# Comparison of Apoptotic Inducing Effect of Zerumbone and Zerumbone-Loaded Nanostructured Lipid Carrier on Human Mammary Adenocarcinoma MDA-MB-231 Cell Line

Authors: Mahnaz Hosseinpour; Ahmad Bustamam Abdul; Heshu Sulaiman Rahman; Abdullah Rasedee; Swee Keong Yeap; Negin Ahmadi; Hemn Hassan Othman; Max Stanley Chartrand
Journal: Journal of Nanomaterials
 (2014)

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## Abstract

This study investigated the anticancer effect of zerumbone (ZER) and zerumbone-loaded nanostructured lipid carrier (ZER-NLC) on the human mammary gland adenocarcinoma (MDA-MB-231) cell line. The effect of ZER and ZER-NLC on MDA-MB-231 cells was determined via electron and fluorescent microscopy and flow cytometry using the Annexin V, cell cycle, and Tdt-mediated dUTP nick-end labeling assays. We demonstrated that ZER and ZER-NLC significantly suppressed the proliferation of MDA-MB-231 cells with an IC50 of 5.96 ± 0.13 and 6.01 ± 0.11 μg/mL, respectively. ZER and ZER-NLC arrested MDA-MB-231 cell cycle at the G2/M phase. The induction of apoptosis by ZER and ZER-NLC was via the intrinsic pathway through the release of cytochrome c and activation of caspase-3 and caspase-9. The treatments also caused the downregulation of antiapoptotic Bcl-2, Bcl-xL proteins, and proliferating cell nuclear protein and upregulation of proapoptotic Bax protein. Therefore, loading of ZER into NLC did not compromise the anticancer effects of ZER on MDA-MB-231 cells. In conclusion, ZER-NLC, which increased the bioavailability of ZER, is an effective agent in the treatment of cancers.


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## Body

## 1. Introduction

The main life-threatening and the most prevalent diseases in women are breast cancers [1]. Development of breast cancers is associated with several risk factors such as age, genes, and family history. There are several treatment methods currently being instituted for breast cancers to include chemotherapy, hormonal therapy using tamoxifen and doxorubicin, radiotherapy, and surgery. Most of these treatment methods are not specific for cancer cells and more often also damage normal cells. Current studies are increasingly providing evidences that therapeutic compounds from natural sources are promising for effective treatment of chronic human diseases including cancers, without significant side-effect [2]. Among these compounds is zerumbone (ZER), extracted from the essential oils ofZingiber zerumbet Smith rhizome.Zerumbone has versatile pharmaceutical properties including anti-inflammatory, antitumor, antioxidant, antimicrobial, and antinociceptive activities [3, 4]. This compound also significantly suppresses activation of nuclear factor κ-light-chain-enhancer in activated B cells (NF-κB) and NF-κB-related gene expression generated by carcinogens in various other cells [5]. Zerumbone has been reported to inhibit proliferation of cervical, ovarian, colon, breast, and liver cancers [6, 7].Although zerumbone has pharmaceutical potentials, like many other drugs and synthetic therapeutic compounds, its application in therapy is hindered by poor water solubility that leads to poor bioavailability and delivery [8]. This property retards zerumbone absorption in the gastrointestinal tract making oral applications relatively ineffective. The poor absorption in the gastrointestinal tract not only reduces bioavailability of zerumbone but also causes mucosal toxicity [9, 10]. One approach to overcome this obstacle is to incorporate drugs into lipid nanocarriers, which would improve solubility and delivery of zerumbone by facilitating transportation across several anatomical barriers. Solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and lipid-drug conjugates are examples of lipid-based carrier systems that can be used to incorporate therapeutic compounds. Compound-loaded lipids nanocarriers exhibit desirable characteristics including low toxicity, controlled release, biodegradable, high drug content, low cost, and easy upscaling [11, 12].Nanostructured lipid carrier, a second generation lipid nanocarrier produced by high-pressure homogenization, is composed of a solid and liquid lipid matrix with disrupted crystalline nanostructure [13, 14]. This lipid nanocarrier, while overcoming some of potential therapeutic application problems associated with SLN, also possesses high loading capacity, low water content of particle suspension, and avoidance of drug expulsion [15, 16]. The purpose of this study was to determine the effect of ZER and zerumbone-loaded nanostructured lipid carriers (ZER-NLC) on the proliferation of a breast cancer (MDA-MB-231) cell line.


## 2. Materials and Methods

### 2.1. Materials

The pure colorless ZER and ZER-NLC were prepared according to the method described by Rahman et al. [10]. MDA-MB-231 cells (human breast adenocarcinoma), MCF-10A (normal breast cell line), RPMI-1640, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from the American Type Culture Collection (Baltimore, MD, USA). Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), penicillin-streptomycin, propidium iodide, acridine orange, trypsin-EDTA, and MTT powder were purchased from Sigma Chemical Co. (St. Louis, MO, USA).


### 2.2. Cytotoxicity Assay

MDA-MB-231 and MCF-10A cells were cultured according to the manufacture’s instruction (ATCC, USA) and each cell line was seeded at a concentration of 2 × 105 cells/mL into a 96-well plate. The cytotoxic effect of 100, 50, 25, 12.5, 6.25, 3.125, 1.562, and 0 μg/mL of ZER and ZER-NLC on these breast cells was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) dye after 24, 48, and 72 hours of incubation [17]. All assays were done in triplicate and 0.1% DMSO was used as the negative control.


### 2.3. Morphological Characterization

MDA-MB-231 cells (2 × 106 cells/mL) were then treated with IC50 concentrations of ZER and ZER-NLC for 24, 48, and 72 hours. At end of each treatment period, the morphology of ZER- and ZER-NLC-treated cells was observed under an inverted microscope (Leica, Tokyo, Japan).


### 2.4. Quantification of Apoptosis

To determine the effect of ZER and ZER-NLC on apoptosis of breast cancer cells, the MDA-MB-231 cells (2 × 106 cells/mL) treated for 24, 48, and 72 hours were double-stained with propidium iodide (PI) and acridine-orange (AO) according to standard procedure and examined under fluorescence microscope (Leica, Tokyo, Japan) [18].


### 2.5. Annexin Assay

Induction of apoptosis in the treated MDA-MB-231 cells was determined using the Annexin V-fluorescein isothiocyanate (FITC) kit (Sigma-Aldrich) according to manufacturer’s instructions and analysed by BD flow cytometer equipped with an argon laser (Cyan ADP; Dako Denmark A/P, Glostrup, Denmark).


### 2.6. Cell Cycle Analysis

The determination of cell cycle distribution of ZER- and ZER-NLC-treated MDA-MB-231 cell was by flow cytometry after staining with PI according to the protocol explained elsewhere [19].


### 2.7. Caspases Assay

The commercial colorimetric assay kit (Genscript Corporation Inc., Piscataway, NJ, USA) was used to determine the activity of caspase-3, caspase-8, and caspase-9 in MDA-MB-231 cells treated with ZER and ZER-NLC for 12, 24, and 48 hours, according to the manufacturer’s instructions.


### 2.8. TUNEL Assay

The Tdt-mediated dUTP nick-end labeling (TUNEL) with the apoptotic detection kit (DeadEnd fluorometric TUNEL system; Promega, Madison, WI, USA) was applied to determine the mode of cell death induced by ZER and ZER-NLC. The assay quantifies fragmented DNA in apoptotic cells using the FACSCalibur flow cytometer (BD) equipped with an argon laser (BD).


### 2.9. Western Blotting Analysis

MDA-MB-231 cells (1 × 106) treated with ZER and ZER-NLC were lysed in the radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) and expressions of cytochrome c, Bcl-2, Bcl-xL and Bax proteins, and proliferating cell nuclear antigen (PCNA) determined by western blotting. Briefly, protein (25 μg) from treated and untreated cells was separated on 10% on sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline with 0.05% Tween-20 for 60 minutes at room temperature. The membrane was probed by incubation with the primary rabbit anti-Bcl-2, -Bcl-xL, -Bax, -cytochrome c, and -PCNA antibodies (Abcam, USA) followed by horseradish peroxidase-conjugated secondary rabbit anti-goat antibody (Abcam, USA). The antibody binding was detected by chemiluminescence system (ECL Western blot substrate; Abcam, Cambridge, UK). The membrane was also probed with rabbit anti-β-actin antibody (Abcam, USA) as a loading control. Finally Image J software was used to quantify the bands.


### 2.10. Statistical Analysis

All experiments were performed in triplicate and results were presented as mean ± standard deviation. Statistical package for the social sciences (SPSS), version 19.0 (SPSS Inc., Chicago, USA), and analysis of variance (ANOVA) were utilized for statistical analysis.


## 2.1. Materials

The pure colorless ZER and ZER-NLC were prepared according to the method described by Rahman et al. [10]. MDA-MB-231 cells (human breast adenocarcinoma), MCF-10A (normal breast cell line), RPMI-1640, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from the American Type Culture Collection (Baltimore, MD, USA). Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), penicillin-streptomycin, propidium iodide, acridine orange, trypsin-EDTA, and MTT powder were purchased from Sigma Chemical Co. (St. Louis, MO, USA).


## 2.2. Cytotoxicity Assay

MDA-MB-231 and MCF-10A cells were cultured according to the manufacture’s instruction (ATCC, USA) and each cell line was seeded at a concentration of 2 × 105 cells/mL into a 96-well plate. The cytotoxic effect of 100, 50, 25, 12.5, 6.25, 3.125, 1.562, and 0 μg/mL of ZER and ZER-NLC on these breast cells was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) dye after 24, 48, and 72 hours of incubation [17]. All assays were done in triplicate and 0.1% DMSO was used as the negative control.


## 2.3. Morphological Characterization

MDA-MB-231 cells (2 × 106 cells/mL) were then treated with IC50 concentrations of ZER and ZER-NLC for 24, 48, and 72 hours. At end of each treatment period, the morphology of ZER- and ZER-NLC-treated cells was observed under an inverted microscope (Leica, Tokyo, Japan).


## 2.4. Quantification of Apoptosis

To determine the effect of ZER and ZER-NLC on apoptosis of breast cancer cells, the MDA-MB-231 cells (2 × 106 cells/mL) treated for 24, 48, and 72 hours were double-stained with propidium iodide (PI) and acridine-orange (AO) according to standard procedure and examined under fluorescence microscope (Leica, Tokyo, Japan) [18].


## 2.5. Annexin Assay

Induction of apoptosis in the treated MDA-MB-231 cells was determined using the Annexin V-fluorescein isothiocyanate (FITC) kit (Sigma-Aldrich) according to manufacturer’s instructions and analysed by BD flow cytometer equipped with an argon laser (Cyan ADP; Dako Denmark A/P, Glostrup, Denmark).


## 2.6. Cell Cycle Analysis

The determination of cell cycle distribution of ZER- and ZER-NLC-treated MDA-MB-231 cell was by flow cytometry after staining with PI according to the protocol explained elsewhere [19].


## 2.7. Caspases Assay

The commercial colorimetric assay kit (Genscript Corporation Inc., Piscataway, NJ, USA) was used to determine the activity of caspase-3, caspase-8, and caspase-9 in MDA-MB-231 cells treated with ZER and ZER-NLC for 12, 24, and 48 hours, according to the manufacturer’s instructions.


## 2.8. TUNEL Assay

The Tdt-mediated dUTP nick-end labeling (TUNEL) with the apoptotic detection kit (DeadEnd fluorometric TUNEL system; Promega, Madison, WI, USA) was applied to determine the mode of cell death induced by ZER and ZER-NLC. The assay quantifies fragmented DNA in apoptotic cells using the FACSCalibur flow cytometer (BD) equipped with an argon laser (BD).


## 2.9. Western Blotting Analysis

MDA-MB-231 cells (1 × 106) treated with ZER and ZER-NLC were lysed in the radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) and expressions of cytochrome c, Bcl-2, Bcl-xL and Bax proteins, and proliferating cell nuclear antigen (PCNA) determined by western blotting. Briefly, protein (25 μg) from treated and untreated cells was separated on 10% on sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline with 0.05% Tween-20 for 60 minutes at room temperature. The membrane was probed by incubation with the primary rabbit anti-Bcl-2, -Bcl-xL, -Bax, -cytochrome c, and -PCNA antibodies (Abcam, USA) followed by horseradish peroxidase-conjugated secondary rabbit anti-goat antibody (Abcam, USA). The antibody binding was detected by chemiluminescence system (ECL Western blot substrate; Abcam, Cambridge, UK). The membrane was also probed with rabbit anti-β-actin antibody (Abcam, USA) as a loading control. Finally Image J software was used to quantify the bands.


## 2.10. Statistical Analysis

All experiments were performed in triplicate and results were presented as mean ± standard deviation. Statistical package for the social sciences (SPSS), version 19.0 (SPSS Inc., Chicago, USA), and analysis of variance (ANOVA) were utilized for statistical analysis.


## 3. Results and Discussion

### 3.1. Zerumbone-Loaded NLC

The ZER-NLC was shown to be physically stable, with a particle size of52.68±0.1 nm, a zeta potential of -25.03±1.24 mV, and a polydispersity index of 0.29±0.0041 μm [10, 14].


### 3.2. Cytotoxic Effect of ZER and ZER-NLC on MDA-MB-231 Cells

Several studies showed that ZER exerts antiproliferative effects on several types of cancer cell while minimally affecting normal cells [20, 21]. The IC50 for ZER on MDA-MB-231 cells were 10.15±0.9, 8.3±0.15, and 5.96±0.13 μg/mL, while for ZER-NLC they were 11.45±0.12, 9.4±0.18, and 6.01±0.11 μg/mL after 24, 48, and 72 hours of incubation, respectively. On the MCF-10A cell, a normal epithelial mammary gland cell line, the IC50 of ZER and ZER-NLC at the same incubation periods were similarly low (Figures 1 and 2). This study also suggests that incorporation of ZER into NLC did not change the cytotoxic effect of the compound on MDA-MB-231 cells, thus maintaining its anticancer activity. This finding is consistent with that reported early in human lymphoblastic leukemia cells [10].Viability of MDA-MB-231 cells treated with (a) ZER and (b) ZER-NLC after 24, 48, and 72 hours.
(a)
(b)Viability of MCF-10A cells treated with (a) ZER and (b) ZER-NLC after 24, 48, and 72 hours.
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(b)


### 3.3. Morphological Characterization Using Normal Inverted Microscope

Both ZER and ZER-NLC treatments suppressed proliferation of MDA-MB-231 cells while the proliferation of untreated cells (control) increased and remained confluent during all incubation periods. Morphologically, MDA-MB-231 cells treated with ZER and ZER-NLC showed irregular shape, membrane blebbing, cell shrinkage, chromatin condensation, and presence of apoptotic bodies (Figure3).Morphology of MDA-MB-231 cells treated with ((d), (e), (f)) ZER and ((g), (h), (i)) ZER-NLC after 24, 48, and 72 hours. (a), (b), and (c) are untreated cells after 24, 48, and 72 hours, respectively. MB = membrane blebbing, NF = nuclear fragmentation, CS = cell shrinkage, and AB = apoptotic body (×400).
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### 3.4. Morphological Characterisation Using Acridine Orange/Propidium Iodide (AO/PI)

Using the AO and PI double-staining method, the breast cancer cells treated with ZER and ZER-NLC showed apoptotic features in a time-dependent manner, while untreated cells showed green nuclear staining suggesting intact nuclear structure (Figure4). Early apoptosis in treated cells indicated DNA fragmentation after 24 hours, as shown by the bright-green color. Chromatin condensation was also observed by the appearance of orange color in the cells indicating formation of apoptotic bodies, characteristic of late apoptosis. On the other hand, necrotic cells, characterised by reddish-orange nuclei, were also observed after 72 hours of treatments.Figure 4
Fluorescent micrograph of AO/PI-stained cells treated with ZER and ZER-NLC. (a) Untreated cells, (b1 and b2) 24-hour treatment, (c1 and c2) 48-hour treatment, and (d1 and d2) 72-hour treatment. VC = viable cells; CC = chromatin condensation; CF = chromatin fragmentation; MB = membrane blebbing; LA = late apoptosis; SN = secondary necrosis.


### 3.5. Apoptosis Detection by Annexin Assay

MDA-MB-231 cells treated with ZER and ZER-NLC showed apoptosis, while viable cells number decreased with period of treatment (Figure5; see Supplementary Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/742738). There was a significant shift (P<0.05) from early to late apoptosis in the distribution of treated MDA-MB-231 cells after ZER and ZER-NLC treatments. This shift in cell distribution is accompanied by decrease in distribution of viable cells, particularly after 48 hours of treatment. The Annexin V assay further confirms that both ZER and ZER-NLC induce apoptosis in MDA-MB-231 cells in a time-dependent manner.MDA-MB-231 cell treated with ZER and ZER-NLC and labeled with FITC-conjugated Annexin V and propidium iodide. ((a), (b), (c)) untreated cells, ((d), (e), (f)) cells treated with ZER, and ((g), (h), (i)) cells treated with ZER-NLC. Cells were treated for 12, 24, and 48 hours.
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### 3.6. Cell Cycle Analysis Using Flow Cytometry

The results demonstrated that ZER and ZER-NLC induced depletion of the G1 phase and S phase of MDA-MB-231 cells with concomitant accumulation of the sub-G0/G1 and G2/M phases at all periods of treatment (Figure6 and Table S2). The study also showed that both ZER and ZER-NLC caused marked arrest of (P<0.05) MDA-MB-231 cells at G2/M phase that increased with duration of treatment. Concurrently, the sub-G0/G1 peak became evident beginning as early as 12 hours of treatment in the ZER-NLC-treated cells. This peak appeared later, at 24 hours with ZER treatment. The results show that both ZER and ZER-NLC are potent in the induction of apoptosis of the MDA-MD-231 cells. Cell cycle arrest is a normal regulatory pathway that allows for tissue maintenance, such as repair of cell damage. Cell cycle arrest is also dependent on availability of essential hormones, growth factors, and nutrients [22]. In disease, gene mutation and tumorigenesis can be induced by defects in these cell cycle checkpoints leading to uncontrolled proliferation. With the treatment of ZER and ZER-NLC, MDA-MB-231 cell cycle arrest at G2/M phase is proposed to occur through increased expression of cyclin B1, Cdk1, Cdc25C, and Cdc25B proteins [23].Cell cycle analysis of MDA-MB-231 cells treated with ((d), (e), (f)) ZER and ((g), (h), (i)) ZER-NLC after 24, 48, and 72 hours. (a), (b), and (c) are untreated cells after 24, 48, and 72 h, respectively.
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### 3.7. Apoptosis Detection Using TUNEL Assay

Nuclear fragmentation was observed in MDA-MB-231 cells treated with either ZER or ZER-NLC. The DNA fragmentation shows that the rate of apoptosis of MDA-MB-231 cells increased gradually and significantly (P<0.05) during the treatment periods (Figure 7). However, untreated cells proliferated without showing apoptosis (Table S3).TUNEL flow cytometric analysis of MDA-MB-231 cells treated with ((d), (e), (f)) ZER and ((g), (h), (i)) ZER-NLC after 24, 48, and 72 hours. (a), (b), and (c) are untreated cells after 24, 48, and 72 h, respectively.
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### 3.8. Detection of Caspase Protease Activity

Caspases are among essential enzymes of the apoptosis-signaling pathway. Caspase-3, initiated by caspase-8, plays a key role in the death receptor pathway, while caspase-9 is a major player in the mitochondrial pathway [24, 25]. Thus these caspases were chosen to determine the mechanism of apoptosis in the MDA-MB-231 cells treated with ZER and ZER-NLC (Figure 8). In this study, the activity of caspase-3 and caspase-9 increased significantly (P<0.05) with time in ZER- and ZER-NLC-treated cells. However, there was no increase in caspase-8 activity in the MDA-MB-cells during the same period of treatment (Table S4). The results suggest that ZER and ZER-NLC induced apoptosis of MDA-MB-231 via the intrinsic pathway.Figure 8
Caspases activity in MDA-MB-231 cells treated with ZER and ZER-NLC for 12, 24, and 48 h.


### 3.9. Western Blotting

Determination of pro- and antiapoptotic proteins is another method of ascertaining the mode of apoptosis in treated cells. In this study, Bax, Bcl-2, and Bcl-xL protein expressions in MDA-MB-231 cells treated with ZER and ZER-NLC were determined. Other proteins, PCNA and cytochrome c, that play roles in cell proliferation and death were also estimated. The treatment of MDA-MB-231 cells with ZER and ZER-NLC caused significant (P<0.05) downregulation of Bcl-2 and Bcl-xL proteins and PCNA, while the Bax protein was upregulated and cytochrome c protein expression increased (Figure 9). The relative density of Bax and cytochrome c bands in both ZER- and ZER-NLC-treated groups increased with duration of treatment, whereas the relative density of Bcl-2, Bcl-xL, and PCNA decreased in comparison with their control (untreated) groups (Tables S5 and S6).Western blot expression apoptosis-regulating proteins in MDA-MB-231 cells treated with (a) ZER and (b) ZER-NLC after 12, 24, and 48 h. The treated cells were compared with untreated cells (control).
(a)
(b)Cytochrome c, released by the mitochondria, is responsible for the activation of caspase-3 and caspase-9. It is suggested that the anticancer effect of ZER and ZER-NLC is through the upregulation of Bax, cleaving poly(adenosine diphosphate-ribose) polymerase (PARP) with concurrent downregulation of Bcl-2 [14]. The apoptosis of MDA-MB-231 cells induced by ZER and ZER-NLC that occurs through the intrinsic pathway is the result of increased permeability of mitochondrial membrane and release of cytochrome c that subsequently activates proapoptotic Bax and inhibits antiapoptotic Bcl-2 and Bcl-xL. These results are consistent with that obtained by a previous study [23]. Therefore, the study shows that ZER and ZER-NLC induced intrinsic apoptotic pathway via downregulation of antiapoptotic and upregulation of apoptotic proteins facilitated the permeabilization of outer mitochondrial membrane and release of cytochrome c. This event activates caspase-9, followed by activation of caspase-3 and cleavage of PARP [23]. Concurrently, ZER and ZER-NLC treatments decreased PCNA resulting in suppression of cancer cell proliferation [25].


## 3.1. Zerumbone-Loaded NLC

The ZER-NLC was shown to be physically stable, with a particle size of52.68±0.1 nm, a zeta potential of -25.03±1.24 mV, and a polydispersity index of 0.29±0.0041 μm [10, 14].


## 3.2. Cytotoxic Effect of ZER and ZER-NLC on MDA-MB-231 Cells

Several studies showed that ZER exerts antiproliferative effects on several types of cancer cell while minimally affecting normal cells [20, 21]. The IC50 for ZER on MDA-MB-231 cells were 10.15±0.9, 8.3±0.15, and 5.96±0.13 μg/mL, while for ZER-NLC they were 11.45±0.12, 9.4±0.18, and 6.01±0.11 μg/mL after 24, 48, and 72 hours of incubation, respectively. On the MCF-10A cell, a normal epithelial mammary gland cell line, the IC50 of ZER and ZER-NLC at the same incubation periods were similarly low (Figures 1 and 2). This study also suggests that incorporation of ZER into NLC did not change the cytotoxic effect of the compound on MDA-MB-231 cells, thus maintaining its anticancer activity. This finding is consistent with that reported early in human lymphoblastic leukemia cells [10].Viability of MDA-MB-231 cells treated with (a) ZER and (b) ZER-NLC after 24, 48, and 72 hours.
(a)
(b)Viability of MCF-10A cells treated with (a) ZER and (b) ZER-NLC after 24, 48, and 72 hours.
(a)
(b)


## 3.3. Morphological Characterization Using Normal Inverted Microscope

Both ZER and ZER-NLC treatments suppressed proliferation of MDA-MB-231 cells while the proliferation of untreated cells (control) increased and remained confluent during all incubation periods. Morphologically, MDA-MB-231 cells treated with ZER and ZER-NLC showed irregular shape, membrane blebbing, cell shrinkage, chromatin condensation, and presence of apoptotic bodies (Figure3).Morphology of MDA-MB-231 cells treated with ((d), (e), (f)) ZER and ((g), (h), (i)) ZER-NLC after 24, 48, and 72 hours. (a), (b), and (c) are untreated cells after 24, 48, and 72 hours, respectively. MB = membrane blebbing, NF = nuclear fragmentation, CS = cell shrinkage, and AB = apoptotic body (×400).
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## 3.4. Morphological Characterisation Using Acridine Orange/Propidium Iodide (AO/PI)

Using the AO and PI double-staining method, the breast cancer cells treated with ZER and ZER-NLC showed apoptotic features in a time-dependent manner, while untreated cells showed green nuclear staining suggesting intact nuclear structure (Figure4). Early apoptosis in treated cells indicated DNA fragmentation after 24 hours, as shown by the bright-green color. Chromatin condensation was also observed by the appearance of orange color in the cells indicating formation of apoptotic bodies, characteristic of late apoptosis. On the other hand, necrotic cells, characterised by reddish-orange nuclei, were also observed after 72 hours of treatments.Figure 4
Fluorescent micrograph of AO/PI-stained cells treated with ZER and ZER-NLC. (a) Untreated cells, (b1 and b2) 24-hour treatment, (c1 and c2) 48-hour treatment, and (d1 and d2) 72-hour treatment. VC = viable cells; CC = chromatin condensation; CF = chromatin fragmentation; MB = membrane blebbing; LA = late apoptosis; SN = secondary necrosis.


## 3.5. Apoptosis Detection by Annexin Assay

MDA-MB-231 cells treated with ZER and ZER-NLC showed apoptosis, while viable cells number decreased with period of treatment (Figure5; see Supplementary Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/742738). There was a significant shift (P<0.05) from early to late apoptosis in the distribution of treated MDA-MB-231 cells after ZER and ZER-NLC treatments. This shift in cell distribution is accompanied by decrease in distribution of viable cells, particularly after 48 hours of treatment. The Annexin V assay further confirms that both ZER and ZER-NLC induce apoptosis in MDA-MB-231 cells in a time-dependent manner.MDA-MB-231 cell treated with ZER and ZER-NLC and labeled with FITC-conjugated Annexin V and propidium iodide. ((a), (b), (c)) untreated cells, ((d), (e), (f)) cells treated with ZER, and ((g), (h), (i)) cells treated with ZER-NLC. Cells were treated for 12, 24, and 48 hours.
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## 3.6. Cell Cycle Analysis Using Flow Cytometry

The results demonstrated that ZER and ZER-NLC induced depletion of the G1 phase and S phase of MDA-MB-231 cells with concomitant accumulation of the sub-G0/G1 and G2/M phases at all periods of treatment (Figure6 and Table S2). The study also showed that both ZER and ZER-NLC caused marked arrest of (P<0.05) MDA-MB-231 cells at G2/M phase that increased with duration of treatment. Concurrently, the sub-G0/G1 peak became evident beginning as early as 12 hours of treatment in the ZER-NLC-treated cells. This peak appeared later, at 24 hours with ZER treatment. The results show that both ZER and ZER-NLC are potent in the induction of apoptosis of the MDA-MD-231 cells. Cell cycle arrest is a normal regulatory pathway that allows for tissue maintenance, such as repair of cell damage. Cell cycle arrest is also dependent on availability of essential hormones, growth factors, and nutrients [22]. In disease, gene mutation and tumorigenesis can be induced by defects in these cell cycle checkpoints leading to uncontrolled proliferation. With the treatment of ZER and ZER-NLC, MDA-MB-231 cell cycle arrest at G2/M phase is proposed to occur through increased expression of cyclin B1, Cdk1, Cdc25C, and Cdc25B proteins [23].Cell cycle analysis of MDA-MB-231 cells treated with ((d), (e), (f)) ZER and ((g), (h), (i)) ZER-NLC after 24, 48, and 72 hours. (a), (b), and (c) are untreated cells after 24, 48, and 72 h, respectively.
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## 3.7. Apoptosis Detection Using TUNEL Assay

Nuclear fragmentation was observed in MDA-MB-231 cells treated with either ZER or ZER-NLC. The DNA fragmentation shows that the rate of apoptosis of MDA-MB-231 cells increased gradually and significantly (P<0.05) during the treatment periods (Figure 7). However, untreated cells proliferated without showing apoptosis (Table S3).TUNEL flow cytometric analysis of MDA-MB-231 cells treated with ((d), (e), (f)) ZER and ((g), (h), (i)) ZER-NLC after 24, 48, and 72 hours. (a), (b), and (c) are untreated cells after 24, 48, and 72 h, respectively.
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## 3.8. Detection of Caspase Protease Activity

Caspases are among essential enzymes of the apoptosis-signaling pathway. Caspase-3, initiated by caspase-8, plays a key role in the death receptor pathway, while caspase-9 is a major player in the mitochondrial pathway [24, 25]. Thus these caspases were chosen to determine the mechanism of apoptosis in the MDA-MB-231 cells treated with ZER and ZER-NLC (Figure 8). In this study, the activity of caspase-3 and caspase-9 increased significantly (P<0.05) with time in ZER- and ZER-NLC-treated cells. However, there was no increase in caspase-8 activity in the MDA-MB-cells during the same period of treatment (Table S4). The results suggest that ZER and ZER-NLC induced apoptosis of MDA-MB-231 via the intrinsic pathway.Figure 8
Caspases activity in MDA-MB-231 cells treated with ZER and ZER-NLC for 12, 24, and 48 h.


## 3.9. Western Blotting

Determination of pro- and antiapoptotic proteins is another method of ascertaining the mode of apoptosis in treated cells. In this study, Bax, Bcl-2, and Bcl-xL protein expressions in MDA-MB-231 cells treated with ZER and ZER-NLC were determined. Other proteins, PCNA and cytochrome c, that play roles in cell proliferation and death were also estimated. The treatment of MDA-MB-231 cells with ZER and ZER-NLC caused significant (P<0.05) downregulation of Bcl-2 and Bcl-xL proteins and PCNA, while the Bax protein was upregulated and cytochrome c protein expression increased (Figure 9). The relative density of Bax and cytochrome c bands in both ZER- and ZER-NLC-treated groups increased with duration of treatment, whereas the relative density of Bcl-2, Bcl-xL, and PCNA decreased in comparison with their control (untreated) groups (Tables S5 and S6).Western blot expression apoptosis-regulating proteins in MDA-MB-231 cells treated with (a) ZER and (b) ZER-NLC after 12, 24, and 48 h. The treated cells were compared with untreated cells (control).
(a)
(b)Cytochrome c, released by the mitochondria, is responsible for the activation of caspase-3 and caspase-9. It is suggested that the anticancer effect of ZER and ZER-NLC is through the upregulation of Bax, cleaving poly(adenosine diphosphate-ribose) polymerase (PARP) with concurrent downregulation of Bcl-2 [14]. The apoptosis of MDA-MB-231 cells induced by ZER and ZER-NLC that occurs through the intrinsic pathway is the result of increased permeability of mitochondrial membrane and release of cytochrome c that subsequently activates proapoptotic Bax and inhibits antiapoptotic Bcl-2 and Bcl-xL. These results are consistent with that obtained by a previous study [23]. Therefore, the study shows that ZER and ZER-NLC induced intrinsic apoptotic pathway via downregulation of antiapoptotic and upregulation of apoptotic proteins facilitated the permeabilization of outer mitochondrial membrane and release of cytochrome c. This event activates caspase-9, followed by activation of caspase-3 and cleavage of PARP [23]. Concurrently, ZER and ZER-NLC treatments decreased PCNA resulting in suppression of cancer cell proliferation [25].


## 4. Conclusion

This study shows that loading of ZER into NLC did not compromise the antiproliferative effect of ZER. Both ZER and ZER-NLC significantly induced apoptosis via the intrinsic pathway in time-dependent manner. The proposed mechanism of apoptosis of cancer cells induced by ZER and ZER-NLC is via activation of caspase-9 and caspase-3, inhibition of antiapoptotic protein, and stimulation of proapoptotic protein expressions. Loading of ZER into NLC will increase the bioavailability of the insoluble ZER in the treatment of cancers.



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*Source: 742738-2014-11-18.xml*