| BACKGROUND:All women would undergo menopause, most of whom will be bothered by menopausal symptoms with impaired health and decreased quality of life during and after midlife. Until now, hormone replacement therapy (HRT) is still the most effective treatment to such symptoms and for prevention from osteoporosis. However, the potential adverse effects related to HRT, e.g. breast cancer and ovarian cancer, are still important issues for both doctors and patients. The WHI study demonstrated that the incidence of breast cancer was increased in the combined arm using CEE and MPA, while the eatradiol only arm was not. Later, the French E3N study found the increase of breast cancer incidence varied with defferent progestogens. Generally, it was considered that progestogens play a role via PR, PR-A and PR-B, which excerted antiproliferative effect. So it is hard to explain the above results. Another study from MWS showed the increased risk of ovarian cancer with HRT. Recently PGRMC1was found in many different tumor tissues and cell lines, including breast cancer and ovarian cancers, indicating there exits a new PGRMC1-dependant progesterone binding pathway for progestogen action, which could be related to tumors. The aime of this study is to uncover the proliferative effect of progestogens and estradiol during HRT on different cell lines regarding breast cancer and ovarian cancer and its probable mechanisms.METHODS:Different breast cancer and ovarian cancer cell lines were stably transfected with expression vector pcDNA3.1containing heme agglutinin-tagged (3HA) wild type(WT) or mutant PGRMC1(S56A, Y179F, Y138F, Y179F/S180A)or only3HA empty vector, resulting in stably transfected MCF7-WT12, S56A, Y179F Y138F, Y179F/S180A and EVC, MDA231-WT1-F8and EVC, SKOV3-WT2and EV2and OVcar29-WT1and EV1respectively with high transfection rates. All the transfected cell lines were stimulated by different progestogens of different concentrations as well as eatradiol alone or combined or sequentially, to detect if there was a difference in proliferative effect among different regimens on the cell lines. To elucidate the probable mechanisms, the expression level of15downstream proteins of the signal transduction pathways were detected with RPPMs, and confirmed by western blot. In the receptor blocking experiment, antagonists of the ERa (fulvestrant), PR (RU486) or PGRMC1(AG205) were used alone and in various combinations to prove the potential involvement of these receptors in the E2/NET-induced proliferation in MCF7-WT12cells. RESULTS:Progestogens and estradiol showed a concentration-dependant effect in MCF7EVC and MCF7-WT12cells. The proliferative effect of MCF7EVC cells was not increased by10-7M progestogens or lower, but an increase of40-50%was seen in MPA、DRSP、DY、 LNG and CMA at a concentration of10-6M. However, the proliferative effect of MCF7-WT12was increased much more significant than that of MCF7EVC, with the most pronounced in NET, even at10-9M. And both10-7M and10-6M of MPA, DRSP, DY, LNG and10-6M CMA induced a two to three-fold increased proliferation of WT12. Neither P4nor NOM would increase the proliferative effect of the cells. No increase was seen in the MCF7EVC cells by E2alone in any of the three concentrations.10-10M E2alone resulted in a significant increased proliferation of MCF7-WT12of about200%, no significant effect was seen at10-12M or10-14M. While in the EP combined or sequential regimen, it was found that no increase was seen in the MCF7EVC cells by any of the HRT regimen and that the proliferation of MCF7-WT12stimulated by10-10M E2with10-7M NET, was similar to that of10-10E2alone, but when it comes to10-12M or10-14M E2combined with NET, the proliferation increased by about170%, similar to NET alone, but more pronounced than the sequential regimen. Regarding the combined or sequential regimen using E2and P4or NOM, the proliferative effect was similar to that of E2alone. In the stimulation experiments of MCF7overexpressing PGRMC1mutant, significant but lower effect was seen by different progestogens and/or E2than that of MCF7-WT12, especially on Y179F/S180A, and no increased proliferative effect was observed in MCF7PGRMC1Y138F except for when DY or DRSP used. The expression level of ERK1/2in MCF7-WTI2was lowered by about40%than that of MCF7according to the result of both RPPMs and western blot. In the receptor blocking experiment, only the presence of fulvestrant was able to block the EP (E2plus NET) combination effect totally. Partial significant inhibitory effects could be found in the presence of AG205, no effect was observed for RU486. No proliferative effect by progestogens or estradiol can be found in any of the parental or transfected MDA231-MB-231, SKOV3or OVcar29cell lines.CONCLUSION:The progesterone-dependant proliferative effect in MCF7was mediated by PGRMC1. And this effect was dependent on the type of the progestegen and its concentration, however, P4and NOM always acted neutrally. In the combined or sequential EP regimen, the proliferation mainly depended on the concentration of E2. But when lower concentrations of E2which did not increase the proliferation, the progestogen became important, such as NET used, it seemed that sequential regimen might be better. The proliferative effects of different progestogens and/or E2on mutant PGRMC1overexpressed MCF7cells were not that high as seen in MCF7-WT12. The PGRMC1-dependant proliferative effect appeared to be mainly mediated by the ERa. However, PGRMC1did not increase the proliferation of MDA-MB-231, SKOV3or OVcar29cells. Thus, breast cancer tumorigenesis may depend on the concentration of estrogen as well as the specific progestogen used. PGRMC1may induce the proliferation of cancer cells during HRT, depending on the type and other characteristics, which is still unknown yet. This effect was different as to different progestogens and cancentrations. It seemed that progestogen showed increased proliferation mainly at a relative lower concentration of E2. P4or NOM may be a better choice in HRT. It is still unknown how PGRMC1acts and ERa may be involved, therefore, more and further studies are needed. |
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