Effects of ethanol-extracted Pueraria mirifica root on the proliferation of normal porcine endometrial cells and RL-95 endometrial cancer cells

Abstract

The aim of this study was to assess the proliferative/anti-proliferative effect of ethanol-extracted Pueraria mirifica (PM) root in normal porcine endometrial cells and RL-95, the human endometrial cancer cells in comparison to daidzein, genistein and estradiol. The endometrial epithelial gland was isolated from immature pig uterus (PE-I) and the cancer cell was RL-95. All cells were treated with PM, daidzein (Di), genistein (Ge) and 17β-estradiol (E₂) at various concentration from 10⁻¹¹ to 10⁻⁴ M. The proliferation was measured by MTT assay at 24, 48 and 72 hours after treated. Level of estrogen receptor protein expression was performed in cells treated with drugs for 72 hours by semiquantitative Western blot analysis. The results showed that PM had no proliferative effect but had anti-proliferative effect at concentrations of 10⁻5 and 10⁻⁴ M at 24, 48 and 72 hrs in PE-I. Other phytoestrogens, Di (10⁻⁴ M) significantly decreased cell numbers of PE-I at 72 hrs. Ge (10⁻⁴M) can significantly decrease cell proliferation of PE-I at 48 and 72 hrs. E₂ (10⁻⁴ M) can significantly decrease PE-I cell proliferation at 24, 48 and 72 hrs. Moreover, E₂ (10⁻⁸ and 10⁻⁷ M) can significantly decrease PE-I cell proliferation at 48 hrs. For the RL-95, PM (10⁻⁶, 10⁻⁵ and 10⁻⁴ M) can significantly decrease cell proliferation at 24, 48 and 72 hrs, while Di and Ge (10⁻⁴ M) significantly decreased cell numbers at 48 and 72 hrs. E₂ at concentration of 10⁻⁴ M can inhibit cell proliferation at 48 and 72 hrs. The effect of PM on inhibition of RL-95 cell proliferation was in a dose-dependent manne (10⁻⁶, 10⁻⁵ and 10⁻⁴ M). Based on the semi-quantitative Western blot analysis, the levels of estrogen receptor type were different between normal endometrial cells and cancer cells. RL-95 had higher level of ERβ expression than ERα; whereas, PE-I had high level of ERα than ERβ. Therefore, the ratio of ERβ to ERα was about 16 in RL-95 and about 0.31 in PE-I. Exposed to PM at 10⁻⁹ M for 72 hrs, the ER ratio was not affected in both cell types, and this concentration had no effect on cell proliferation. Interestingly, the 10⁻⁶ M PM can alter ER ratio of both cells to 1.32 and 1.26 (PE-I and RL-95, respectively), this was caused by that the PM can increase ERα level. It should be noted that this concentration contained anti-proliferative effect in RL-95 but no effect on PE-I. It is possible that PM can modulate the expression of ER and therefore inhibit cell proliferation in cancer cell. Unfortunately, we cannot compare this effect to other phytoestrogens or estradiol because the vehicle used to dissolve phytoestrogens and estradiol (DMSO and ethanol, respectively) can alter the ER expression. Thus, the relationship between anti-proliferative effect and ER expression could not be evaluated. In conclusion, the current study revealed that normal and cancer cells are differed in term of ERs and ratio of ER. The PM can reduce cancer cells proliferation and this is in accordance to the increase in ERα expression and the decrease in ERβ to ERα ratio. These findings can be used as further information for using Pueraria mirifica (PM) as an estrogen supplement or cancer treatment. Moreover, the effects of PM on the modulation of ER protein expression level in cancer cells may be beneficial for developing the potent cancer therapy.