EGTA, 1 mol/L EDTA, 20 mmol/L NaF, 100 mmol/L Na3VO4, 0.5% NP40, 1% Triton X-100, and 1 mol/L phenylmethylsulfonyl fluoride (pH 7.4) with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem). The lysates were collected and cleared by centrifugation, and the supernatants were aliquoted and stored at −80°C. The protein contents in the lysates were measured by BCA protein assay (Pierce, Rockford, IL, USA), as per the manufacturer’s protocol. Western immunoblotting was done essentially as described elsewhere [28]. Briefly, aliquots of the lysates containing the same quantity of proteins were boiled for 5 min in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 5% β-mercaptoethanol, electrophoresed on 10% SDS-PAGE, and transferred to PVDF membranes. After transfer, the membranes were incubated with primary antibody against tested proteins, followed by incubation with a secondary horseradish peroxidase-conjugated antibody. Antibodies were purchased from Sigma. The membranes were developed by the enhanced chemiluminescence (ECL) detection kit (Amersham). In all experiments, the blots were stripped with stripping buffer (62.5 mM Tris, pH 6.7, 2% SDS, and 90 mM 2-mercaptoethanol) and reprobed with anti-β-actin (Spring Bioscience) antibody as a control for protein loading. Signals were detected with an enhanced chemiluminescence detection kit (Amersham).


### 2.12. Statistical Analyses

All experiments were done at least three times independently and in triplicate. The results were presented as mean ± standard deviation (SD) for continuous variables. Differences between samples were analyzed with by one-way analysis of variance (ANOVA), followed by post hoc test using SPSS16.0 software (Fiddler L, Hecht L, Nelson EE, Nelson EN, Ross J. SPSS for Windows 16.0: A Basic Tutorial. Social Science Research and Instruction Center. California State University. Accessed 25/8/2011. Available athttp://www.ssric.org/trd/spss16). Results with a P value <0.05 were considered statistically significant. Univariate analyses with linear regression model were used to estimate the probabilities of suppression of malignant cells. Standardized coefficients (Beta) and their 95% CIs were computed for variables in the model.


## 2.1. Preparation and Phytochemical Examination of Crude Flavonoid and Alkaloid Extracts

A crude alkaloid extract ofR. stricta leaves was prepared essentially as described elsewhere [19]. Briefly, air-dried leaves ofR. stricta (350 g) were soaked in 80% methanol (1 L) at ambient temperature for seven days after which the methanolic extract was evaporated in a rotatory evaporator and the remaining residue was suspended in water and filtered. The aqueous extract was then acidified with 10% glacial acetic acid and extracted with chloroform. This chloroform fraction contained weakly basic alkaloids and neutral compounds. The remaining aqueous solution was alkalinized using NaOH and the pH was adjusted to 11. The alkaline aqueous layer was extracted with chloroform to yield a chloroform fraction enriched in strongly basic alkaloids. The chloroform layer was evaporated to dryness to obtain a crude extract of alkaloids. Before use, the stock was further diluted in DMSO to give the final indicated concentrations and termed as crude alkaloid extract ofR. stricta (CAERS).For preparation of crude flavonoid extract fromZ. officinale, a rhizome of the herb was purchased from local market and powdered. The dried powder was extracted by cold percolation with 70% (2 L) ethanol for 72 h at room temperature and then filtered. The extraction was repeated twice. The combined filtrates were concentrated in a vacuum evaporator to afford a syrupy brown residue. This residue was suspended in 250 mL hot water (60°C), for h, filtered, and defatted by using petroleum ether (250 mL × 3). The aqueous portion was then separated, collected, and fractionated with N-butanol saturated water (250 mL × 3). The aqueous portion was discarded and the N-butanol portion was then separated, collected before being fractionated with 1% KOH. The KOH portion was then fractionated with dilute HCl (2%) and N-butanol saturated water. The dilute HCl portion was discarded. The N-butanol portion was then separated, collected, and dried to obtain a crude extract of flavonoids. Before use, the stock was further diluted in DMSO to give the final indicated concentrations and termed as crude flavonoid extract ofZ. officinale (CFEZO).Phytochemical examination for testing the presence of alkaloids in CAERS was carried out using Dragendorff’s test and Mayer’s test. Few quantity of each portion was stirred with 5 mL of 1% aqueous HCl on water bath and then filtered. Of the filtrate, 1 mL was taken individually into 2 test tubes. To the first portion, few drops of Dragendorff’s reagent (solution of potassium bismuth iodide) were added; occurrence of orange-red precipitate was taken as positive. To the second 1 mL, Mayer’s reagent (potassium mercuric iodide) was forming a buff-colored precipitate indicating the presence of alkaloids [26].Phytochemical examination for testing the presence of flavonoids in CFEZO was carried out using sodium hydroxide test. Few quantity of the each portion was dissolved in water and filtered; to this 2 mL of the 10% aqueous sodium hydroxide was later added to produce a yellow colouration. A change in colour from yellow to colourless on addition of dilute hydrochloric acid was an indication of the presence of flavonoids [26].


## 2.2. Cell Culture

The human GBM, cancer breast (MCF-7), cervical carcinoma (HeLa), and nonmalignant human foreskin fibroblast (HF-5) cell lines were obtained from King Fahd Center for Medical Research, King Abdulaziz University, Saudi Arabia. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Promega) containing 10% fetal bovine serum (FBS, Promega) and 1% penicillin-streptomycin antibiotics (Promega) in tissue culture flasks under a humidifying atmosphere containing 5% CO2 and 95% air at 37°C. The cells were subcultured at 3-4-day interval.


## 2.3. Cell Viability and Clonogenic and Soft Agar Colony-Forming Assays

Cell viability and the effects of CAERS and/or CFEZO on the growth of U251 cells were assessed by tetrazolium salt WST-1 kit (Cayman Chemicals, USA) following the manufacturer’s instructions. Briefly, U251 cells were seeded, at a density of 104/well in 96 wells plate for 24 h; then cells were treated with the indicated concentrations of the extracts and incubated for 24, 48, or 72 h. After the specified time of treatment, 10 μL of freshly prepared WST-1 solution was added to each well. Culture medium and WST-1/solution were added in an empty well as a blank for the microtiter plate reader. The absorbance of the treated and untreated samples was measured after 2 hours by (BioTek Synergy HT, USA) micro plate reader, at 450 nm with a reference wavelength 630 nm to avoid the interference of cell layer absorbance that blocks light passing through. In all experiments, the effects of CAERS and/or CFEZO on growth inhibition were assessed as percent cell viability, where nontreated cells were taken as 100% viable. For these studies, all experiments were repeated three or more times in triplicate.For clonogenic assay, approximately 500 cells were seeded into six-well plates in triplicate and allowed to adhere overnight. Thereafter, cell culture media were changed and cells were treated with indicated concentrations of CAERS and/or CFEZO extract(s). The cells were allowed to incubate at 37°C in the incubator undisturbed for 15 days. During this period each individual surviving cell would proliferate and form colonies. On day 15, the colonies were washed with cold phosphate buffer saline and fixed with −20°C cold methanol before being stained with 0.1% Coomassie Brilliant Blue. The colonies that had >50 cells/colony were counted and expressed as percent control.For soft agar colony-forming assay, U251 cells were seeded at 5,000 cells per well in 0.35% top agarose (Promega) with a base agarose of 0.7% agarose supplemented with complete medium. Cultures were treated with indicated concentrations of CAERS and/or CFEZO extract(s) and incubated in a humidified incubator at 37°C for 3 weeks. Cells were then stained with 0.5 mL of 0.0005% crystal violet, and colonies were counted visually. All experiments were done in triplicate with two independent experiments.


## 2.4. Assessment of Cell Morphological Changes

The nuclear morphological changes associated with apoptosis were analyzed using DAPI staining. Briefly, cells (2
×
10
4) were plated on coverslips, allowed to attach overnight, and exposed to indicated concentrations of the CAERS and/or CFEZO extract(s) for 48 h. The cells were washed with PBS and fixed with 3.7% paraformaldehyde (Sigma-Aldrich, USA) in PBS for 10 min at room temperature. Fixed cells were washed with PBS and stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) solution for 10 min at room temperature. The cells were washed two more times with PBS and analyzed. The cells with condensed and fragmented DNA (apoptotic cells) were scored under a fluorescence microscope (Carl Zeiss, Germany) using the magnification indicated in the figures.


## 2.5. Photomicrograph Images

Cells (2
×
10
4) were plated on coverslips, allowed to attach overnight, and exposed to indicated concentrations of the CAERS and/or CFEZO extract(s) for 48 h. After incubation, cells were trypsinized and centrifuged for 5 min at room temperature. Then, the supernatant was decanted and pellets were dried and fixed with 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Then, fixed samples were washed in 0.1 M sodium cacodylate buffer (pH 7.2) and postfixed in 1% osmium tetroxide in the same buffer. The cells were dehydrated in a graded ethanol series and propylene oxide. The resin infiltration was performed with a 1 : 1 mixture of propylene oxide and epon for 5 h, followed by 100% epon for another 5 h. Next, the material was embedded, followed by 48 h of polymerization. Thin sections were produced using an ultramicrotome (LEICA EM UC6) and these were stained with toluidine blue.


## 2.6. Flow Cytometer

The ability of CAERS and/or CFEZO treatments to induce apoptosis in U251 cells was determined using an Annexin V-FITC Apoptosis Detection Kit (Sigma), according to manufacturer’s instructions. Briefly, U251 cells were seeded in 6-well plate (20
×
10
4 cells/well) treated with the indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h, harvested, washed in PBS, centrifugated at 12,500 ×g for 5 min, and resuspended in binding buffer. Then, 5 μL of Annexin V-FITC conjugate and 10 μL of propidium iodide (PI) solutions were added to the cell suspension and the cells were incubated at room temperature for 10 minutes protected from light. The FITC/PI fluorescence intensity was measured by flow cytometry to differentiate between viable (annexin V-negative and PI-negative), early apoptotic (annexin V-positive, PI-negative), and late apoptotic (annexin V-positive and PI-positive) cells. The extent of apoptosis was quantified as percentage of annexin V-positive cells.


## 2.7. DNA Fragmentation Assay

DNA gel electrophoresis was used to determine the presence of internucleosomal DNA cleavage as described previously [23]. Briefly, U251 cells (3
×
10
6 cells/100 mm dish) treated with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h were collected, washed in PBS, and centrifugated at 12,500 ×g for 5 min. Cell pellets were then lysed in 600 μL lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100), kept on ice for 30 min, and centrifuged at 12,500 ×g for 20 min. The supernatant from the lysate was treated with 2 μL RNase A (20 mg/mL) at 37°C for 1 h, followed by proteinase K digestion, 2 μL proteinase K (20 mg/mL) at 56°C for 15 min and then phenol chloroform extraction and isopropanol precipitation. After centrifugation at 12,500 ×g for 20 min, the DNA pellets were dissolved in 20 μL TE buffer (10 mM Tris (pH 7.4) and 1 mM EDTA (pH 8.0)) and the concentration of DNA was determined spectroscopically. Then, DNA was resolved by electrophoresis on 1.5% agarose gel at 80 100 V, stained with ethidium bromide, and visualized by a UV transilluminator (BIO-RAD).


## 2.8. Single-Cell Gel Electrophoresis (Comet Assay)

CAERS and/or CFEZO-induced DNA damage was determined using the comet assay. Cells were treated with 25 and 50μg/mL EENS for 24 h in complete medium, and the comet assay was done as described earlier [27]. Briefly, after treatment with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h, the cells were harvested and resuspended in ice-cold PBS. Approximately, 10 × 103 cells in a volume of 75 μL of 0.5% (w/v) low-melting-point agarose were pipetted onto a frosted glass slide coated with a thin layer of 1.0% (w/v) agarose, covered with a coverslip, and allowed to set on ice for 10 min. Following removal of the coverslip, the slides were immersed in ice cold lysis buffer containing 2.5 mol/L NaCl, 10 mmol/L Tris, 100 mmol/L Na2-EDTA, and 1% (w/v) N-lauroylsarcosine, adjusted to pH 10.0, and 1.0% Triton X-100 was added immediately before use. After 2 h at 4°C, the slides were placed into a horizontal electrophoresis tank filled with buffer (0.3 mol/L NaOH, 1 mmol/L EDTA (pH 13)) and subjected to electrophoresis for 30 min at 300 mA. Slides were transferred to neutralization buffer (0.4 mol/L Tris-HCl) for 3 to 5 min washes and stained with ethidium bromide for 5 min. After a final wash in double-distilled water, the gels were covered with glass coverslips. To prevent additional DNA damage from visible light, all the steps described above were conducted under a dimmed light. Slides were viewed and nuclei images were visualized and captured at 400x magnifications with an Axioplan 2 fluorescence microscope (Zeiss) equipped with a CCD camera (Optronics). Hundreds of cells were scored to calculate the overall percentage of comet tail-positive cells.


## 2.9. RNA Extraction and qRT-PCR

Cells were seeded (250 × 103/well) onto 6-well plates and treated with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h. After this period, floating and adherent cells were collected (with care being taken that none of the floating cells were lost during washes) and pelleted by centrifugation (700 g, 5 min). RNA extraction and reverse transcriptase-PCR were done as previously described [19]. Briefly, total RNA was extracted using SV Total RNA Isolation System (Promega) before being reverse-transcribed and amplified by PCR using GoTaqR 1-Step RT-qPCR System (Promega) according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was done using gene-specific primers. The primer sequences for Bcl-2, survivin (BIRC5), Noxa, XIAP, and GAPDH were described earlier [27]. Amplification products obtained by PCR were separated electrophoretically on 1% agarose gels and visualized by ethidium bromide (0.5 μg/mL) staining.


## 2.10. Preparation of Mitochondrial Cytosolic Extracts and Nuclear Extracts

To detect cytc release by western immunoblotting, mitochondrial and cytosolic extracts were obtained as described previously [19]. Briefly, cells were seeded (250
×
10
3/well) onto 6-well plates, treated with the indicated concentrations of CAERS and/or CFEZO extract(s), and incubated for 24 h. After this incubation, the cells were collected by centrifugation, washed twice with cold PBS, and resuspended in 500 μL of ice-cold cytosol extraction buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) containing a protease inhibitor cocktail (1 mM PMSF, 1% aprotinin, 1 mM leupeptin, and 1 μg of pepstatin A/mL). After 30 min incubation on ice, the cells were homogenized in the same buffer using a dounce homogenizer (30 strokes) and centrifuged (1000 ×g, 10 min, 4°C). The supernatant was collected and centrifuged again (14,000 ×g, 30 min) to collect the mitochondria-rich (pellet) and cytosolic (supernatant) fractions. The supernatant was used as cytosolic lysate while the pellet was suspended in lysis buffer (137 mM NaCl, 20 mM Tris, pH 7.9, 10 mM NaF, 5 mM EDTA, 1 mM EGTA, 10% (v/v) glycerol, and 1% Triton X-100) supplemented with a protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem) before being centrifuged to obtain the mitochondrial lysate. Proteins concentrations were determined with a BCA protein assay kit (Pierce) and equal amounts of protein fractions were subjected for further analyses as described below. For preparation of nuclear extracts, treated cells were collected by centrifugation and washed twice with cold PBS, and nuclear extract was prepared using Pierce BCA Protein Assay Kit (Thermo Scientific) following the manufacture’s procedure.


## 2.11. Western Blot Analysis

The western blot analyses were carried out as detailed previously [19]. Briefly, cells were seeded (250 × 103/well) onto 6-well plates, treated with the indicated concentrations of CAERS and/or CFEZO extract(s), and incubated for 24 h. The cells were washed three times with PBS and lysed in cold lysis buffer containing 0.05 mmol/L Tris-HCl, 0.15 mmol/L NaCl, 1 mol/L EGTA, 1 mol/L EDTA, 20 mmol/L NaF, 100 mmol/L Na3VO4, 0.5% NP40, 1% Triton X-100, and 1 mol/L phenylmethylsulfonyl fluoride (pH 7.4) with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem). The lysates were collected and cleared by centrifugation, and the supernatants were aliquoted and stored at −80°C. The protein contents in the lysates were measured by BCA protein assay (Pierce, Rockford, IL, USA), as per the manufacturer’s protocol. Western immunoblotting was done essentially as described elsewhere [28]. Briefly, aliquots of the lysates containing the same quantity of proteins were boiled for 5 min in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 5% β-mercaptoethanol, electrophoresed on 10% SDS-PAGE, and transferred to PVDF membranes. After transfer, the membranes were incubated with primary antibody against tested proteins, followed by incubation with a secondary horseradish peroxidase-conjugated antibody. Antibodies were purchased from Sigma. The membranes were developed by the enhanced chemiluminescence (ECL) detection kit (Amersham). In all experiments, the blots were stripped with stripping buffer (62.5 mM Tris, pH 6.7, 2% SDS, and 90 mM 2-mercaptoethanol) and reprobed with anti-β-actin (Spring Bioscience) antibody as a control for protein loading. Signals were detected with an enhanced chemiluminescence detection kit (Amersham).


## 2.12. Statistical Analyses

All experiments were done at least three times independently and in triplicate. The results were presented as mean ± standard deviation (SD) for continuous variables. Differences between samples were analyzed with by one-way analysis of variance (ANOVA), followed by post hoc test using SPSS16.0 software (Fiddler L, Hecht L, Nelson EE, Nelson EN, Ross J. SPSS for Windows 16.0: A Basic Tutorial. Social Science Research and Instruction Center. California State University. Accessed 25/8/2011. Available athttp://www.ssric.org/trd/spss16). Results with a P value <0.05 were considered statistically significant. Univariate analyses with linear regression model were used to estimate the probabilities of suppression of malignant cells. Standardized coefficients (Beta) and their 95% CIs were computed for variables in the model.


## 3. Results

### 3.1. Cytostatic Effects of CAERS and CFEZO

Initially, we examined the sensitivity of U251 cells to different doses of both extracts. So, cells were treated with increasing concentrations of CAERS (0, 50, 100, 150, and 200μg/mL) or CFEZO (0, 50, 100, 150, and 200 μg/mL) for 24, 48, and 72 h. The cytotoxicity assessed by WST-1 kit showed that CAERS or CFEZO efficiently inhibited cell viability in dose- and time-dependent manners (Figure 1(a)). Calculated IC50 values (IC50, the concentration of test compound that inhibits 50% of the cell growth) for CAERS were 200, 135, and 80 μg/mL after 24, 48, and 72 h, respectively, and for CFEZO were 150, 115, and 75 μg/mL after 24, 48, and 72 and 150 μg/mL, respectively. To find whether CAERS and CFEZO have synergistic antiproliferative potentialities, U251 cells were incubated with increasing doses of CAERS (0, 10, 20, 40, and 50 μg/mL) and CFEZO (0, 10, 20, 40, and 50 μg/mL) in combination. As shown in Figure 1(a) combination of CAERS and CFEZO worked synergistically to inhibit viability of U251 cells. Notably, lower IC50 value indicated higher synergistic effect of the combination of extracts; this because IC50 values after 24, 48, and 72 h of combined treatments were 40, 20, and 15 μg/mL. Although we found a lower IC50 value of combination of 40 μg/mL CAERS and 40 μg/mL CFEZO than combination of 20 μg/mL CAERS and 20 μg/mL CFEZO (Figure 1), we did not want to continue with the combination of 40 μg/mL CAERS and 40 μg/mL CFEZO that showed too much cytotoxicity (necrotic cells) as we found in thein situ Wright staining (data not shown). Therefore, we selected 150 μg/mL CAERS and 100 μg/mL CFEZO for single treatment and 20 μg/mL CAERS and 20 μg/mL CFEZO for combined treatment in all subsequent experiments.Combination of CAERS and CFEZO acted synergistically to inhibit cell proliferation and colony formation in U251 cells. The U251 cells were seeded, at a density of 104/well in 96-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for displayed time intervals. The inhibition of cell proliferation was assessed by the tetrazolium salt WST-1 kit as detailed in Section 2. The experiments were repeated five times in triplicate, and cell viabilities at each dose of extract(s) were expressed in terms of percent of control and reported as the mean ± SD. (b) Left histogram: multiple comparisons for effect of different doses’ categories on U251 cells using post hoc test (Dunnett T3). All reportedP values are 2-tailed; **correlation is significant at the 0.01 level (2-tailed); ***correlation is significant at the 0.001 level (2-tailed);a= significant differences compared to control;b = significant differences compared to the lowest dose, 10 μg/mL. Compared to control, doses in 10, 20, 40, and 50 μg/mL showed a significantly lower percentage of viable U251 cells (P
=
0.002**, P
=
0.000***, P
=
0.000***, and P
=
0.000***, resp.). We observed another significant variation between dose 10 μg/mL and doses 40 and 50 μg/mL, with a stronger suppression of GBM cells with the two higher doses (40 and 50 μg/mL) (P
=
0.008**, and P
=
0.001***, resp.). Right histogram: multiple comparisons for effect of different durations on U251 viable cells using post hoc test (LSD). All reportedP values are 2-tailed. *Correlation is significant at the 0.05 level (2-tailed). Compared to one-day duration, three-day duration of treatment showed a significantly lower percentage of viable U251 cells (P
=
0.02*). The table (on the right) shows regression of dose and duration with percent U251 viable cells. A multiple linear regression analysis was used (Enter Selection Procedure).***Correlation is significant at 0.001 level (2-tailed). Dose had a stronger negative impact than that of duration, in predicting the % U251 viable cells, (Beta = −0.84*** andBeta = −0.34*** resp.). TheR
2 indicates that 81.3% of the variation in % U251 viable cells could be explained by these two variables (dose and duration). (c) The MCF-7, HeLa, and HF-5 cell lines were seeded and treated with the indicated concentrations of CAERS and/or CFEZO for displayed time intervals. The experiments were repeated five times in triplicate, and cell viabilities at each dose of extract(s) were expressed in terms of percent of control and reported as the mean ± SD. (d) U251 cells were seeded onto a 6-well plate at 1000 cells/well and treated with the indicated concentrations of CAERS and/or CFEZO as detailed in Section 2. The colonies were counted under a dissection microscope and the experiment was repeated three times. (e) CAERS and/or CFEZO acted synergistically to inhibit anchorage-independent growth in U251 cells in growth in soft agarose assays. U251 cells were plated, in triplicate, in 0.35% soft agarose and treated with CAERS (25 μg/mL) or CFEZO (25 μg/mL) and a combination of CAERS (5 μg/mL) and CFEZO (5 μg/mL). After 2 weeks, the colonies were stained with 0.0005% crystal violet and photographed using a digital camera coupled to a Carl Zeiss inverted microscope. Representative images of colonies in soft agar are shown.
(a)
(b)
(c)
(d)
(e)To find out whether the antiproliferative potentialities of CAERS and CFEZO are restricted to U251 cells, the above experiments were repeated using human breast and cervical carcinoma cell lines, MCF-7 and HeLa, respectively, as models. The findings depicted in Figure1(b) demonstrate combined treatments of CAERS and CFEZO consistently retaining their growth-inhibitory potentialities in the contexts of MCF-7 and HeLa cells, in dose- and time-dependent manners. These observations indicated that antiproliferative potentiality of CAERS and CFEZO treatments is not cell-type-specific. Finally, we assessed the effects of CAERS and CFEZO treatments on viability of nonmalignant human foreskin fibroblasts, HF-5 cells. We noticed that the HF-5 cell line was significantly more resistant to growth inhibition by the CAERS or CFEZO (Figure 1(b)), suggesting that CAERS and CFEZO selectivity target transformed cells. Taken together, these results indicated that the single and combined treatments of CAERS and CFEZO obviously inhibited the growth of U251 cells.Having established growth inhibiting potentialities of CAERS and CFEZO in the U251 cells, we next determined the effects of CAERS and CFEZO on colony formation (also referred to as clonogenicity) in U251 cells. This assay measures the ability of tumor cells to grow and form foci in a manner unrestricted by growth contact inhibition as is characteristically found in normal, untransformed cells. As such, clonogenicity provides an indirect estimation of the tendency of tumor cells to undergo neoplastic transformation. To measure clonogenicity, U251 cells at a given cell density were plated onto multiple well tissue culture dishes, with and without addition of increasingdoses of CAERS or CFEZO. Control and treated cells were maintained in culture for an additional 14 days to allow formation of colonies. Size and number of colonies were visually inspected by fixing and staining in 0.1% Coomassie Blue. Figure1(c) shows that single and combined treatments with CAERS and CFEZO were able to reduce both numbers and sizes of growing colonies.To further validate effects of CAERS and CFEZO extracts on U251 colony formation, we carried out Soft Agar Colony Formation Assay. This assay is used to measure the ability of cells to grow in soft agar in an anchorage-independent manner, which is considered the most stringent assay for detecting malignant transformation of cells. In this assay cells are plated in a soft agar media matrix where they are unable to attach to an underlying substrate. If cells are able to proliferate they will grow in clumps forming colonies. As seen in Figure1(d), growth of U251 cells in soft agar was noticeable; on the other hand, after treatments with CAERS and/or CFEZO extracts inhibited colony growth of U251 cells. These data suggest that these extracts have ability to inhibit anchorage-dependent and - independent growth of U251 cancer cells.


### 3.2. CAERS and CFEZO Treatments Induced Features of Apoptotic Cell Death

It is generally believed that the induction of apoptosis is the primary cytotoxic mechanism of phytochemicals and the major goal of cancer chemotherapy is to commit tumor cells to apoptosis following exposure to anticancer agents [29]. To determine whether CAERS and CFEZO inhibited the cell growth of U251 cells by inducing apoptosis, we examined the effects of CAERS and CFEZO alone and in combination on cells leading to induction of apoptosis. In the beginning of apoptosis, phosphatidylserine inside of the cell membrane is inverted to the outside. Thereby, incident of apoptosis was investigated by measuring the externalization of phosphatidylserine on the plasma membrane (a marker for early stages of apoptosis) using FITC-annexin-V and propidium iodide (PI) double staining by flow cytometry. The U251 cells were treated with CERS and CFEZO in single and double treatments for 24 h and the intensity of fluorescent-tagged annexin V and PI assayed by flow cytometry. The results are shown in Figure 2(c); viable cells were negative for both PI and annexin V (left bottom quadrants); early apoptotic cells were positive for annexin V and negative for PI (right bottom quadrants) whereas late apoptotic/necrotic cells displayed both high annexin V and PI labeling (right top quadrants); nonviable cells undergoing necrosis were positive for PI and negative for annexin V (left top quadrants). As seen in the Figure the amounts of early apoptotic cells (annexin-V positive and PI negative) were 5.37, 5.37, 5.18, and 8.23% for control, single treatment of CAERS, single treatment of CFEZO, and combined treatment of CAERS and CFEZO, respectively. The amounts of late apoptotic cells (annexin-V positive and PI positive) were 3.87, 12.31, 13.73, and 13.17% for control, single treatment of CAERS, single treatment of CFEZO, and combined treatment of CAERS and CFEZO, respectively. The figure demonstrates too that the amount of necrotic cells (left top quadrants) were much lower in treated cells than in control ones, indicating that CAERS and CFEZO agents may not only induce apoptotic cell death, but they also suppress necrotic cell death. Collectively, these results demonstrate that the CAERS and CFEZO treatments not only may inhibit growth of HT116 cells but also induce apoptosis of the cells.CAERS and CFEZO treatments induced apoptotic cell death. (a) Flow cytometric analyses of apoptosis and necrosis using Annexin V-FITC/PI staining. Panels I, II, III, and IV represent cells treated with vehicle, CAERS (100μg/mL), CFEZO (100 μg/mL), and combined treatment of CAERS (20 μg/mL) and CFEZO (20 μg/mL), respectively. Cells in left lower quadrants represent viable population (annexin V-negative and PI-negative); cells in right lower quadrants represent early apoptotic population (annexin V-positive, PI-negative); cells in right top quadrants represent late apoptotic population (annexin V-positive and PI-positive) and cells in left top quadrants represent necrotic population (annexin V-negative and PI-positive). (b) Microphotographs showing CAERS and CFEZO treatments induced morphological features of apoptosis in U251 cells. The cells were treated with the indicated concentrations of CAERS and/or CFEZO for 48 h. Then, the photographs were taken directly from culture plates using a phase contrast microscope. Magnification of micrographs was as follows: I: 20x; II: 40x; and III: 63x. (c) Toluidine blue-stained semithin sections. The cells were treated and stained with toluidine blue, as detailed in Section 2. Depicted results are representative for independent experiments with almost identical observations.
(a)
(b)
(c)Next, cells were treated with increasing concentrations of CAERS and CFEZO alone and in combination for 48 h, and frequency of apoptotic cell death was assessed by light microscopy. As seen under an inverted phase microscope, untreated U251 cells grew well to form confluent monolayer with a homogenous morphology containing lightly and evenly stained nuclei (Figure2). In contrast, in CAERS or CFEZO monotherapy, U251 cells slightly experienced apoptotic nuclear features such as aggregation and marginality of chromatin and nuclear and cytoplasmic condensation. In combination treatment, the morphologic changes were much more severe than the monotherapy, a number of nuclei displayed condensed appearance, and many were fractured and scattered out of the cells, even the bud-formation of the apoptotic nucleus.  At higher concentrations, cells of single and combined treatments were much more readily detached, relative to control cells, and exhibited a rounded-up, balloon-like shape.


### 3.3. CAERS and CFEZO Treatments Induced DNA Damage

Apoptotic cell death is accompanied by the shrinkage and fragmentation of both cells and their nuclei and extensive degradation of chromosomal DNA into nucleosomal units [6]. Therefore, apoptotic cells, treated with CAERS and CFEZO extracts, were visualized by DAPI staining. As depicted in Figure 3(a), DAPI staining revealed the occurrence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic bodies in U251 cultures treated with CAERS and CFEZO, but not in control cultures.Combination of CAERS and CFEZO induced an early biochemical feature of apoptosis. U251 cells were treated with indicated concentrations of CAERS and for 24 h and assayed for existence of apoptotic cell death. Depicted results are representative for independent experiments with almost identical observations. (a) DAPI staining showing combination of CAERS and CFEZO induced nuclear condensation, DNA fragmentation, and perinuclear apoptotic bodies in U251 cells (arrows). (b) Agarose gel showing CAERS and CFEZO induced DNA fragmentation in U251 cells. Lane “M” indicates the DNA marker ladder. (c) Comet assay showing formation of DNA tail in CAERS- and CFEZO-treated U251 cells. Nuclei with damaged DNA have the appearance of a Comet with a bright head and a tail, whereas nuclei with undamaged DNA appear round with no tail. In the panel denoted with 24 h, cells were treated with 20μg CAERS and 20 μg CFEZO for 24 h; then treatment medium was discarded, cells were washed and grown in CAERS- and CFEZO-free medium for 24 before being harvested and assayed for comet analysis. The histogram displays percentage of cells with comet tail being analyzed in 50 cells for one slide. The bar denoted with 24, at the top, represents cells treated for 24 h, which then were grown in CAERS- and CFEZO-free medium for 24.
(a)
(b)
(c)DNA fragmentation to yield DNA ladders is a characteristic feature of apoptosis [30]. To examine whether CAERS or CFEZO might induce such fragmentation in U251 cells, genomic DNA from U251 cells treated with CAERS and/or CFEZO was extracted and separated by agarose gel electrophoresis. Figure 3(b) shows that there was clear DNA fragmentation ladders in samples from cells treated with all concentrations of CAERS or CFEZO. To substantiate DNA laddering findings, we carried out comet assay. This assay is a sensitive method for monitoring single strand (ss) DNA breaks at the single cell level and used as a biomarker of apoptosis [31]. As shown in Figure 3(c), combined treatment of U251 cells with CAERS and CFEZO (total 25 μg/mL) for 24 hours resulted in significant DNA damage (P
<
0.001) compared with control cells. Furthermore, persistence of DNA damage can be observed after growing cells for 24 hours in CAERS- and CFEZO-free medium. Thus, these independent methods (DAPI staining, DNA laddering, and comet assays) of assessing apoptosis provided similar results, suggesting that the antiproliferative potentials of CAERS and CFEZO are linked to their abilities to induce apoptosis in U251 cells.


### 3.4. CAERS and CFEZO Treatments Trigger Mitochondrial-Dependent Apoptotic Pathway

Apoptosis is tightly regulated by Bcl-2 family of proteins (such as Bcl-2, Bax), executed by caspases and, in most physiological and pathological situations, triggered by mitochondrial pathway with eventual release of mitochondrial cytochromec to the cytoplasm [4]. Therefore, we employed western blotting to examine the changes in expression of these proteins following CAERS and CFEZO treatments. Initially, we separated the cytosolic and mitochondrial fractions and we examined expression levels of the cytochrome c in both fractions. The data in Figure 4(a) reveal that treatments of cells with CAERS and CFEZO most dramatically caused mitochondrial release of cytochrome c into the cytosol. Anticytochrome oxidase IV (COXIV) (a marker for mitochondria) was not detected in the cytosol, indicating that cytosolic fractions were not contaminated with mitochondrial proteins.CAERS and CFEZO treatments trigger mitochondrial-dependent apoptotic pathway. U251 cells were treated with indicated concentrations of CAERS, CFEZO, and combination of CAERS CFEZO for 24 h and assayed as detailed in Section2. (a) Immunoblots showing CAERS and/or CFEZO treatments mediated mitochondrial cytoc release, activation of caspases 9 and 3, and PARP-1 cleavage. The mitochondrial marker, anticytochrome oxidase IV (COXIV), shows the purity of the cytoplasmic fraction and equal loading of the mitochondrial fraction. (b) CAERS and/or CFEZO treatment(s) altered expression ratio of Bax/Bcl-2 at protein and mRNA levels, in favor of apoptosis. The histogram depicts the Bcl-2 (dark bars) and Bax (light bars) mRNA ratio measured by using densitometric analysis. In all western blot analyses, the membranes were stripped and reprobed with antiactin antibody as a loading control.
(a)
(b)Next, we determined activation of the cysteine proteases, caspase-3 and -9. Combination therapy most impressively activated caspase-9, the central player of intrinsic pathway, and caspase-3, the executioner caspase (Figure4(a)). We further examined the proteolytic activity of caspase-3 in the fragmentation of the nuclear DNA repair enzyme poly(ADP-ribose) polymerase (PARP-1). Fragmentation of PARP-1 further confirmed an increase in caspase-3 activity for induction apoptosis (Figure 4(a)). Next, we examined the effect of the CAERS and CFEZO treatments on expression levels of the Bcl-2 (antiapoptotic) and Bax (proapoptotic); as shown in Figure 3(b), combination therapy highly increased expression of Bax and concomitantly reduced expression of Bcl-2 so as to favor apoptotic death. All these observations are related to CAERS and CFEZO treatments, since almost uniform expression ofβ-actin, the cytosolic protein loading control, was observed in all cases. To substantiate the above finding, we carried out qRT-PCR analysis to assess expression levels of Bcl-2 and Bax gene products following CAERS and CFEZO treatments. We observed that CAERS and CFEZO treatments downregulated transcript of Bcl-2 and, on the other hand, upregulated transcript of Bax.


### 3.5. CAERS and CFEZO Treatments Downregulated Molecules Involved in Cell Survival and Proliferation

To further explore the molecular mechanisms underlying the effect of CAERS and CFEZO treatments on U251 cells, we quantified changes in the expression levels of key molecules controlling cell survival and proliferation pathways. NF-κB is responsible for the transactivation of various target genes that are implicated in cell survival and constitutive activation of proteins in the NF-κB signaling pathway is evidenced in glioblastoma cells [32, 33]. So, we monitored expression levels of NF-κB and p53 after CAERS and CFEZO treatments. We found that CAERS and CFEZO treatments markedly downregulated the expression level of the NF-κB (Figure 5(a)). To further investigate these findings, we monitored expression levels of the NF-κB in the nuclear and cytosolic fractions. We observed that a decrease in the expression level of the NF-κBp65 in the nuclear fraction; in contrast the expression level of the cytosolic NF-κBp65 remained steady after CAERS and CFEZO treatments. These data suggest that the reduction of the total NF-κBp65 pool was due to a decrease in the quantity of the nuclear NF-κBp65. On the other hand, p53 is a tumor suppressor protein and its activation can initiate either cell cycle arrest and DNA repair or apoptosis [34]. On the other hand, p53 is a tumor suppressor protein and its activation can initiate either cell cycle arrest and DNA repair or apoptosis [34]. The CAERS and CFEZO treatments resulted in upregulation of the expression level of p53 (Figure 5(a)). We next examined the effects of CAERS and CFEZO on expression and activities of the genes known to be downstream effectors of the NF-κB and p53 and involved in cell survival or apoptosis pathways, including survivin, XIAP, cyclin D1, p21, and Noxa. Treatments of cells with CAERS and CFEZO apparently decreased mRNA expression levels of survivin, XIAP, and cyclin D1 but increased expression levels of the p21 and Noxa (Figure 5(b)). Overall, these findings indicate that CAERS and CFEZO treatments modulated several molecular and cellular parameters relevant to glioblastoma carcinogenesis. They modulated expression levels of the survival and cell cycle regulatory proteins through a possible synergistic effect leading to inhibition of the U251 cell growth.(a) CAERS and/or CFEZO treatment(s) modulated the expression of the NF-κBp65 and p53 proteins. The U251 cells (20 × 104 cells/well) were seeded onto 6-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for 24 h. Subsequently, 20 μg of whole cell (or nuclear) protein extract was isolated from treated cells and subjected to SDS-PAGE in 10% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with antibodies against the depicted proteins. The immunoblots showed that CAERS and/or CFEZO treatment(s) downregulated expression level of NF-κBp65 protein found in whole cell or nuclear extract but did not alter level of NF-κBp65 found in the cytosolic extract. The immunoblot showing nuclear NF-κBp65 was stripped off and reprobed with anti-PCNA antibody as an internal loading control for nuclear extract. The CAERS and/or CFEZO treatment(s) upregulated expression level of p53 protein. Representative blots from several independent experiments are shown. (b) CAERS and/or CFEZO treatment(s) modulated the expression of the displayed antiapoptotic and proapoptotic gene products. After CAERS and/or CFEZO treatment(s) total RNA was then isolated, reverse-transcribed, and subjected to PCR with gene-specific primers. The PCR products of the genes were then subjected to electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide. GAPDH was used as the internal control, M, DNA ladder. The data are representative of three separate experiments.
(a)
(b)


## 3.1. Cytostatic Effects of CAERS and CFEZO

Initially, we examined the sensitivity of U251 cells to different doses of both extracts. So, cells were treated with increasing concentrations of CAERS (0, 50, 100, 150, and 200μg/mL) or CFEZO (0, 50, 100, 150, and 200 μg/mL) for 24, 48, and 72 h. The cytotoxicity assessed by WST-1 kit showed that CAERS or CFEZO efficiently inhibited cell viability in dose- and time-dependent manners (Figure 1(a)). Calculated IC50 values (IC50, the concentration of test compound that inhibits 50% of the cell growth) for CAERS were 200, 135, and 80 μg/mL after 24, 48, and 72 h, respectively, and for CFEZO were 150, 115, and 75 μg/mL after 24, 48, and 72 and 150 μg/mL, respectively. To find whether CAERS and CFEZO have synergistic antiproliferative potentialities, U251 cells were incubated with increasing doses of CAERS (0, 10, 20, 40, and 50 μg/mL) and CFEZO (0, 10, 20, 40, and 50 μg/mL) in combination. As shown in Figure 1(a) combination of CAERS and CFEZO worked synergistically to inhibit viability of U251 cells. Notably, lower IC50 value indicated higher synergistic effect of the combination of extracts; this because IC50 values after 24, 48, and 72 h of combined treatments were 40, 20, and 15 μg/mL. Although we found a lower IC50 value of combination of 40 μg/mL CAERS and 40 μg/mL CFEZO than combination of 20 μg/mL CAERS and 20 μg/mL CFEZO (Figure 1), we did not want to continue with the combination of 40 μg/mL CAERS and 40 μg/mL CFEZO that showed too much cytotoxicity (necrotic cells) as we found in thein situ Wright staining (data not shown). Therefore, we selected 150 μg/mL CAERS and 100 μg/mL CFEZO for single treatment and 20 μg/mL CAERS and 20 μg/mL CFEZO for combined treatment in all subsequent experiments.Combination of CAERS and CFEZO acted synergistically to inhibit cell proliferation and colony formation in U251 cells. The U251 cells were seeded, at a density of 104/well in 96-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for displayed time intervals. The inhibition of cell proliferation was assessed by the tetrazolium salt WST-1 kit as detailed in Section 2. The experiments were repeated five times in triplicate, and cell viabilities at each dose of extract(s) were expressed in terms of percent of control and reported as the mean ± SD. (b) Left histogram: multiple comparisons for effect of different doses’ categories on U251 cells using post hoc test (Dunnett T3). All reportedP values are 2-tailed; **correlation is significant at the 0.01 level (2-tailed); ***correlation is significant at the 0.001 level (2-tailed);a= significant differences compared to control;b = significant differences compared to the lowest dose, 10 μg/mL. Compared to control, doses in 10, 20, 40, and 50 μg/mL showed a significantly lower percentage of viable U251 cells (P
=
0.002**, P
=
0.000***, P
=
0.000***, and P
=
0.000***, resp.). We observed another significant variation between dose 10 μg/mL and doses 40 and 50 μg/mL, with a stronger suppression of GBM cells with the two higher doses (40 and 50 μg/mL) (P
=
0.008**, and P
=
0.001***, resp.). Right histogram: multiple comparisons for effect of different durations on U251 viable cells using post hoc test (LSD). All reportedP values are 2-tailed. *Correlation is significant at the 0.05 level (2-tailed). Compared to one-day duration, three-day duration of treatment showed a significantly lower percentage of viable U251 cells (P
=
0.02*). The table (on the right) shows regression of dose and duration with percent U251 viable cells. A multiple linear regression analysis was used (Enter Selection Procedure).***Correlation is significant at 0.001 level (2-tailed). Dose had a stronger negative impact than that of duration, in predicting the % U251 viable cells, (Beta = −0.84*** andBeta = −0.34*** resp.). TheR
2 indicates that 81.3% of the variation in % U251 viable cells could be explained by these two variables (dose and duration). (c) The MCF-7, HeLa, and HF-5 cell lines were seeded and treated with the indicated concentrations of CAERS and/or CFEZO for displayed time intervals. The experiments were repeated five times in triplicate, and cell viabilities at each dose of extract(s) were expressed in terms of percent of control and reported as the mean ± SD. (d) U251 cells were seeded onto a 6-well plate at 1000 cells/well and treated with the indicated concentrations of CAERS and/or CFEZO as detailed in Section 2. The colonies were counted under a dissection microscope and the experiment was repeated three times. (e) CAERS and/or CFEZO acted synergistically to inhibit anchorage-independent growth in U251 cells in growth in soft agarose assays. U251 cells were plated, in triplicate, in 0.35% soft agarose and treated with CAERS (25 μg/mL) or CFEZO (25 μg/mL) and a combination of CAERS (5 μg/mL) and CFEZO (5 μg/mL). After 2 weeks, the colonies were stained with 0.0005% crystal violet and photographed using a digital camera coupled to a Carl Zeiss inverted microscope. Representative images of colonies in soft agar are shown.
(a)
(b)
(c)
(d)
(e)To find out whether the antiproliferative potentialities of CAERS and CFEZO are restricted to U251 cells, the above experiments were repeated using human breast and cervical carcinoma cell lines, MCF-7 and HeLa, respectively, as models. The findings depicted in Figure1(b) demonstrate combined treatments of CAERS and CFEZO consistently retaining their growth-inhibitory potentialities in the contexts of MCF-7 and HeLa cells, in dose- and time-dependent manners. These observations indicated that antiproliferative potentiality of CAERS and CFEZO treatments is not cell-type-specific. Finally, we assessed the effects of CAERS and CFEZO treatments on viability of nonmalignant human foreskin fibroblasts, HF-5 cells. We noticed that the HF-5 cell line was significantly more resistant to growth inhibition by the CAERS or CFEZO (Figure 1(b)), suggesting that CAERS and CFEZO selectivity target transformed cells. Taken together, these results indicated that the single and combined treatments of CAERS and CFEZO obviously inhibited the growth of U251 cells.Having established growth inhibiting potentialities of CAERS and CFEZO in the U251 cells, we next determined the effects of CAERS and CFEZO on colony formation (also referred to as clonogenicity) in U251 cells. This assay measures the ability of tumor cells to grow and form foci in a manner unrestricted by growth contact inhibition as is characteristically found in normal, untransformed cells. As such, clonogenicity provides an indirect estimation of the tendency of tumor cells to undergo neoplastic transformation. To measure clonogenicity, U251 cells at a given cell density were plated onto multiple well tissue culture dishes, with and without addition of increasingdoses of CAERS or CFEZO. Control and treated cells were maintained in culture for an additional 14 days to allow formation of colonies. Size and number of colonies were visually inspected by fixing and staining in 0.1% Coomassie Blue. Figure1(c) shows that single and combined treatments with CAERS and CFEZO were able to reduce both numbers and sizes of growing colonies.To further validate effects of CAERS and CFEZO extracts on U251 colony formation, we carried out Soft Agar Colony Formation Assay. This assay is used to measure the ability of cells to grow in soft agar in an anchorage-independent manner, which is considered the most stringent assay for detecting malignant transformation of cells. In this assay cells are plated in a soft agar media matrix where they are unable to attach to an underlying substrate. If cells are able to proliferate they will grow in clumps forming colonies. As seen in Figure1(d), growth of U251 cells in soft agar was noticeable; on the other hand, after treatments with CAERS and/or CFEZO extracts inhibited colony growth of U251 cells. These data suggest that these extracts have ability to inhibit anchorage-dependent and - independent growth of U251 cancer cells.


## 3.2. CAERS and CFEZO Treatments Induced Features of Apoptotic Cell Death

It is generally believed that the induction of apoptosis is the primary cytotoxic mechanism of phytochemicals and the major goal of cancer chemotherapy is to commit tumor cells to apoptosis following exposure to anticancer agents [29]. To determine whether CAERS and CFEZO inhibited the cell growth of U251 cells by inducing apoptosis, we examined the effects of CAERS and CFEZO alone and in combination on cells leading to induction of apoptosis. In the beginning of apoptosis, phosphatidylserine inside of the cell membrane is inverted to the outside. Thereby, incident of apoptosis was investigated by measuring the externalization of phosphatidylserine on the plasma membrane (a marker for early stages of apoptosis) using FITC-annexin-V and propidium iodide (PI) double staining by flow cytometry. The U251 cells were treated with CERS and CFEZO in single and double treatments for 24 h and the intensity of fluorescent-tagged annexin V and PI assayed by flow cytometry. The results are shown in Figure 2(c); viable cells were negative for both PI and annexin V (left bottom quadrants); early apoptotic cells were positive for annexin V and negative for PI (right bottom quadrants) whereas late apoptotic/necrotic cells displayed both high annexin V and PI labeling (right top quadrants); nonviable cells undergoing necrosis were positive for PI and negative for annexin V (left top quadrants). As seen in the Figure the amounts of early apoptotic cells (annexin-V positive and PI negative) were 5.37, 5.37, 5.18, and 8.23% for control, single treatment of CAERS, single treatment of CFEZO, and combined treatment of CAERS and CFEZO, respectively. The amounts of late apoptotic cells (annexin-V positive and PI positive) were 3.87, 12.31, 13.73, and 13.17% for control, single treatment of CAERS, single treatment of CFEZO, and combined treatment of CAERS and CFEZO, respectively. The figure demonstrates too that the amount of necrotic cells (left top quadrants) were much lower in treated cells than in control ones, indicating that CAERS and CFEZO agents may not only induce apoptotic cell death, but they also suppress necrotic cell death. Collectively, these results demonstrate that the CAERS and CFEZO treatments not only may inhibit growth of HT116 cells but also induce apoptosis of the cells.CAERS and CFEZO treatments induced apoptotic cell death. (a) Flow cytometric analyses of apoptosis and necrosis using Annexin V-FITC/PI staining. Panels I, II, III, and IV represent cells treated with vehicle, CAERS (100μg/mL), CFEZO (100 μg/mL), and combined treatment of CAERS (20 μg/mL) and CFEZO (20 μg/mL), respectively. Cells in left lower quadrants represent viable population (annexin V-negative and PI-negative); cells in right lower quadrants represent early apoptotic population (annexin V-positive, PI-negative); cells in right top quadrants represent late apoptotic population (annexin V-positive and PI-positive) and cells in left top quadrants represent necrotic population (annexin V-negative and PI-positive). (b) Microphotographs showing CAERS and CFEZO treatments induced morphological features of apoptosis in U251 cells. The cells were treated with the indicated concentrations of CAERS and/or CFEZO for 48 h. Then, the photographs were taken directly from culture plates using a phase contrast microscope. Magnification of micrographs was as follows: I: 20x; II: 40x; and III: 63x. (c) Toluidine blue-stained semithin sections. The cells were treated and stained with toluidine blue, as detailed in Section 2. Depicted results are representative for independent experiments with almost identical observations.
(a)
(b)
(c)Next, cells were treated with increasing concentrations of CAERS and CFEZO alone and in combination for 48 h, and frequency of apoptotic cell death was assessed by light microscopy. As seen under an inverted phase microscope, untreated U251 cells grew well to form confluent monolayer with a homogenous morphology containing lightly and evenly stained nuclei (Figure2). In contrast, in CAERS or CFEZO monotherapy, U251 cells slightly experienced apoptotic nuclear features such as aggregation and marginality of chromatin and nuclear and cytoplasmic condensation. In combination treatment, the morphologic changes were much more severe than the monotherapy, a number of nuclei displayed condensed appearance, and many were fractured and scattered out of the cells, even the bud-formation of the apoptotic nucleus.  At higher concentrations, cells of single and combined treatments were much more readily detached, relative to control cells, and exhibited a rounded-up, balloon-like shape.


## 3.3. CAERS and CFEZO Treatments Induced DNA Damage

Apoptotic cell death is accompanied by the shrinkage and fragmentation of both cells and their nuclei and extensive degradation of chromosomal DNA into nucleosomal units [6]. Therefore, apoptotic cells, treated with CAERS and CFEZO extracts, were visualized by DAPI staining. As depicted in Figure 3(a), DAPI staining revealed the occurrence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic bodies in U251 cultures treated with CAERS and CFEZO, but not in control cultures.Combination of CAERS and CFEZO induced an early biochemical feature of apoptosis. U251 cells were treated with indicated concentrations of CAERS and for 24 h and assayed for existence of apoptotic cell death. Depicted results are representative for independent experiments with almost identical observations. (a) DAPI staining showing combination of CAERS and CFEZO induced nuclear condensation, DNA fragmentation, and perinuclear apoptotic bodies in U251 cells (arrows). (b) Agarose gel showing CAERS and CFEZO induced DNA fragmentation in U251 cells. Lane “M” indicates the DNA marker ladder. (c) Comet assay showing formation of DNA tail in CAERS- and CFEZO-treated U251 cells. Nuclei with damaged DNA have the appearance of a Comet with a bright head and a tail, whereas nuclei with undamaged DNA appear round with no tail. In the panel denoted with 24 h, cells were treated with 20μg CAERS and 20 μg CFEZO for 24 h; then treatment medium was discarded, cells were washed and grown in CAERS- and CFEZO-free medium for 24 before being harvested and assayed for comet analysis. The histogram displays percentage of cells with comet tail being analyzed in 50 cells for one slide. The bar denoted with 24, at the top, represents cells treated for 24 h, which then were grown in CAERS- and CFEZO-free medium for 24.
(a)
(b)
(c)DNA fragmentation to yield DNA ladders is a characteristic feature of apoptosis [30]. To examine whether CAERS or CFEZO might induce such fragmentation in U251 cells, genomic DNA from U251 cells treated with CAERS and/or CFEZO was extracted and separated by agarose gel electrophoresis. Figure 3(b) shows that there was clear DNA fragmentation ladders in samples from cells treated with all concentrations of CAERS or CFEZO. To substantiate DNA laddering findings, we carried out comet assay. This assay is a sensitive method for monitoring single strand (ss) DNA breaks at the single cell level and used as a biomarker of apoptosis [31]. As shown in Figure 3(c), combined treatment of U251 cells with CAERS and CFEZO (total 25 μg/mL) for 24 hours resulted in significant DNA damage (P
<
0.001) compared with control cells. Furthermore, persistence of DNA damage can be observed after growing cells for 24 hours in CAERS- and CFEZO-free medium. Thus, these independent methods (DAPI staining, DNA laddering, and comet assays) of assessing apoptosis provided similar results, suggesting that the antiproliferative potentials of CAERS and CFEZO are linked to their abilities to induce apoptosis in U251 cells.


## 3.4. CAERS and CFEZO Treatments Trigger Mitochondrial-Dependent Apoptotic Pathway

Apoptosis is tightly regulated by Bcl-2 family of proteins (such as Bcl-2, Bax), executed by caspases and, in most physiological and pathological situations, triggered by mitochondrial pathway with eventual release of mitochondrial cytochromec to the cytoplasm [4]. Therefore, we employed western blotting to examine the changes in expression of these proteins following CAERS and CFEZO treatments. Initially, we separated the cytosolic and mitochondrial fractions and we examined expression levels of the cytochrome c in both fractions. The data in Figure 4(a) reveal that treatments of cells with CAERS and CFEZO most dramatically caused mitochondrial release of cytochrome c into the cytosol. Anticytochrome oxidase IV (COXIV) (a marker for mitochondria) was not detected in the cytosol, indicating that cytosolic fractions were not contaminated with mitochondrial proteins.CAERS and CFEZO treatments trigger mitochondrial-dependent apoptotic pathway. U251 cells were treated with indicated concentrations of CAERS, CFEZO, and combination of CAERS CFEZO for 24 h and assayed as detailed in Section2. (a) Immunoblots showing CAERS and/or CFEZO treatments mediated mitochondrial cytoc release, activation of caspases 9 and 3, and PARP-1 cleavage. The mitochondrial marker, anticytochrome oxidase IV (COXIV), shows the purity of the cytoplasmic fraction and equal loading of the mitochondrial fraction. (b) CAERS and/or CFEZO treatment(s) altered expression ratio of Bax/Bcl-2 at protein and mRNA levels, in favor of apoptosis. The histogram depicts the Bcl-2 (dark bars) and Bax (light bars) mRNA ratio measured by using densitometric analysis. In all western blot analyses, the membranes were stripped and reprobed with antiactin antibody as a loading control.
(a)
(b)Next, we determined activation of the cysteine proteases, caspase-3 and -9. Combination therapy most impressively activated caspase-9, the central player of intrinsic pathway, and caspase-3, the executioner caspase (Figure4(a)). We further examined the proteolytic activity of caspase-3 in the fragmentation of the nuclear DNA repair enzyme poly(ADP-ribose) polymerase (PARP-1). Fragmentation of PARP-1 further confirmed an increase in caspase-3 activity for induction apoptosis (Figure 4(a)). Next, we examined the effect of the CAERS and CFEZO treatments on expression levels of the Bcl-2 (antiapoptotic) and Bax (proapoptotic); as shown in Figure 3(b), combination therapy highly increased expression of Bax and concomitantly reduced expression of Bcl-2 so as to favor apoptotic death. All these observations are related to CAERS and CFEZO treatments, since almost uniform expression ofβ-actin, the cytosolic protein loading control, was observed in all cases. To substantiate the above finding, we carried out qRT-PCR analysis to assess expression levels of Bcl-2 and Bax gene products following CAERS and CFEZO treatments. We observed that CAERS and CFEZO treatments downregulated transcript of Bcl-2 and, on the other hand, upregulated transcript of Bax.


## 3.5. CAERS and CFEZO Treatments Downregulated Molecules Involved in Cell Survival and Proliferation

To further explore the molecular mechanisms underlying the effect of CAERS and CFEZO treatments on U251 cells, we quantified changes in the expression levels of key molecules controlling cell survival and proliferation pathways. NF-κB is responsible for the transactivation of various target genes that are implicated in cell survival and constitutive activation of proteins in the NF-κB signaling pathway is evidenced in glioblastoma cells [32, 33]. So, we monitored expression levels of NF-κB and p53 after CAERS and CFEZO treatments. We found that CAERS and CFEZO treatments markedly downregulated the expression level of the NF-κB (Figure 5(a)). To further investigate these findings, we monitored expression levels of the NF-κB in the nuclear and cytosolic fractions. We observed that a decrease in the expression level of the NF-κBp65 in the nuclear fraction; in contrast the expression level of the cytosolic NF-κBp65 remained steady after CAERS and CFEZO treatments. These data suggest that the reduction of the total NF-κBp65 pool was due to a decrease in the quantity of the nuclear NF-κBp65. On the other hand, p53 is a tumor suppressor protein and its activation can initiate either cell cycle arrest and DNA repair or apoptosis [34]. On the other hand, p53 is a tumor suppressor protein and its activation can initiate either cell cycle arrest and DNA repair or apoptosis [34]. The CAERS and CFEZO treatments resulted in upregulation of the expression level of p53 (Figure 5(a)). We next examined the effects of CAERS and CFEZO on expression and activities of the genes known to be downstream effectors of the NF-κB and p53 and involved in cell survival or apoptosis pathways, including survivin, XIAP, cyclin D1, p21, and Noxa. Treatments of cells with CAERS and CFEZO apparently decreased mRNA expression levels of survivin, XIAP, and cyclin D1 but increased expression levels of the p21 and Noxa (Figure 5(b)). Overall, these findings indicate that CAERS and CFEZO treatments modulated several molecular and cellular parameters relevant to glioblastoma carcinogenesis. They modulated expression levels of the survival and cell cycle regulatory proteins through a possible synergistic effect leading to inhibition of the U251 cell growth.(a) CAERS and/or CFEZO treatment(s) modulated the expression of the NF-κBp65 and p53 proteins. The U251 cells (20 × 104 cells/well) were seeded onto 6-well plates and treated with the indicated concentrations of CAERS and/or CFEZO for 24 h. Subsequently, 20 μg of whole cell (or nuclear) protein extract was isolated from treated cells and subjected to SDS-PAGE in 10% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with antibodies against the depicted proteins. The immunoblots showed that CAERS and/or CFEZO treatment(s) downregulated expression level of NF-κBp65 protein found in whole cell or nuclear extract but did not alter level of NF-κBp65 found in the cytosolic extract. The immunoblot showing nuclear NF-κBp65 was stripped off and reprobed with anti-PCNA antibody as an internal loading control for nuclear extract. The CAERS and/or CFEZO treatment(s) upregulated expression level of p53 protein. Representative blots from several independent experiments are shown. (b) CAERS and/or CFEZO treatment(s) modulated the expression of the displayed antiapoptotic and proapoptotic gene products. After CAERS and/or CFEZO treatment(s) total RNA was then isolated, reverse-transcribed, and subjected to PCR with gene-specific primers. The PCR products of the genes were then subjected to electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide. GAPDH was used as the internal control, M, DNA ladder. The data are representative of three separate experiments.
(a)
(b)


## 4. Discussion

Current anticancer chemotherapeutic agents for GBM have not significantly improved the survival of glioblastoma patients during the past ten years [1]. Therefore, there is an unmet need to develop novel chemotherapeutic agents that target multiple molecular pathways to inhibit prosurvival signals and induce apoptosis in the GBM cells. Considerable data indicate that combinations of dietary agents are more effective than a single agent [8]. However, the challenge is to identify an effective combination, with chemopreventive agents working through similar or different mechanisms to produce an additive or synergistic chemopreventive effect. We have previously reported that crude extracts ofZ. officinale andR. stricta independently suppressed proliferation and induced apoptosis in human breast cancer cell lines, MCF-7 and MB-MDA-231 [35, 36]. In light of these earlier studies, we carried out the current work to evaluate the beneficial effect of a combination of crude alkaloid extract isolated fromR. stricta and crude flavonoid extract fromZ. officinale in anin vitro model of GBM, U251 cells. We hypothesized that, since alkaloids and flavonoids have substantially different biochemical characteristics, a combinational approach may simultaneously target multiple molecular and cellular pathways involved in the process of GBM carcinogenesis. In line with this hypothesis, the results in this study demonstrate that the crude extracts of alkaloids (CAERS) and flavonoids (CFEZO), alone and in combination, inhibited cell proliferation and colony formation in U251 cells, in a dose- and time-dependent manner. Significantly, the IC50 values of cell viability assays revealed that a combination of low doses (at which single agent, CAERS or CFEZO, induced a minimal growth suppression) inhibited proliferation and colony formation in U251 cells and induced apoptosis more effectively than high doses of the single agent. Thus, the chemical entities found in the CAERS and CFEZO formula represent an effective combination for exerting synergistic pharmacodynamic actions and neutralizing the adverse effects and toxicities of specific individual chemicals. Interestingly, CAERS and CFEZO treatments recapitulated their growth-suppression potentiality in the contexts of MCF-7 and HeLa cells indicating that their cytotoxic effect is not cell-type specific. On the other hand, these treatments exerted minor effect on the growth of nonmalignant human fibroblasts HF-5, which raises a possibility that these treatments may selectively target malignant, but not normal, cells. However, the assays herein cannot exclude the possibility that there are tissue-specific differences between HF-5 fibroblasts and glioblastoma U251 cells. A further proof of synergistic action of alkaloids (inR. stricta) and flavonoids (inZ. officinale) was concluded from the observations of the soft agar colony formation assay. This assay is a sensitive parameter of toxicity because the number of colonies formed is assessed when the cells are in a state of proliferation. Thus, in the present study, we have identified a novel combination of two natural dietary agents, CAERS and CFEZO, which exhibit highly synergistic action to target growth of U251 cells and provide an experimental basis for effective chemotherapy of GBM.Accumulating evidences demonstrate that a characteristic feature of primary GBM, as well as several phenotypes of cancers, is resistant to commit apoptotic cell death [37]. At the onsetof apoptosis, phosphatidylserine (PS) inside of the cell membrane is inverted to the outside due to loss of membrane integrity. Since annexin V binds to PS with high affinity, therefore, binding of PS to a fluorescent-tagged annexin V can be used for detecting apoptosis at its earlier stages with a fluorescent-detecting system, such as flow cytometer or fluorescent microscopy. The data generated from flow cytometric analysis explain that the percentages of cells stained with annexin V (positive cells) were higher in cells treated with CAERS and/or CFEZO than in control cells, indicating the loss of membrane integrity with a subsequent externalization of PS, a major characteristic of cell death by apoptosis [6].To further confirm apoptogenic effects of CAERS and CFEZO, we looked for some morphologic and biochemical hallmarks of apoptosis. The hallmarks of apoptosis comprise shrinkage of the cell and the nucleus as well as condensation of nuclear chromatin into sharply delineated masses. Later on, the nucleus progressively condenses and breaks up (karyorrhexis) [6]. Eventually, DNA is fragmented at the internucleosomal linker regions forming DNA ladder; degradation of DNA into nucleosomal units has been observed in cells undergoing apoptosis induced by a variety of agents and is widely used as biochemical markers of apoptosis [30]. Consistent with these hallmarks, light microscopy investigation demonstrated that control U251 cells assumed an epithelial morphology and attached to the substrate; on the other hand, CAERS- and/or CFEZO-treated cells appeared much more readily detached and exhibited a rounded-up, balloon-like shape, a loss of cell viability, cell shrinkage, and irregularity in cellular shape. In addition, DAPI stain exhibited occurrence of nuclear condensation, karyorrhexis, and perinuclear apoptotic bodies in CAERS- and/or CFEZO-treated cells. And DNA purified from CAERS- and/or CFEZO-treated cells exhibited laddering appearance in agarose gel electrophoresis. Finally, comet assay demonstrated formation of comet tail after CAERS- and/or CFEZO treatments. It is noteworthy that comet assay is more sensitive than DNA ladder assay in detecting DNA damage and distinguishes apoptosis from necrosis making it a reliable assay for detection of apoptotic cell death [38]. Therefore, results of comet assay add a further proof that CAERS and/or CFEZO treatments mediated DNA damage in U251 cells. Since in response to DNA damage, cells with damaged DNA could undergo apoptosis if damaged DNA is hardly to be repaired; therefore, the observation of these assays suggests that CAERS and/or CFEZO treatments might trigger events leading to DNA damage and initiation of apoptotic cascade, which may contribute, at least in part, to reduction of U251 cell viability.Most dietary bioactive agents trigger apoptosis by activation of the intrinsic (mitochondrial) apoptotic pathway [29]. A pivotal event in the intrinsic pathway is the release of cytochrome c from the mitochondrial intermembrane space. The release of cytochrome c is controlled by two opposing groups of proteins. These are the prosurvival Bcl-2 protein family (Bcl-2, Bcl-XL, Mcl-1, and death agonists) that constrain cytochrome release, while Bax and other proapoptosis proteins precipitate the release, and the Bcl-2 : Bax ratio determines the relative sensitivity or resistance of the cells to various apoptotic stimuli [4]. Once cytochrome c is released into the cytosol, it mediates activation of caspase-9, and the latter then activates caspase-3 [4]. Caspase-3 is a prevalent cysteine protease ultimately responsible for the majority of apoptotic processes and mediates the cleavage or degradation of several important substrates, including PARP-1. The data fit well with the scenario of apoptotic cascade, since there was an increase in the level of the cytochrome c in the cytosolic fraction and its decrease in the mitochondrial fraction after treatment of cells with the CAERS and/or CFEZO extracts, indicating that the mitochondrial cytochrome c released into cytoplasm. Additionally, we found increase in activities of the caspases 9 and 3 and PARP-1 cleavage. Intact PARP-1 can help cells to maintain their viability, but cleavage of PARP-1 facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis. Therefore, cleavage of PARP-1 might be the key for the ultimate apoptotic death of U251 cells induced by CAERS and/or CFEZO treatments. CAERS- and/or CFEZO-induced apoptosis was further confirmed by measuring the levels of Bax and Bcl-2 expression. The analysis revealed an increase in Bax expression and a decrease in Bcl-2 expression indicating that CAERS and/or CFEZO treatments tipped balance of Bax : Bcl-2 ratio in favor of apoptosis. This is an interesting finding, since upregulation of the prosurvival proteins Bcl-2 and Bcl-2XL has been described in recurrent GBMs independent of treatment [39] and overexpression of Bcl-2 or Bcl-XL not only leads to resistance to apoptosis but also has been linked to increased GBM cell motility [40]. On the other hand, overexpression of Bax induced apoptosis in glioma cell lines and increased their sensitivity toward radiation therapy [41, 42]. Therefore, these results support the idea that the CAERS and/or CFEZO treatments induced mitochondrial-dependent apoptotic pathway in U251 cells.Aberrant constitutive activation of the NF-κB signaling pathway is a characteristic feature of glioblastoma cells [32, 33]. In nonstimulated tumor cells, there are two pools of NF-κBp65, an inducible cytoplasmic pool and a basal pool that drives the transcription of antiapoptotic genes; the presence of NF-κB in the nucleus is critical for the maintenance of a malignant phenotype of GBM cells [32]. The data herein demonstrate that CAERS and CFEZO downregulated expression level of the nuclear (but not cytosolic) NF-κBp65. These findings suggest that CAERS and CFEZO specifically targeted the NF-κBp65 counterpart that drives transcription of antiapoptotic genes, which could be a part of their proapoptotic scenarios to inhibit growth of U251 cells. Although the distinct mechanism underlying NF-κBp65 downregulation necessitates further investigation, it is possible that CAERS and CFEZO treatments provoked ubiquitination-mediated NF-κBp65 degradation events. Recent reports have shown that ubiquitin- and proteasome-dependent degradation of the nuclear NF-κBp65 has been suggested to be a candidate mechanism for degradation of nuclear NF-κBp65 to prevent its excessive activation [43]. Of note, downregulation of NF-κBp65 by other dietary agents has been cited [44, 45]. Significantly, downregulation of the nuclear NF-κBp65 by CAERS and CFEZO was associated with repression of its transcriptional targets, survivin, XIAP, and cyclin D1 [46]. These findings deserve attention since survivin and XIAP are antiapoptotic proteins and mediate the resistance of proapoptotic signals induced by chemotherapeutic agents [37]. In particular, it has been found that GBM cells exhibit intrinsic apoptosis resistance related, in part, to overexpression of survivin [47, 48], knockdown of survivin precipitated apoptosis [49], and inhibited cell invasion, angiogenesis, and tumorigenesis of GBM cells [50]. Cyclin D1 is a principal player in cell cycle progression [51] and a key molecular target of NF-κB [46]. Cyclin D1 overexpression plays a central role in inhibition of apoptosis in GBM [51]. Therefore, one of the seminal findings in the current study the CAERS and/or CFEZO treatments targeted survival signaling pathway regulated by NF-κB and its effectors, survivin and XIAP and cyclin D1, which could be partially responsible for their apoptosis-setting-off activities leading to growth inhibition of U251 cells.The p53 protein plays a central role in the response to a wide range of cellular stresses including DNA damage. Induction of DNA damage initiates a cascade of signaling leading to p53 activation and subsequent transcriptional activation of p53 response genes (including p21, Bax, PUMA, and Noxa), thus provoking cell cycle arrest and/or apoptosis [52]. Mutational inactivation of the p53 has been reported in 63–65% of high-grade gliomas [53–55]. Our data demonstrate that CAERS and/or CFEZO treatments resulted in a significant induction of p53 protein level, which hint at the point that the induction of p53 is responsible, at least in part, for the combination of CAERS and/or CFEZO treatment-induced apoptosis in U251 cells. Consistently, induction of p53 was correlated with the concomitant upregulation of its transcriptional targets, p21 and Noxa [52]. The p21 is a universal inhibitor of cell cycle progression, and deregulated expression of p21 plays a critical role in the pathogenesis of many human tumors [56]. The data, herein, showing CAERS and/or CFEZO treatments increased expression level of p21 gene suggest that elevated p21 could inactivate the cyclin D1-CDK4/6 complexes, which might, in turn, induce cell cycle arrest and reduction of U251 cell number. Noxa is an antiapoptotic protein and contributes to p53-dependent and -independent apoptotic responses [57, 58]. The findings showing upregulation of Noxa by CAERS and/or CFEZO treatments have an important implication in molecular targeted therapy, which implement BH3 mimetics such as ABT-737. The ABT-737 is a small-molecule antagonist of Bcl-2 protein family members developed to sensitize cells to apoptosis. However, ABT-737 has been found to selectively inhibit antiapoptotic proteins, Bcl-2, Bcl-xL, and Bcl-w, but not Mcl-1 [59]. Furthermore, GBM cells express high level of Mcl-1 and the role of Mcl-1 in the resistance of GBM cells to ABT-737 is well established [60]. Meanwhile, studies confirmed that only Noxa, but not other BH3-only family members, appears to be crucial in fine-tuning cell death decisions by targeting the Mcl-1 for proteasomal degradation [61]. Since the data here showed CAERS and/or CFEZO treatments upregulated expression level of Noxa, therefore, these treatments might represent a promising strategy to increase the therapeutic efficacy of ABT-737 to induce apoptosis in GBM.


## 5. Conclusion

In conclusion, our current investigation clearly showed that both CAERS and CFEZO extracts acted synergistically to control growth and to induce apoptosis in human GBM cells. Apoptosis was evaluated by flow cytometry and mediated by cytochromec release, activation of caspase-3 and -9, cleavage of PARP-1, and increasing Bax : Bcl-2 ratio. CAERS and CFEZO treatments downregulated expression level of the nuclear NF-κBp65, which was accompanied by repression of its effectors survivin, XIAP, and cyclin D1. On the other hand, CAERS and CFEZO treatments enhanced expression levels of p53 and its downstream targets, p21 and Noxa. This study provides a useful foundation for studying and developing new antiproliferative substances based on these extracts for the treatment of GBM. Furtherin vivo studies will validate the role of CAERS and CFEZO as new agents for the chemoprevention of GBM.



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*Source: 260210-2014-07-22.xml*