Down-regulated PI3K Pathway Might Induce Metabolic Reprogramming In Breast Cancer By PdpaMn

Objective:There is an assumption that glycolysis is upregulated and oxidative phosphorylation（OXPHOS）capacity is damaged in all cancer cells.This notion is being challenged by recent studies to advocate that mitochondrial metabolism is not impaired.Tumor cells that uses aerobic glycolysis cohabit with cancer cells with active OXPHOS.The impact of phosphoinositide-（3）-kinase（PI3K）pathway onto fatty acid synthase（FASN）has an effect on cellular metabolism.However,the influence and repercussion activities from FASN-mediated glycolysis on oxidative phosphorylation via PI3K are still indefinable.Perchance,inhibition of FASN-mediated glycolysis could restore and elevate OXPHOS.At present,we explored the anti-breast cancer mechanism and the induction of metabolic reprogramming via PI3k signaling pathway,providing a new strategy for the improvement of anti-breast cancer drugs.Methods:In vitro,four types of cancer cells:human breast cancer cell（MCF-7）,mouse breast cancer cells（4T1）,triple-negative breast cancer cell（MDA-MB-231）and human gastric carcinoma cell（BGC-823）were selected and treated with series concentrations of PdpaMn in an increasing order for 24 h.Cell viability was measured 24 h later by MTT and the 50%inhibitory concentrations(IC₅₀)were calculated to detect the inhibitory effect of PdpaMn.Then,the viability of MCF-7,4T1 and MDA-MB-231 alongside normal breast epithelial cells MCF-10A compared after treatment with PdpaMn to detect selective cytotoxicity of PdpaMn.FASN activity was tested in the three kinds of breast cancer cells after 24 h treatment of PdpaMn.Western blot to detect the expression of M-pyruvate kinase 2（PKM2）and LDHA to check some glycolysis related enzymes.Western blot was used to detect the expression of LDHA in three breast cancer cells and in normal mammary breast epithelial cells MCF-10A.To detect glycolysis variance,lactate assay kit used to detect intracellular lactic acid content.Adenosine triphosphate（ATP）detection assay kit was used to investigate the intracellular ATP content.Nicotinamide Adenine dinucleotide（NADH）assay kit was used to detect the content level of NADH.Pyruvate level assay kit used to investigate glycolytic pyruvate levels.Scratch test experiments were undertaken to detect the ability of cell migration and invasion.Western blot used to detect the protein expression of PI3K pathway to indicate its involvement in this process.To illustrate the transformation in OXPHOS,western blot was used to detect the protein expression of PDH and PDH assay kit used to detect PDH level,Acetyl coenzyme A assay kit used to ascertain Acetyl-Co A content level and Clark oxygen electrode was used to measure mitochondrial oxygen consumption after 24 h treatment with PdpaMn.In vivo,a total of 30 female BALB/c mice（6–8 weeks old）were adopted to establish the mouse breast cancer transplantation model using 4T1 mouse breast cancer cells.The mice were randomly divided into three groups,ten mice per group and the mice were treated with saline and 10 mg/kg or 20 mg/kg PdpaMn respectively.The body weight and tumor size of the mice were recorded after the tumor volume of the mice reached a certain size,and liver and spleen indexes calculated to evaluate the anti-tumor effect and toxic effects of the compound.The expression level of FASN was detected by western blot and Immunohistochemical staining to detect fold expression of FASN in tumor tissues.Lactate assay kit and ATP detection kit were used to detect the lactate level and intracellular ATP content respectively in the mice serum to check glycolysis discrepancy.The Enzyme lactate dehydrogenase A（LDHA）expression in tumor tissues was detected by western blot to check glycolysis variance.The expression of PI3K pathway was detected by western blot.Results:In vitro,MTT results illustrate that PdpaMn can inhibit the viability of a variety of cancer cells.The IC₅₀ values were MCF-7 26.24μmol/L,4T1 34.67μmol/L,BGC-823 58.48μmol/L and MDA-MB-231 51.35μmol/L respectively.PdpaMn had no significant effect on normal mammary epithelial cell MCF-10A at the same dose,indicating that PdpaMn was fairly selective to cancer cells.Furthermore,it was found that PdpaMn decreased intracellular FASN levels in all cancer cells.Western blot reveals that the protein expression of glycolysis related enzyme LDHA decreased with increased dose of PdpaMn in breast cancer cells.However,glycolytic enzyme PKM2 proteins level were not affected.Lactate acid content decreased in all the three kinds of breast cancer cells.The Intracellular ATP level steadily amplified with increasing drug dose treatment.The NADH content was down-regulated in the breast cancer cells with increased concentration of PdpaMn.These results illustrates that glycolysis was down-regulated in all breast cancer cells by PdpaMn.The glycolytic pyruvate content level increased significantly with increasing concentration of PdpaMn treatment illustrating the alteration in glycolysis of the cancer cells.The scratch test experiment indicated that the number of cells invading into the lower chamber was less with the increase of PdpaMn concentration indicating that PdpaMn could inhibit cell migration of all the breast cancer cells.The western blot shows that the protein expression of PI3K in tumor cells significantly decreased with the increase of drug dose.These results indicates that PdpaMn down-regulated glycolysis level in all the cancer cells.PdpaMn increased oxygen（O₂）consumption in mitochondria.The expression of PDH and acetyl-Co A increased with the increase of PdpaMn concentration.It showed that PdpaMn can up-regulate the oxidative phosphorylation of breast cancer cells.Finally,the regulation of all breast cancer cells energy metabolism by PdpaMn may be related to the up-regulation of PDH and down-regulation of PI3K.In vivo,the in-situ model of breast cancer was successfully established.The inhibition rates of treatment groups were 33.03%（PdpaMn 10 mg/kg）and 68.11%（PdpaMn 20 mg/kg）.Treatment with PdpaMn showed no significant changes on the body weight of the mice.Liver,thymus and spleen index were not affected significantly,and the color of liver was normal.PdpaMn significantly decrease the expression of intracellular FASN and FASN protein articulation in tumor tissues.At the same time,with the increase of PdpaMn concentration significantly reduced serum lactic acid content.The expression of LDHA decreased ominously with the increase of PdpaMn concentration in tumor mice.However,western blot showed that glycolytic enzyme PKM2proteins level were not affected with the treatment of PdpaMn.The intracellular ATP content increased significantly at lower concentration of PdpaMn but sharply decreased with increased drug dose.By western blot,protein expression of PI3K significantly decreased with the increase of drug dose.Conclusion:PdpaMn can obstruct the expression of FASN via down-regulating the expression of PI3K pathway.PdpaMn inhibits the enzyme LDHA and reduce lactate production while maintaining increase ATP content indicating change in metabolic glycolysis.PdpaMn elevated Oxidative Phosphorylation,increased acetyl-Co A content and amplified PDH level,reflecting the enhancement of mitochondrial function.In conclusion,PdpaMn could inhibit proliferation and metastasis of breast cancer cells through down regulating FASN via PI3K adjusting the energy metabolism by improving OXPHOS capacity in breast cancer.