European Journal of Cancer Prevention:
Research Paper: Gynaecological Cancer
Resveratrol induces cell death in cervical cancer cells through apoptosis and autophagy
García-Zepeda, Sihomara P.a; García-Villa, Enriquea; Díaz-Chávez, Joséb; Hernández-Pando, Rogelioc; Gariglio, Patricioa
aDepartment of Genetics and Molecular Biology, Center for Research and Advance Studies, National Polytechnic Institute
bUnit of Biomedical Research in Cancer, Institute of Biomedical Research, UNAM/National Cancer Institute
cDepartment of Experimental Pathology, National Institute for Medical Sciences and Nutrition Salvador Zubiran, Mexico City, Mexico
All supplementary digital content is available directly from the corresponding author.
Correspondence to Patricio Gariglio, PhD, Department of Genetics and Molecular Biology, Center for Research and Advance Studies, National Polytechnic Institute, IPN Av. 2508, PO Box 07360, Mexico City, Mexico Tel: +52 555 747 3337; fax: +52 555 747 3931; e-mail: firstname.lastname@example.org
Received August 15, 2012
Accepted February 11, 2013
Cervical neoplasia is one of the most frequent cancers in women and is associated with high-risk human papillomavirus (HPV) infection. Resveratrol, a natural polyphenolic phytochemical, has received considerable interest on the basis of its potential as a chemopreventive agent against human cancer. In this work, we analyzed the type of cell death induced by resveratrol in several cervical cancer cell lines. Resveratrol treatment (150–250 µmol/l) for 48 h increased cell cycle arrest at the G1 phase in C33A (with mutation in p53) and HeLa cells (HPV18 positive), as well as in CaSki and SiHa cell lines (HPV16 positive). Resveratrol treatment induced apoptosis in all cell lines, particularly in CaSki cells, as measured by Annexin-V flow cytometry analysis. There was a decrease in the mitochondrial membrane potential (apoptosis) in HeLa, CaSki, and SiHa cells and an increased lysosomal permeability (autophagy) in C33A, CaLo (HPV18 positive), and HeLa cell lines. Furthermore, when we used the IC50 of each line, we found that resveratrol produces a similar effect, suggesting that this effect is not dependent on the concentration of resveratrol. Interestingly, after resveratrol treatment, the expression of p53 was decreased in HPV18-positive cell lines (CaLo and HeLa) and increased in HPV16-positive cell lines (CaSki and SiHa) and C33A cells. The expression of p65 (an NF-κB subunit) was decreased after treatment in all cell lines except SiHa cells. These data indicate that resveratrol uses different mechanisms to induce cell death in cell lines derived from cervical cancer.
Worldwide, cervical cancer is the second most common malignancy in women (Phongsavan et al., 2010). Human papillomaviruses (HPVs) are associated with cervical cancer, particularly a subgroup of HPVs designated as the ‘high-risk’ subgroup, which includes HPV16 and 18 (zur Hausen, 2000). As part of their carcinogenic mechanism, these high-risk human papillomaviruses (HR-HPVs) encode E6 and E7 viral oncoproteins that interfere with the function of the tumor suppressor proteins p53 and retinoblastoma, respectively (Narisawa-Saito and Kiyono, 2007).
Resveratrol is a phytoalexin present in more than 70 plant species, including a wide variety of fruits and vegetables such as grapes, berries, peanuts, and various herbs (Kundu and Surh, 2008; Bishayee, 2009). In the first report of resveratrol as a possible cancer chemopreventive agent, Jang et al. (1997) observed that this compound exerts antitumor properties at all three stages of skin carcinogenesis. Resveratrol has been reported to inhibit growth and induce apoptosis in cancer cell lines, including promyelocytic leukemia, breast, prostate, lung, rhabdomyosarcoma, and colon cancer cells (Chow et al., 2005; Alkhalaf, 2007; Kundu and Surh, 2008; Bishayee, 2009; Mann et al., 2009; Malhotra et al., 2011; Leon-Galicia et al., 2012).
There are various types of cell death, namely apoptosis, autophagy, mitotic catastrophe, and necrosis. Apoptosis is characterized by common morphological and biochemical alterations (Le Bras et al., 2006). Autophagy, a dynamic process involving the bulk degradation of cytoplasmic organelles and proteins, participates in cell death under various conditions (Trincheri et al., 2007; Tsuchihara et al., 2009). In addition, it has been proposed in some cases that mitochondrial membrane permeabilization might constitute a common phenomenon that would mark the point of integration and nonreturn of the lethal signal transduction cascade (Chalah and Khosravi-Far, 2008).
Although resveratrol exerts proapoptotic activity in various cancer cell types, the influence of HPV in this process has not been evaluated. Some studies have analyzed the effects of resveratrol on cellular viability and the cell cycle in cervical cancer cell lines when it is combined with radiation or the compound roscovitine (Zoberi et al., 2002; Kramer and Wesierska-Gadek, 2009). The aim of this study was to analyze the type of cell death induced by resveratrol in several cervical cancer cell lines and to find a possible association with the type of HPV infection. We found that resveratrol caused an increase in cell accumulation at the G1 phase and augmented cell death independent of the presence or the type of HPV. However, different cell death pathways seem to be related to particular HPV types. For example, lysosomal rupture participated in cell death induced by resveratrol in HPV18-positive cells and in C33A cells, whereas mitochondrial damage was more evident in cells containing HPV16. These findings suggest that resveratrol acts through different mechanisms to induce cell death in different cell lines derived from cervical cancer.
Materials and methods
Cell culture and treatment
The cervical cancer cell lines C33A (HPV negative), CaLo and HeLa (HPV18 positive), and CaSki and SiHa (HPV16 positive) were obtained from ATCC (Rockville, Maryland, USA), except CaLo, which was donated by Dr Alberto Monroy and Dr Benny Weiss. Cells were cultured in DMEM supplemented with 10% fetal bovine serum with 100 U/ml penicillin and 100 mg/ml streptomycin. Cultures were maintained in 5% CO2 in a humidified atmosphere at 37°C. Resveratrol was purchased from Sigma-Aldrich (St Louis, Missouri, USA), dissolved in ethanol to a concentration of 3 mmol/l, and stored at −20°C. Cells were incubated for 48 h or 5 days with resveratrol concentrations from 10 to 300 µmol/l. Culture media containing fresh resveratrol or ethanol (negative control) were changed daily.
Cell proliferation assay
Cervical cancer cell lines were seeded in a 24-well plate at a density of 50 000 cells/well. After attachment, cell viability was determined using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After resveratrol treatment, MTT (0.5 mg/ml) was added for 30 min. The formazan dye crystals were solubilized with 500 µl of acid isopropanol and absorbance was measured at 570 nm wavelength (Bio-Rad Laboratories, Benicia, California, USA).
Cell cycle distribution analysis
Cells were harvested after their respective treatments and washed with PBS (pH 7.4) before being fixed with 70% ethanol for 1 day at −70°C. Subsequently, cells were centrifuged and residual alcohol was aspirated. Cells were washed with PBS and resuspended with propidium iodide (PI) solution (Sigma-Aldrich) (30 µg/ml) containing RNase (100 µg/ml) and 0.05% Triton X-100 and then incubated at room temperature in the dark for 40 min. The cellular DNA content was then analyzed by flow cytometry evaluating 10 000 events per sample.
Cells were seeded at a density of 200 000 cells/well and treated with different concentrations of resveratrol (0–250 µmol/l in ethanol) for 48 h. Following treatment, the cells were harvested, washed with PBS, and resuspended in 100 µl binding buffer. The cell suspension was mixed with 5 µl of Annexin-V–FITC and 5 µl PI (50 µg/ml) for 10 min. Apoptosis was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, California, USA). Analysis was carried out on 10 000 events using the Summit V4.3 software (Beckman Coulter Inc., Fullerton, California, USA).
Mitochondrial membrane potential detection assay
After treatment with resveratrol or ethanol (negative control) for 48 h, cells were incubated with tetramethylrhodamine ethyl ester (Sigma-Aldrich) at a final concentration of 200 ng/ml for 15 min at 37°C. After washing with PBS, fluorescence intensity was monitored at 582 nm.
Lysosome integrity assessment
Cells treated with resveratrol or ethanol for 48 h were incubated with 5 µg/ml of acridine orange (AO) for 15 min at 37°C. Red lysosomal fluorescence was determined by flow cytometry, and Summit V4.3 software was used for data acquisition and analyses of 10 000 events.
CaLo and HeLa cells were treated for 48 h with 250 µmol/l resveratrol, washed with PBS, and fixed in 10% glutaraldehyde dissolved in cacodylate buffer for 4 h at 4°C, followed by a second fixation with osmium tetroxide fumes. Fixed cells were then dehydrated with a graded ethyl alcohol series and embedded in Epon resins. Thin sections (70 nm) were placed on copper grids, contrasted with uranium salts and lead citrate (Electron Microscopy Sciences, Fort Washington, Pennsylvania, USA), and examined using an M-10 Zeiss electron microscope (Karl Zeiss, Jena, Germany).
Western blot analysis
Cervical cancer cell lines were treated for 48 h with ethanol or resveratrol. Cells were lysed with RIPA lysis buffer. Proteins were separated using 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. After 1 h of blocking with 5% nonfat milk, the membranes were incubated with an anti-nuclear factor κB (NF-κB) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) or anti-p53 monoclonal antibody (Santa Cruz Biotechnology) overnight at 4°C. α-Actin was detected in the same membrane using anti-actin monoclonal antibody (Santa Cruz Biotechnology). We then used an anti-mouse secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich). Immune reactivity was visualized using ECL western blotting detection reagents (Pierce, Thermo Scientific, Rockford, Illinois, USA) and then analyzed by scanning densitometry using the Syngene Image Software (Syngene International Ltd, Iselin, New Jersey, USA).
Results are expressed as the mean±SD of at least three independent experiments. One-way analysis of variance was used for statistical analyses. P values less than 0.05 were considered statistically significant.
Human cervical cancer cell lines show different sensitivity to resveratrol-mediated inhibition of proliferation independent of the presence or the type of human papillomavirus
To analyze the antiproliferative cytostatic effect of resveratrol, we determined the production of MTT in C33A, CaLo, HeLa, CaSki, and SiHa cell lines treated with different concentrations of resveratrol (10–300 µmol/l) for 48 h. A dose-dependent inhibitory effect on the proliferation of cervical cancer cell lines was induced by resveratrol treatment, with inhibitory concentration 50 (IC)50 values in the range of 50–200 µmol/l (C33A 194.6 µmol/l, CaLo 203.9 µmol/l, HeLa 137.1 µmol/l, CaSki 53.5 µmol/l, and SiHa 198.5 µmol/l) (Fig. 1). Despite this varying sensitivity to resveratrol, the cell lines showed no correlation with the presence or type of HPV. To determine whether the reduction in the biotransformation of MTT to formazan was because of cell proliferation arrest (cytostaticity) or cell death (cytotoxicity), we evaluated the effect of resveratrol on the progression of the cell cycle in exponentially dividing cultures of the cell lines mentioned. The data obtained after 48 h of incubation with resveratrol are summarized in Table 1, which shows that 150 µmol/l resveratrol increased the G1 phase and induced a corresponding decrease in S and G2–M phases in CaSki cells, whereas C33A, CaLo, and SiHa cell lines showed an increase in the S phase. An increase in the G1 phase was observed in almost all cell lines with the higher concentration of resveratrol (250 µmol/l), with the exception of CaLo cells, in which no significant changes in cell cycle distribution were observed. Thus, at low resveratrol concentrations, most cell lines are blocked in the S phase, but at higher concentrations, this compound induced an accumulation of cells in the G1 phase in almost all cell lines.
Given that the cell cycle alterations did not completely explain the observed reduction in cell proliferation, we then determined the induction of cell death by resveratrol treatment. Thus, resveratrol-mediated apoptosis in the cancer cell lines was determined by flow cytometry using the Annexin-V method. Because lower resveratrol concentrations (50 µmol/l) required longer exposure times, cervical cancer cell lines were treated with either ethanol (0.3%) or resveratrol (150 or 250 µmol/l) for 48 h. Considering that each cell line shows different sensitivity, we decided to use these concentrations to analyze the effect of resveratrol. We observed a higher increase in the induction of apoptosis in HeLa and CaSki cell lines compared with C33A, CaLo, and SiHa cells at 150 µmol/l resveratrol. When cell lines were treated with 250 µmol/l resveratrol for 48 h, only the C33A cell line failed to show an increase in apoptosis. CaSki cells showed the highest induction of apoptosis compared with the other cell lines (Fig. 2), which was consistent with the previously described reduction in cell viability (Fig. 1). Thus, resveratrol exerts proapoptotic effects on all human cervical cancer cell lines in vitro and the CaSki cell line was the most sensitive to the effects of this compound.
Mitochondrial and lysosomal permeability changes induced by resveratrol in human cervical cancer cell lines
To further elucidate apoptosis mechanisms in cervical cancer cell lines, we considered the perturbation of mitochondrial function as a target. The changes in mitochondria that are related to apoptosis signaling are, among others, decreased membrane potential (ΔΨm) and increased permeability, including cytochrome c release. As a possible mechanism of apoptosis induction, we tested whether resveratrol could decrease the mitochondrial membrane potential in cervical cancer cells. A similar reduction in this potential was observed in HeLa, CaSki, and SiHa cells after 48 h of resveratrol treatment (Fig. 3a), but this reduction was not evident in C33A or CaLo cell lines. To rule out a differential cytotoxic effect of resveratrol because of the different sensitivity shown by the tested cell lines, we used the IC50 of each line. Under these conditions, we again detected a reduction in mitochondrial membrane potential only in HeLa, CaSki, and SiHa cells. This reduction was also observed in CaSki and HeLa cells at low concentrations (30 and 50 µmol/l) after 5 days of treatment. Thus, resveratrol induced mitochondrial membrane permeability in some but not all human cervical cancer cell lines. Interestingly, the HPV16 cervical cancer cell lines (CaSki and SiHa) showed higher mitochondrial membrane permeability than the HPV18 cell lines (CaLo and HeLa).
We observed that resveratrol induces a dose-dependent reduction in cell viability, but we did not observe a corresponding induction of apoptosis. Therefore, we decided to determine whether these cells were dying by another type of cell death. Autophagy can be an early event to trigger apoptosis or an independent mechanism of cell death. Therefore, we studied whether resveratrol could modify the permeability of lysosomal membranes by analyzing the lysosomal retention of AO. Our results showed an increase in lysosomal permeability induced by resveratrol in C33A, CaLo, and HeLa cells (Fig. 3b), whereas the cell lines CaSki and SiHa infected with HPV16 showed lower or null increase in lysosomal permeability. When we used the IC50 concentrations, as above, only C33A, CaLo, and HeLa showed an increase in lysosomal permeability. Concentrations of 30 and 50 µmol/l resveratrol also induced lysosomal permeability in C33A, CaLo, and HeLa after 5 days of treatment. In addition, we observed autophagosomes by electron microscopy, lysosomal swelling, and degranulation in CaLo and HeLa cells treated with resveratrol, in agreement with the results of AO retention (Fig. 4). Thus, it seems that cervical cancer cell lines with HPV18 (HeLa and CaLo) showed the highest lysosomal permeability among the cervical cancer cell lines tested.
The expression of p53 and nuclear factor κB transcription factors is affected by resveratrol in human cervical cancer cell lines
It has been described that p53 plays a central role in the cell cycle and apoptosis, and it has been found that one of resveratrol’s antitumor effects is mediated through the stabilization and activation of the p53 protein (Kundu and Surh, 2008; Bishayee, 2009). Thus, we analyzed the effects of this phytoalexin on p53 in cervical cancer cell lines. We observed that resveratrol increased the expression of the p53 protein in C33A (with a mutation in p53) and in CaSki and SiHa cell lines (both HPV16 positive) (Fig. 5). In contrast, resveratrol treatment downregulated p53 expression in CaLo and HeLa cells (both HPV18 positive). These results suggest that HPV18-positive cell lines could die by a p53-independent mechanism.
However, considering that many natural compounds, including resveratrol, block oncogenic antiapoptotic NF-κB activity (Benitez et al., 2009; Brunelli et al., 2010), we decided to analyze whether resveratrol also affects the expression of the p65 subunit of NF-κB. This subunit was downregulated in most cervical lines analyzed, except in SiHa cells, suggesting that this effect was not dependent on the HPV type (Fig. 5).
The current study extends the knowledge of the participation of resveratrol in the induction of cell death in cervical cancer cell lines. Our results show that resveratrol has selective effects on mitochondrial and lysosomal permeability. In particular, resveratrol mainly increased the mitochondrial permeability in HPV16-positive cervical cancer cells and the lysosomal permeability in HPV18-positive and HPV-negative cancer cell lines. We also observed different sensitivities to resveratrol treatment among the different cell lines analyzed, with CaSki cells being the most sensitive. The differences in the resveratrol-mediated induction of apoptosis among the cervical cancer cell lines observed in this study are in agreement with other reports. For example, Hougardy et al. (2005) reported that CaSki and HeLa cells are more sensitive to Fas-mediated apoptosis than C33A and SiHa cells. In addition, levels of the proapoptotic protein Bid were easily detectable in CaSki and HeLa cells, but levels were very low in SiHa cells (Hougardy et al., 2005). Furthermore, Espinosa et al. (2004) reported that mRNA expression of the proapoptotic Smac/Diablo gene was present in CaSki and HeLa cell lines, but was absent in SiHa and CaLo cells.
Recently, it has been reported that autophagy precedes apoptosis in two cervical cancer cell lines (HeLa and Cx) after resveratrol treatment (Hsu et al., 2009). These authors suggested that resveratrol might cause apoptotic cell death through autophagy in all cervical cancer cell lines. However, we found that upon resveratrol treatment, not all cervical cancer cell lines showed increased lysosomal membrane permeabilization. In particular, HPV16-positive cervical cancer cell lines did not show increased lysosomal membrane permeabilization after resveratrol treatment, whereas both HPV18-positive cell lines used in this study (CaLo and HeLa) showed autophagy, which was clearly shown by electron microscopy.
In addition, we observed an increased expression of p53 in HPV16-positive cell lines following resveratrol treatment. It was shown previously that resveratrol upregulates p53 and induces apoptosis in cells that express this tumor suppressor protein (Bishayee, 2009). Other studies indicated that resveratrol can also induce apoptosis in p53-deficient cells (Kundu and Surh, 2008). It is noteworthy that this phytoalexin increases the concentration of p53 in C33A cells that contain a mutation in its DNA-binding domain at residue 273. In agreement with this result, it was reported that resveratrol causes an increase in the cellular p53 content and serine-15 phosphorylation of p53 in a prostate cancer cell line containing a mutant version of p53 (Lin et al., 2002).
In contrast, resveratrol downregulated p53 in HPV18-positive cervical cancer cell lines, suggesting that it can induce cell death independent of p53 in these cell lines. Consistent with these results is the demonstration that SIRT1 activation by resveratrol reduces both cisplatin-mediated p53 acetylation and the expression of p53-responsive genes such as PUMA and BAX (Kim et al., 2011). This finding could explain in part why HPV18-positive cell lines die principally by autophagy. It has also been reported that the inactivation of p53 by deletion, depletion, or inhibition at the protein level can trigger autophagy, and it has been suggested that p73, a member of the p53 family, transactivates target genes that modulate autophagy positively (Rosenbluth and Pietenpol, 2009). Because resveratrol can induce the upregulation of p73 (Roccaro et al., 2008), it is possible that this gene is also upregulated by resveratrol in HPV18-positive cell lines.
Inhibition of antiapoptotic NF-κB by natural compounds promotes both apoptosis and autophagy (Zhang et al., 2009). Woo et al. (2004) reported that NF-κB activity is significantly reduced by treating CaSki cells with 50 µmol/l resveratrol. Interestingly, here, we show for the first time that resveratrol can downregulate NF-κB protein expression and not only NF-κB translocation to the nucleus as reported previously (Benitez et al., 2009; Brunelli et al., 2010). Furthermore, the CaSki cell line, which was the most sensitive cell line to resveratrol, showed both an upregulation of p53 and downregulation of NF-κB.
Importantly, the data obtained for resveratrol in animal models are very promising and encourage the use of this compound in human clinical trials. In this respect, there are several reports of clinical trials using resveratrol in patients with colon cancer (Nguyen et al., 2009; Patel et al., 2010) and women at an increased risk of breast cancer (Zhu et al., 2012). Nguyen et al. (2009) reported that after 14 days of resveratrol administration, the expression of Wnt target genes was reduced in human normal mucosa, suggesting that low concentrations of resveratrol (80 mg/day) may play a beneficial role in colon cancer prevention. In addition, a recent report on 20 colorectal cancer patients indicates that resveratrol treatment for 8 days before surgical resection led to a significant decrease in the proliferation of colorectal tissue (Patel et al., 2010). Recently, Zhu et al. (2012) suggested a novel mechanism for the chemopreventive effect of resveratrol in the breast tissue of women at an increased risk of breast cancer. In this study, 39 adult women with an increased risk of breast cancer were treated with placebo, 5, or 50 mg resveratrol twice daily for 12 weeks in a randomized double-blind study. They observed a decrease in promoter methylation of the tumor suppressor gene RASSF-1a with increasing levels of resveratrol and resveratrol-glucuronide in the circulation in these patients (Zhu et al., 2012). Interestingly, Howells et al. (2011) reported that resveratrol can reach potentially active concentrations in human tissues that are distant to the gastrointestinal tract. Given that resveratrol may be distributed to many tissues (Juan et al., 2010; Howells et al., 2011) with a possible intracellular presentation as free resveratrol, we might consider it for use as a cancer chemopreventive agent.
Our results suggest that resveratrol induces cell death through different mechanisms. Autophagy was the predominant form of cell death in C33A (HPV negative), CaLo, and HeLa cells (HPV18 positive), whereas CaSki and SiHa (HPV16 positive) cells died by apoptosis.
The authors thank Victor Hugo Rosales-Garcia, Elizabeth Alvarez-Rios and Juan Carlos Leon for their technical assistance, Dr Libia Vega, Dr Javier Hernández-Sánchez from CINVESTAV-IPN, and Dr Apolinar Maya-Mendoza from the University of Manchester for participating in helpful discussions. This work was supported by grants from CONACyT to P. Gariglio and J. Díaz-Chávez (83597 and 168896). S.P. García-Zepeda is grateful to CONACyT for a scholarship (173186).
Conflicts of interest
There are no conflicts of interest.
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cell death; cervical cancer; lysosomal permeability; mitochondrial membrane potential; resveratrol
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