Journal of Medical Virology 88:127–134 (2016)
Gallic Acid Induces Apoptosis in Human Cervical Epithelial Cells Containing Human Papillomavirus Type 16 Episomes Lin Shi,1,2 Yanjun Lei,1 Ranjana Srivastava,2 Weihua Qin,3 and Jason J. Chen2,4* 1
Department of Immunology and Microbiology, Xi’an Jiaotong University Health Science Center, Xi’an, China Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA 3 SoonFast Pharma Science & Technology, Guangzhou, China 4 Cancer Research Center, Shandong University School of Medicine, Jinan, Shandong, China 2
The high-risk human papillomaviruses (HPV) that infect the anogenital tract are strongly associated with the development of cervical carcinoma, which is the second most common cancer in women worldwide. Therapeutic drugs specifically targeting HPV are not available. Polyphenolic compounds have gained considerable attention because of their cytotoxic effects against a variety of cancers and certain viruses. In this study, we examined the effects of several polyphenols on cellular proliferation and death of the human cervical cancer cells and human cervical epithelial cells containing stable HPV type 16 episomes (HPVep). Our results show that three polyphenols inhibited proliferation of HeLa cells dose-dependently. Furthermore, one of the examined polyphenols, gallic acid (GA), also inhibited the proliferation of HPVep cells and exhibited significant specificity towards HPV-positive cells. The anti-proliferative effect of GA on HPVep and HeLa cells was associated with apoptosis and upregulation of p53. These results suggest that GA can be a potential candidate for the development of anti-HPV agents. J. Med. Virol. 88:127–134, 2016. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
human papillomavirus; antiviral; exifone; propyl gallate; gallic acid
INTRODUCTION Human papillomavirus (HPV) are small DNA viruses that replicate in the stratified layers of skin and mucosa and give rise to benign lesions such as warts or papillomas. Infection of the high-risk HPVs is strongly associated with the development of cervical carcinoma and a subset of oropharyngeal cancer [zur Hausen, 2002; Mehanna et al., 2013]. Genital C 2015 WILEY PERIODICALS, INC.
warts are highly transmissible and affect all races and socioeconomic groups. Over 5% of 18- to 59-year old participants of the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2004 have been diagnosed with genital warts [Dinh et al., 2008]. There are more than 200 genotypes of HPV, a subset of which are associated with the development of malignant lesions and are classified as “high-risk” because of its cancer-promoting ability. Reportedly, HPV is present in 99.7% of all cervical neoplasma [Munoz et al., 2003; Moody and Laimins, 2010]. Ninety percent of cervical cancers are caused by nine types of HPV in which type 16 is responsible for about 50% of cases. Worldwide, approximately, 500,000 new cases of cervical cancer are diagnosed each year and half of which are fatal [Thaxton and Waxman et al., 2015]. However, there is no specific medical treatment available that target HPV. Cutaneous, genital warts and advanced cervical dysplasia are mainly treated by destroying or removing the infected tissue by cytotoxic agents or surgery [Beutner and Ferenczy, 1997; von Krogh et al., 2011]. Although physically ablative therapies are often effective in short term, the recurrence rate is as high as 25%–39% [Maw, 2004]. Though prophylactic HPV vaccines have been available, it offers no benefit to millions of people who are already infected by HPV.
Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81471944; 81201284.; Grant sponsor: National Cancer Institute; Grant number: R01CA119134. Conflict of interest: None. Correspondence to: Jason J. Chen, Cancer Research Center, Shandong University School of Medicine, No. 44, Wenhua West Rd Jinan, Shandong 250012, China. E-mail:
[email protected] Received in original form 26 January 2015; revised form 23 May 2015; Accepted 30 May 2015 DOI 10.1002/jmv.24291 Published online 25 June 2015 in Wiley Online Library (wileyonlinelibrary.com).
128
Shi et al.
An effective and specific anti-HPV agent is in urgent need. In addition to the primary antioxidant activity, some naturally existing polyphenols and analogues have a wide variety of biological functions, including the anti-carcinogenic activity and the anti-viral effect [Inoue et al., 1995; Rosl et al., 1997; Reddy et al., 2001; Fiuza et al., 2004; Yokoyama et al., 2004; Prusty and Das, 2005; Prusty et al., 2005; Savi et al., 2005; Divya and Pillai, 2006; Uozaki et al., 2007; Kratz et al., 2008a, 2008b; Choi et al., 2010; Han et al., 2010; Sagara et al., 2010; Singh et al., 2010; Siegel et al., 2010; Tomita et al., 2010; You and Park, 2011; Di Domenico et al., 2012]. Several herbal extracts containing polyphenols can selectively suppress host cellular transcription factors such as AP-1 and NF-kB in HPV-infected cells [Rosl et al., 1997; Reddy et al., 2001; Prusty and Das, 2005; Prusty et al., 2005; Divya and Pillai, 2006]. The selective suppression and the compositional alteration of these factors are associated with down-regulation of HPV gene expression and induction of apoptosis in the infected cells [Divya and Pillai, 2006]. Recently, an inverse association was observed between antioxidant and HPV persistence, squamous intraepithelial lesions, and cervical neoplasia risk, suggesting the protective effect of natural extracts against HPV persistence and cervical dysplasia [Siegel et al., 2010; Tomita et al., 2010]. Exifone possesses potent antiradical properties [Bentue-Ferrer et al., 1989], and previous findings demonstrated its beneficial effects on age-related cognitive disorders [Porsolt et al., 1987]. Gallic acid (GA) and propyl gallate (PG), two natural plant polyphenols that are used as antioxidant additives in food, cosmetics, and pharmaceutical industry, have exhibited anti-tumor activities against a variety of tumor cell lines [Inoue et al., 1995; Fiuza et al., 2004; Becker 2007; Raina et al., 2008; Chen et al., 2009; Ji et al., 2009; Han et al., 2010; You and Park, 2011]. In the present study, three representative polyphenolic compounds: exifone, PG and GA were evaluated for their ability to inhibit proliferation of HPVexpressing cells. We then focused on the most active anti-HPV polyphenolic compound (GA) and studied the mechanism by which it inhibits the growth of HPV-positive cells. MATERIALS AND METHODS Chemicals Exifone, PG and GA (Fig. 1) were obtained from Sigma Chemicals Co. (St. Louis). Exifone and PG were dissolved in dimethyl sulfoxide (DMSO) and diluted in cell culture media to the final concentration of 0.1%. GA was dissolved in distilled water and filtered through a 0.22-mm filter. All chemicals used in this study were analytical grade unless otherwise specified. J. Med. Virol. DOI 10.1002/jmv
Fig. 1. Chemical structures of polyphenols. (A) Exifone (3,4,5,20 ,30 ,40 - hexahydroxybenzophenone); (B) Pentyl gallate (3,4,5-trihydroxybenzoic acid propyl ester); (C) Gallic acid (3,4,5-trihydroxybenzoic acid).
Cell Culture Primary human keratinocytes (PHKs) were derived from neonatal human foreskin epithelium obtained from the University of Massachusetts Hospital as described previously [Liu et al., 2007]. PHKs and human cervical epithelial cell line containing stable HPV-16 episomes (HPVep) [Sprague et al., 2002; Berger et al., 2006] were grown in keratinocyteserum free medium (K-SFM) supplemented with 5 ng/ ml epidermal growth factor and 50 mg/ml bovine pituitary extract (GIBCO-BRL, Gaithersburg). Retinal pigment epithelial cells (RPE1) [Uetake et al., 2004] were originally maintained in a 1:1 diluted medium of DMEM and Ham’s F12 (GIBCO-BRL, Gaithersburg), plus 10% fetal calf serum (FBS) (GIBCO-BRL, Gaithersburg). HeLa cells (HPV-18 positive human cervical carcinoma cell line) were originally purchase from ATCC, recently identified by Genetica DNA Laboratories, and maintained in a DMEM medium plus 10% FBS. Cytotoxicity Assay The anti-proliferative effects of polyphenolic compounds against above mentioned cell lines (PHKs, HPVep, HeLa, and RPE1) were detected by the 1 CellTiter-Glo luminescent cell viability assay (Promega, Madison), a method that counts viable cells in a culture tube based on quantitation of ATPs present. This assay relies on the property of a thermostable luciferase, which generates stable glow-type luminescent signals in the presence of ATP and the intensity of the signals is directly proportional to cell viability. This assay was performed according to the manufacturer’s instructions. Briefly, cells were seeded in triplicate in opaque 96well plates at 1 103 cells per well. Twenty-four hours after seeding the cells, media were changed, and compounds were added at the final concentrations of 0, 12.5, 25, 50, 75, and 100 mM respectively. DMSO was used as the negative control. After 48 h incubation at 37˚C, the substrate was added. The plates were incubated at room temperature for 10 min to stabilize luminescent signals, and then the LUMs (cps) were measured using an EnVision 2102 Multilabel Reader (Perkin Elmer, Waltham). The 50% growth inhibitory concentration (IC50) was calculated for each compound from the dose response curve (48 h of incubation).
Gallic Acid Induces Apoptosis in HPV-positive Cells
129
PE Annexin V and 7-AAD Stain
Statistical Analysis
The HPVep and HeLa cells at 80% confluence were treated with GA at 25 to 100 mM at 37˚C. As long-term culture resulted in too much cell debris, the assay was limited to 24 h. At the end of incubation, both floating and adherent cells were harvested. The cells were washed twice with cold phosphate buffer solution (PBS) and resuspended in 1 binding buffer (0.01 M Hepes/NaOH, 0.14 M NaCl, 2.5 mM CaCl2, pH 7.4) at a concentration of 1 106 cells/ml. Then 100 ml of cell suspension (1 105 cells) was transferred to a 5-ml tube, and 5 ml of PE-Annexin V (BD Pharmingen, San Diego, USA) and 5 ml of 7-AAD (BD Pharmingen, San Diego) were added. The cell suspension with PEAnnexin V and 7-AAD was incubated at room temperature for 20 min in dark. Fluorescence-activated cell sorting (FACS) was then carried out on a LSR II flow cytometer (BD Bioscience, San Jose). The cells without staining were used as negative controls, and the cells with single staining were used as compensation controls. The FACS data were analyzed by FlowJo software (Tree Star Inc. Ashland).
The data are expressed as mean standard deviation (SD). Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Bonferroni as the post hoc test. P < 0.05 was considered as significant.
Hoechst 33342 Nuclear Stain Apoptotic cells were also detected by staining with hoechst 33342 (Sigma, St. Louis), a DNAspecific fluorescent dye, which stains the condensed chromatin of apoptotic cells more brightly compared to that of normal cells. The HPVep cells growing on glass cover slips treated with GA for 24 h and 48 h were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After two washings in ice-cold PBS, hoechst 33342 dye (5 mg/ml in PBS) was added and the cells were incubated for 30 min at room temperature in dark. Cell morphology was examined with a Nikon ECLIPSE E400 microscope and photographed with a SPOT digital camera. Western Blot Analysis HPVep and HeLa cells were treated with various concentration of GA (0, 50, 100, and 150 mM) for 24 h. cells were harvested and rinse with PBS followed by extraction in 200 ml RIPA lysis buffer in the presence of protease inhibitor. Equal amount of each sample were separated by 12% Tris-Glycine gels then transferred to PVDF membranes (GE Whatman, Little Chalfont, UK). Membrane was blocked using blocking buffer (5% dry milk in TBST), followed by incubation with primary antibody against p53 (Santa Cruz, Dallas, USA) and GAPDH (Santa Cruz, Dallas). Membrane was analyzed after being incubated with horse-radish peroxidase-conjugated secondary antibodies (Beyotime, Beijing, China), followed by use of a BeyoECL Plus chemiluminescent protein detection kit (Beyotime, Beijing, China).
RESULTS AND DISCUSSION Cytotoxic Effect of Polyphenols on HPV-Positive Cells To examine the cytotoxic effect of polyphenols on HPV-positive cells, HeLa, and HPVep cells, along with control PHKs that are negative for HPV, were treated with various concentrations of polyphenolic compounds. Cell proliferation was examined by the 1 CellTiter-Glo assay. As shown in Figure 2, three polyphenols (exifone, PG and GA) inhibited cell proliferation in a dose-dependent manner. The IC50 values for the examined polyphenolic compounds are presented in Table I. Exifone has anti-tumor activity against HeLa cells with IC50 values of 12.3 mM. However, it also exhibited a cytotoxic effect on both control PHKs and HPVep cells. Therefore exifone is not a good candidate for HPV inhibitor. Similarly, PG had a cytotoxic effect on PHKs. Surprisingly, its IC50 value for HPVep was relatively high (82.9 mM). Therefore PG is also not a good candidate for HPV inhibitor. Interestingly and importantly, GA exhibited HPV-specific cytotoxicity; its IC50 values were low for both HeLa and HPVep cells (10.2 mM and 12.2 mM, respectively), whereas the value was high for the normal control PHKs (102.4 mM). These results demonstrate that GA is a potential lead for the development of HPV inhibitors. Several previous studies have examined the effect of GA on HeLa cells. In these studies, the inhibitory effect varied. For example, in one study, the IC50 value of GA on HeLa cells was 35.9 mM [Inoue et al., 1995]. In another study, the IC50 value was relatively high (80 mM) [You et al., 2011]. The reason for the difference in the IC50 values from different studies is not clear, the experimental condition may account for some of the differences. Specificity of GA on HPV-Positive Cells Noticeably, our results indicated that GA not only has the significant anti-tumor efficacy on HeLa cells containing integrated HPV18 genome, but also inhibits the proliferation of HPVep cells containing stable HPV-16 episomes. GA maybe has the potential to specifically inhibit the proliferation of HPV-positive cells. However, the selective cytotoxicity of GA on HPV-positive cells could simply be a result of its effect on cell proliferation, as both HeLa and HPVep cells proliferate much faster than PHKs (data not shown). This raised the possibility that GA is inhibitory to cells that proliferate efficiently. J. Med. Virol. DOI 10.1002/jmv
130
Shi et al.
Fig. 2. Cytotoxic effects of polyphenols on HPV-positive cells. Cells were treated with various concentrations of polyphenols for 48 h, the cell growth inhibitory rates were determined with CellTiter-Glo1 Luminescent Cell Viability Assay. (A) PHKs; (B) HeLa cells (C) HPVep cells. The cell growth inhibitory rates are averaged from at least three independent experiments. Data are represented as meanSD.
To rule out this possibility, we determined the IC50 of RPE1 cells, which proliferate at a similar rate as HeLa and HPVep cells (data not shown). As shown in Figure 3, the IC50 of GA on RPE1 cells was 116.2 16.3 mM, which is similar to that of PHKs (102.4 1.0 mM) (P > 0.05), although the latter proliferates much slower. In contrast, although HPVep and RPE1 cells proliferate at similar rates, the IC50 for HPVep cells treated with GA was much lower (12.2 2.1 mM) (P < 0.01). These results demonstrate that the inhibitory activity of GA on HPV-positive cells is not related to the proliferation rate. Therefore, we tentatively conclude that GA exhibits certain specificity on HPV-positive cells. The anti-viral activity of GA may not be limited to HPV containing cells. It was reported that GA exhibited cytotoxicity towards Vero cells containing HSV-1, although the IC50 was significantly higher than what we detected for HPV containing cells (57.1 mM vs. 10 mM) [Kratz et al., 2008b]. Therefore J. Med. Virol. DOI 10.1002/jmv
GA may have a broad-spectrum antiviral activity against cells containing viruses. It will be interesting to characterize viruses that might be inhibited by GA in future studies.
TABLE I. The IC50 Values of the Tested Compounds on PHKs, HeLa and HPVep Cell Lines. Compound Exifone Propyl gallate Gallic acid a
IC50(mM) PHK 41.1 14.7 43.2 7.1 102.4 1.0
a,b
HeLa 12.3 4.9 23.9 1.6 10.2 2.8
HPVep 37.6 1.5 82.9 3.9 12.2 2.1
IC50 values were determined with luminescent cell viability assay after 48 h treatment. Data are mean values of three experiments and are reported as mean standard error of the mean (SEM). b The control cells with DMSO did not show any statistical differences from the control cells without it. P < 0.05, P < 0.01, as compared to PHK.
Gallic Acid Induces Apoptosis in HPV-positive Cells
Fig. 3. The IC50 values of GA on PHKs, HPVep and RPE1 cells. IC50 values were determined with luminescent cell viability assay after 48 h of GA treatment. P < 0.01, as compared to RPE1 and PHK.
Apoptosis is a Putative Mechanism by Which GA Inhibits HPVep Cell Proliferation HPVep cells are human cervical keratinocyte cells containing stable HPV-16 episomes, and exhibite
131
episomally replicating HPV-16 genomes at an estimated copy number between 10 to 50 genomes per cell. HPVep cells express HPV genes E6 and E7 stably. Compared to normal keratinocytes, HPVep cells show partial but incomplete degradation of p53 [Sprague et al., 2002; Berger et al., 2006]. HPVep cells display features that are similar to HPV infected human keratinocytes and appear to be a suitable model for study the mechanism by which GA reduces the viability of HPV-positive cells. To determine the mechanism by which GA reduces the viability of HPVep cells, the GA-treated HPVep cells were detected for apoptosis. Specifically, the treated cells were stained with Annexin-V/7-AAD and subjected to flow cytometry analysis. As shown in Figure 4, GA treatment increased apoptosis of HPVep cells significantly in a dose dependent manner, but did not increase necrosis of the cells. Similar results were also obtained in GA treated HeLa cells (Fig. 5). These results indicated that prompting apoptosis is a mechanism by which GA induces HPV positive cell death. The induction of apoptosis by GA in HPVep cells was further demonstrated by hoechst 33342 fluorescent staining. Consistent with the results of the AnnexinV/7-AAd stain, an increased number of cells with chromatin condensation and fragmentation was
Fig. 4. GA induced the apoptosis of HPVep cells. HPVep cells were treated with GA for 24 h and detected by PE-Annexin-V and 7-AAD double staining. (A) untreated (% of apoptosis: 2.88%); (B) treated with 25 mM GA (% apoptosis: 7.34%); (C) treated with 50 mM GA (% apoptosis: 19.9%); (D) treated with 100 mM GA (% apoptosis: 25.5%). A representative of three independent experiments is shown.
J. Med. Virol. DOI 10.1002/jmv
132
Shi et al.
Fig. 5. GA induced apoptosis of HeLa cells. HeLa cells were treated with GA for 24 h and detected by PEAnnexin-V and 7-AAD double staining. (A) untreated (% of apoptosis: 0.300%); (B) treated with 25 mM GA (% apoptosis: 7.20%); (C) treated with 50 mM GA (% apoptosis: 14.6%); (D) treated with 100 mM GA (% apoptosis: 28.9%). A representative experiment of three is shown.
observed after treatment with GA (25 mM: 10.3% cells showed chromatin condensation/fragmentation, 439 cells examined; 50 mM: 13.5% cells showed chromatin condensation/fragmentation, 438 cells; 100 mM: 22.1% cells showed chromatin condensation/fragmentation, 435 cells.) as compared with untreated HPVep cells (7.2% cells showed chromatin condensation/fragmentation, 607 cells examined) (Fig. 6a). A representative GA-treated HPVep cell with condensed chromatin is shown in Figure 6b. Although it was reported that GA has anti-viral activity against several other viruses [Savi et al., 2005; Uozaki et al., 2007; Kratz et al., 2008a, 2008b; Choi et al., 2010], the mechanism of GA’s anti-viral activity is not known. To the best of our knowledge, this is the first study demonstrating the anti-viral activity of GA involving apoptosis. Upregulation of p53 Correlates With GA Treatment in HPV Containing Cells Several mechanisms have been identified regarding GA induced apoptosis in cancer cells, including: eliciting reactive oxygen species (ROS) [Inoue et al., J. Med. Virol. DOI 10.1002/jmv
1995; Chuang et al., 2010]; via both caspase-dependent and caspase-independent pathways [Sakaguchi et al., 1998; Raina et al., 2008; Ji et al., 2009; Reddivari et al., 2010]; down-regulation of antiapoptosis-related proteins and Akt/mTOR pathway [Faried et al., 2007]; inhibiting ribonucleotide reductase (RR) [Madlener et al., 2007]; induction of p53 and Fas/FasL [Hsu et al., 2007; Chuang et al., 2010]. Our results showed that exifone and PG (a propylderivative of GA) do not have specific cytotoxicity to HPVep cells. Because all three compounds have the potential to elicit ROS [Inoue et al., 1995; Urios et al., 2006; Han et al., 2009; Chuang et al., 2010], the primary mechanism of GA inducing HPVep cell apoptosis could not be the generation of ROS. GA may have a structure specific effect to induce HPV positive cell apoptosis. Alternatively, other polyphenols may have issues with cell permeability, metabolism, etc. HPVep cells contain stable HPV-16 episomes and express viral early oncoproteins E6 and E7 [Sprague et al., 2002; Berger et al., 2006]. The best known function of HPV-16 E6 is to target and degrade the tumor suppressor protein p53 through the ubiquitin pathway [Werness et al., 1990;
Gallic Acid Induces Apoptosis in HPV-positive Cells
133
Scheffner et al., 1990; 1993]. We hypothesized that GA may increase the p53 expression that counteracts E6’s function and thereby induces apoptosis. To test this possibility, we examined the steady-state levels of p53 in HPVep and HeLa cells after treatment with GA. As shown in Figure 7, p53 level went up in response to GA treatment in a dose-dependent manner. In summary, we demonstrated that GA has the inhibitory effect on HPV containing cells; inducing apoptosis is a mechanism by which GA kills cells containing HPV genome, and p53 may play a role in this process. These results suggest that GA can be a potential candidate for the development of anti-HPV agents. Further studies are needed to explore detailed mechanism by which GA induces apoptosis in HPV containing cells and to develop GA as a new lead for HPV infection therapy. ACKNOWLEDGMENTS This work was supported by Grant Numbers 81471944 and 81201284 from the National Natural Science Foundation of China and Grant Number R01CA119134 from the National Cancer Institute. REFERENCES Fig. 6. Increased apoptosis in HPVep cells treated with GA.
HPVep cells growing on glass slip treated with GA for 24 h were fixed in paraformaldehyde and stained with Hoechst 33342. (A) Increased % of cells with chromatin condensation and fragmentation were observed after treatment with GA. (B) Representative images of GA treated HPVep cells were shown, untreated cells served as control. Arrow indicates a cell with condensed and fragmented chromatin.
Fig. 7. GA treatment increased p53 level in HPVep and HeLa cells. HPVep and HeLa cells were treated with various concentration of GA for 24 h. p53 levels were determined by Western blot analysis. GAPDH were used as loading control. (A) HPVep cells. (B) HeLa cells.
Becker L 2007. Final report on the amended safety assessment of Propyl Gallate. Int J Toxicol 26:89–118. Bentue-Ferrer D, Philouze V, Pape D, Reymann JM, Allain H, Van den Driessche J. 1989. Comparative evaluation of scavenger properties of exifone, piracetam and vinburnine. Fundam Clin Pharmacol 3:323–328. Berger KL, Barriga F, Lace MJ, Turek LP, Zamba GJ, Domann FE, Lee JH, Klingelhutz AJ. 2006. Cervical keratinocytes containing stably replicating extrachromosomal HPV-16 are refractory to transformation by oncogenic H-Ras. Virology 356:68–78. Beutner KR, Ferenczy A. 1997. Therapeutic approaches to genital warts. Am J Med 102:28–37. Chen HM, Wu YC, Chia YC, Chang FR, Hsu HK, Hsieh YC, Chen CC, Yuan SS. 2009. Gallic acid, a major component of Toona sinensis leaf extracts, contains a ROS-mediated anti-cancer activity in human prostate cancer cells. Cancer Lett 286: 161–171. Choi HJ, Song JH, Bhatt LR, Baek SH. 2010. Anti-human rhinovirus activity of gallic acid possessing antioxidant capacity. Phytother Res 24:1292–1296. Chuang CY, Liu HC, Wu LC, Chen CY, Chang JT, Hsu SL. 2010. Gallic acid induces apoptosis of lung fibroblasts via a reactive oxygen species-dependent ataxia telangiectasia mutated-p53 activation pathway. J Agric Food Chem 58:2943–2951. Di Domenico F, Foppoli C, Coccia R, Perluigi M. 2012. Antioxidants in cervical cancer: Chemopreventive and chemotherapeutic effects of polyphenols. Biochim Biophys Acta 1822:737–747. Dinh TH, Sternberg M, Dunne EF, Markowitz LE. 2008. Genital warts among 18- to 59-year-olds in the United States, National Health and Nutrition Examination Survey, 1999–2004. Sex Transm Dis 35:772–773. Divya CS, Pillai MR. 2006. Antitumor action of curcumin in human papillomavirus associated cells involves downregulation of viral oncogenes, prevention of NFkB and AP-1 translocation, and modulation of apoptosis. Mol Carcinog 45:320–332. Faried A, Kurnia D, Faried LS, Usman N, Miyazaki T, Kato H, Kuwano H. 2007. Anticancer effects of gallic acid isolated from Indonesian herbal medicine, Phaleria macrocarpa (Scheff.) Boerl, on human cancer cell lines. Int J Oncol 30:605–613. Fiuza SM, Gomes C, Teixeira LJ, Gir~ ao da Cruz MT, Cordeiro MN, Milhazes N, Borges F, Marques MP. 2004. Phenolic acid derivatives with potential anticancer properties?a structure-
J. Med. Virol. DOI 10.1002/jmv
134 activity relationship study. Part 1: Methyl, propyl and octyl esters of caffeic and gallic acids. Bioorg Med Chem 12: 3581–3589. Han YH, Moon HJ, You BR, Kim SZ, Kim SH, Park WH. 2010. Propyl gallate inhibits the growth of HeLa cells via caspasedependent apoptosis as well as a G1 phase arrest of the cell cycle. Oncol Rep 23:1153–1158. Hsu CL, Lo WH, Yen GC. 2007. Gallic acid induces apoptosis in 3T3-L1 pre-adipocytes via a Fas- and mitochondrial-mediated pathway. J Agric Food Chem 55:7359–7365. Inoue M, Suzuki R, Sakaguchi N, Li Z, Takeda T, Ogihara Y, Jiang BY, Chen Y. 1995. Selective induction of cell death in cancer cells by gallic acid. Biol Pharm Bull 18:1526–1530. Ji BC, Hsu WH, Yang JS, Hsia TC, Lu CC, Chiang JH, Yang JL, Lin CH, Lin JJ, Suen LJ, Gibson Wood W, Chung JG. 2009. Gallic acid induces apoptosis via caspase-3 and mitochondriondependent pathways in vitro and suppresses lung xenograft tumor growth in vivo. J Agric Food Chem 57:7596–7604. Kratz JM, Andrighetti-Fr€ ohner CR, Leal PC, Nunes RJ, Yunes RA, Trybala E, Bergstr€ om T, Barardi CR, Sim~ oes CM. 2008a. Evaluation of anti-HSV-2 activity of gallic acid and pentyl gallate. Biol Pharm Bull 31:903–907. Kratz JM, Andrighetti-Fr€ ohner CR, Kolling DJ, Leal PC, CirneSantos CC, Yunes RA, Nunes RJ, Trybala E, Bergstrom T, Frugulhetti IC, Barardi CR, Simoes CM. 2008b. Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate. Mem Inst Oswaldo Cruz 103:437–442. Liu Y, Heilman SA, Illanes D, Sluder G, Chen JJ. 2007. P53-independent abrogation of a postmitotic checkpoint contributes to HPV E6-induced polyploidy. Cancer Res 67:2603–2610. Madlener S, Illmer C, Horvath Z, Saiko P, Losert A, Herbacek I, Grusch M, Elford HL, Krupitza G, Bernhaus A, Fritzer-Szekeres M, Szekeres T. 2007. Gallic acid inhibits ribonucleotide reductase and cyclooxygenases in human HL-60 promyelocytic leukemia cells. Cancer Lett 245:156–162. Maw R 2004. Critical appraisal of commonly used treatment for genital warts. Int J STD AIDS 15:357–364. Mehanna H, Beech T, Nicholson T, El-Hariry I, McConkey C. 2013. Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and neck cancer--systematic review and metaanalysis of trends by time and region. Head Neck 35: 747–755. Moody CA, Laimins LA. 2010. Human papillomavirus oncoproteins: Pathways to transformation. Nat Rev Cancer 10:550–560. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. 2003. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518–527. Porsolt RD, Lenegre A, Avril I, St eru L, Doumont G. 1987. The effects of exifone, a new agent for senile memory disorder, on two models of memory in the mouse. Pharmacol Biochem Behav 27:253–256. Prusty BK, Das BC. 2005. Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin. Int J Cancer 113:951–960. Prusty BK, Husain SA, Das BC. 2005. Constitutive activation of nuclear factor -kB: Preferntial homodimerization of p50 subunits in cervical carcinoma. Front Biosci 10:1510–1519. Raina K, Rajamanickam S, Deep G, Singh M, Agarwal R, Agarwal C. 2008. Chemopreventive effects of oral gallic acid feeding on tumor growth and progression in TRAMP mice. Mol Cancer Ther 7:1258–1267. Reddivari L, Vanamala J, Safe SH, Miller JC, Jr. 2010. The bioactive compounds alpha-chaconine and gallic acid in potato extracts decrease survival and induce apoptosis in LNCaP and PC3 prostate cancer cells. Nutr Cancer 62:601–610. Reddy VG, Khanna N, Singh N. 2001. Vitamin C augments chemotherapeutic response of cervical carcinoma HeLa cells by stabilizing P 53. Biochem Biophys Res Commun 282:409–415.
J. Med. Virol. DOI 10.1002/jmv
Shi et al. Rosl F, Das BC, Lengert M, Geletneky K, zur Hausen H. 1997. Antioxidant-induced changes of the AP-1 transcription complex are paralleled by a selective suppression of human papillomavirus transcription. J Virol 71:362–370. Sagara Y, Miyata Y, Nomata K, Hayashi T, Kanetake H. 2010. Green tea polyphenol suppresses tumor invasion and angiogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-induced bladder cancer. Cancer Epidemiol 34:350–354. Sakaguchi N, Inoue M, Ogihara Y. 1998. Reactive oxygen species and intracellular Ca2þ, common signals for apoptosis induced by gallic acid. Biochem Pharmacol 55:1973–1981. Savi LA, Leal PC, Vieira TO, Rosso R, Nunes RJ, Yunes RA, Creczynski-Pasa TB, Barardi CR, Sim~ oes CM. 2005. Evaluation of anti-herpetic and antioxidant activities, and cytotoxic and genotoxic effects of synthetic alkyl-esters of gallic acid. Arzneimittelforschung 55:66–75. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. 1993. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75:495–505. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. 1990. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63: 1129–1136. Siegel EM, Salemi JL, Villa LL, Ferenczy A, Franco EL, Giuliano AR. 2010. Dietary consumption of antioxidant nutrients and risk of incident cervical intraepithelial neoplasia. Gynecol Oncol 118:289–294. Singh M, Tyagi S, Bhui K, Prasad S, Shukla Y. 2010. Regulation of cell growth through cell cycle arrest and apoptosis in HPV 16 positive human cervical cancer cells by tea polyphenols. Invest New Drugs 28:216–224. Sprague DL, Phillips SL, Mitchell CJ, Berger KL, Lace M, Turek LP, Klingelhutz AJ. 2002. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology 301:247–254. Thaxton L, Waxman AG. 2015. Cervical Cancer Prevention: Immunization and Screening 2015. Med Clin North Am 99:469–477. Tomita LY, Longatto Filho A, Costa MC, Andreoli MA, Villa LL, Franco EL, Cardoso MA. 2010. Brazilian Investigation into Nutrition and Cervical Cancer Prevention (BRINCA) Study Team. Diet and serummicronutrients in relation to cervical neoplasia and cancer among low-income Brazilian women. Int J Cancer 126:703–714. Uetake Y, Sluder G. 2004. Cell cycle progression after cleavage failure: Mammalian somatic cells do not possess a “tetraploidy checkpoint”. J Cell Biol 165:609–615. Uozaki M, Yamasaki H, Katsuyama Y, Higuchi M, Higuti T, Koyama AH. 2007. Antiviral effect of octyl gallate against DNA and RNA viruses. Antiviral Res 73:85–91. Urios A, Largeron M, Fleury MB, Blanco M. 2006. A convenient approach for evaluating the toxicity profiles of in vitro neuroprotective alkylaminophenol derivatives. Free Radic Biol Med 40:791–800. von Krogh G, Lacey CJ, Gross G, Barrasso R, Schneider A. 2000. European course on HPV associated pathology: Guidelines for primary care physicians for the diagnosis and management of anogenital warts. Sex Transm Infect 76:162–168. Werness BA, Levine AJ, Howley PM. 1990. Association of human papillomavirus types 16 and1 8 E6 proteins with p53. Science 248:76–79. Yokoyama M, Noguchi M, Nakao Y, Pater A, Iwasaka T. 2004. The tea polyphenol, ()-epigallocatechin gallate effects on growth, apoptosis, and telomerase activity in cervical cell lines. Gynecol Oncol 92:197–204. You BR, Park WH. 2011. The effects of mitogen-activated protein kinase inhibitors or small interfering RNAs on gallic acidinduced HeLa cell death in relation to reactive oxygen species and glutathione. J Agric Food Chem 59:763–771. zur Hausen H 2002. Papillomaviruses and cancer: From basic studies to clinical application. Nat Rev Cance 2:342–350.