| ObjectiveInterferon is an important cytokine which has the effects of anti-virus, anti-tumor and immunomodulation. MCF-7 breast cancer cells resist to the apoptosis by Fas/FasL pathway induced with interferon-γ. Even if the concentration of IFN-γis 10 times higher, MCF-7 breast cancer cells still survived.This study was to investigate if interferon-α2b has the reversing effects on the resistence to apoptosis by Fas/FasL pathway induced with interferon-γin MCF-7 breast cancer cells.Methods1.MCF-7 breast cancer cells line was used as the trail materials and the treated time was 24h, 48h, 72h.2.Groups: Groups included control group and experimental groups. Apoptosis-inducing experiments: experimental group 1 was treated by 500U/mlIFN-α2b,experimental group2 was treated by 500U/mlIFN-γ,experimental group3 was treated by both of them,the control group was the normal MCF-7 breast cancer cells.FasL blocking experiments:the control group1 was the normal MCF-7 breast cancer cells,the control group 2 was treated by both of 500U/mlIFN-α2b and 500U/mlIFN-γ,the experimental group was MCF-7 breast cancer cells which was treated by the antibody of FasL before it was dealed with combinated both of 500U/mlIFN-α2b and 500U/mlIFN-γ,treated time was 72h.3.Treatment:Apoptosis-inducing experiments:The control group and experimental group was dealed with 24h,48h,72h after the groups treated by interferon.Detect apoptosis of MCF-7 breast cancer cells line by flow cytometry (FCM).Observe the proliferative inhibition of MCF-7 breast cancer cells line through MTT test.Detect the expression of Fas/FasL protein of MCF-7 breast cancer cells line by flow cytometry.FasL blocking test:Detect the apoptosis of MCF-7 breast cancer cells of the control group1,the control group2 and the experimental group by flow cytometry. 4.Statistical analysis:All the datas were mean±standard deviation (±S).statistical process was used by SPSS11.5 of statistical software.analysis of variance was used by factorial design.Tukey or Dunnett t test was used to compared with each other.(P<0.05) was considered statistically different between of themResults1.Apoptosis-inducing experiments: The percentage of apoptotic cells of the experimental group1,experimen tal group2 and experimental group3were 2.39%,2.35%,2.95%after treated 24h;the percentage of apoptotic cells of the experim ental group1,experiment- al group2 and experimental group3were 2.29%,1.93%,2.00%treated 48h;the percentage of apoptotic cells of the experim ental group1,experimental group 2 and experimental group3were 4.02%,2.25%,5.33%after treated72h;the per- centage of apoptotic cells of the control groups were2.11%,1.82%,2.27% tre ated24h,48h,72h;in the time of 24h and 48h,the difference was not statistical- ly significant compared with each other between the groups(P>0.05);in the ti- me of 72h,the difference was statistically significant respectively comparedwi- th the control group,experimental group2(P<0.05);the difference was signi- ficant statistically compared with group of 24h treated by interferon-α2b (P< 0.05), the difference was not significant statistically compared with each group of 24h,48h,72h treated by interferon-γ(P>0.05).the difference was significant statistically compared with group of 24h and 48h treated by interf-eron-γ+α2b (P<0.05).The percentage of apoptotic cells of the experimental group 1,experimental group 2 and experimental group 3were 1.73%,2.06%,1.64% after treated 24h;the percentage of apoptotic cells of the experimental group 1,experimental group 2 and experimental group 3were 2.04%,4.08%,7.02% after treated 48h;the percentage of apoptotic cells of the experimental group1,experimental group2 and experimental group3were 3.32%,6.76%,28.61% after treated 72h;the percentage of apoptotic cells of the control groups were1.51%,1.80%,3.09%after treated 24h,48h,72h;In the time of 24h the difference was not statistically significant compared with each other between the groups(P>0.05);In the time of 48h the difference was statistically significant respectively compared with the control group,experimental group2(P<0.05), In the time of 72h the difference was statistically significant respectively compared with the control group,experimental group1,experimental group2 and experimental group2 compared with the control group,experimental group1(P<0.05);the difference was significant statistically compared with group of 24h and 48h treated by interferon-α2b(P<0.05),the difference was significant statistically compared with group of 24h treated by interferon-γ(P<0.05),the difference was significant statistically compared with each group of 24h,48h,72htreated by interferon-γ+α2b(P<0.05). Detect the proliferation of MCF-7 breast cancer cells byMTT:The OD values of the experimental group1,experimental group2 and ex perimental group3were0.92,1.08,1.01after treated 24h;the OD values of the experimental group1,experimental group2 and experimental group3 were 0.69,0.81,0.69after treated 48h;the OD values of the experimental group1,experimental group2 and experimental group3 were0.91,1.09,0.38after tre ated 72h;the OD values of the control groups were1.00,1.00,1.00 after trea ted24h,48h,72h;In the time of 24h the difference was statistically significant compared with control group (P<0.05);in the time of 48h the differ- rence was statistically significant respectively compared with the control gro- up (P< 0.05),in the time of 72h the difference was statistically significant respe- cttiv ely compared with the control group,experimental group1,experimental gr oup2(P<0.05);the difference was significant statistically compared with gro- up of 24h and 72h treated by interferon-α2b (P<0.05),the difference was signi- ficant statistically compared with group of 24h and 72h treated by interferon-γ(P<0.05) the difference was significant statistically compared with each one group of 24h,48h,72h treated by interferon-γ+α2b(P<0.05).Expression of Fas/FasL protein: The percentage of expression of FasL protein of the experimental group1,experimental group2 and experimental group3were4.09%,2.76%,3.89% after treated 24h;the percentage of expression of FasL protein of the experimental group1,experimental group2 and experimental group3were3.63%,1.25%,1.20% after treated 48h;the percentage of expression of FasL protein of the experimental group1,experimental group2 and experimental group3were 7.21%,2.91%,7.23% after treated 72h;the percentage of expression of FasL protein of the control groups were2.30%,1.42%,2.43%after treated 24h,48h,72h.In the time of 24h the difference was not statistically significant compared with each other between the groups(P>0.05);in the time of 48h,the difference was not statistically significant respectively compared with each other between the groups(P>0.05),in the time of 72h the difference was statistically significant respectively compared with the control group,experimental group2(P<0.05),the difference was significant statistically compared with group of 24h treated by interferon-α2b(P<0.05),the difference was not significant statistically compared with each group of 24h,48h,72h treated by interferon-γ(P>0.05),the difference was significant statistically compared with group of 24h,48h treated by interferon-γ+α2b(P<0.05).The difference of the percentage of expression of Fas protein of the experimental group1,experimental group2 and experimental group3 compared control group was not significant statistically(P>0.05).2.FasL blocking experiments:The percentage of apoptotic cells of the experimental group was 7.15%,the percentage of apoptotic cells of the control group1and control group 2 were 3.48% and 10.89%,the differences were statistically significant between the groups,(P<0.05).The percentage of apoptotic cells of the experimental group was 9.11%%,the percentage of apoptotic cells of the control group1and control group 2 were3.55% and 27.65%,the differences were statistically significant between the groups(P<0.05).Conclusion Interferon-α2b can reverse the resistence of apoptosis by Fas/FasL pathway induced with interferon-γin MCF-7 breast cancer cells.Its mechanism maybe involve upregulated expression of FasL protein in MCF-7 breast cancer cells,strengthened link of Fas/FasL,increased the sensitivity of Fas/FasL and increased concentration of interferon-γintracellular or increased sensitivity to the apoptosis induced by interferon-γin the cells. |
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