Mechanism Of Lapatinib-induced Cell Tolerance From The Perspective Of Metabolic Changes In ErbB2Positive Breast Cancer Cells

Objective:To investigate the effect of lapatinib on the metabolism of ErbBl/ErbB2positive breast cancer cells, a small molecule inhibitor of the tyrosine kinase receptor of ErbB2/ErbB1, and to explore the metabolic regulation mechanisms of lapatinib effects. At the same time, to provide insight to reveal the mechnism of the apoptosis lapatinib induced of ErbB2/ErbBl positive breast cancer cells and the cell tolerance mechanisms. Try to find new targets of medicine for the combination therapy to overcome its resistance.Methods:The breast cancer cell lines was chosen of BT474(ErbB2+), SKBR3(ErbB2+) and MDA-MB-231(ErbB1+). And two Lapatinib groups of the time gradient and the concentration gradient were setted. Use Real-time PCR technology to detect mRNA expression level, andthe relative proteins expression involved in metabolic regulating signal pathways were detected by Western Blot. Using mass spectrometry and two-dimensional electrophoresis to detect metabolic changes in drug-resistant strains and maternal of protein levels, and mRNA levels were detected in the use of qPCR technology.Results:After added of lapatinib, the expression of ErbB2in BT474cell was decreased in low concentrations and increased in high concentrations, and AMP kinase (AMPK) increased. Proliferation signals Ras/MAPK and PI3K/AKT growth-promoting signals in the downstream were significantly inhibited. BT474cell Primarily makes the glucose uptake by overexpress glucose transporter1(GLUT1), and enhanced glycolytic pathway.2,6-diphosphate kinase (PFK2) expression in BT474cell was suppressed in low concentrations, and was enhanced in high concentrations, but the pyruvate was used mainly into the mitochondria for aerobic oxidation. P53phosphorylation was significantly inhibited in BT474, resulting in the pentose phosphate pathway was inhibited and fatty acid synthesis and oxidation were suppressed. Furthermore, NF-κB pathway is inhibited, resulting in intracellular reductase were inhibited in cytoplasmic (PRDX1, PRDX2, PRDX6), mitochondrial (Mn-SOD, PRDX3), endoplasmic reticulum (PRDX4), but overexpression in peroxisome(CAT, PRDX5). Mitochondrial stress response of (GRP75, VDAC1) is at slow level, and unsustainable, ultimately leading to cell dysfunctional metabolism due to ROS produced in mitochondrial can not be cleared.then endoplasmic reticulum stress the ROS caused significantly enhanced, at last apoptosis was induced by the endoplasmic reticulum pathway. Drug targets in SKBR3cells was enhanced, and its downstream signal such as proliferational P-ERK was inhibited, otherwise growth-promoting signals P-AKT slightly was enhanced. And AMP kinase (AMPK) significantly was enhanced, resulting in cell glucose transporter (GLUT1), glycolysis and pentose phosphate pathway significantly were significant enhanced. Pentose phosphate pathway slightly enhanced resulting in increased synthesis and oxidation of fatty acid. The activation of NF-κB signaling pathway in SKBR3cell increased reductase expression of cytoplasm, endoplasmic reticulum, peroxisome and mitochondria to prevent against ROS damage. However, the mitochondrial stress protection unable to cope with mitochondrial apoptosis which induced by JNK. MDA-MB-231cells, the expression of drug targets ErbB1slightly reduced after cultured with GW2974, and downstream proliferative signal P-ERK was significantly inhibited, and a significant increase in the growth promoting signals P-AKT. AMP kinase (AMPK) increased slightly. What Resulting in cell glucose uptake (GLUT4) increased significantly, and PKM2and PFK2in glycolytic pathway were significantly enhanced and lactic acid increased significantly. P-P53increases resulting in enhancement of the pentose phosphate pathway and fatty acid metabolism, the load is greatly increased mitochondria. The NF-κB signaling pathway’s activation increased intracellular reductase expression in cytoplasm, endoplasmic reticulum, peroxisomes and mitochondria, which can resist a certain ROS damage. However, the protection of its stress response is not sustainable, not antagonize mitochondrial apoptosis induced by JNK pathway. Resistant strains rBT474in metabolic changes compared to BT474, mainly increased glucose uptake (by GLUT4), enhanced the metabolic pathways of glycolysis. the pentose phosphate pathway, oxidative metabolism and fatty acid synthesis were increased. Intracellular reductases of Mn-SOD, CAT and PRDX family are different degrees of enhancement. Mitochondria and endoplasmic reticulum stress response greatly increased protection and contribute to a higher tolerance to oxidative stress.Conclusion:P-AKT, P-P53and the AMPK regulates cell glucose uptake. And Ras/MAPK and PI3K/AKT regulates glycolysis, P53regulating metabolism of pentose phosphate pathway, lactate dehydrogenase (LDHA) regulate the anaerobic glycolytic pathway metabolism, changes in fatty acid synthesis and oxidation, improve the efficiency and level of aerobic oxidation of mitochondrial energy metabolism. NF-κB’s activation resulted in intracellular reductase and protection of mitochondrial reticulum stress response enhanced, to resist excessive mitochondrial ROS, and ultimately decide the fate of cells according to the level of intracellular accumulation of ROS. BT474has low capacity of metabolic regulation, its ROS induced apoptosis directly through the endoplasmic reticulum apoptosis pathway. and in SKBR3and MDA-MB-231cell, metabolic regulation were increased to some extent, and decreased the ability to generate excess ROS, which activate JNK signaling pathway to induce mitochondria apoptosis. Resistant strains increase metabolic efficiency and reducing ability to allow the reservoir to reduce ROS and survival.