ANTICANCER RESEARCH 24: 2649-2656 (2004)

Apoptosis in Cervical Cancer Cells: Implications for Adjunct Anti-Estrogen Therapy for Cervical Cancer

DAZHI CHEN1, TIMOTHY H. CARTER1,2,3 and KAREN J. AUBORN1,3

1North Shore-Long Island Jewish Research Institute, 350 Community Drive, Manhasset, NY 11030; 2Department of Biological Sciences, St. John’s University, Jamaica, NY 11530; 3Department of Otolaryngology, Long Island Jewish Medical Center, The Long Island Campus of Albert Einstein College of Medicine, New Hyde Park, NY 11040, U.S.A.

Abstract. Background: Many tumors show dependence on from the observation that these tumors typically arise in the estrogen for growth and establishment of drug resistance. We most estrogen-responsive tissue, the "tranformation examined the effects of estrogen on cervical cancer cells zone"(3). This restricted localization occurs in spite of the exposed to apoptotic agents including drugs used for treatment. fact that the viral co-factors for cervical cancer, certain Materials and Methods: We tested the effect of estradiol on highly oncogenic strains of human papillomavirus (HPV), apoptosis in three cervical cancer cell lines. Apoptosis was are commonly found in cells from all regions of the cervix, measured by endonucleolytic degradation of DNA. Bcl-2 was in surrounding tissues and in the male genitalia (4,5). A measured by Western analysis. Results: Estradiol reduced the clear indication of the involvement of estrogen in cervical percentage of cells undergoing apoptosis after exposure to the cancer was that mice with HPV16 transgenes develop DNA-damaging agents UVB, mitomycin-C and . cervical cancer when given estradiol chronically (6). Protection against taxol-induced apoptosis was marginal. The mechanism by which estrogen accomplishes this Protection was independent of HPV gene expression, and not effect is not fully understood. Estrogen stimulates the specific to apoptosis induced by DNA damage, since estradiol growth of many tissues, such as breast and cervix (2,7,8), significantly reduced the number of apoptotic cells produced including anchorage-independent growth (2,9). However, after exposure to indole-3-carbinol (I3C), a non-genotoxic the situation is more complex than simple growth phytochemical effective in preventing HPV-induced tumors. stimulation by estradiol. An interplay between estrogen and Higher concentrations of I3C overcame the anti-apoptotic HPVs occurs, each apparently amplifying the other’s effects. effect of estradiol. Treatment with I3C resulted in loss of the In the transformation zone, estradiol has a high rate of survival protein Bcl-2, and estradiol partially reversed this conversion to 16a-hydroxyestrone (1), a metabolite that effect. Conclusion: Estrogen protects cervical cancer cells prolongs estrogen effects (10,11), and is genotoxic (2,9). treated with DNA-damaging agents; UVB, mitomycin-C and This conversion is dramatically increased when cervical cells cisplatin, from apoptotic death. For I3C, which induces are immortalized with HPV16 (1). Whereas the virus apoptosis and is anti-estrogenic, the amount of apoptosis increases estrogenic activity, the expression of integrated versus survival and the level of Bcl-2 depend on the HPV oncogenes in transformed cervical cell lines is itself I3C/estradiol ratio. stimulated by estrogen (12,13). In highly oncogenic strains of HPV, these genes encode proteins that inactivate the Induction and progression of cervical cancer are both cellular tumor suppressor proteins p53 (14) and Rb (15), estrogen-enhanced processes (1,2). In fact, one of the first respectively, abrogating normal cell-cycle checkpoints and indications that cervical cancer is estrogen-dependent came leading to uncontrolled cell division. Another potential mechanism by which estrogen could stimulate the development of cervical cancer involves interference with the process of programmed cell death, or Correspondence to: Karen J. Auborn, PhD, Head Phytochemical apoptosis. Cell growth, whether in tumors, tissues, or cell Research, North Shore-Long Island Jewish Research Institute, culture, is now generally recognized to represent a balance BoasMarks Biomedical Science Research Building, 350 Community Drive, Manhasset, NY 11030, USA. Tel: 516 562-1184, between two actively regulated processes: cell division and Fax: 516 562-1022, e-mail: [email protected] cell death (16). All cells are thought to have the potential to kill themselves in a highly regulated fashion in response to Key Words: Cervical cancer, estrogen, apoptosis, indole-3-carbinol. appropriate developmental or distress signals. For example,

0250-7005/2004 $2.00+.40 2649 ANTICANCER RESEARCH 24: 2649-2656 (2004) during the course of normal differentiation, keratinocytes phosphate-buffered saline (PBS) and examined microscopically. undergo many of the morphologic changes associated with The percentage of cells excluding trypan blue was determined by apoptosis (17,18). Fragmentation of DNA is one of the major counting 500 - 1500 cells. cytopathologic hallmarks of apoptosis (17, reviewed by 19). UV irradiation. For cells growing in monolayer the medium was In the pathological case, cells exposed to DNA damaging replaced with PBS, and the cultures were exposed for 15sec to 576 agents such as UVB will enter the apoptotic pathway via a J/m2 of UVB from a G30T8 30 watt germicidal lamp (General mechanism involving p53 (20,21,22). Although the signaling Electric). The UVB output was monitored with a shortwave pathways leading to apoptosis are diverse, they have several ultraviolet measuring Meter (Blak-rat J-225, UVP, Inc., Upland, points of convergence, including the requirement that an CA, USA) at a distance of 25 cm. After exposure, culture medium apoptosis suppressor function be absent or inactivated. One was replaced, and the cells were grown for 18 h. of the most-studied of these apoptosis suppressors is a family Protein was quantitated with the MicroBCA kit (Pierce, Inc. of proteins that includes the bcl-2 gene and its protein Rockford, IL, USA) using a BSA standard. product, Bcl-2 (reviewed by 16). A reduction in Bcl-2 protein, or its complete absence, often accompanies apoptosis. Western blotting. Cells treated with I3C or vehicle controls, with or Investigators have reported that estrogen is anti-apoptotic in without estradiol, were lysed at room temperature in buffer breast cancer cells, and that estrogen treatment is containing 10 mM NaHPO4, 2% triton X-100, 1.2% SDS, 1.0% accompanied by increased Bcl-2 expression (23,24). Likewise, DOC supplemented just before use with 2 ÌM aprotinin, 100 ÌM estrogen increases Bcl-2 in human kerationcytes (25) and phenylmethylsulfonyl fluoride (PMSF) and 1mM EDTA, boiled for 2 min, and centrifuged for 10 min at 12,000 x g at 4ÆC. neuronal cells (26), preventing oxidative stress and glutamate- Supernatant solutions were stored at -80ÆC until use. One induced apoptosis, repectively. Alternately, estrogen hundred Ìg of extract protein in sample buffer (125 mM Tris-HCl, starvation in breast cells provokes apotosis with only marginal pH6.8, 1%SDS, 2% ‚-mercaptoethanol and 0.01% bromophenol changes in Bcl-2-related proteins (27). blue) was loaded onto a 12% SDS-polyacrylamide gel. After However, unlike most cervical cancers, the breast cancer electrophoresis at 32V for 3 h at room temperature, protein bands cells used in many of the studies contain a functional p53 were transferred to an Immobilon-P membrane (Millipore Corp., and are not infected by HPV. We report here that estrogen Bedford, MA USA) by electroblotting overnight in Transfer Buffer (192mM glycine, 25mM Tris and 20% methanol). Before also protects cervical cancer cells from apoptosis, suggesting incubation with antibodies, the membrane was blocked with that interference with apoptosis, possibly through a TBST/milk (20mM Tris-HCl, 137mM NaCl, 1.5% nonfat dry milk regulatory mechanism that bypasses p53 and increases Bcl- and 0.1% Tween20, pH7.6) for 1h. Bcl-2 was detected with mouse 2 expression, may be a general pathway by which estrogen monoclonal anti-Bcl-2 antibody (Santa Cruz Biotechnology, Inc., stimulates tumor cell growth in hormone-responsive Santa Cruz, CA, USA) by incubating for 1h. After washing in cancers. We show and discuss the possible use of an anti- TBST/milk, the filters were incubated with HRP-conjugated anti- estrogen in overcoming the anti-apoptotic effect of mouse IgG antibody (Santa Cruz) at 1/2000 dilution for 1h at room temperature. Antibody bound to protein was detected using estrogen. the enhanced chemiluminescence system (Amersham LIFE Science, Piscataway, NJ, USA). Materials and Methods TUNEL Assay used a kit (in situ cell death detection kit, POD, Reagents. 17‚- estradiol (E2), indole-3-carbinol, and from Boehinger Mannheim Inc. Indianapolis, IN, USA). Cells were were purchased from Sigma (St. Louis, MO, USA). Cis- grown for 24 h in 8-well chamber slides seeded with 105 cells per diamminedichloroplatinum was purchased from Aldrich Chemical well, treated and incubated of 37ÆC, for 1 or 2 days depending on Co. Inc. (Milwaukee, WI, USA). the treatment. The slides were washed in PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Fixed cells Cell lines and cell culture. The cervical cancer cell lines CaSki were washed in PBS, permeabilized with sodium citrate buffer (containing multiple copies of integrated HPV16 DNA) and C33A containing 0.1% Triton X-100 for 2 min on ice, and then incubated (HPV-negative, mutant p53) were obtained from the American with terminal deoxynucleotidyl transferase for 1 h at 37ÆC. After Type Culture Collection (ATCC, Manassas, VA, USA). C33AE6 rinsing with PBS, the slides were treated with converter-POD cells were stably-transfected with the HPV16 E6 gene (conjugated with horseradish peroxidase) at 37ÆC for 30 min and (pLXSN16E6 from D. Galloway, Fred Hutchinson Cancer mounted with a glass coverslip. At least 200 cells/well were Research Center, Seattle, WA, USA) and express low levels of E6 evaluated for staining. transcripts. All cells were grown in monolayer at 37ÆC, 5% C√2 in RPMI medium with glutamine and bicarbonate (GIBCO-BRL, DNA fragmentation was assessed directly by gel electrophoresis. Gaithersburg, MD, USA), supplemented with 10% fetal bovine After treatment, the cells were harvested, centrifuged at 500xg for serum, penicillin and streptomycin. 5 min and washed with PBS. The cell pellet was lysed in 200 Ìl of lysis buffer containing 50 mM Tris-HCl, 20 mM EDTA and 1% Cell viability was assessed by trypan blue exclusion. Trypsinized NP-40, and centrifuged at 1600xg for 5 min. The supernatant cells were incubated with 0.2% trypan blue (GIBCO-BRL) in solution was incubated with (5 Ìg/ml) RNAse A for 2 h at 56ÆC

2650 Chen et al: Estrogen Inhibits Apoptosis in Cervical Cancer Cells

Figure 1. Effect of estradiol on cell survival after UV or mitromycin C exposure. Panel A: CaSki cells growing in monolayer were exposed to the indicated doses of UVB and then grown for 18 h with and without various concentrations of estradiol. Viable cells were determined by trypan blue exclusion. Panel B. Cells were treated with mitomycin C for 24 h in the presence or absence of estradiol at indicated concentrations, and viability determined as in panel A. P≤0.5 for treatment and 10mM E2 (A and B).

and then digested with proteinase K (2.5 Ìg/ml) at 37ÆC for 16 h. Because cell death after moderate UVB exposure is DNA was precipitated with an equal volume of 10M ammonium known to be apoptotic, we next asked whether increased acetate and 2.5 volumes of ethanol. The precipitates were rinsed survival of CaSki cells after irradiation was reflected by a with 70% ethanol, air dried, dissolved in TE buffer (10 mM Tris- decrease in the number of cells undergoing apoptosis. Cell HCl, pH 7.5, 1 mM EDTA), electrophoresed through a 1.5% agarose gel, stained with ethidium bromide and photographed on a cultures were irradiated and then exposed to varying UV transilluminator. concentrations of estradiol as before. DNA fragmentation was assessed by agarose gel electrophoresis 18 h after Results irradiation (Figure 2A) and also by TUNEL assay (Figure 2B). Nucleosomal "laddering" of cell DNA characteristic of Estradiol increases cell survival and decreases apoptosis after UV apoptosis was clearly observed in irradiated cells (Figure 2A, irradiation. Ultraviolet light at 254nm (UVB) induces lane 3), and degradation was decreased in a dose-dependent apoptosis in many cell types, including keratinocytes (22). This manner by E2 (Figure 2A, lanes 4-6). No laddering was process results in a rate of cell killing greater than that observed in DNA from estradiol-treated, un-irradiated cells expected from the genotoxic effects of UV. We asked whether (Figure 2A, lane 7). These results were confirmed more estradiol could protect cells from killing by UVB (Figure 1a). quantitatively by TUNEL assay (Figure 2B). At 10 ÌM E2, When CaSki cells growing in monolayer were UV-irradiated, the fraction of cells containing multiple DNA strand breaks cell death (measured by trypan blue exclusion) was dependent 18 h after UV exposure (16%) was significantly reduced on the intensity of UV exposure, with 40% survival 18 h after compared to irradiated, untreated cells (22%). E2 did not at a standard dose of 576 Joules/m2. After treatment with 10 alter the percentage of un-irradiated cells undergoing ÌM estradiol (E2) immediately following UV irradiation, 18-h apoptosis (7%). Because the anti-apoptotic effect of E2 in survival was increased to nearly 60%. Even at E2 CaSki cells might have been due to its effect on expression of concentrations as low as 100 nM, cell survival was significantly the extensive HPV DNA sequences resident in this cell line improved. Estradiol did not alter the proportion of viable cells (28), we tested the ability of E2 to protect UVB-induced in un-irradiated cultures. The effect of E2 on cell survival after apoptosis in C33A, another cervical cancer cell line believed treatment with another genotoxic apoptotic inducer, to be void of HPV sequences. The results were identical to mitomycin C, was qualitatively similar to its effect on UV- those for CaSki cells except that the C33A cells were more irradiated cells (Figure 1b). sensitive to UVB-induced apoptosis (results not shown).

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Figure 2. Effect of estradiol on DNA fragmentation after U.V. exposure. Panel A: DNA from cells grown and treated as in Fig.1 was analyzed by gel electrophoresis and ethidium bromide staining. Panel B. Cells from the same experiment were immunostained for DNA strand breaks by TUNEL assay. P≤0.5 for treatment and 10mM E2.

Effect of estradiol on apoptosis induced by cisplatin and taxol. Table I. Effect of estradiol on apoptosis induced in C33A cells by cis- Two anti-cancer drugs in current clinical use, cisplatin and platinum and taxol. taxol, have been shown to induce apoptosis in a number of tumor cell lines in vitro (29-32). In particular, taxol induces Cis-platinum Taxol Estradiol % Apoptotic Cells (± s.d.) apoptosis in breast tissue which, like the cervix, is estrogen- responsive. We tested the effect of E on apoptosis in the 2 0 0 0 9.7 ± 1.7 C33A cervical cancer cell line treated with each of these drugs (Table I). E2 inhibited the induction of apoptosis by 10 ÌM 0 0 19.6 ± 2.1 cisplatin to a small but significant extent. Although the 10 ÌM 0 0.1 ÌM 19.6 ± 2.5 systematic error (p=0.054) in the TUNEL assay did not 10 ÌM 0 1.0 ÌM 15.8 ± 1.0 allow us to conclude that E2 reduces taxol-induced 10 ÌM 0 10.0 ÌM 13.6 ± 0.6 apoptosis in CaSki cells, a trend in the E dose response in 2 0 10.0 ÌM 0 22.3 ± 3.3 taxol-treated cells suggests that E2 may affect this case also. Estradiol reduces apoptosis of cervical cancer cells induced 0 10.0 ÌM 0.1 ÌM 17.8 ± 1.2 by indole-3-carbinol in vitro. We have determined that 0 10.0 ÌM 1.0 ÌM 18.1 ± 1.5 indole-3-carbinol (I3C) is a potent apoptotic inducer in 0 10.0 ÌM 10.0 ÌM 16.3 ± 2.5 human cervical cancer cell lines in vivo and in vitro using 0 0 0.1 ÌM 9.3 ± 1.4 multiple indicators of apoptosis (33). Others have observed 0 0 1.0 ÌM 9.0 ± 1.4 that I3C induced apoptosis in breast cancer cell lines (34, 35). Unlike UV, cisplatin, taxol and mitomycin C, however, 0 0 10.0 ÌM 9.8 ± 1.8 I3C does not damage DNA directly. We therefore tested C33A cells were treated for 24 h with cis-platinum or taxol, with and whether E2 could also counteract the apoptotic effect of without estradiol, and apoptosis was evaluated by TUNEL. Results are I3C. In this experiment, we used CaSki, C33A and C33AE6 expressed as mean ± SD.

2652 Chen et al: Estrogen Inhibits Apoptosis in Cervical Cancer Cells

Table II. Effect of relative amounts of indole-3-carbinol and estradiol on apoptosis.

Indole-3- Estradiol % Apoptotic carbinol Cells (± s.d.)

0 0 13.2 ± 1.8 50 ÌM 0 24.6 ± 1.8 100 ÌM 0 28.6 ± 3.2 50 ÌM 10 ÌM 21.6 ± 4.7 100 ÌM 1 ÌM 25.8 ± 4.7 100 ÌM 10 ÌM 23.9 ± 1.2 100 ÌM 100 ÌM 22.3 ± 1.3 200 ÌM 10 ÌM 27.5 ± 2.8 0 0 14.4 ± 4.2 0 0 15.4 ± 3.3 0 0 15.8 ± 4.0

C33A cells were treated with varying concentrations of indole-3- carbinol and estradiol for 48 h. Apoptosis was determined by TUNEL. Results are expressed as mean ± SD.

(p≤0.05). HPV sequences did not contribute either to the induction of apoptosis by I3C or to the protective effect of E2. However, C33A cells, without the viral oncogenes, were the most sensitive to induction of apoptosis by I3C.

Relative amounts of I3C and estradiol determine the amount of I3C-induced apoptosis. I3C induces multiple anti- estrogenic activities (13,36-38) separate from inducing apoptosis. Therefore, two opposing activities (anti- estrogenic versus estrogenic and apoptotic versus anti- apoptotic) occur when both I3C and estrogen coexist. We then asked about how the relative amounts of I3C and estradiol affect apoptosis (Table II). Higher concentrations of I3C partially abrogated the protective effect of estradiol. Conversely, higher concentrations of estradiol are more Figure 3. Effect of estradiol on DNA fragmentation induced by indole-3- carbinol. C33A, C33AE6 or CaSki cells in monolayer were treated with effective in preventing the apoptotic effect caused by I3C. I3C for 48 h with or without added estradiol at various concentrations. DNA strand breaks were analyzed microscopically by TUNEL. Estradiol partially restores Bcl-2 levels in indole-3-carbinol- treated cervical cancer cells. Apoptosis in a number of cell types and circumstances correlates with reduced (cells stably transfected HPV16-E6 gene). As shown in intracellular levels of the Bcl-2 gene product. We found that Figure 3, exposure to I3C for 24 h (untreated controls) I3C reduces Bcl-2 expression in cervical cancer cell lines increased the percentage of cells scoring positive in the (33). It was therefore reasonable to ask whether E2 could TUNEL assay for all of the cell lines (C33A, 35% > restore Bcl-2 levels in I3C-treated CaSki cells, and this C33AE6, 21%, > CaSki, 13%). E2 partially blocked this I3C- proved to be the case (Figure 4). Whereas the steady-state induced apoptosis (28%, 18% and 9% for C33A, C33AE6 content of Bcl-2 determined by Western analysis was and CaSki, respectively). The inhibition was dependent on reduced approximately 10-fold by 200 ÌM I3C, 100 nM E2 the dose of E2 (not shown), and the effect of E2 at 10 ÌM restored Bcl-2 levels to 50 % of untreated controls, and 1 ÌM was significant for C33A and C33AE6 but not CaSki E2 raised Bcl-2 levels even further.

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I3C-induced apoptosis and, conversely, increased concentrations of I3C could abrogate the inhibitory effect of E2 on I3C-induced apoptosis. The level of protection from estrogen was consistent with the overall increase in the cell survival protein Bcl-2, whose levels increase after estrogen treatment (38,39). This supports the idea that most, if not all, of the protective effect of E2 might be due to its effect on Bcl-2. As with apoptosis, I3C decreased the level of Bcl-2 and E2 could abrogate this effect of I3C. Interesting, perhaps because of its anti-estrogenic activities, I3C has become a treatment strategy for both laryngeal papillomatosis (benign tumors with an HPV etiology) (40,41) and, more recently, for cervical dysplasia (42). In vitro, high Figure 4. Effect of estradiol on Bcl-2 expression in C33A cells treated with indole-3-carbinol. Cells were treated with I3C, or left untreated, for 48 h concentrations of I3C need to be used since this with or without added estradiol, and their Bcl-2 content was measured by compound is slowly converted into active condensation Western analysis. Membranes were stained with fast green to determine products, e.g. diindolymethane, compounds rapidly relative amounts of protein. formed in acid environments as in the stomach (43) whereas in vivo benefits for cervical disease occur at concentrations obtainable from eating cruciferous vegetables (6, 42). Discussion Interference with apoptosis by E2 probably occurs downstream from or independently of the DNA damage- This study not only identifies a mechanism whereby E2 sensing pathway that involves p53, because the could increase transformation of cervical cells, but also has phytochemical I3C has not been shown to damage DNA. important implications for the treatment of cervical cancer Additionally, I3C inactivates many carcinogens by with cisplatin or other agents that induce apoptosis. inducing phase I and phase II enzymes, and directly Our data clearly show that E2 significantly diminished reduces DNA damage by reducing free radicals (reviewed the apoptotic response in three human cervical cancer by 44,45). The I3C-induced apoptosis in breast cancer cell lines exposed to a variety of agents, including UVB, does not depend on p53 (34). This conclusion is mitomycin-C, cisplatin and I3C. Inhibition of the taxol- consistent with the fact that one of the HPV early genes induced apoptosis was highly suggestive but not expressed in two of the cell lines (C33AE6 and CaSki) statistically significant. Our results were generally in would inactivate p53, and the other cell line, C33A, accord with those obtained for MCF-7 breast cancer cells, contains a mutant p53. Inactivation of p53 should except for taxol, where E2 was highly effective for abrogate the normal UVB-stimulated apoptotic response inhibiting taxol-induced apoptosis (24). If the taxol result pathway. Our results with cervical cancer cell lines are represents an underlying tissue type difference, this may generally in accord with those obtained in breast cancer have therapeutic significance for the use of taxol for lines, and the protective action of E2 in breast cancer cervical cancer. Separately, it is worth noting that a cells (23,24) might also be independent of p53 status, substantial background of apoptotic cells was observed in although this remains to be determined. Finally, our untreated cultures. The lack of effect of E2 on this studies suggest that HPV genes, which are expressed in population of cells suggests that a different mechanism most cervical cancers, may make them more resistant to may be at work to induce untreated cells in culture to apoptosis. However, the E2 inhibition of apoptosis is undergo apoptotic cell death. clearly independent of the HPV status. Since apoptotic agents such as cisplatin are used as Clearly, the results of this study indicate the need to for cervical cancer and many other consider estrogen status in the treatment of cervical cancer, cancers, our data suggest that anti-estrogens should be since estrogen has the potential to interfere with the an adjunct treatment in order to prevent abrogation of effectiveness of certain treatment modalities. apoptosis by estrogen and survival of tumor cells. Because the phytochemical I3C is an anti-estrogenic Acknowledgements compound as well as an inducer of apoptosis, it is logical that the relative amounts of E2 and I3C would affect This work was supported by Grant No. 6RO1CA73385 (KJA) from apoptosis in cervical cells (Figures 4,5). E2 could inhibit the National Cancer Institute, USA.

2654 Chen et al: Estrogen Inhibits Apoptosis in Cervical Cancer Cells

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