Guben Yiliu Ⅱ And Its Disassembled Prescriptions Can Interfere With Autophagy And PI3K Signaling Pathway In Breast Cancer MCF-7 Cells

Breast cancer is the second leading cause of cancer-related deaths among women in China.Currently there are treatments of surgery,chemotherapy,radiation therapy,targeted therapy and endocrine therapy.Chinese medicine believes that breast cancer is mainly due to the organs deficiency,phlegm resistance,bleeding off resistance,toxin-induced stagnation.Some clinical studies have found the presence of advanced cancer patients in stasis or should be tonifying qi and activate blood and detoxify therapy.Gubenyiliu Formula Ⅱ(固本抑瘤 Ⅱ 号)is an effective drug,which in our Hospital,often combined with chemotherapy.Several studies suggest that the Gubenyiliu Formula Ⅱ,to some extent,can inhibit tumor cell growth and induced apoptosis in MCF-7 cells,there may be autophagy.The present study aimed to investigate the inhibitory effects of Gubenyiliu Formula Ⅱ and its prescriptions on the growth of MCF-7 human breast cancer,and explore apoptosis and autophagy in cells and xengrafts in nude mice.Part 1:Inhibitory effects and autophagy observed of Gubenyiliu Formula Ⅱ and its disassembled recipes medicated serums in MCF-7 cellsObjective:To investigate the inhibitory effects of the GF Ⅱ and its disassembled recipes medicated serums,and observe autophagy induced by GFⅡ on MCF-7 breast cancer cell.Methods:20%、10%and 5%GFⅡ and its disassembled recipes medicated serums ware prepared.The MCF-7 cells inhibition was measured by methyl thiazolyl tetrazolium(MTT)assay.Microtubule associated protein 1 light chain 3(LC3 Ⅱ)expression was observed by Western blotting and immunofluorescence.Results:20%serums:Q,H,J group have higher inhibition rate(27.39%,24.97%,16.83%).10%serums:Q,B,H,J group can inhibit MCF-7 cell proliferation,Q,H group have higher inhibition rate(7.08%,10.17%).5%serum:Q,H,J group can inhibit MCF-7 growth,B group can promote cell proliferation in 6h,In the 12h,The LC3 protein expression has increased in the Q,B,H,J group(P<0.05).When 24h,Q,B,H,J group had autophagy,Q group has similar levels as 12h,H,J group bite from higher levels,after applying 3-MA,the autophagy levels in each group were decreased.In 6h and 12h,The LC3Ⅱ bands of Q,Y,H,J group have gradually thickening,LC3 Ⅱ/Ⅰ ratio have increased,but no significant difference(P>0.05).When 24h,The ratio of LC3 Ⅱ/Ⅰ,Q,H group is greater than 1,and H were significantly higher than before,since the higher level of macrophages(P<0.05).Conclusion:10%and 20%serum of GF Ⅱ and blood activation prescriptions can inhibit breast cancer MCF-7 cell proliferation.Consolidate certain inhibitory GF Ⅱ,and its serum induced autophagy in the MCF-7 cells,but different groups of inducing autophagy level.3-MA can inhibits autophagy.Blood activation prescriptions induced higher levels of autophagy,may be involved in inhibition of MCF-7 cell growth.Part 2:Inhibitory effects of Gubenyiliu Formula Ⅱ and its prescriptions on the growth of MCF-7 human breast cancer xengrafts in nude miceObjective:The present study aimed to investigate the inhibitory effects of Gubenyiliu Formula Ⅱ(GFⅡ)and its prescriptions on the growth of MCF-7 human breast cancer xengrafts in nude mice,and explore their mechanisms of action.Methods:After the establishment of the MCF-7 human breast cancer xenograft model in nude mice,the mice in the GFⅡ(6.56 g/ml),tonifying qi prescriptions(B)3.50g/ml,blood activation prescriptions(H)1.65 g/ml and detoxify prescriptions(J)1.40g/ml were administered with by gavage for 28 days,respectively.The tumor volume and weight were measured twice a week throughout the treatment period.Apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.The expression of microtubule associated protein 1 light chain 3(LC3)was examined by immunohistochemistry,and western blotting analysis was performed to detect the expression of anti-apoptotic protein Bcl-2 andLC3 Meanwhile,the effects on PI3K/Akt/mTOR signaling pathway were also determined by western blotting analysis.Results:Compared with the control group,GFⅡ group and its blood activation prescriptions could significantly inhibit the growth of MCF-7 human breast cancer xenografts in nude mice.The expression of Bcl-2 protein was lower in the GFⅡ and blood activation groups than in the control group,whereas both the percentage of apoptotic cells and LC3-Ⅱ/LC3-Ⅰ ratio were higher than in the control group.In addition,a significantly reduced expression of phospho-Akt,phospho-mTOR and mTOR was observed in the blood activation group(P<0.05).Conclusions:To some extent,GFⅡ and its blood activation prescriptions can exert their inhibitory effect on the growth of MCF-7 human breast cancer xenografts by inducing the cell apoptosis and autophagy.In addition to the induction of cell apoptosis,we also found that the blood activation prescriptions of GFⅡ could induce cell autophagy by inhibiting of PI3K/AKT/mTOR signaling pathway,and then suppress the breast cancer cell growth.