Mifepristone(RU486) Inhibits The Growth Of MCF-7 Breast Cancer Cells By Down-regulating Progesterone Induced Blocking Factor

Background and objectivesProgesterone induced blocking factor(PIBF) is secreted by maternal lymphocytes and mediates the immunological effects of progesterone during pregnancy. After taking RU486, PIBF levels in pregnant women declined and fetal abortion rate increased. PIBF was expressed in some tumor cell lines and tumor tissues. RU486 could inhibit the growth of breast cancer cells, but it is also unclear whether the change of biological behavior is related with PIBF. In this paper, by treating PR-positive MCF-7 cells with RU486, we study the biological behavior of cancer cells and explore changes of PIBF in cells to provide new clues for the anti-tumor mechanism of RU486.MethodsAfter RU486 treatment, morphological changes of breast cancer MCF-7 cells cells was observed.Then cells were treated with different concentration RU486 for 24 h, 48 h and 72 h, cell viability was assessed with CCK-8 assay respectively. Then we selected 0μM, 10μM, 20μM, 30μM of RU486 for 72 h to conduct subsequent tests. Then cell cycle and apotosis was detected by flow cytometry. RNA and protein were extracted from cancer cells selectively after RU486 treatment. PIBF m RNA was detected by RT-q PCR. Protein levels of PIBF, WNT1, p21, Bax and cyclin A was detected by western blot.Results1. RU486 cause morphological changes in MCF-7 cell linesAfter 72 h, cells in treated groups presented small cell body, elongated or rounded shape, spindle-like shape, loosely adhered compared with control. With the increasing of RU486 concentration, morphological changes were more obvious.2. RU486 inhibits proliferation of MCF-7 cellsRU486 inhibited cell proliferation in a concentration-dependent manner. After 24 h, 40μM RU486 inhibited cell proliferation compared to 20μM. When treated with 30μM and 40μM RU486, number of cells significantly reduced at 48 h compared with 0μM and 10μM respectively. 40μM RU486 also significantly inhibited cell proliferation compared with 20μM RU486. When treated with 20μM, 30μM and 40μM RU486, MCF-7 cells were inhibited, and there were statistically significant difference between different concentrations.RU486 also inhibited cell proliferation in a time-dependent manner. When treated with 10μM RU486, cell proliferation at 48 h and 72 h was inhibited compared with 24 h, but no difference between 24 h and 48 h. When treated with 20μM, 30μM and 40μM RU486, number of cells in 48 h and 72 h were also significantly decreased compared with 24 h. When cells was treated with 40μM RU486, there was also significant difference between 48 h and 72 h.IC50 values, the half inhibitory concentration, were 41.15μM, 35.47μM and 25.74μM for 24 h, 48 h, 72 h respectively.3. RU486 induced G1 arrest in MCF-7 cellsFlow cytometry showed that cell ratio of G0/G1 phase increased after RU486 treatment, while the proportion of cells in S phase and G2/M phase decreased. In G0/G1 phase, cell ratio in 20μM and 30μM RU486 increased by 10% and 27% respectively compared with 0μM; 30μM increased by 21% compared with 10μM and 17% compared with 20μM. In S phase, cell ratio in 30μM reduced by 20% compared with 0μM and 17% compared with 10μM. In G2/M phase, cell ratio in 30μM decreased by 7% compared with 0μM.4. RU486 promoted apoptosis in MCF-7 cellsFlow cytometry showed that the number of apoptotic cells increased after RU486 treatment. Total cell apoptosis rate of 30μM RU486 increased 6.2% compared with 0μM and 6% compared with 10μM.5. RU486 down-regulated PIBF expressionRT-q PCR showed that PIBF m RNA levels in treating groups were decreased compared with 0μM after 72 hours, and any of the two groups were statistically significant.Western blot showed that PIBF protein levels in treating groups were decreased compared with 0μM after 72 hours. 20μM and 30μM had a significant difference compared with 0μM, so did 10μM and 30μM.6. RU486 down-regulated WNT1 expressionAfter 72 hours, with the increase of drug concentration, expression of WNT1 protein in treating groups were decreased compared with 0μM after 72 hours, and any of the two groups were statistically significant.7. RU486 up-regulated p21 and Bax,and also down-regulated cdk2 and cyclin AAfter 72 hours, expression of p21 protein gradually increased. 30μM RU486 had a significantly higher level compared with 0μM RU486. Levels of cdk2 protein gradually decreased. 30μM RU486 also decreased significantly compared with 0μM and 10μM respectively. Expression of cyclin A protein gradually decreased. 20μM and 30μM RU486 decreased significantly compared with 0μM RU486 respectively. Levels of Bax protein gradually increased. 10μM and 30μM RU486 had a significant higher level compared with 0μM RU486 respectively. 30μM RU486 also had a lower level compared with 10μM.ConclusionsRU486 may play an anti-tumor effect by down-regulating PIBF in MCF-7 cells by these mechanisms:(1) inhibited the proliferation of cells by up-regulating p21,(2)induced G1 phase arrest by down-regulating cdk2 and cyclin A,(3) promoted apoptosis by up-regulating Bax.