The Mechanism Underlying Interaction Of PKC ζ With Ⅱ A HDACs Contributions To The Warburg Effect In Prostate Cancer Cells

BackgroundMetabolic changes, hallmark of cancer, are associated with the progression of cancer. Tumor cells, whether in aerobic or anaerobic conditions, must consume glucose via glycolysis to meet the demand of energy for rapid growth. The metabolic characteristic of tumor cells which was called Warburg effect is the common phenomenon of tumor cells. Warburg effect is not limited to glycolysis and the changes of the Krebs cycle, fatty acid, glutamine, serine, one carbon unit, and many other metabolic pathways in cancer cells undergo the metabolic reprogramming. Moreover, current results about metabolic diseases such as obesity, diabetes also show that these metabolic diseases are closely associated with the development of cancer. Therefore, further elucidating the Warburg effect and its role in progression of tumors not only help to reveal the correlation of tumor metabolic changes with cell growth and development of tumor, but also identify highly specific metabolic markers for tumor clinical diagnosis as well as new strategy for target therapy.Aerobic glycolysis of tumor cells, namely the Warburg effect makes mitochondrial oxidative phosphorylation (oxidative phosphorylation, OXPHOS) switch to aerobic glycolysis and pentose phosphate (pentose phosphate pathway, PPP) forming the nucleotide metabolic pathways. This abnormal glucose metabolism switch not only provides energy (ATP), biological macromolecular precursors (amino acid and nucleotide, etc.) and coenzyme (nicotinamide adenine dinucleotide phosphate NADPH) for tumor cells proliferation, but also form the acidification microenvironment through lactate secretion, which is beneficial to the growth and metastasis of tumor cells. In addition, the tumor cells reduce reactive oxygen species (reactive oxygen species, ROS) through the switch to glycolysis metabolism from the mitochondrial oxidative phosphorylation, which reduces the toxicity of ROS to the tumor cell. For this reason, metabolic abnormalities in the tumors are classified into one of eight new hallmarks of tumors (including the tumor proliferation, apoptosis resistance, the replication of unlimited potential and insensitivity against growth signals, persistent angiogenesis, invasion and metastasis ability of organization, and escape immune surveillance).Many factors lead to the Warburg effect in tumor cells, and mainly include: mutations of oncogene, loss of tumor suppressor genes, etc. Change of key enzymes in glycolysis pathway or related gene expression; Respiratory chain lack or oxidative phosphorylation efficiency caused by Mitochondrial function of mtDNA mutations of mitochondrial; Adaptation to hypoxic microenvironment, overexpression of HIF-laactivate the downstream of glycolystic related target genes and lactic acid secretion transport protein expression. Although the Warburg effect is one of the most important features of the tumor, but how the other regulatory molecules and key signaling pathways in cancers contributions to the Warburg effect of tumor cells remain unclear.PKC belongs to a family of serine/threonine protein kinases which are activated by tyrosine kinase Receptor (Receptor Tyrosine kinase, RTK) and the G protein coupled Receptor (G protein coupled Receptor, GPCR), including three subfamilies, namely Ca2+ and DAG dependent typical PKC (PKC alpha, beta, gamma); DAG-dependent and Ca2+ nondependent PKC (PKC-delta,-epsilon, eta, theta); both DAG and Ca2+ nondependent atypical PKC (PKC-zeta,-tau). PKC family play an important role in cell growth, metabolism, proliferation and the remodeling of the cytoskeletons. PKC zeta is one of the atypical form of PKC family, which contributes to the integration of extracellular signal stimulation, the regulation of cell growth, metabolism, cell polarity and relevant signal transduction. Current studies have shown that PKC zeta can promote tumor proliferation, invasion and metastasis, Moreover, PKC zeta gene deletion can promote tumor cell metabolic reprogramming, thus effectively use glutamate through serine synthesis instead of glucose uptake in glucose starvation.Existing research shows that epigenetic changes regulated by histone acetylation enzyme (HDACs) play an important role in tumor proliferation, migration, the stability of the genome, angiogenesis and tumor apoptosis. Histone acetylation enzyme (HDACs) consist of three type of Class I, Class II and Class IE. Recently, the metabolic changes of HDACs in tumor cells began to attract more and more interesting. However, whether the Class II HDACs associated with the tumor proliferation and progress of tumors contribution to the tumor metabolism especially glucose metabolism are still not clear.ObjectivesThis study is to explore the role and mechanism underlying the interaction of PKC zeta and II class HDACs contributions to the glcolysis, glycolytic related gene expression and cell growth in prostate cancer cells. The study not only helps to understand the functional role and mechanism of PKC zeta and II class HDACs in the growth of prostate cancer, but also provide basis for further identification metabolic target in tumor cells.MethodsThis research mainly explore the role and mechanism underlying the interaction of PKC zeta and II class HDACs contributions to the glcolysis, glycolytic related gene expression and cell growth in prostate cancer cells through the overexpression of plasmids or interference (si RNA) strategy. By immunofluorescence staining and immunoprecipitation method confirmed that PKC zeta and II a HDACs colocalized and interacted with each other in the nucleus, regulation of prostate cancer cells aerobic glycolysis and the tumor growth.Results1. The PKC zeta promotes the tumor cell growth Warburg effect in prostate cancer DU145 cells.Overexpression of PKC zeta promotes the growth of prostate cancer DU145 cells, glucose uptake and the secretion of lactic acid, On the contrary, knockdown of endogenous PKC zeta expression by siRNA-PKC zeta transfection significantly reduce the growth of prostate cancer DU145 cells, glucose uptake and the secretion of lactic acid.2. The PKC zeta regulates the Warburg effect related protein expression in prostate cancer cells.Real time RT-PCR and Western blot test showed that overexpression PKC zeta promotes glycolytic related gene expression (HKII, PFKP, MCT4, CD 147) in prostate cancer DU145 cells. In contrast, knockdown of endogenous PKC zeta expression by siRNA-PKC zeta transfection redcues glycolytic related gene expression (HKII, PFKP, MCT4, CD147) in prostate cancer DU145 cells. The above results suggest PKC may contribute to the Warburg effect and tumor cell growth through regualation of the glycolystic related protein expression in prostate cancer cells3. Overexpression of II class HDACs(HDAC4,5,7) reduces the growth of prostate cancer DU145 cells, glucose uptake and lactic acid production.Research has shown that there is a feedback loop between HDACs and cell metabolism effect. In order to explore the effect of II class HDACs (HDAC4,5,7) on tumor cell growth and tumor glycolysis, we transfected HDAC4,5,7 respectively into prostate cancer DU145 cells. Firstly, we detected the effect of expression of HDAC4,5,7 on prostate cancer cell growth, the results showed that the overexpression of HA-HDAC4,5,7 can remarkably decrease DU145 cell growth and survival, but knockdown of endogenous HDAC7 expression by siRNA HDAC7 transfection promote the growth of DU145 cells. Secondly, alpha CHCA, a lactic acid transporter (MCT4) inhibitor, can antagonize the cell growth promoting effect induced by knockdown of endogenous HDAC7 expression in DU145 cells. Finally, we further explore the effect of II class HDACs on the glucose uptake and lactic acid production, the results showed that the overexpression of HA-HDAC4,5,7 can reduce glucose uptake and lactic acid secretion both in DU145 cells and PC-3M cells. These results suggested that II class HDACs may inhibit tumor cell growth through negative regulation of glycolysis in prostate cancer cells.4. Overexpression of II class HDACs decrease the related protein expression of Warburg effect in prostate cancer DU145 cells.Real time RT-PCR showed that Overexpression of HA-HDAC4,5,7 obviously decrease protein expression involved in glycolysis, glucose uptake and lactic acid transport (HKâ…¡, PFKP, MCT4, CD 147) in prostate cancer cells.Western blot further showed that overexpression of HA-HDAC4,5,7 not only reduced protein expression involved in glycolysis, glucose uptake and lactic acid transport, but also inhibited key glycolytic enzyme (LDHA, PDH) and hypoxia inducing factor (HIF-1 alpha) expression.5. PKC zeta and â…¡ a HDACs colocalized and interacted with each other in the nucleus, knockdown of PKC zeta expression can reduce the key phosphorylation site of â…¡ a HDACs associated with exit from nucleus.Immunofluorescence staining showed that endogenous PKC zeta colocalized with â…¡ a HDACs HDAC4,5,7 within the nucleus. Immunoprecipitation further showed that each of HDAC4,5,7 can interact with PKC zeta. In addition, knockdown of endogenous PKC zeta expression can obviously reduce an reduce the key phosphorylation site ofâ…¡a HDACs associated with exit from nucleus. These results suggested that PKC zeta may regulate â…¡ a HDACs exit from nucleus through key phosphorylation site of â…¡ a HDACs, thus remove inhibition of glycolystic related gene expression triggered by â…¡ a HDACs.6. HDAC7 can antagonize promoting-growth effect of PKC zeta on DU145 cells.Cell growth and cell survival showed that knockdown of endogenous PKC zeta expression can remarkably inhibit the DU145 cells growth, while knockdown of endogenous HDAC7 expression can remarkably promote the DU145 cells growth. Cotransfection of PKC zeta siRNA and HDAC7 siRNA showed that knockdown of endogenous HDAC7 expression can antagonize the cell growth inhibition induced by PKC zeta siRNA transfection.ConclusionThis results indicated that PKC zeta contribute to glycolystic related gene expression and the Warburg effect through interaction with â…¡a HDACs, thus promote glucose uptake, lactic acid secretion and prostate cancer cells growth. This study will provide the basis for the intrinsic association of glucose metabolic alterations with tumor growth and progression of prostate cancer, and also provide potential metabolic targets for prostate cancer diagnosis and treatment.