| Objective: To investigate the relationship between the expression of estrogen receptor α and β subtype and endocrine therapy in breast cancer and the underlying mechanisms.Methods: 1. Human breast cancer cell lines MCF-7 and MDA-MB-231 were used in this section. Semi-quantitative RT-PCR was used to examine the m RNA levels of ERα and ERβ. Western blot was used to assess the protein levels of ERα and ERβ. After incubation for 24 h, 48 h, 72 h, 96 h, MTT test was used to assess the ability of cell proliferation. 2. MCF-7 cells with different expression of ERα or ERβ [M/siα(ERαlow/ERβhigh), M/siβ(ERαhigh/ERβlow)] were used in this section. MTT test was used to assess the effect of 0.1, 0.5, 1, 5, 10 μmol/L TAM alone, or in combination with 0.5 or 100 ng/m L human recombine IFN-γ(rh IFN-γ) on cell proliferation. Semi-quantitative RT-PCR and Western blot were used to assess the effect of 0.5 ng/m L rh IFN-γ on the expression of ERβ in human breast cancer cells. 3. MCF-7, M/HK, M/siα and M/siβ were used in this section. Semi-quantitative RT-PCR was used to examine the m RNA levels of drug-resistance related genes(MDR1, TOPOII, LRP, GST-π). Western blot was used to assess the protein levels of p-Akt and p-ERK in MAPK and PI3K/Akt, which were drug-resistance related signal pathways.Results: 1. Semi-quantitative RT-PCR results showed that the m RNA expression of ERβ in MCF-7 was higher than MDA-MB-231 [(0.191 ± 0.005) vs(0.152 ± 0.003), P<0.05]. Western blot results showed that the protein of expression of ERβ was lower than MDA-MB-231 [(0.462 ± 0.001) vs(0.584 ± 0.004), P<0.05]. MTT analysis results revealed that, after 24 h, 48 h, 72 h incubation, there was no significantly difference about the proliferation rate between MCF-7 and MDA-MB-231 [(1.121 ± 0.002) vs(1.204 ± 0.062),(1.236 ± 0.012) vs(1.287 ± 0.054),(1.374 ± 0.028) vs(1.401 ± 0.033), P>0.05]; after 96 h incubation, the proliferation rate of MCF-7 was higher than MDA-MB-231 [(2.218 ± 0.07) vs(1.463 ± 0.011), P<0.05].2. MTT analysis results revealed that, compared with the control group MCF-7, high expression of ERβ in MCF-7 could increase the inhibitory effect of 1, 5 or 10 μmol/L TAM on cell proliferation in a dose-dependent manner [(45.788 ± 1.641) % vs(24.288 ± 1.170) %;(57.899 ± 1.583) % vs(31.499 ± 1.978) %;(59.853 ± 1.648) % vs(38.039 ± 1.482) %, P<0.05]. Compared with the control group TAM, rh IFN-γ(0.5, 100 ng/m L) could increase the inhibition effect of TAM on MCF-7, M/siα, M/siβ cells [(29.911 ± 1.775) %,(40.223 ± 1.083)% vs(25.876 ± 1.122) %;(49.098 ± 1.504)%,(52.2 ± 2.223) % vs(46.556 ± 1.288) %;(34.434 ± 2.002) %,(50.134 ± 1.554) % vs(20.677 ± 1.724) %, P<0.05]. Compared with the negative control, Semi-quantitative RT-PCR results showed that rh IFN-γ(0.5 ng/m L) significantly increased the m RNA expression levels of ERβ [(0.248 ± 0.0002) vs(0.184 ± 0.0006), P < 0.05]; Western Blot results showed that rh IFN-γ(0.5 ng/m L) significantly increased the protein expression levels of ERβ [(0.521 ± 0.003) vs(0.356 ± 0.019), P<0.05]. 3. In ERβ up-regulated MCF-7 cells, the m RNA expression levels of drug-resistance genes, including MDR1, TOPOII, LRP, could be inhibited significantly [(0.431 ± 0.032) vs(0.932 ± 0.083);(0.234 ± 0.008) vs(0.391 ± 0.002);(0.47 ± 0.028) vs(0.586 ± 0.036); P<0.05]; the protein levels of p-Akt and p-ERK might be down-regulated significantly [0.224 ± 0.006 vs 0.437 ± 0.007, 0.367 ± 0.015 vs 0.756 ± 0.039; P<0.05].Conclusion: 1. After incubation for 96 h, the proliferation rate of MCF-7 cells were significantly higher than MDA-MB-231, and found that it may be related to different ER subtype, which ERα could increase the proliferation ability of breast cancer cells, and ERβ could decrease the proliferation ability of cells. 2. The expression of ERβ in human breast cancer cells could increase the inhibition effect of TAM on cells. IFN-γ could promote ERβ expression and upregulate the sensitivity to TAM of MCF-7 cells. 3. ERβ may alter the resistance of human breast cancer cells to TAM, possibly, at least partially, through down-regulating the expression of drug-resistance genes(MDR1, TOPOII, LRP, GST-π) and activating PI3K/ AKT and MAPK signalpathways. |
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