Expression Of Pyruvate Dehydrogenase Kinase 1 In Human Colon Cancer And The Effect On Cell Proliferation And Chemotherapy Of Its Inhibition

Background and aimsColorectal cancer is the one of the most common malignant tumor in the world, and the change of people's lives and diet lead to the high morbidity and mortality of colorectal cancer. It is reported that the morbidity increases 4.2% every year. Now the effctive treatment includs operation and chemotherapy, which ease the pains of patients partly. However, the current treatment still has deficiency. The efficacy of surgery and chemotherapy is not significant for advanced stage patients.Therefore,other adjuvant therapies are required for colorectal cancer and enhanced effect of chemotherapy.Early in the 1920s, Otto Warburg proposed that cancer cells obtained energy from glycolysis in hypoxia enviroment. Some cancer cells depended on glycolysis even in the presence of available oxygen, and such an enhancement of glycolysis becomes known as the"Warburg effect". The most striking biochemical phenotype of malignant tumor cells is their elevated glycolysis,which is one of the main energy sources of ATP. Based on Warburg effect,positron emission tomography(PET)is widely used in the clinical diagnosis of cancers.Importantly,the therapeutic strategies targeting tumor bioenergetics are being explored.Among the four mammalian pyruvate dehydrogenase kinase 1(PDK1)types(I-IV), which catalyze the pyruvate dehydrogenase (PDH), which is one of the most important steps of glycolysis. PDK1 is predominantly overexpressed in the most poorly differentiated malignant tumors.In such normal tissues as cardiac and skeletal muscles,and pancreatic island, PDK1 often maintains very low levels. And PDK1 is quite important in the process of PDH phosphorylation, and regulates PDH in long term. It is repoted that PDK1 could be regulated by HIF-1α,which is associated with the hypoxia enviroment of tumor.Recent studies have shown that PDK1 plays important roles in both glucose metabolism and cellular apoptosis,and inhibition of PDK1 could induce apoptosis of malignant tumor cells. In vitro study, it has been found that HIF-1αand PDK1 over express in head and neck squamous cancer, Non-Small-Cell Lung Cancer and brest cancer. It has not been reported whether high HIF-1αor PDK1 activity was found in in human colorectal cancer tissues, or high PDK1 in cultured colorectal cancer cells and animal models of experimental colorectal cancer. As well as the expression of PDK1, its mechanisms and their therapeutic significance are not clear in colorectal cancer.The result of chemotherapy is poor although it is a major treatment. It is known that colon cancer cells are not very sensitive to single dose chemotherapy. PDK1 integrates energy metabolism of tumor cell with control of apoptosis,which may contribute to treatment, and inhibitiong of PDK1 may enhance the effectiveness of current chemotherapy. Regulation of mitochondria influences the growth,proliferation and apoptosis of cells.It was indeedly demonstrated that the structure and function of mitochondria changes in cancer cells which resulted in incomplete of mitochondria membrane and high resting membrane potential of mitochondria membrane. As a result, the proliferation is inhibited and apoptosis is induced. It is proposed that inhibition of PDK1 regulates the membrane structure and function and decreases the membrane potential. Whether it is related to the proliferation and apoptosis with colorectal cancer remain be studied.The aim of this study is to exam the expression and distribution of HIF-1αand PDK1 in human colorectal cancer tissue, as well as the expression of PDK1 in four colorectal cancer cell lines. Also, we observed the effect of PDK1 inhibition on cell proliferation, apoptosis and chemotherapeutic treatment in colorectal cancer cells. Furthermore, the effect of mitochondria function was also analyzed .Methods1,The expression and distribution of hypoxia-inducible factor-1α(HIF-1α) and PDK1 in human colorectal cancer tissue were detected in human colon carcinoma tissue by immunohistochemistry using streptavidin/peroxidase(SP) and double-label immunofluorescence methods. And the relationship with the certain clinicopathological features, such as tumor location, differentiation degrees, Dukes'stages and lymphatic invasion were analyzed.2,The mRNA and protein expression of PDK1 in human colorectal cancer tissue and four kinds of human colon cancer cell lines were detected by immunohistochemistry, RT-PCR, real-time PCR , immunofluorescence and Western blot analysis respectively.2,The effect of PDK1 inhibitor(Dichloroacetate,DCA)on colon cancer cell proliferation,apoptosis and chemotherapeutic sensitivity were analyzed via MTT assay, BrdU uptake test, Hoechst 33258 stain and Annex-V staining respectively.3,The mechanism that DCA induced cellular apoptosis were assessed by means of exploring mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration.4,In vivo, LoVo cells were inoculated in the right front armpits of the mice, then the effect of DCA and OXA on the mass of tumor was observed the tumors were measured and weighed after a 4-week follow-up period. Then,the expression of Caspase-3 and PCNA were detected by immunohistochemistry.Results1,Positive expression of HIF-1αans PDK1 was found in 78.04% and 76.20% of cases of colorectal cancer respectively,which were much different than that in normal samples(P<0.01).The positive expression of PDK1 correlated with the Duke's stages of cancer cells and lymph node metastasis(P<0.05),but not related to either patient's gender,age,tumor location, tumor size or Dukes'stage(P>0.05). Association analysis displays that the expression of HIF-1αprotein is correlated significantly with PDK1.2,High mRNA and protein expression of PDK1 were observed in colorectal cancer tissue and four kinds of human colon cancer cells(LS174T,LoVo,HT-29 and SW-620) respectively. And LoVo expression is the highest.2,Treated with DCA at different concentrations(0-90mmol/L)for 48h, there were obvious growth inhibition of LoVo,LS174T,HT-29 and SW620 cells respectively. Combined with low dose of DCA(2.5mmol/l,5 mmol/l)for 48h, the inhibition rate of 5-fluorouracil(5FU,5,10μg/ml) and Oxaliplatin(OXA,0.5,1μg/ml) is higher than 5FU or OXA alone on LoVo cell proliferation. After analysis of data, it is showen that DCA has synergetic effect with 5FU or OXA.3,When cells are treated with DCA at different concentrations(0-30mmol/L), cellular apoptosis is found via Hoechst 33258 stain and flow cytometry. Combined with low dose of DCA(2.5mmol/l,5 mmol/l)for 48h, the apoptosis rate of 5-fluorouracil(5FU,5,10μg/ml) and Oxaliplatin(OXA,0.5,1μg/ml) is higher than 5FU or OXA alone on LoVo cell apoptosis. And the decreasment of mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration is lower than 5FU and OXA alone.4,In nude mice experiment, three groups of LoVo transplantable tumor were treated with DCA or OXA or combination. The average tumor volume and weight were significant lower than those of the control respectively(both,P<0.01).As compared with control, proliferation indexes of the LoVo cells treated with three groups of drugs were significant lower, and their apoptosis indexes were significant higher.Conclusion1,Overexpression and coexpression of HIF-1αandPDK1 protein do exist in human colorectal cancer tissue.The expression of HIF-1αandPDK1 is not correlated with gender, age,location,and differentiation degree.But the expression of HIF-1αand PDK1 at different Dukes'stages and whether involved in lymphatic invasion shows a significant difference. Association analysis displays that the expression of HIF-1αprotein is correlated significantly with PDK1.2,PDK1 inhibitor DCA induced colorectal cancer cells apoptosis via decreasing the mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration, and increasing mitochondrial membrane permeability.3,Inhibition of PDK1 increases the chemosnsitivity of colorectal cancer LoVo cells via decreasing the mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration, and increasing mitochondrial membrane permeability.