Dr. Weeks’ Comment: If you have cancer, consider progesterone!
Progesterone induces apoptosis and up-regulation of p53 expression in human ovarian carcinoma cell lines
Article first published online: 29 SEP 2000
Progesterone (PROG) has been shown to reduce the risk of developing ovarian carcinoma in postmenopausal women who have undergone estrogen and progestogen replacement therapy, and it has been clinically used to treat some types of ovarian tumors. It is not yet clear whether or not the antitumor activity of progestogen is due to its ability to induce apoptosis in precarcinomatous and carcinomatous ovarian cells. The apoptosis-related genes p53, bcl-2, and c-myc have important roles in the regulation of programmed cell death, and thus may be involved in the process of the suspected PROG-induced apoptosis.
Antiproliferation effects of PROG on 3AO and AO ovarian carcinoma cells were determined by 3H-thymidine incorporation. Apoptosis of the PROG-treated cells was determined by DNA laddering analysis and was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide. Cell cycle analysis was also performed by flow cytometry. The expression of p53, bcl-2, and c-myc after 72 hours of PROG treatment was detected by Northern blot analysis.
In both 3AO and AO cell lines, cells proliferation was maximally inhibited by PROG after 72 hours of treatment at 10 Î¼M concentration. Under the same conditions, more than 50% of PROG-treated cells had undergone apoptosis, whereas less than 3% of the cells were apoptotic in untreated cell cultures. After exposure to PROG for 72 hours, cells were arrested in the G1 phase of the cell cycle, and the levels of p53 mRNA were remarkably increased in both cell lines. No changes in expression of bcl-2 or c-myc were detected.
PROG significantly inhibited cell proliferation and induced apoptosis in both of the ovarian carcinoma cell lines tested in this study. PROG treatment markedly up-regulated p53 expression in these cells, indicating involvement of p53 in PROG-induced apoptosis. Cancer 1997; 79:1944-50. © 1997 American Cancer Society.
Ovarian carcinoma is one of the most common fatal gynecologic malignancies in the world.1 The incidence of this disease rises after women reach menopause due to lower levels of sex steroids.2 Estrogen replacement therapy in postmenopausal women does not reduce ovarian carcinoma risk.3 However, the risk may be reduced by use of combination-type oral contraceptives (COCs),4 which contain estrogen and a high dose of progestogen. In addition, progestogen has been widely used in the clinical treatment of ovarian carcinomas.5 However, the molecular mechanism of the anticancer effect of progestogen is not yet fully understood.
It has been reported that many anticancer agents exert at least part of their effects by triggering programmed cell death,6 and the induction of apoptosis in tumor cells has become a therapeutic objective. For example, recent data show that N-(4-hydroxyphenyl)-all-trans-retinamide, a member of the superfamily of nuclear receptor legends that includes progestogen and estrogen, is effective against human ovarian carcinoma transplanted in mice.7 The retinoid in vitro suppresses human breast carcinoma cell growth by inducing apoptosis.8 Therefore, induction of apoptosis may be one of the key mechanisms mediating the therapeutic effect of progesterone (PROG) in treatment of ovarian carcinoma.
Apoptosis, which plays a key role in normal development and oncogenesis, is a process genetically controlled by a number of genes,9including p53, bcl-2, and c-myc.10, 11 Among them, p53 is one of the most frequently investigated tumor-suppressing genes.12 It has been shown that wild-type p53 can induce apoptosis in variety of cell types,13 and any reagent that induces overexpression of wild-type p53 might promote apoptosis. p53 protein has a critical role in G1 cell cycle arrest,14, 15 and p53-mediated apoptosis and cell cycle arrest have been used as indicators of wild-type and functional p53.16 In this study, our objective was to determine whether PROG could induce apoptosis in ovarian carcinoma cells and whether any apoptosis-related genes were involved in the process.
MATERIALS AND METHODS
Cell Cultures and Drug Treatment
Human ovarian carcinoma cell lines, 3AO and AO, are estrogen- and progestogen-dependent. They were obtained from the Cell Bank at the Chinese Academy of Sciences for this study.17-19 Both cell lines were grown in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Evergreen, Hang Zhou, China), 100 units/mL penicillin, 100 Î¼g/mL streptomycin, and 2 mM glutamine. Exponentially growing 3AO and AO cells (5 Ã— 104 cells/mL) were treated with different concentrations of PROG (Sigma, St. Louis, MO) for 48 hours and 72 hours, respectively.
Assay for Inhibition of 3H-Thymidime Incorporation
3AO and AO cell lines were plated in sextuplicate wells of 96-well microtest plates are treated as described.20 At various intervals after PROG was added, the plates were pulsed with 2 Î¼Ci 3H-thymidine/well (specific activity, 22 Ci/mmol; Shanghai Institute of Nuclear Sciences, Chinese Academy of Sciences), trypsinized, and harvested on strips of fiberglass filter paper with the use of multiple automated sample harvesters. The radioactivity of individual samples was measured in a liquid scintillation counter.
Analysis of DNA Fragmentation by Gel Electrophoresis
Drug-treated and untreated 3AO and AO cells (1 Ã— 106) were washed twice with phosphate-buffered saline (PBS) and resuspended in 25 Î¼L PBS. The cells were lysed by the addition of 25 Î¼L lysis buffer (60 mM Tris, pH 7.4; 50 mM ethylene diamine tetraacetic acid; and 1.6% sodium lauryl sarcosine) containing 1 mg/mL proteinase K, incubated for 3 hours at 50 °C, and digested with 200 Î¼g/mL DNAse-free RNAse A for an additional 20 minutes. DNA from the cell lysates was then analyzed on a 1.2% agarose gel containing ethidium bromide, and visualized and photographed under ultraviolet light.21
Quantitative Analysis of Apoptosis
After being treated with 10 Î¼m PROG and all-trans-retinoic acid (ATRA, Sigma, St. Louis, MO) for 72 hours, cells were centrifuged, and the pellets were gently resuspended in propidium iodide solution (PI; 50 Î¼g/mL in 0.1% sodium citrate plus 0.15 Triton X-100; Sigma, St. Louis, MO).22 Random fields of each treated cell culture were observed under a microscope through a Ã—40 objective lens in fluorescent mode. Apoptotic cells had condensed nuclei, and the percentage of apoptotic cells was calculated by counting approximately 500 cells.
PROG-treated and untreated 3AO and AO cells (2 Ã— 106) were washed twice with PBS containing 0.1% glucose and then fixed in 1 mL ice-cold ethanol overnight at 4 °C. The fixed cells were pelleted and resuspended in 0.5 mL of PBS containing 0.1% glucose, 30 Î¼g/mL PI, and 1 mg/mL RNAse A (Sigma, St. Louis, MO). The DNA contents of the cell were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA) as described.23
Cell Cycle Analysis
Cell cycle distribution was determined by DNA content, as assayed by propidium iodide staining. The percentage of cells in each phase of the cell cycle was determined with the Cellfit software provided by Becton-Dickinson (San Jose, CA) as described.24
Northern Blot Analysis
Extraction of total RNA and Northern blot analysis were performed as described.25 The human DNA probes used in this study were from the Pst I cDNA insert of pMG-WAF1 plasmid for wild-type p53,26 the EcoRI/Hind III cDNA insert of plasmid pFL1 for bcl-2,27 and the EcoRI cDNA insert of plasmid pGDSV7 for c-myc.28 Thirty Î¼g of total RNA were loaded in each lane of a 1% agarose gel containing 3% formaldehyde and transferred to nylon membranes. Blots were hybridized to the probes radiolabelled to specific activity of 1-2 Ã— 109 cpm/Î¼g with Î±-32P dATP (Amersham, Buckinghamshire, United Kingdom). Then the blots were exposed to X-ray films (Kodak, New York, NY) for 3-5 days. The membranes were rehybridized with a Î²-actin cDNA probe to serve as an internal control.
PROG Inhibition of Cell Proliferation
Cell proliferation was dramatically inhibited by PROG treatment in both ovarian carcinoma cell lines (Fig. 1). After 72 hours, 1 Î¼M PROG inhibited 3H-thymidine incorporation by 23% and 21% and 10 Î¼M PROG by 69% and 73% in 3AO and AO cell lines, respectively. In contrast, the inhibitions after 48 hours of treatment with both concentrations of PROG were less significant than those after 72 hours of treatment. Therefore, PROG treatment at 10 Î¼M for 72 hours was chosen for the subsequent experiments.
Analysis of PROG-Induced Apoptosis
After 72 hours of 10 Î¼M PROG treatment, as shown in Figure 2, agarose gel electrophoresis of DNA from the apoptotic cells showed the characteristic DNA fragmentation ladder. Under the same conditions, the percentage of apoptotic cells reached 70 ± 9% in 3AO cells and 49 ± 7% in AO cells, as measured by the counting method (Fig. 3). Under the same conditions, cell apoptosis was approximately 76 ± 6% in 3AO cells and 55 ± 6% in AO cells, as determined by the sub-G1 peak in the flow cytometry histograms (Fig. 4). The two independent assays in this study gave similar results, and thus clearly demonstrated that PROG could indeed promote apoptosis in the ovarian carcinoma cells tested. The ability of PROG to induce apoptosis is apparently higher than that of ATRA (also at 10 Î¼M for 72 hours), which is reported to induce apoptosis of breast carcinoma cells.29
Cell Cycle Analysis
The nuclear DNA content of individual cells in each population was determined by flow cytometry. The results are presented in Figure 5. Seventy-two hours after treatment with PROG, the peak representing 3AO cells with G1 DNA content had increased from 37.0 to 72.5%, and from 53.5 to 73.8% in AO cells, whereas the fraction of cells in S-phase had decreased from 31.9 to 11.3% in 3AO cells and from 26.6 to 13.4% in AO cells.
PROG Induces Up-Regulation of p53 Expression
Among the apoptosis-related genes we tested, after the cells were treated with 10 Î¼M PROG for 72 hours, only the level of p53 mRNA markedly increased in both 3AO and AO cells as detected by Northern blot analysis (Fig. 6). The mRNA levels of bcl-2 and c-myc, however, were not significantly changed by the PROG treatment in the two cell lines (data not shown).
In the clinical treatment of ovarian carcinoma, PROG is usually used at a concentration 10-100 times higher than its physical concentration during luteal phase (about 50 nM).30 Molecular mechanisms of PROG antitumor activity, though commonly believed to induce differentiation or growth inhibition, are not yet fully understood. In this study, our data clearly established that PROG can promote apoptosis in ovarian carcinoma cells besides its inhibition effects on cell growth. PROG at 10 Î¼M is more potent in inducing the apoptosis than at 1 Î¼M, indicating its dose-dependent manner.
It was previously reported that the use of COCs to treat postmenopausal women reduces ovarian carcinoma risk during estrogen replacement therapy.4 Our results suggest that antitumor activity of COCs may come from its main component, progestogen, since PROG has been shown to induce apoptosis in the ovarian carcinoma cells here.
Retinoids, which belong to the same superfamily of nuclear receptor ligands, have been also widely used in the clinical treatment of some types of cancers. Recently, there have been some reports that retinoids can induce apoptosis in different cell lines, such as leukemia,31breast carcinoma,32 and neuroblastoma cells lines.29 Our study shows that ATRA can induce apoptosis in both ovarian carcinoma cell lines tested, suggesting that the retinoids may be applied to treat ovarian carcinomas clinically. However, the apoptotic effect of ATRA, in the parallel experiments in this study, was not so powerful as that of PROG. This is probably because both 3AO and AO cells are estrogen- and progestogen-dependent, so they are more sensitive to PROG than to ATRA.
As reported, the programmed cell death or apoptosis is a physiologic and genetically controlled multistep process.33 Among the genes which regulate apoptosis, p53 and c-myc are primary apoptosis-promoting genes, and bcl-2 is a major apoptosis-suppressing gene. A pivotal role for p53 in the control of apoptosis has been demonstrated by experiments that the wild-type form of this protein induces rapid programmed cell death in leukemic cells.34, 35 Studies with thymocytes of p53 knockout mice further reveal the existence of two apoptotic pathways, one initiated by DNA damage, which requires p53, and the other stimulated by glucocorticoids and Ca2+ ionophores, which are p53-independent.36 Wild-type p53 can trigger G1 cell cycle arrest37, 38 and regulate a set of genes playing a role in the passage from G1 to S.39In this study, the level of p53 mRNA increased in response to PROG, whereas that of c-myc and bcl-2 mRNA did not change. Our results thus suggest that p53 may be involved in PROG-induced apoptosis in 3AO and AO cells.
The authors thank Wei Hu, Lu Pu, Yan-Ping Wang, Yong-Qin Wu, You-Ji Feng, and Cong-Jian Xu for technical help.
Does HRT modify risk of gynecological cancer? Int J Fertil Menopausal Stud 1995; 40(Suppl 1): 40–53., .
Definition and incidence of apoptosis: an historical perspective. In: TomeiLD, CopeFO, editors. Apoptosis: the molecular basis of cell death. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1991:5., .
Gonadotropins stimulate the proliferation of human epithelial ovarian cancer cells. Chin J Obstet Gynecol 1996; 3: 166–8., , .
rhTNF effect on c-erbB-2 protein expression in the human ovarian 3AO cencer cell line. Chin J Obstet Gynecol 1996; 3: 181–2., , .