Anti-breast Cancer Effect And The Mechanism Of Novel Manganese Complex PdpaMn

Aims of study:This study sought to evaluate the effects of a novel manganese complex PdpaMn?[?Pdpa?MnCl₂]?on fatty acid synthesis and mitochondrial function in breast cancer.The hypothetical mechanisms of anti-breast cancer on fatty acid synthase?FASN?and glycolysis investigated in order to develop a"double-effect"metal antitumor drug that can regulate fatty acid synthesis and mitochondrial function.Materials and Methods:Four different cancer cell lines including MCF-7,SMMC-7721,HCT116 and 4T1 cells treated by PdpaMn for 24 h.The Cancer cells proliferations detected by MTT assay and the 50%inhibitory concentrations(IC₅₀)were calculated.To compare the inhibition rate of PdpaMn on normal breast epithelial cells MCF-10A and breast cancer cells MCF-7 distinguished.At the same time,4T1 mouse breast cancer cells was used to establish a mouse xenograft tumor model,which were treated by PdpaMn and 5-Fu and then measure tumor volume and organ index.The expression of PCNA analyzed by H&E staining and immunohistochemistry to detect the inhibition of PdpaMn on tumors.The expression of FASN in cells and tumor tissues detected by western blot.FASN activity in cells detected by fatty acid synthase kit.Free fatty acid kit used to detect free fatty acids in serum.The change of cell nucleus observed by Hoechst 33342 staining.The rates of cell apoptosis detected by staining with AnnexinV/PI.Western blot used to analyze releasing of Cytochrome C?Cyto C?from mitochondria and expressions of cleaved-caspase-9 and PCNA protein in cells.The mitochondria functions were analyzed:mitochondrial membrane potential was detected by JC-1 staining,ATP content in cells and tumor tissues was detected by ATP assay kit,O₂ consumption was measured by Clark oxygen electrode,generation of reactive oxygen species?ROS?in cells was detected by DCFH-DA staining.Glucose transporter 1?GLUT1?,pyruvate kinase M2?PKM2?,and lactate dehydrogenase?LDHA?expression were further measured by western blot.The lactic acid assay kit used to measure the content of lactic acid in the culture medium.The expression of LDHA in tumor tissue and serum also detected.The activation of the PI3K/Akt pathway in the cells investigated by using FASN specific inhibitor G28UCM and PdpaMn to verify the relationship between FASN and glycolysis.Results:The manganese complex PdpaMn can significant inhibit the proliferation of a variety of tumor cells with IC₅₀ values around 30-56?mol/L.PdpaMn dose-dependently inhibited the proliferation of breast cancer cells MCF-7 observed by MTT assay and microscopy,however it has no significant effect on breast epithelial cells MCF-10A.The results showed that PdpaMn can effectively inhibit the growth of tumors and has limited effect on normal organ function.H&E staining and the decrease of PCNA level in the tumor tissue demonstrated that PdpaMn inhibited tumor proliferation.In the meantime,it was discovered that this selectivity identified with FASN levels in tumor cells.PdpaMn can decrease intracellular FASN movement,protein articulation,and serum free unsaturated fats.PdpaMn can prompt apoptosis in tumor cells by inhibiting the expression of FASN:the nucleus shrinks and the number of apoptotic cells increases significantly,whereas the expression of intracellular PCNA decreases and the expression of Cyto C and cleaved-caspase-9 in cells increases.In addition,we found that PdpaMn could damage mitochondrial function:mitochondrial membrane potential reduced in both breast cancer cells,ATP creation clined and Pdpa Mn can incite intracellular ROS age expanded,which can be abolish after given NAC as pre-protection.On the other hand,we found that PdpaMn can also inhibit the expression of LDHA in cells and tumor tissues and decrease the production of lactic acid,but it has no critical impact on GLUT1 and PKM2.The impact of PdpaMn on PI3K/Akt pathway additionally distinguished after FASN specific inhibitor G28UCM treated.It was detected that PdpaMn and G28UCM has a comparative inhibitory impact on FASN and from that point onward,LDHA expression and PI3K and phosphorylated Akt expression were stifled.It was additionally discovered that hindrance of FASN may down-manage PI3K/Akt pathway and in this way diminish glycolysis levels.Conclusions:PdpaMn apply great and specific anticancer action that might be identified with the articulation level of FASN in various cells.Out of the blue,this examination found that PdpaMn could harm mitochondrial function and induce ROS-mediated apoptosis in breast cancer cells.In addition,PdpaMn found for the first time to inhibit the expression of fatty acid synthase and reduce fatty acid synthesis.The inhibition of FASN expression reduces the activity of the PI3K/Akt pathway and inhibits glycolysis.PdpaMn,could damage mitochondria and remove the vital energy source of breast cancer,exerts an effect on"double effect"anti-tumor,and its mechanism required further discussion.