Diisopropylamine Dichloroacetate Inhibits Malignant Tumors Through Autophagy:In Vitro And In Vivo

Background and ObjectiveMalignant tumors severely jeopardize the human being currently and various kinds of therapies are emerging due to the refractory results of treatment.Metabolic reprogramming is one of the hallmarks of cancer.Autophagy is a process of degradation of damaged or redundant proteins and organelles to maintain the cellular homeostasis and turnover,which is utilized by the tumor cells to against the stress in order to sustain the survival.Targeting altered metabolism or atuophagy are becoming potent therapeutic strategies in cancer therapy.Dichloroacetate(DCA),an inhibitor of pyruvate dehydrogenase kinase(PDK),can inhibit tumor growth via blocking aerobic glycolysis.Diisopropylamine dichloroacetate(DADA)is an effective clinical hepatoprotective agent,which is synthesized by DCA and Diisopropylamine(DIA).The present study is to explore the antitumor efficacy of DADA in malignant tumors.We also systemically investigated the antitumor efficacy of DADA in comparison with DCA.In addition to its inhibitory effect on aerobic glycolysis,we found for the first time that DADA effectively blocked autophagic flux resulted in incompleted autophagy,which was contributed by DIA.Our results provide new insights for treatment of malignant tumors using DADA,which can be readily translated into clinics.Materials and Methods1.Detection of cell inhibition effect:1)Human hepatocarcinoma cell strains 97H,7492,LM3,human cholangiocellular carcinoma cell strain QBC939,human pancreatic carcinoma cell strain PANC-1,human gastric carcinoma cell strain SGC-7901,human breast cancer cell strains MDA-MB-231 and MCF-7 are exposed to the DADA and DCA at increasing concentration for 48h respectively followed by determination of cell viability and inhibition rate via MTT assay.2)Human breast cancer cell strains MCF-7 and MDA-MB-231 are selected to determine the IC50 of DADA and DCA 48h after exposure to agents with gradient concentration via MTT assay.2.Detection of suppression effect in vivo:5×106 cells are injected into right back of 6?8-week-old female BALB/c nude mice followed by observation of the xenograft tumor growth.About one week after the cell transplantation the volume of graft tumors reach 100?150mm 3 and the mice are divided into groups.1)The mice are divided into three groups as control group,low dose DADA group(15 mg/kg)and high dose DADA group(100 mg/kg),and are performed with intragastrical administration of normal saline or DADA.The growth curve is made by the determination of the volume of tumor every three days.2)The mice are divided into three groups as control group,DADA group(100mg/kg)and DC A group(100 mg/kg)with intragastrical administration of normal saline,DCA and DADA respectively.The growth curve is made by the determination of the volume of tumor every three days.3.Detection of the expression of autophagy associated protein:1)The expression of p62 and LC3 are determined via western blot analysis 24h or 48h after the MDA-MB-231 and MCF-7 cells exposure to the DADA or DCA with gradient time and concentration.2)The expression of p62 and LC3 are determined via western blot analysis 24h after the MDA-MB-231 cells exposure to DADA combined with Chloroquine(CQ)and DADA combined with BEZ235.4.Detection of related metabolism:1)The supernatants are harvest some time after the MDA-MB-231 cells exposed to the DADA or DCA with gradient time and concentration to measure the lactic acid production and consumption of glucose and made the curve.5.Detection of autophagy:1)The plasmids expressed the GFP-LC3 are transfected into cells for 24h followed by addition of DADA for another 24h,then distribution of the LC3 is observed by confocal microscopy after staining of the cells.2)The typical ultramicro-structure of double membranes vesicles are observed by transmission electron microscope in the MCF-7 cells treated with DADA for 24h.Results1.DADA can inhibit the proliferation of several malignant cell strains in vitro and suppress the growth of xenograft tumor in vivo with dose of 100 mg/kg.Compared with DCA,DADA is more effective on the inhibition of tumor cell proliferation and tumor growth although the DCA also has the suppression effect.The low expression of Ki67 is found in the DADA group via immunohistochemistry detection instead of in DCA group.2.The potential mechanism that DADA is more effective than DCA on inhibition cell proliferation ascribes to:1)The DADA suppresses the production of lactic acid and uptake of glucose of tumor cells more effectively than DCA in the tumor metabolism;2)DADA and DCA both induce the autophagy,while the DADA induces the incomplete autophagy which leads to autophagic cell death instead of DCA.3.DADA induces autophagy which is evidenced by:massive vesicles in the tumor cells but not in normal cells;the autophagic marker lapidated LC3 can be detected via western blot in the tumors treated with DADA;the autophagic puncta can be monitored by LC3 expressing green fluorescent protein distributes on the surface of vesicles using confocal microscopy;and the double-layered membranes can be observed by transmission electron microscope.4.DADA blocks autophagic flux resulted in incompleted autophagy:1)The autophagic receptor protein SQSTM1/p62 and lapidated LC3 are both increase at late stage after DADA treatment(over 24h),which indicates blocked autophagic flux;2)Accumulation of p62 instead of degradation together with autolysosomes is observed at late stage by confocal microscope via transfection of RFP-p62 plasmid;3)The combination of DCA and DIA gets the same results of induction of autophagy with DADA instead of them alone.Conclusions1.DADA inhibits the proliferation of tumor cells in vitro and suppresses the growth of xenograft tumor in vivo,which is more effective than DCA.2.The mechanisms that DADA is more effective than DCA include the more effective inhibition of lactic acid production and uptake of glucose and induction of incomplete autophagy.3.The vesicles induced by DADA in tumor cells are demonstrated to be autophagosomes.And these increased vesicles are due to blocked autophagic flux induced by DADA.The blocked autophagic flux is contributed by DIA,which enhances antitumor effect.