Effects Of Th1/Th2-type Cytokines On The Biological Behaviors And ER Expression Of Human Breast Cancer Cell Line MCF-7

Objective: To study the effects of Th1-type cytokine IFN-γ and Th2-type cytokine IL-4 on the biological behaviors and the expression of estrogen receptor(ER) subtypes of human breast cancer cell line MCF-7.Methods: 1. MCF-7 was treated with different concentration of rh IFN-γ or rh IL-4 for 24, 48, 72 and 96 h, and then cell proliferation was detected using MTT assay. MCF-7 cells were treated with 100ng/m L rh IFN-γ(M/IFN-γ) or with10ng/m L rh IL-4(M/IL-4) for 96 h in the following tests. Flow cytometry technique was used to detect the cell cycle distribution. RT-PCR was used to analyse the m RNA expression of apoptosis suppressor gene Bcl-2, Bcl-xl and XIAP.2. Double layer soft-agar clony formation test and Matrigel adhesion assay were used to detect the abilities of cell tumorigenesis and adhesion. RT-PCR and Western blot were conducted to detect the expression of VEGF, MMP-2 and MMP-9. Western blot was used to detect the phosphorylation level of Erk and Akt in PI3K/Akt, Ras/Raf/MEK/ERK signal pathway. 3. Western blot was used to detect the protein expression of ERα and ERβ. Then, ELISA was conducted to detect the IFN-γ and IL-4 levels secreted by MCF-7. MCF-7 was treated with rh IFN-γ or rh IL-4 which is equivalent to autocrine level for 96 h. RT-PCR and Western blot were then conducted to detect the m RNA and protein levels of ERα and ERβ.Results: 1. Compared with the blank control group, the proliferation of MCF-7 was inhibited significantly after treated with 100ng/m L IFN-γ for 96h(1.07±0.05 vs 0.87±0.05, P<0.01). The proportion of G0~G1 increased significantly in M/ IFN-γ cells(55.42±2.28% vs 66.98±0.88%, P<0.01), while the proportion of Sreduced(27.61±3.11% vs 15.36±0.94%, P<0.01). On the other hand, the proliferation of MCF-7 was improved significantly after treated with 10ng/m L IL-4 for 96h(1.07±0.05 vs 1.38±0.06, P<0.01). The percentage of cells in G0~G1 phase was decreased(55.42±2.28% vs 37.77±5.93%, P<0.01) but in S phase was increased(27.61±3.11% vs 45.45±4.15%, P<0.01). And the apoptosis suppression gene Bcl-2(1.35±0.02 vs 2.25±0.03, P<0.01), Bcl-xl(1.33±0.01 vs 2.27±0.02, P<0.01) and XIAP(0.35±0.01 vs 0.60±0.03, P<0.01) expression were increased. 2. Compared with the blank control group, the number of M/IFN-γ colony forming was decreased(112.00±3.95 vs 74.12±1.11, P<0.01), while no significant difference was observed in cell adhesion between M/IFN-γ and blank control group. The VEGF m RNA(0.71±0.03 vs 0.45±0.02, P<0.01) and protein(1.12±0.08 vs 0.78±0.03, P<0.01) levels of M/IFN-γ were decreased. MMP-9 m RNA(0.29±0.03 vs 0.09±0.00, P<0.01) and protein(1.00±0.10 vs 0.81±0.03, P<0.01) levels were also decreased. And the phosphorylation level of Erk was decreased(1.33±0.01 vs 0.88±0.02, P<0.01). Compared with the blank control group, the number of M/IL-4 colony forming(112.00±3.95 vs 144.23±4.89, P<0.05) and the ability of M/IL-4 cell adhesion(100.00±0.01% vs 128.57±5.97%, P<0.05) were increased. The VEGF(m RNA: 0.71±0.03 vs 1.11±0.07; protein: 1.12±0.08 vs 1.36±0.11), MMP-2(m RNA: 0.11±0.00 vs 0.52±0.12; protein: 0.35±0.02 vs 1.21±0.12) and MMP-9(m RNA: 0.29±0.03 vs 0.67±0.07; protein: 1.00±0.10 vs 1.21±0.13) expression of M/IL-4 were increased(P<0.01). And the phosphorylation level of Erk(0.82±0.05 vs 1.53±0.01) and Akt(1.33±0.01 vs 2.42±0.05) were increased(P<0.01). 3. Western blot results showed that no significant difference was observed in ERα protein expression when Compared with the blank control group. ERβ protein expression of M/IFN-γ was increased(0.69±0.08 vs 1.10±0.11, P<0.05) while ERβ protein expression of M/IL-4 was decreased(0.69±0.08 vs 0.42±0.02, P<0.05). IFN-γ and IL-4 secreted by MCF-7 were 0.15±0.01ng/m L(<0.5ng/m L) and 0.32±0.03ng/m L(<0.5ng/m L) respectively. ERβ m RNA expression of MCF-7 was improved significantly(0.28±0.01 vs 0.32±0.01, P<0.01) aftertreated with 0.5ng/m L IFN-γ for 96 hours. ERα and ERβ m RNA expression of MCF-7 remains unchanged after treated with 0.5ng/m L IL-4 for 96 hours. ERα and ERβ protein expression did not change significantly.Conclusion: 1. Our results suggest that Th1/Th2 balance can affect the biological behaviors of MCF-7. Th1-type cytokine IFN-γ can inhibit the proliferation, tumorigenesis, invasion and metastasis of MCF-7. While Th2-type cytokine IL-4 can promote the proliferation of MCF-7. Thus, regulating Th1/Th2 balance may be a new treatment strategy for breast cancer. 2. The results also show that Th1/Th2 balance can affect ERβ expression of MCF-7 without affecting the ERα expression. Th1-type cytokine IFN-γ can promote ERβ expression while Th2-type cytokine IL-4 can inhibit ERβ expression. Low level of IFN-γ, which is equivalent to autocrine level, secreted by MCF-7 can also regulate the ERβ expression. Thus, the effect of IFN-γ on ERβ may improve the sensibility to endocrine treatment for breast cancer.