Autophagy Related Genes MTORC1、mTORC2 And Beclin1 Expression And Relationship With Molecular Mechanism Of Multidrug Resistance And Targeting Therapies In Colorectal Carcinoma

Objective:Evaluate the relationship between the expressions of autophagy-related genes mTORC1, mTORC2 and Beclinl and the expression of Multidrug Resistance gene MDR-1 in colorectal cancer (CRC) patients. Explore the correlation of autophagy with MDR in colorectal carcinoma. At the same time, explore the effect and mechanism of mTORC1, mTORC2 and Beclinl in colorectal carcinoma and discuss the correlations between mTORC1, mTORC2, Beclinl and MDR-1 and their effects on targeted therapy and chemotherapy in colorectal carcinoma taking the colorectal cancer multidrug resistance cell line HCT-8/5-FU as the object of the study.Materials and MethodsA total of 279 CRC tissue samples were collected from the Department of Pathology of Binzhou Medical University Hospital between January 2006 and January 2010. All CRC patients were clinically and pathologically proven to have not received preoperative chemotherapy or radiotherapy. Immunohistochemistry was employed to detect the expressions of mTOR, mTORC1 core molecule Raptor,mTORC2 core molecule Rictor, Beclinl, LC3, and MDR-1 in 279 CRC specimens.Patients were followed-up annually by telephone or at an outpatient clinic.χ2 test was used to analyze the correlation between genes and clinical pathological parameters.Kaplan Meier method was used to analysis the factors which affected the prognosis of colorectal cancer patients.shRNAs targeting Beclinl were transfected into HCT-8/5-FU cells. HCT-8/5-FU cells were treated with rapamycin, KU0063794, and 5-FU, respectively. The RNA and protein expression levels of LC3-II, NF-kB, MDR-1, Bcl-2, Bax, Cleavage Caspase-3, and CyclinD1 in all groups were evaluated through RT-PCR and Western blot. Autophagic cell death was evaluated through monodansylcadaverine (MDC)Staining. Apoptotic cell death was confirmed by Hoechst33342 staining. Cell proliferation activity was investigated through colony formation and CCK-8 assays.Cell migration and invasion activity were determined using Transwell and Matrigel assays. Healthy BALB/c nude mice were selected, subcutaneously implanted with tumor cells, and observed for the growth of the xenografted tumors. The mice were intraperitoneally injected with rapamycin, KU0063794, and 5-FU every day and sacrificed after 4 weeks. RNA and protein expression levels of LC3-Ⅱ, NF-kB and MDR-1 in the xenografted tumors were evaluated through Immunohistochemistry,RT-PCR and Western blot.ResultsThe expressions of Beclinl, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC(90.32%、87.09%、46.23%、42.65%、62.72% and 72.75%) were significantly higher than in adjacent tissues. LC3 expression in poorly differentiated CRC was higher than that in well differentiated CRC, and the expressions of mTOR, Raptor, Rictor, and LC3 in lymph node metastasis were higher than that obtained in the absence of lymph node metastasis. The expression of LC3 was positively correlated with those of Beclinl and Rictor, and negatively correlated with Raptor and mTOR in CRC. The expression of Raptor was negatively correlated with Rictor. The expression of MDR-1 was positively correlated with those of Beclinl, LC3, and Rictor, and negatively correlated with Raptor and mTOR. Kaplan-Meier analysis revealed that the 5 year survival rate of patients without lymph node metastasis, positive expression of Rictor,Beclinl and LC3, and negative expression of Raptor, mTOR were higher than those with these characteristics.The shRNA directed against Beclinl effectively inhibited the endogenous mRNA and protein expression of Beclinl, significantly downregulated the expression of LC3-Ⅱ, Bax and Cleavage Caspase-3, and upregulated the expression of Bcl-2.Furthermore, the simultaneous inhibition of Beclinl evidently increased tumor cell proliferation activity, migration, and invasion potential, while reduced apoptosis. The cells attained reduced sensitivity to 5-FU after Beclinl was inhibited. KU0063794 effectively inhibited mTORC1 and mTORC2, significantly upregulated the expression of LC3-Ⅱ, and downregulated the expression of NF-kB, MDR-1 and CyclinD1. Furthermore, the simultaneous inhibition of mTORC1 and mTORC2 evidently reduced tumor cell proliferation activity, migration, and invasion potential,while increased apoptosis. The cells attained increased sensitivity to 5-FU after the genes of mTORC1 and mTORC2 were inhibited.Conclusion1. The high expressions of Beclinl, Raptor, and Rictor were related to the development, progress, metastasis and survival rate of colorectal carcinoma and MDR,and were significant to judge the prognosis of colorectal carcinoma patients.2. The expression of MDR-1 was closely related to Beclinl, LC3, Raptor, and Rictor. The combined detection of Beclinl、 Raptor and Rictor was important for patients with colorectal cancer in prognosis and optimal therapy.3. The inhibition of mTORC1 and mTORC2, and the activation of Beclinl could reduce tumor cell proliferation activity, migration, and invasion potential, and increase apoptosis, and inhibit the development and progress of CRC.4. The inhibition of mTORCl and mTORC2, and the activation of Beclinl could reverse MDR of CRC, increase the cooperative influence of chemotherapy, and enhance the therapy effect of CRC.