Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor
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Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southern China. Although there are conventional therapies, the side effects and toxicities are not always tolerable for patients. Recently, the tumoricidal effect of ginsenosides on different cancer cells has been studied. This study aims to investigate the anti-cancer effect of ginsenosides on NPC cells and their underlying mechanism.
The cytotoxicity of ginsenosides on NPC cell line HK-1 was measured by MTT assay. Apoptosis was detected by propidium iodide staining followed by flow cytometry. A xenograft tumor model was established by injecting nude mice with HK-1 cells. The activation of caspases and apoptosis-inducing factor (AIF) were evaluated by Western blot analysis. Nuclear translocation of AIF was also studied by immunofluorescence staining. Mitochondrial membrane potential was measured by JC-1 dye using flow cytometry.
Four ginsenosides, 20 (S)-Rh2, compound K (CK), panaxadiol (PD) and protopanaxadiol (PPD), induced apoptotic cell death in HK-1 cells in a concentration-dependent manner. CK inhibited HK-1 xenograft tumor growth most extensively and depleted mitochondrial membrane potential depolarization and induced translocation of AIF from cytoplasm to nucleus in HK-1 cells. In addition, depletion of AIF by siRNA abolished CK-induced HK-1 cell death.
Ginsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo.
KeywordsMitochondrial Membrane Potential Ginsenosides Human Astrocytoma Cell FV1000 Confocal Scanning Laser Microscope Nuclear Extraction Buffer
Nasopharyngeal carcinoma (NPC) is a head and neck cancer with a distinctive ethnic and geographic distribution. In Southern China, NPC has a high incidence of about 25–30 per 100,000 persons per year, in contrast to the low incidence of less than 1 per 100,000 persons per year was recorded in Western countries . The common treatment for NPC is radiotherapy. Besides the undesirable side effects of radiotherapy, the location of the tumor also leads to complications after treatment. Chemotherapy is an alternative in treating NPC but resistance to conventional drugs is a challenge. Therefore, new multiple regimens such as radiochemotherapy and combination treatments with adjuvant drugs are being studied .
Ginsenosides are a group of saponin glycosides, which contribute to the pharmacological effects of ginseng . More than 40 ginsenosides have been separated and identified from ginseng [4, 5] and can be classified into three groups: protopanaxadiols (PPD) (e.g., Rb2, Rc, Rd, Rg3, and Rh2), protopanaxatriols (PPT) (e.g., Re, Rf, Rg1, Rg2, and Rh1), and oleanolic acid derivatives [6, 7]. Structure-activity relationship studies on different ginsenosides and their anti-cancer effects have been demonstrated that ginsenosides with a sugar moiety at C-6 (PPT-type) exhibit less cytotoxicity than those without a sugar moiety at C-6 (PPD-type) . In the last few years, ginsenosides were reported to be responsible for the vasorelaxation, antioxidation, anti-inflammation, anti-angiogenesis and anti-cancer effects of ginseng [9, 10]. Ginsenosides PPD and Rh2 exhibited anti-proliferative effects on intestinal and glioma cell models [11, 12, 13]. Apoptosis induction by different ginsenosides was also demonstrated on human astrocytoma cells, human epidermal carcinoma cells, HeLa cells, and HT-29 colon cells [14, 15, 16, 17, 18]. Compound K (CK) is the major metabolite of PPD-type ginsenosides, and is transformed by intestinal bacteria . CK is rapidly absorbed in the gastrointestinal tract and is retained for a long time in rat plasma [20, 21]. The anti-angiogenic effect of CK was also reported [14, 16, 22]. Although the anti-cancer effects of ginsenosides have been studied extensively in other cancer models, the effect of ginsenosides on NPC is unknown. Several natural compounds extracted from plants could induce apoptosis in NPC through the mitochondria-dependent pathway. For example, capsaicin (EC50 ~ 300 μM) from hot chili peppers , aloe emodin (EC50 100 μM) , and rhein (EC50 180 μM)  isolated from the rhizome of rhubarb, induced depletion of mitochondrial membrane potential and subsequent AIF release in NPC-derived cell lines. However, ginsenosides and especially CK are more potent (EC50 15 μM) than these natural compounds. Although caspase-dependent apoptosis induced by CK was reported in other cancer cell-lines [26, 27, 28], cell type-specific intracellular signaling might account for the discrepancy observed. The adjuvant effect of ginsenosides has been demonstrated by increasing chemotherapy efficacy  and patient survival rates [29, 30].
This study aims to investigate the anti-cancer effects and action mechanism of ginsenosides on NPC cells.
High-performance liquid chromatography-purified ginsenosides as standard compounds (purity >98%) were purchased from Fleton Natural Products (Chengdu, China). Stock solutions of PPD (20 mM), CK (40 mM), and 20 (S)-Rh2 (40 mM) were prepared in dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louise, MO, USA), while PD (20 mM) was prepared in absolute ethanol.
Cell culture and drug treatments
NPC cell line HK-1 was maintained in RPMI 1640 medium (Gibco, Grand Island , NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin and streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO2. HK-1 cells were starved in medium with 1% FBS for 24 h before drug treatment. Cells were treated with indicated concentrations of ginsenosides for different times in medium supplemented with 1% FBS.
Cell viability assay
Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HK-1 cells (1 × 104 cells/well) were seeded onto 96-well plates and incubated overnight. Cells were starved in medium with 1% FBS for 24 h and then subjected to different treatments for another 24 h. After that, MTT solution (USB, Cleveland, OH, USA) was added into each well to a final concentration of 0.5 mg/mL and incubated for 3 h. The culture medium was then removed and DMSO was added to solubilize the purple formazan product. Absorbances at wavelengths of 540 and 690 nm were measured by a microplate reader (ELx800, Biotek, Winooski, VT, USA).
Cell cycle analysis
HK-1 cells (1.5 × 105 cells/well) were seeded onto 6-well plates and incubated overnight. Cells were starved in medium with 1% FBS for 24 h and then treated with different ginsenosides for 24 h. Cells were harvested, washed with PBS (Gibco) twice, and fixed in 70% ethanol at −20°C. The cells were then stained with propidium iodide solution (Sigma-Aldrich) containing RNase A (1 mg/mL) (Roche, Mannheim, Germany). Cell-cycle analysis was performed with the FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and the data were analyzed with the Cell Quest and the Modfit LT Version 3.0 software (Verity Software House, Topsham, Maine, USA).
Western blot analysis
After drug treatment, cytosolic and nuclear lysates were extracted with the NE-PER Nuclear Protein Extraction Kit (Millipore, Bedford, MD, USA) according to the manufacturer’s protocol. The cytosolic fraction was extracted with cytoplasmic lysis buffer (1× cytoplasmic lysis buffer, 0.5 mM DTT, 1:1000 dilution of inhibitor cocktail) while the nuclear fraction was extracted with nuclear extraction buffer (1× nuclear extraction buffer, 1:1000 dilution of inhibitor cocktail). Protein concentrations were determined with the Bio-Rad Dc protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then probed with primary antibodies (anti-AIF) (Cell Signaling, Beverly, MA, USA) and subsequently incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG) (Invitrogen). After washing with 0.1% TBS-T (USB), the membrane was visualized by an enhanced chemiluminescence detection system (Bio-Rad). For the cytosolic fraction, protein expression was compared with β-actin (Sigma-Aldrich). For the nuclear fraction, lamin A/C (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for normalization.
Xenografts in nude mice
Male BALB/c nude mice were purchased from the Animal Services Centre of Chinese University of Hong Kong. For the animal study, HK-1 cells were harvested and washed twice with PBS. For each site of injection, 3 × 106 HK-1 cells were suspended in 100 μL serum-free RPMI-1640 culture medium and mixed with Matrigel in a 1:1 ratio (BD Biosciences). The cell-matrigel mixture was inoculated subcutaneously into the left and right flanks of 6–7 week-old nude mice. When the tumors were palpable (8 days after injection), the tumor-bearing animals were randomly divided into two groups (four mice per group). In group 1, mice were treated with 10 mg/kg/day CK orally (CK was mixed in a 0.5% carboxymethyl cellulose [CMC] suspension). In group 2, mice were treated with 0.5% CMC orally as the control. Tumor sizes were measured daily and calculated using the formula (L × W2)/2 mm3 (L = length; W = width). The experiment was performed according to the Animals (Control of Experiments) Ordinance (Hong Kong) and followed the Hong Kong Baptist University’s guidelines on animal experimentation. The tumor inhibition (%) was calculated as follows:
Tumor inhibition (%) = (Tumor volume of control group − Tumor volume of CK-treated group)/(Tumor volume of control group).
Detection of mitochondrial membrane potential
HK-1 cells were incubated with 5 μg/mL JC-1 dye (Invitrogen) for 30 min. After that, cells were trypsinized and resuspended in PBS for flow cytometry analysis. JC-1 monomers and J-aggregates were detected by a flow cytometer on the FL1 and FL2 channels, respectively. The mitochondrial membrane potential is presented by the 580/530 nm ratio.
HK-1 cells were seeded on a glass coverslip at a density of 2 × 105 cells/well in a 6-well plate and incubated overnight. Cells were starved with 1% FBS medium for 24 h and then treated with or without CK for another 8 and 24 h. The medium was then removed and the glass cover slips were washed with PBS. After that, cells were fixed with 4% paraformaldehyde (Sigma Aldrich) for 10 min followed by washing with PBS three times. Cells were permeabilized with 0.2% of Triton X-100 for 10 min followed by washing with PBS. Cells were probed with anti-AIF antibody in 3% BSA (Sigma Aldrich) overnight at 4°C and then secondary antibody (PE-conjugated goat-anti-rabbit IgG) (Invitrogen) for 2 h at room temperature. After washing with PBS, the coverslip was incubated with DAPI (0.5 g/mL) (Invitrogen) for 5 min. Coverslips were mounted with fluorescence mounting medium on slides and were subjected to examination and image capture by an Olympus FV1000 confocal scanning laser microscope (Essex, UK).
Transfection of small interference RNA (siRNA)
HK-1 cells were seeded onto 6-well plates overnight, cells were then transfected with AIFM1 specific siRNA (50 nM) (Ambion, Austin, TX, USA) using Lipofectamine RNAiMAX transfection reagent (Invitrogen) in antibiotic-free RPMI-1640 culture medium. Drug treatment was performed 48 h after transfection.
All data were presented as mean ± standard deviation (S.D). Comparisons were subjected to Student’s t-test or Kruskal-Wallis One Way Analysis of Variance (ANOVA) followed by Dunnets post hoc test for multiple comparisons. Statistical significance was accepted at P < 0.05.
Ginsenosides 20(S)-Rh2, CK, PD, and PPD exhibited cytotoxicities towards HK-1 cells
Ginsenosides induced apoptosis in HK-1 cells
Ginsenosides induced caspase activation in HK-1 cells
CK attenuated HK-1 xenograft tumors in vivo and induced caspase-independent apoptosis
CK induced apoptosis-inducing factor (AIF) translocation and mitochondrial membrane depolarization
Ginsenosides were reported to exhibit anti-proliferative, anti-metastatic, and anti-angiogenic activities in different in vitro and in vivo tumor models [10, 32, 33, 34, 35]. However, different ginsenosides induced diverse biological effects on different models due to structural differences. The number of sugar moieties were found to mediate ginsenosides activity by altering hydrophilicity. Moreover, aglycone ginsenosides (i.e., CK, PPD, and PPT) showed higher cytotoxicity than glycosides. This property of ginsenosides also mediated their affinity towards different molecular targets. CK is the major metabolite of all PPD-type ginsenosides in both rat and human plasma [20, 21, 31]. Aside from its tumoricidal effects, CK was shown to have neuroprotective, hypoglycemic, and antidepressant-like effects in mice, and enhancement of type I procollagen levels in ultraviolet-A-irradiated fibroblasts [36, 37, 38, 39]. In the present study, HK-1 cells had a similar response towards 20(S)-Rh2, CK, PD, and PPD, and ginsenoside CK showed the most potent sub-G1 phase induction.
Apoptosis is a common type of cell death induced by anti-cancer drugs. Ginsenosides can induce apoptosis in different cancer models including human astrocytoma cells, HT-29 colon cells, A431 cells, and HeLa cells [14, 17, 18, 40]. Apoptosis is mainly induced by a caspase cascade or translocation of AIF . There are two pathways of caspase activation, which are the cell surface death receptor pathway (extrinsic) and mitochondria-initiated pathway (intrinsic). Caspase-3 is the “execute” caspase for the apoptotic induction, while caspase-8 and caspase-9 are the critical caspases and signify the activation of the extrinsic and intrinsic pathways, respectively . In our study, we demonstrated apoptosis induction and caspase activation of ginsenosides in NPC cells. And pretreatment with caspase inhibitors did not reverse the cell death of CK-treated cells. This indicated that CK-induced cell death was caspase-independent. Besides inducing apoptosis, caspase activation was involved in other cellular responses, such as differentiation or cell migration . Therefore, the CK-activated caspase cascade did not participate in the apoptotic execution.
Ginsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo.
The present work is supported by the Area of Excellence Scheme (AoE/M-06/08) by the Research Grant Council of Hong Kong SAR Government; the Faculty Research Grant (FRG2/09-10/068) of the Hong Kong Baptist University, and Dr. Gilbert Hung Ginseng Laboratory Fund. We also thank Mr Yuk-Kit Chan for technical assistance.
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