The Activities Of PI3K Inhibitor ZSTK474 On Breast Cancer MCF-7 Cell And The Molecular Mechanism

Objective:ZSTK474 is a pan PI3 K inhibitor, which can inhibit four different subtypes of type?PI3K. Previous studies showed that ZSTK474 exhibited good antineoplastic activities aganist 39 human cancer cell lines. This paper further invesigated the multifaceted activities of ZSTK474 on MCF-7 cells, including cell cycle arrest and authphagy and the mechanisms involved in cell growth inhibition. The effect of autophagy induced by ZSTK474 on tumour proliferation was also studied by us.These results will provide guide and theory for clinical study.Methods:1. MTT assay was used to study the effect of different concentrations of ZSTK474 on the proliferation of MCF-7 cells.2. Flow cytometry was used to analyze the effect of ZSTK474 on cell cycle distribution and apoptosis.3. MDC staining was used to identify autophagy.4. Transmission electron microscopy was used to observe the micro-morphology of autophagy.5. MRFP-GFP-LC3 plasmid was transfected into MCF-7 cells to observe the effect of ZSTK474 on autophagy.6. Western Blot assay was used to detect the expresss level of cell cycle related protein p27, cyclin D1, p Rb and p-GSK-3?, the autophagy markers LC3-?/?,p62, Atg5 and PI3K/Akt/m TOR signaling pathway related molecules after ZSTK474 treatment.7. MTT method was used to detect the effect of application of autophagy inhibitor3-MA and chloroquine on anti-tumor activity of ZSTK474.Result:1. MTT assay showed that ZSTK474 inhibited the proliferation of MCF-7 cells time and concentration dependently. Flow cytometry analysis showed that ZSTK474 induced MCF-7 cell cycle arrest in G1 phase, but did not induce apoptosis in MCF-7.2. MDC staining showed that after ZSTK474 treatment, numbers of bright green fluorescent vesicles appeared in cytoplasm, it was also observed autophagosomes with double membrane formed in the cytoplasm by electron microscopy, besides, autophagic flux was high in m RFP-GFP-LC3 plasmid transfected MCF-7 cells,which manifested that ZSTK474 induced autophagy in MCF-7 cells.3. After treatment with ZSTK474, the expression level of p27 increased, while the levels of cyclin D1, phosphorylated Rb and phosphorylated GSK-3? decreased in a concentration-dependent manner.4. After treatment with ZSTK474, the conversion of LC3 B ? to ? and the expression of Atg5 increased, while the expression of p62 decreased. Meanhile,phosphorylation of PDK1, Akt, m TOR, as well as p70S6 K was inhibited, while no significant changes in Akt expression.5. Pharmacological blockade of autophagy by 3-MA or chloroquine increased viability of the ZSTK474-treated MCF-7 cells.Conclusion:1. MCF-7 cell proliferation was inhibited by ZSTK474 in a dose-dependent manner with a low concentration, showing a strong anti-tumor activity of ZSTK474.2. ZSTK474 inhibited the proliferation of MCF-7 cells by inducing G1 cell cycle arrest, its mechanism may involved the regulation of p27, cyclin D1, p-Rb and p-GSK-3? expression. Our result indicated that ZSTK474 did not induce obvious apoptosis in MCF-7 cells.3. ZSTK474 induced autophagy via blocking of PI3K/Akt/m TOR pathway and the autophagy have inhibitory effect on cancer cell proliferation, which contribute to its antitumour activity.