Effects Of ¹³¹I-trastuzumab On Pi3k/akt Pathway And Expression Of Apoptosis-related Proteins In Her2 Overexpressing Breast Cancer Cells

Objective:(1) To investigate the Lethal effects of 131I-Trastuzumab in HER2 overexpressing breast cancer cells, as well as analyse its mechanisms.(2)To explore effects of 131I-Trastuzumab on the activation of PI3K/Akt pathway and the expression of apoptosis-related proteins in HER2 overexpressing breast cancer cells.Methods: The expression levels of HER2 on the surface of three different breast cancer cell lines were detected by immunofluorescence. Trastuzumab was labeled with 131I using the Iodogen method and 131I-Trastuzumab was isolated by ultrafiltration membrane, then the labeling efficiency, radiochemical purity and immunoreactivity were measured. The effect of 131I, Trastuzumab and 131I-Trastuzumab on viability of BT474 cells were evaluated by CCK-8(Cell Counting Kits-8) assay. The levels of total-Akt, phosphorylated Akt(p-Akt)(Ser473) and Bcl-x L were detected by Western blot analysis. Pearson correlation analysis, One-way ANOVA(analysis of variance), ANOVA for factorial designed data, and Bonferroni correction were used for data analysis.Results: The expression level of HER2 in BT474 cells was overexpressing, and significantly higher than those of MDA-MB-231 and MCF-7 cells. The labeling efficiency, radiochemical purity and immunoreactivity of 131I-Trastuzumab were(89.71±2.93)%,(91.80±1.43)% and(58.84±3.35)% respectively. 131I, Trastuzumab and 131I-Trastuzumab exhibited a dose-dependent cytotoxicity against BT474 cells(r=-0.964、r=-0.912、r=-0.618, all P <0.05), the cell viability of 131I-Trastuzumab(4.625 MBq/m L) was significantly lower than 131I(4.625 MBq/m L) and Trastuzumab(125 μg/m L)(t=10.373, P<0.01; t=8.180, P<0.01); and 131I-Trastuzumab shown a positive multiplicative interaction between 131I and Trastuzumab(F=9.226, P<0.05; CDI=0.929). Western blot results shown that no significant difference of total-Akt expression was found among the four groups(F=0.208, P>0.05), yet p-Akt(Ser473) expression in both Trastuzumab and 131I-Trastuzumab groups were much lower than those of control and 131I groups(t=12.524, t=15.984, t=7.347, t=10.807, all P<0.01), while there was no significant difference of p-Akt(Ser473) expression between Trastuzumab and 131I-Trastuzumab(t=3.460, P>0.05). There was a significant difference of Bcl-x L expression among the four groups(F=10.362, P<0.05), meanwhile the Bcl-x L expressions of both Trastuzumab and 131I-Trastuzumab groups were much lower than those of control and 131I groups(t=3.860, t=3.977, t=3.906, t=4.023, all P<0.01), while there was no significant difference of Bcl-x L expression between Trastuzumab and 131I-Trastuzumab(t=0.046, P>0.05).Conclusions: 131I-Trastuzumab not only has biological effects of 131I and Trastuzumab;but also enhance the radiosensitivity by inhibiting the activation of PI3K/Akt pathway and accelerate 131I-induced apoptosis with the down-regulation Bcl-x L expression. thus exerts the synergetic effect between 131I and Trastuzumab, and so kills HER2 overexpressing breast cancer cells more effectively than Trastuzumab.