| Background and aim:The incidence of colorectal carcinoma is upgrading with the change of residents’ diet and lifestyle in our country, however, the therapeutic is less effective.The cancer drug resistance of the colorectal carcinoma could be improved after the level of autophagy elevating in colorectal carcinoma, and then it could help cancer cells continue to grow. Autophagy plays a positive role in the development of colorectal carcinoma. Hence, to explore the molecular mechanisms of regulation of autophagy in colorectal carcinoma is beneficial to realize the impact factors of autophagy and to find a new therapy target for the treatment of colorectal carcinoma. ART1 and PARP-1, which can transfer the single ADP-ribose moiety or ADP-ribose polymers to certain protein amino acids of target proteins, catalyze the mono-ADP-ribosylation or poly-ADP-ribosylation respectively. In our previous studies, we had studied that both ART1 and PARP-1 were involed in the development of colorectal carcinoma. The inhibition of ART 1 and PARP-1 could restrain the proliferation and invasion of colorectal carcinoma cells. ART1 and PARP1 even had a synergistic effect on CDDP induced apoptosis in CT26 cells. We speculate that ART1 could regulate PARP-1 derived from mono-ADP-ribosylation maybe the basis of poly-ADP-ribosyaltion which is reported in some studise. Research has showed that PARP-1 could participate in the regulation of autophagy through the mTOR pathway. In this study,we will detect the role and concrete mechanism of ART1 in starvation-induced autophagy of CT26 cells, and further study the effect of starvation-induced autophagy which promoted by ART1 on proliferation and apotosis of CT26 cells.Methods:This research includes three parts.l.The effect of ART1 on the formation of autophagy in starvation-induced CT26 cells. (1) CT26 cells was disposed with D-Hanks solution for inducing the autophagy. To obseve the formation of autophagosome regulated by ART1 in starvation-induced CT26 cells, the formation of autophagosome in ART1-shRNA CT26 cells, Vector CT26 cells,GFP-ART1 CT26 cells and untransfected CT26 cells with starvation-induced or not was detected by electron microscopy, acridine orange stainin and flow cytometry. (2)To observe the effect of autophagy labelled protein LC3A and LC3B regulated by ART1, RT-PCR and western blot were used to detect the change of mRNA and protein level of LC3A andLC3 B in starvation-induced ART1-shRNA CT26 cells, Vector CT26 cells,GFP-ART1 CT26 cells and untransfected CT26 cells and subcutaneous tumor in BALB/c mice respectively.2.The research about concrete mechanism of starvation-induced autophagy regulated by ART1 in CT26 cells. (1) To detect the relationship between ART1 and integrina7/Racl/NF-kB/PARP-l,the expression and correlation of PARP-1 and ART1 in colorectal carcinoma were detected by immunochemistry assay. PARP-1 and ART1 double-label immunofluoresence staining revealed the change of expressions of PARP-1 and ART1 in CT26 cells with the inhibitor 5-AIQ or MIB. Co-immunoprecipitation assay was used to detect the interaction of ART1 and integrina7 in starvation-induced GFP-ART1 CT26 cells and Vector CT26 cells. The expression of LC3A and LC3B in starvation-induced GFP-ART1 CT26 cells with inhibitor of PARP-1 or Racl which was detected by western blot. Expressions of Racl and PARP-1 in starvation-induced ARTl-shRNA CT26 cells, Vector CT26 cells,GFP-ART1 CT26 cells, untransfected CT26 cells and subcutaneous tumor in BALB/c mice were detected by western blot respectively. For further research to determine the pathway of the regulation of ART1 in PARP-1, the change of expressions of Racl,PARP-1 and NF-κB in starvation-induced GFP-ART1 CT26 cells with inhibitor of PARP-1 or Racl were detected by western blot respectively. (2) To detect the relationship between ART1 and LKB1/AMPK/mTOR,expressions of LKB1, p-AMPK and p-p70S6 in starvation-induced ART1-shRNA CT26 cells, Vector CT26 cells, GFP-ART1 CT26 cells, untransfected CT26 cells and subcutaneous tumor in BALB/c mice were detected by western blot respectively. For further research to determine the pathway of the regulation of ART1 in starvation-induced autophagy is rely on LKB1/AMPK/mTOR regulated by Racl and PARP-1, the change of expressions of LKBl,p-AMPK and p-p70S6 in starvation-induced GFP-ART1 CT26 cells with inhibitor of PARP-1 or Racl was also detected by western blot respectively.3. The effect of starvation-induced autophagy regulated by ART1 on proliferation and apotosis in CT26 cells.(1) To observe the effect of starvation-induced autophagy regulated by ART1 on proliferation, the change of proliferation in starvation-induced GFP-ART1 CT26 cells with 3-MA inhibitor was detected by CCK-8, soft agar assay, flow cytometry; the ability of tumor fromation was observed in GFP-ART1 CT26 subcutaneous transplanted tumor of starvation-induced BALB/c injected with 3-MA. (2) To observe the effect of starvation-induced autophagy regulated by ART1 on apotosis, the change of apotosis in starvation-induced GFP-ART1 CT26 cells with 3-MA inhibitor was detected by flow cytometry, Hochest33342 and caspase3 activity assay; the change of caspase3 activity was also observed in GFP-ART1 CT26 subcutaneous transplanted tumor of starvation-induced BALB/c injected with 3-MA.Results:1.The effect of ART1 on the formation of autophagy in starvation-induced CT26 cells.(1) Autophagosome were not observed in the absence of starvation ARTl-shRNA CT26 cells, Vector CT26 cells,GFP-ART1 CT26 cells and untransfected CT26 cells. Starvation-induced GFP-ART1 CT26 cells displayed some autophagosomes, which were more numerous than in the starvation-induced untransfected and starvation-induced Vector CT26 cells. Autophagosomes were hardly observed and many apoptotic cells were detected in starvation-induced ARTl-shRNA CT26 cells.2) The autophagosome with acridine orange staining are labeled in red florescence and the red florescence intensity correlates with the level of autophagy. There was no obvious red florescence in each group when CT26 cells without starvation. The red florescence in starvation-induced GFP-ART1 CT26 cells was higher than in other groups. By contrast, the red florescence was lower in starvation-induced ART1-shRNA CT26 cells, compared to other groups.3) Comparison of the intensity of the red florescence with flw cytometry showed that the red florescence was stronger in the starvation-induced GFP-ART1 group and weaker in the starvation-induced ART1-shRNA CT26 cells, compared with control group(P< 0.01). (2) The protein and mRNA levels of LC3A and LC3B in starvation-induced ART1-shRNA CT26 cells, Vector CT26 cells,GFP-ART1 CT26 cells, untransfected CT26 cells and subcutaneous tumor in BALB/c mice were measured by western blot or RT-PCR, respectively, to assess the level of autophagy under starvation-induced conditions. The mRNA level of LC3A and LC3B, the protein ratio of LC3B to LC3A were all increased in the starvation-induced GFP-ART1 group and decreased in starvation-induced ART1-shRNA group, compared with control groups in vitro and in vivo, respectively (P<0.05)2. The research about concrete mechanism of starvation-induced autophagy regulated by ART1 in CT26 cells. (1)The relationship between ART12 and integrina7/Racl/NF-KB/PAPR-1.1) In the human colorectal carcinoma, the positive ratio of PARP-1 and ART1 was signifiantly higher than that in the control colonic mucosa samples (P<0.01). A positive correlation was observed in the expression of PARP-1 and ART1 in the colorectal carcinoma tissues (P<0.01).The positive ratio of PARP-1 and ART1 also has correction with lymph node metastasis of tumor(P<0.05).2) The result of double-label immunofluoresence staining showed that a signifiant decrease in PARP and ART1 expression was observed in the CT26 cell with MIBG, compared to the untreated CT26 cells (P<0.05). In the CT26 cells with 5-AIQ, the expression of PARP was lower than that in untreated CT26 cells (P<0.05); however, the expression of ARTl did not differ between CT26 cells with PARP-1 inhibitor and CT26 cells without PAPR-1 inhibitor (P<0.05).3) To determine whether ART1 interacts with integrin a7, co-immunoprecipitation was performed on extracts of starvation-induced ART1-GFPCT26 cells and vector CT26 cells. The ARTl has interaction with integrin α7 in starvation-induced CT26 cells. 4) To provide additional evidence for regulation of starvation-induced autophagy by ART1 via PARP-1 and Racl, the effect of inhibitors of Racl or PARP-1 on expression of LC3A and LC3B in starvation-induced GFP-ART1 cells was investigated. The protein ratio of LC3B to LC3A decreased in starvation-induced GFP-ART1CT26 cells with inhibitor (P< 0.01).5) Expressions of Racl and PARP-1 were lower in starvation-induced ARTl-shRNA CT26 cells and subcutaneous transplanted ARTl-shRNA CT26 tumor than those in control groups (P<0.05). Expressions of Racl and PARP-1 were higher in starvation-induced GFP-ART1 CT26 cells and subcutaneous transplanted GFP-ART1 CT26 tumor than those in control groups (P<0.01).6) To explore whether ART1 could regulate PARP-1 through Racl and NF-kB, starvation-induced GFP-ART1 CT26 cells were treated with NSC23766 or 5-AIQ. Compared to the starvation-induced GFP-ART1 CT26 cells without inhibitor, expressions of NF-kB (nucleus) and PARP-1 decreased in starvation-induced GFP-ART1 CT26 cells treated with NSC23766 (P< 0.05). There was no signifiant difference in the expression of Raclbetween starvation-induced GFP-ART1 CT26 cells with 5-AIQ and starvation-induced GFP-ART1 CT26 cells without 5-AIQ (P>0.05). (2) The relationship between ART1 and LKBl/AMPK/mTOR.1) Western blotting was used to measure the expression of proteins LKB1 p-AMPK and p-p70S6k in vitro and in vivo. Expressions of LKB1 and p-AMPK were lower in starvation-induced ART1-shRNA CT26 cells and subcutaneous transplanted ART1-shRNA CT26 tumor than in control groups (P<0.05). Expressions of LKB1 and p-AMPK were higher in starvation-induced GFP-ART1 groups than in control groups (P<0.01). The expression of p-p70S6k declined in starvation-induced GFP-ART1 group (P<0.01) but was elevated in the starvation-induced ARTl-shRNA group, compared with control groups (P<0.01).2) To explore whether ART1 could regulate starvation-induced autophagy via AMPK/mTOR regulated by Rac1 and PARP-1, starvation-induced GFP-ART1 CT26 cells were treated with NSC23766 or 5-AIQ. Compared to starvation-induced GFP-ART1 CT26 cells without inhibitor, the expression of p-AMPK decreased in starvation-induced GFP-ART1 CT26 cells treated with NSC23766 or 5-AIQ(P<0.05). However, the expression of p-p70S6K was enhanced in starvation-induced GFP-ART1 CT26 cells with inhibitor of Racl or PARP-1 (P<0.05).3. The effect of starvation-induced autophagy regulated by ART1 on proliferation and apotosis in CT26 cells.(1) The research about the effect of starvation-induced autophagy regulated by ART1 on proliferation.1) Starvation-induced GFP-ART1 CT26 cells treated with 3-MA at different concentrations were detected by CCK-8. This results showed that the proliferation of starvation-induced GFP-ART1 CT26 cells was decreased with certain increasing concentrations of 3-MA (P<0.05). The inhibition efficiency of starvation-induced GFP-ARTl CT26 cells treated with 5 mM 3-MA was higher than starvation-induced GFP-ART1 CT26 cells treated with 1 mM or 3 mM 3-MA respectively(P<0.05). There is no difference between starvation-induced GFP-ART1 CT26 cells treated with 5 mM 3-MA and starvation-induced GFP-ART1 CT26 cells treated with 7 mM 3-MA (P>0.05).2) Soft agar assay assay showed that the growth ability of starvation-induced GFP-ART1 CT26 cells was diminished when starvation-induced GFP-ART1 CT26 cells with 3-MA, compared with starvation-induced GFP-ART1 CT26 cells without 3-MA (P<0.05) 3) The results of flow cytometry showed that the distribution ratio in cell cycle was changed (the proportion of S phase and G2 phase was less) and proliferation index was reduced in starvation-induced GFP-ART1 CT26 cells with 3-MA, compares with the cells without 3-MA (P<0.05).4) The volume and weight of subcutaneous transplanted GFP-ART1 CT26 tumor with 3-MA were both less than those in subcutaneous transplanted GFP-ART1 CT26 tumor without 3-MA (P<0.05). (2)The research about the effect of starvation-induced autophagy regulated by ART1 on apotosis. 1) Flow cytometry was used to detect the rate of apoptosis. The results showed that the rate of apoptosis was higher in starvation-induced GFP-ART1 CT26 cells with 3-MA than in cells without 3-MA(P<0.01).2) Apoptotic cells were detected with Hoechst33342 staining. Comparatively, there were more cells with apoptotic hallmarks in starvation-induced GFP-ART1 CT26 cells with 3-MA than in starvation-induced GFP-ART1 cells without 3-MA (P<0.01).3) Caspase3 activity assay was used to detect the activity of caspase3 in starvation-induced GFP-ART1 CT26 cells and subcutaneous transplanted GFP-ART1 CT26 tumors. The results showed that the activity of caspase-3 was higher in GFP-ART1 group with 3-MA than in group without 3-MA (P< 0.05).Conclusion:1. ART1 could regulate starvation-induced autophagy in CT26 cells. Overexpression of ART1 could promote sarvation-induced autophagy in CT26 cellsc; Inhibition of ART1 could restrain starvation-induced autophagy in CT26 cells.2. ART1 may interact with integrin α7 and then participate in the regulation of expression or activity of Racl and NF-κB. NF-κB could affect the expression of PARP-1 and, therefore, inflence the activity of LKB1, AMPK and mTOR, leading to changes in autophagy.3.Inhibition of starvation-induced autophagy mediated by ART1 could restrain proliferation and promote apotosis of colorectal tumor cells. A potential role of ART1 serve as a new therapy in the treatment of colon carcinoma. |
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