| In recent years,people found that pyruvate dehydrogenase kinase(PDK1)I was overexpressed in ovarian tumor tissues,at the same time,studies had shown that PDK1 is a potential anti-tumor target.Previous computer analysis showed that coumarin compounds might be potential effective PDK1 inhibitors.Dicumarol is a kind of coumarin compounds and is used as anticoagulant drugs in clinical application more than 60 years,which was recently found to inhibit tumor growth.However,anticancer mechanism of dicumarol was still unclear.In our research,we discovered that it could combine lipoamide site on the PDK1 through the computer analysis.Due to this fact,we speculated that dicumarol could inhibit PDK1 to promote ovarian tumor cell apoptosis and would be a novel,safe and effective kind of biological targeting drug against ovarian tumor in the future.Purpose: In this paper,we want to observe the changes in PDK1 activity,glucose metabolism,reactive oxygen species level,mitochondrial membrane potential level and apoptosis rate of ovarian cancer cells which were given by dicoumarol.Apoptosis rate of the animal model was also concerned by measuring tumor volume and weight,conducting TUNEL staining assay and testing Caspase3 and PARP expression level.Therefore,we mainly investigated the potential use of dicumarol against ovarian cancer cells and its possible antitumor mechanism.Method: 1.We detected whether there existed a combination between dicumarol and PDK1 and regulation of PDK1 activity by dicoumarol through taking computer analysis,enzyme linked immunosorbent assay(ELISA)and western blot method measuring the expression of p-PDHA1 in ovarian cancer cells.MTT colorimetry,Hoechst 33342 immunofluorescence staining and Annexin V/PI flow cytometry and western blot method to detect Caspase3 and poly(adp ribose polymerase(PARP)expression were used in the evaluation of dicumarol influence on cell apoptosis.2.Glucose quantitative kit and lactic acid determination kit were used to survey the change of glucose consumption and lactic acid production in ovarian cancer cells after dicoumarol treatment.Application of time-resolved fluorescence probe was taken to detect real-time change of oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)in ovarian cancer cells and to make assessment of glucose metabolic remodelling in ovarian cancer cells handled by dicumarol.Determinations of mitochondrial reactive oxygen species(ROS)level and the level of mitochondrial membrane potential(MMP)were carried out to evaluate oxidation reduction level and the effects of mitochondrial function in ovarian cancer cells treated with dicumarol.3.Constructing subcutaneous human SKOV3 xenografts model was conducted to analyze tumor growth influence on this kind of animal model coped with dicumarol intraperitoneal injection.SKOV3 xenografts were randomly divided into 5 groups,saline group and solvent control group,positive control group,low dose dicumarol group and high dose dicumarol group.By means of measuring tumor size and tumor weight,operating TUNEL and p-PDHA1 immunohistochemical(IHC)experiments among those five groups,we evaluated safety and efficacy of dicumarol intraperitoneal injection in subcutaneous ovarian tumor nude mice.Results: 1.The computer software analysis showed us that dicumarol could combind with lipoamide site on pyruvate dehydrogenase kinase I.We found dicoumarol could inhibit PDK1 activity via ELISA kit assay and the half-maximal inhibitory concentration(IC50)was 19.42 ±0.032 micromole.Western blot method found that dicumarol could increase the expression of Caspase3 and PARP in ovarian cancer cells.Dicumarol could also promote ovarian cancer cell apoptosis rate determined by MTT colorimetry,Hoechst staining and Annexin V/PI flow cytometry.2.Glucose consumption increased and lactate generation decreased in ovarian cancer cells treated with dicumarol after 24 h.Time-resolved fluorescence real-time detection showed oxygen consumption rate enhanced and extracellular acidification rate declined in ovarian cancer cells under the effect of dicumarol,which reversed the Warburg effect of ovarian cancer cells and transferred aerobic glycolysis into oxidative phosphorylation.Flow cytometry test elucidated that ROS level in the mitochondria increased and MMP level decreased under the action of dicumarol in ovarian cancer cells.3.Nude mice weight change is not obvious,but tumor size and tumor weight were smaller than normal saline control group in subcutaneous ovarian tumor nude mice after intraperitoneal injection of dicumarol.Immunohistochemical study found dicoumarol could mitigate p-PDHA1 expression,which indirectly reflected its ability of downregulating PDK1 activity.TUNEL staining assay discovered that dicoumarol can promote apoptosis of ovarian tumor cells.Conclusion: In this article,we used computer analysis technology for the first time to find that dicumarol was a novel potential pyruvate dehydrogenase kinase inhibitor.At the same time,we used ELISA technique to determine dicumarol could combine with PDK1 and restrain the activity of PDK1.Western blot method and immunohistochemical experiments verified dicumarol could cut the expression of p-PDHA1 both on a cellular and tissue level,indirectly reflecting that dicumarol inhibited the activity of PDK1.The observation that glucose consumption and ROS level in the mitochondria enhanced while lactate production and MMP level reduced had shown dicumarol could reconstruct glucose metabolism of ovarian tumor cells,promoting the transformation from aerobic glycolysis to oxidative phosphorylation.Determined by MTT colorimetry,Hoechst staining,Annexin V/PI,TUNEL staining,Caspase3 and PARP immunoblot assays suggested that dicumarol could facilitate apoptosis of ovarian cancer cells in different levels.In conclusion,the activity of pyruvate dehydrogenase kinase I inhibiting by dicumarol accelerated ovarian tumor cell apoptosis.Not only we added a new member for the family of PDK1 inhibitors,supplemented dicoumarol antitumor mechanism,but also enriched the proof that PDK1 is the crucial molecule to reverse tumor cell glucose metabolism and to promote tumor cell apoptosis... |
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