The role of non‐genomic actions of progesterone and its membrane receptor agonist in ovarian cancer cell death

Abstract Background Progesterone therapy is a relatively inexpensive treatment option for endometrial and breast cancers, with few side effects. Two signaling pathways usually mediate the physiological effects of progesterone, namely genomic and non‐genomic actions. Genomic action occurs slowly via the nuclear progesterone receptor (PR), whereas the membrane progesterone receptor (mPR) induces rapid non‐genomic action. Aims We investigated the effects of progesterone and various PR agonists on ovarian cancer cells. Methods and Results PR expression of six serous ovarian cancer cell lines was examined by western blotting, and mPR expression was examined by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). PR‐negative and mPR‐positive ovarian cancer cells were exposed to progesterone and seven types of PR agonists (medroxyprogesterone acetate [MPA], dehydroepiandrosterone, dienogest, levonorgestrel, drospirenone, pregnenolone, and allopregnanolone) at 10–400 μM, and viable cell counts after exposure for 30 min were measured using the water‐soluble tetrazolium (WST‐1) assay. Ovarian cancer cell lines were exposed to 100 μM progesterone, and the expression of BAX, a pro‐apoptotic protein, after 1–5 min was examined by western blotting. Western blotting detected no PR expression in the six serous ovarian cancer cell lines. In contrast, RT‐qPCR detected mPR expression in all six serous ovarian cancer cell lines. Progesterone and MPA‐induced cell death in all tested ovarian cancer cell lines in a concentration‐dependent manner, whereas no effect was observed for other PR agonists. Western blotting revealed that pro‐apoptotic protein BAX expression occurred 1 min after exposure to progesterone, suggesting that the cytocidal effects are mediated by rapid non‐genomic action. Conclusion Progesterone and MPA exhibited a rapid cytocidal effect on PR‐negative ovarian cancer cells through non‐genomic action. Progesterone and MPA could be novel adjuvant therapies for ovarian cancer.

Conclusion: Progesterone and MPA exhibited a rapid cytocidal effect on PR-negative ovarian cancer cells through non-genomic action.Progesterone and MPA could be novel adjuvant therapies for ovarian cancer.

K E Y W O R D S
cell death, genomics, medroxyprogesterone acetate, ovarian cancer, progesterone, progesterone receptor

| INTRODUCTION
In the United States, ovarian cancer is the fifth leading cause of cancer-related deaths in women.It is estimated that approximately 20 000 women will develop ovarian cancer and approximately 13 000 women will die of this disease every year. 1 More than half of the patients are diagnosed with progressive disease with peritoneal dissemination and massive ascites because the early stage of ovarian cancer is asymptomatic.The standard treatments for advanced ovarian cancer are debulking surgery and adjuvant chemotherapy with platinum and taxane, which initially achieve a complete response in approximately 80% of patients.However, the antitumor effects are usually transient, and many patients experience abdominal recurrence with reduced chemosensitivity, ultimately leading to death due to disease progression. 2,3Recently, several novel molecular targeted agents, including bevacizumab and poly adenosine diphosphate-ribose polymerase (PARP) inhibitors, have been applied clinically. 4Although these drugs exert some antitumor effects, adverse side effects and economic burdens are challenges that need to be addressed.Therefore, more cost-effective novel therapeutic strategies are required.
Progesterone is a steroid hormone that is primarily synthesized in the corpus luteum of the ovary.It forms a complex with the intracellular progesterone receptor (PR) proteins.This complex binds to the DNA promotor sites in the nucleus, thereby regulating the expression of various genes. 5For cancer therapy, progesterone is applied to endometrial and breast cancers as an inexpensive treatment option with few side effects. 6,7In 2003, the membrane progesterone receptor (mPR) family was identified by Zhu et al. 8 These mPRs exert rapid non-genomic action, whereas the commonly known genomic action occurs more slowly through the nuclear PR. 9 Our group previously reported that the incidence of the p53 signature in fallopian tubes was significantly lower in pregnant and parous women. 10Another report suggested that progesterone from follicular fluid during ovulation induces necroptosis in p53 mutated tubal epithelial cells and prevents carcinogenesis. 11Progesterone may prevent early-phase serous carcinogenesis and suppress ovarian cancer cell growth.
In the present study, we evaluated the antitumor effects of progesterone and its derivatives (PR agonists) in ovarian cancer cell lines to obtain some clues for developing a novel therapeutic strategy.

| Quantitative reverse transcription PCR (RT-qPCR)
Cellular mRNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

| PR expression
Firstly, PR expression was evaluated using western blot analysis.As shown in Figure 1, PR expression was not detected in any of the tested ovarian cancer cell lines, suggesting that the genomic action does not occur in these cells.The human breast adenocarcinoma cell line MCF-7 was used as the positive control.

| mPR expression
Next, the expression of five types of mPRs (α, β, γ, δ, and ε) was evaluated using RT-qPCR.All tested ovarian cancer cell lines expressed various mRNAs with various values (Figure 2).These results suggest that progesterone could show non-genomic actions through mPRs in these ovarian cancer cells.

| Effects of progesterone and PR agonists
The cytocidal effect of progesterone and PR agonists was examined in each ovarian cancer cell line.As shown in Figure 3, progesterone and one of its derivatives (MPA) induced distinct cell death in all tested ovarian cancer cell lines in a concentration-dependent manner.However, PR agonists other than MPA did not exhibit cytocidal effects on any ovarian cancer cell lines.

| BAX expression
The expression of one of the pro-apoptotic proteins, BAX, was evaluated by western blotting.BAX protein expression was detected at a position corresponding to a molecular weight of 20 kDa in all tested cell lines exposed to progesterone for 1-5 min (Figure 4).Upregulation of BAX protein was noted 1 min after exposure to progesterone, suggesting that this prompt response may result from the nongenomic action of progesterone via the mPR.

| DISCUSSION
In the present study, nuclear PR was not expressed in the six ovarian serous carcinoma cell lines.In contrast, all six ovarian cancer cell lines expressed various mPRs.Progesterone and one of its derivatives, MPA, exerted concentration-dependent cytocidal effects.No cytotoxic effects were observed for the other PR agonists.In addition, the expression of the pro-apoptotic protein BAX increased 1 min after exposure to progesterone.
PR is physiologically expressed in organs, such as the mammary glands, endometrium, and ovaries.Regarding malignancies, PR is expressed in breast and endometrial cancers, whereas most ovarian cancers lack its expression. 15In this study, no PR expression was identified in all the examined ovarian cancer cell lines.
F I G U R E 3 Cytocidal effects of progesterone and PR agonists on ovarian cancer cell lines.The viable cell count was measured by the WST-1 assay after 30 minutes' exposure to each drug at the concentrations of 10-400 μM.The data were presented as a percentage ratio to the count of the control untreated with drugs.P4, progesterone; MPA, medroxyprogesterone acetate; DHEA, dehydroepiandrosterone sulfate; DNG, dienogest; LNG, levonorgestrel; DRSP, drospirenone; PREGN, pregnenolone; ALLO, allopregnanolone.Genomic action via transcriptional regulation by activated nuclear PR occurs slowly, whereas non-genomic action via mPR is rapidly induced.There are five types of mPRs, namely mPR-α, β, γ, δ, and ε. 9 mPR expression in ovarian cancer was previously reported. 16mPR expression was identified in all six cell lines examined in this study.
Progesterone and MPA displayed concentration-dependent cytocidal effects.However, no cytocidal effects were observed for the other PR agonists.This cytocidal effect appears to be mediated by non-genomic actions via mPRs because it was observed in PR-negative and mPR-positive ovarian cancer cells within a short time (30 min).Pang et al. 17  BAX is a member of the Bcl-2 family that accelerates apoptosis.
Recently, BAX has been reported to be associated with other programmed cell death pathways, including pyroptosis and ferroptosis. 18,19arles et al. 16 demonstrated that 24 h progesterone stimulation induced BAX overexpression in PR-negative and mPR-positive ovarian cancer cells using comprehensive genetic analysis with a qPCR super array.In the present study, BAX expression was rapidly induced 1 min after progesterone exposure.This prompt response may result from the non-genomic action of progesterone via the mPR.
Recently, novel molecular targeted agents, including bevacizumab and PARP inhibitors, have been applied in clinical practice, especially in maintenance therapy for ovarian cancer. 20Although these drugs exhibit antitumor effects to some extent, side effects such as hypertension, proteinuria, and gastrointestinal perforation are associated with bevacizumab treatment, while severe anemia, thrombocytopenia, and renal dysfunction are associated with PARP inhibitors.Another challenge is the economic burden owing to the high cost of these novel drugs.Progesterone and MPA are clinically used for threatened premature birth, and their cost is relatively low. 21Therefore, progesterone and its derivatives are safer and less expensive than chemotherapeutic agents and molecular targeted drugs.
In this research, progesterone and MPA exerted cytocidal effects against ovarian cancer cells at concentrations of 10-400 μM.This concentration range is much higher than those used in pregnant women or patients with cancer taking MPA, and approximates the concentration in follicular fluid under physiological conditions. 22Progesterone is rapidly metabolized in vivo by various hydroxylases synthesized in the liver.The serum concentration of progesterone increases in patients with congenital adrenal hyperplasia who lack these hydroxylases. 23When applying progesterone and its derivatives in ovarian cancer treatment, a combination of some hydroxylase inhibitors might be useful.Further investigations including in vivo experiments are required to establish a novel therapeutic strategy targeting mPRs.
In conclusion, MPA and progesterone exhibited a rapid cytocidal effect on PR-negative ovarian cancer cells through non-genomic action.Progesterone and its derivative MPA could be new treatment modalities for ovarian cancer.

F I G U R E 2
Confirmation of the expression of membrane progesterone receptor (mPR).mPR expression was identified in all six ovarian cancer cell lines by RT-qPCR.mPR, membrane progesterone receptor.mPRsare membrane-bound receptors with high affinity and specificity for progesterone and adiponectin.mPRs induce non-genomic actions by activating non-canonical intracellular signaling pathways.
demonstrated that PR agonists (MPA not included) possess a significantly lower binding affinity for mPR than progesterone.Most PR agonists may not affect ovarian cancer cell survival because of their low binding affinity for mPR.