| ObjectiveStudies have shown that people with type 2 diabetes have a higher incidence of colon cancer,and type 2 diabetes mellitus has been reported to associate with the development and progression of colorectal cancer.Hence,explore the mechanism which influences the development of colon carcinoma patients with T2 DM may help to find a therapeutic target to inhibit the occurrence of colon carcinoma patients with T2 DM.Arginine-specific mono-ADP-ribose transferase 1(ART1),an important mono-ADP ribosyl transferase,is involved in a variety of biological behaviors of cells.Our previous study has found that the expression of ART1 in colorectal cancer tissue of patients with type 2 diabetes was higher than that of non-type 2 diabetes colorectal cancer tissues,which suggested that ART1 may have a regulatory role in colon carcinoma patients with T2 DM.In addition,studies have reported that ?-caryophyllene(BCP)regulates glucose-stimulated insulin secretion by ADP-ribosylation factor 6(Arf6)in MIN6 islet cells,and BCP can inhibit the growth of various tumor cells.In high glucose environment,whether BCP inhibits glycolysis mediated by ART1 and proliferation of colon cancer cells,and the relationship between BCP and ART1 is unclear.This topic is based on previous research,investigating whether BCP inhibited the glycolysis promoted by ART1 and affected the proliferation and apoptosis of colorectal cancer.Then,the molecular mechanism of BCP inhibiting the glycolysis promoted by ART1 was explored.Provide a certain experimental basis for the choice of targeted therapy in diabetes with colorectal cancer.MethodThis study is divided into two parts.1: ?-Caryophyllene inhibits glycolysis promoted by ART1 and affects the proliferation and apoptosis of colorectal cancer(1)Effect of ART1 on glycolysis,proliferation and apoptosis in colorectal cancer CT26 cells with high glucose 1)Effect of ART1 on lactic acid and ATP content in colorectal cancer CT26 cells with high glucoseThe lactate content in the culture supernatant and intracellular ATP content of ART1-cDNA CT26 cells and ART1-sh RNA CT26 cells cultured in high glucose were detected by Lactic acid assay kit and ATP detection kit,respectively,and LV-control CT26 cells and Un-transfected CT26 cells were used as control groups.2)Effect of ART1 on the growth of colorectal cancer CT26 cells allograft transplanted tumors of DM Balb/c miceDiabetic Balb/c mouse model was established by intraperitoneal injection of streptozotocin(STZ)and nicotinamide(NA).Four groups of CT26 cells were inoculated subcutaneously in the right armpits of diabetic BALB/c mice to establish a transplanted tumor model.We observed the effect of ART1 on transplanted tumor of diabetic mice,and detected the expression of Cleaved caspase3 and Bax,which are apoptotic proteins,by Western blot in all groups.(2)Effect of BCP on glycolysis,proliferation and apoptosis in ART1-cDNA CT26 cells treated with high glucose 1)Effect of BCP on the generation of ATP and Lactic acid in ART1-cDNA CT26 cellTo observe the effect of BCP on promoted by ART1 lactate and intracellular ATP levels,ART1-cDNA CT26 cells incubating with high-glucose(25 mM)was set as control group,and ART1-cDNA CT26 cells incubating with high-glucose(25 mM)and 50 ?M BCP was set as experimental group.2)Effect of BCP on the proliferation and apoptosis of ART1-cDNA CT26 cell in high-glucoseCCK8 assay,flow cytometry,and Hoechst33258 apoptosis staining were used to detect the effect of BCP on the proliferation and apoptosis of ART1-cDNA CT26 cells.3)Effect of BCP on the growth of ART1-cDNA CT26 cells allograft transplanted tumors in DM Balb/c mice:DM Balb/c mice which were inoculated ART1-cDNA CT26 cells were treated with BCP,and the effect of BCP on the growth of the transplanted tumor was observed.Western blot was used to detect the expression of Cleaved caspase3 and Bax proteins in both groups of transplanted tumors.2: The molecular mechanism of ?-caryophyllene inhibiting glycolysis promoted by ART1:(1)Effect of ART1 on glycolytic enzymes and related factors of CT26 cells in high-glucose 1)The effect of BCP on the expression of AKT,P-AKT,MTOR,P-MTOR,and C-MYC and the glycolytic enzymes PDK1 and LDHA in CT26 cells were detected by Western blot,after cells were treated with high glucose.2)The Effect of expression of MTOR,P-MTOR,PDK1 and LDHA proteins in CT26 cells of transplanted tumors of diabetic mice were detected by Western blot.(2)Effect of BCP on ART1,glycolytic enzymes and related factors of colorectal cancer ART1-cDNA CT26 cells in high-glucose 1)Western blot was used to detect the effect of BCP on ART1 expression.2)Western blot was used to detect the expression of AKT,P-AKT,MTOR,P-MTOR,C-MYC,PDK1,and LDHA in ART1-cDNA CT26 cells treated with BCP.3)Effect of BCP on the expressions of MTOR,P-MTOR,PDK1,LDHA in ART1-cDNA CT26 transplanted tumor were detected by Western blot.Result 1.?-Caryophyllene inhibits glycolysis promoted by ART1 and affects the proliferation and apoptosis of colorectal cancer(1)Effect of ART1 on glycolysis,proliferation and apoptosis in colorectal cancer CT26 cells treated with high glucose 1)Effect of ART1 on lactic acid and ATP content in CT26 cells with high glucoseCompared to with Un-transfected group and LV-control group,the lactic acid and ATP content of ART1-cDNA group were significant increase,and these in ART1-shRNA group were both decrease in high-glucose(P<0.05).2)Effect of ART1 on the growth of CT26 cells allograft transplanted tumors of DM Balb/c miceCompared with Un-transfected group and LV-control group,the weight and volume of transplanted tumor were both statistically increased in the ART1-cDNA group and decreased in the ART1-sh RNA group(P<0.01).The expressions of Bax and Cleaved caspase3 in four groups of transplanted tumor tissues were both decreased in ART1-cDNA CT26 cells and increased in the ART1-shRNA group,compared with Un-transfected group and LV-control group(P<0.05).(2)Effect of BCP on glycolysis,proliferation and apoptosis in ART1-cDNA CT26 cells treated with high glucose 1)Effect of BCP on the generation of ATP and Lactic acid in ART1-cDNA CT26 cellCompared with ART1-cDNA CT26 cells without BCP,the concentration of ATP and Lactic acid was decreased in ART1-cDNA CT26 cells treated with BCP(P<0.05).2)Effect of BCP on the proliferation and apoptosis of ART1-cDNA CT26 cellThe results of CCK8 assay showed that with the increase of BCP concentration and time,the proliferation activity of ART1-cDNA CT26 cells was decreased.The flow cytometry detected that cell populations at the G1 phase and PI decrease in ART1-cDNA CT26 cells treated with BCP(P<0.05).The results of Hoechst 33342 staining showed that apoptosis rate was increased in ART1-cDNA CT26 cells treated with BCP than ART1-cDNA CT26 cells without BCP(P<0.05).3)Effect of BCP on the growth of ART1-cDNA CT26 cells allograft transplanted tumors in DM Balb/c miceAfter transplantation tumor model treated with BCP,the results showed that the weight and volume both statistically decreased(P<0.05).Simultaneously,the expression of apoptosis-related proteins(BAX and Cleaved caspase3)increased in ART1-cDNA CT26 transplanted tumor treated with BCP(P<0.05).2.The molecular mechanism of ?-Caryophyllene inhibiting glycolysis promoted by ART1(1)Effect of ART1 on glycolytic enzymes and related factors of colorectal cancer CT26 cells in high-glucose 1)After four groups cells were treated with high glucose,compared with Un-transfected group and LV-control group,the expressions of P-AKT,P-MTOR,C-MYC,PDK1 and LDHA were both increased in the ART1-cDNA group,and these in ART1-shRNA were decreased(P<0.05).However,the expressions of AKT and MTOR showed no statistic difference in these four groups(P>0.05).2)The expression of P-MTOR,PDK1,LDHA in CT26 cells transplanted tumor of DM Balb/c mouse were detected by Western blot,the expression of P-MTOR,PDK1,LDHA in ART1-cDNA CT26 cells were increased,while in ART1-shRNA CT26 cells were decreased,compared with Un-transfected group and LV-control group(P<0.05).The expression of MTOR showed no statistic difference in these groups(P>0.05).(2)Effect of BCP on ART1,glycolytic enzymes and related factors of colorectal cancer ART1-cDNA CT26 cells in high-glucose 1)Western blot showed that: the level of ART1 decreased in ART1-cDNA CT26 group treated with BCP in vitro and vivo(P<0.05),and PDTC also could down-regulate the expression of ART1(P<0.05).2)After ART1-cDNA CT26 cells were treated with BCP and high-glucose,the expression P-AKT,P-MTOR,C-MYC,PDK1 and LDHA were all decreased,compared to these in ART1-cDNA CT26 cells without BCP(P<0.05).Meanwhile,the expression of AKT and MTOR showed no significant difference between ART1-cDNA CT26 cells were treated with BCP or not(P>0.05).3)The expression of P-MTOR,PDK1 and LDHA also showed decreased in ART1-cDNA CT26 transplanted tumor treated with BCP(P<0.05),the expression of AKT and MTOR showed no significant difference(P>0.05).ConclusionBCP can inhibit the proliferation and apoptosis of colorectal cancer in high glucose environment,which may be achieved by inhibiting ART1 and then inhibiting glycolysis mediated by ART1 promoted by AKT/MTOR/C-MYC pathway.ART1 plays an important role in the glycolysis of colon cancer,and BCP may play an important role in the treatment of diabetic with colorectal cancer patients.The detailed mechanism needs further study. |
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