The Mechanical And Clinical Studies Of Midkine-induced Epithelial-mesenchymal Transition To Promote Resitance To Chemotherapy In CRC Cells

[Objective and background]Tumor heterogeneity, mainly in the biological behavior of tumor cells, pathological features, and sensitivity to chemotherapeutic drugs, ultimately produced resistant tumor cell clones, making part of the tumor cells acquire resistance, which was a fatal factor to leading to part of radically postoperative cancers remains for recurrence and metastasis after chemotherapy. Currently it is considered that the tumor microenvironment plays an important role in mediating tumor cells to acquire resistance and resist to apoptosis. A special niche in the tumor microenvironment may provide a "refuge" for cloned subsets of tumor cells, so that it provides an advantage for tumor cell survival. Studies have shown that epithelial-mesenchymal transformation not only plays a key role in tumor cell invasion and metastasis, and was closely related with the tumor cell resistance, but the mechanism is unclear. Midkine is a glycosaminoglycan-binding molecule, as a unique cytokine in the tumor microenvironment, may play an importantly regulatory role in making tumor cell obtain chemoresistance. Therefore, this paper focuses on that the impact of the midkine inducing colorectal cancer cell to acquire resistance to the conventional chemotherapeutic drugs; profoundly discuss the molecular mechanisms of resistance to chemotherapy which is promoted by midkine-induced epithelial-mesenchymal transformation in CRC cells:and analyzes clinical significance of the expression of midkine and its downstream target protein in colorectal cancer tissues.[Methods]1. Midkine induced colorectal cancer cell tolerance of chemotherapy drugs in vitro.(1) Analyze growth inhibition rate of SW480and HCT116treating with5-FU. L-OHP. CPT-11and sorafenib by MTT assay, and calculate half inhibitory concentration (50%) of various chemotherapy drugs in different groups of CRC cells.(2) Analyze growth inhibition rate of SW480and HCT116treating with5-FU, L-OHP, CPT-11and sorafenib by MTT assay in pretreatment with or without midkine, and calculate50%of various chemotherapy drugs in different groups of CRC cells.(3) Inhibition test:IKKa inhibitor was used to pretreat colorectal cancer cells, and detected growth inhibition rate of CRC cells acting by the IC50of variety chemotherapy drugs after treating with or without midkine through MTT assay.2. The mechanical study of signal transduction that midkine induced epithelial-mesenchymal transformation to promoting chemoresistance in CRC cells in vitro.(1) Cell morphology was observed in the stimulation of midkine in CRC cells by a microscope.(2) Western blot was used to analyze the expression of EMT markers, P-ERK1/2, NF-kappaB (P65), Bcl-2and P-gp in treatment with or without midkine in CRC cells.(3) The expression and distribution of EMT markers and NF-kappa B (P65) were detected by Immunofluorescence staining.(4) Inhibition test:ERK inhibitor or IKKa inhibitor was used to pretreat colorectal cancer cells, and detected the expression and distribution of EMT markers, ERK1/2, NF-kappaB (P65), Bcl-2and P-gp in treatment with or without midkine in SW480and HCT116of CRC by Western blot and Immunofluorescence. 3. The expression and clinical significance of MK, NF-κB (P65) and P-gp in CRC tissues.(1)Detect the expression of MK, NF-kappaB (P65) and P-gp in CRC tissues and normal mucosa by immunohistochemistry.(2)Correlation with the expression of MK and NF-kappaB (P65) and P-gp in CRC tissues was analyzed by Spearman.(3)Survival rates of CRC patients were analyzed by Kaplan-Meier according to different expression of MK and NF-kappaB (P65).[Results]1. In the treatment with or without midkine,5-FU IC50of SW480cells (3.50±0.17) μg/mL vs.(7.92±0.21) ug/mL,5-FU IC50of HCT116cells (3.20±0.15) μg/mL vs.(6.80±0.19) μg/mL (P<0.01); CPT-11IC50of SW480cells (14.24±0.22) μM vs.(28.58±0.26) μM, CPT-11IC50of HCT116cells (20.67±0.26) μM vs.(46.72±0.31) μM (P<0.01): L-OHPI C50of SW480cells (0.86±0.09) μM vs.(0.92±0.13) μM, L-OHP IC50of HCT116cells (0.93±0.12) μM vs.(0.98±0.18) μM (P>0.05); Sorafenib IC50of SW480cells (4.85±0.19) μM vs.(5.12±0.23) μM, SorafenibIC50ofHCTl16cells (5.40±0.16) μM vs.(5.87±0.17) μM (P>0.05).2. IKKa inhibitor could reverse the role of midkine to5-FU and CPT-11resistance in CRC cells. In the treatment with or without IKK inhibitor, MK+5-FU (IC50) of SW480cell viability was78.4±5.7%, Bay11-7082+MK+5-FU (IC50) of SW480cell viability was14.8±1.8%; MK+CPT-11(IC50) of SW480cell viability was83±5.6%, Bayll-7082+MK+CPT-l1(IC50) of SW480cell viability was38.0±3.7%; MK+5-FU (IC50) of HCT116cell viability was76.3±6.5%. Bay11-7082+MK+5-FU (IC50) of HCT116cell viability was22.3±4.6%; MK+CPT-11(IC50) of HCT116cell viability was78.5±7.1%; Bay11-7082+MK+CPT-l1(IC50) of HCT116cell viability was47.3±7.6%. The statistical significance was found between the two groups (P <0.01).3. MK could induce epithelial-mesenchymal transformation in SW480and HCT116cells. We found that most of cells changed from spindle-shaped to elongated morphology, and loosed association and occasional misorientation compared to the closely contacted monolayer, polygon cobblestone-like cells. E-cadherin downregulated, but vimentin and beta-caienin upregulated by Western bolt and Immunofluorescence.4. MK activated ERK1/2-NF-kappaB signaling pathway, induced NF-kappaB (P65) transferring to the nucleus, mediated the occurrence of EMT, and promoted the upregulation of target protein Bcl-2and P-gp. P-ERK1/2. NF-kappa B (P65). Bcl-2and P-gp were upregulated in CRC cells in treatment with midkine. and NF-kappaB (P65) was mainly distributed in the nucleus:P-ERK1/2. NF-kappa B (P65), Bcl-2and P-gp were downregulated in CRC cells of pretreatment with PD98059; in treatment with BAY11-7082, the expression of NF-kappa B (P65), Bcl-2and P-gp was downregulated. but P-ERK1/2had no significant change, in addition. NF-kappaB (P65) was still largely remain in the cytoplasm.5. The positive rates of MK. NF-κB (P65) and P-gp were68.5%(χ2=80.821, P=0.000),61.9%(χ2=81.612, P=0.000) and63.5%(χ2=78.906,P=0.000) respectively in CRC cancerous tissues; MK and NF-kappa B (P65) expression were positively correlated (r=0.631. P=0.000), MK and p-gp expression were positively correlated (r=0.537.P=0.000); the average overall survival were90.0months and92.7months respectively in CRC patients without MK or NF-kappa B (P65) expression, but the average overall survival were76.2months and76.5months respectively which were positive (MK log rank=5.98, P<0.05:NF-kB (p65) log rank=6.82, P<0.01).[Conclusion]Midkine not only could induce colon cancer cells resistance to the conventional chemotherapy drugs in colorectal cancer, but also induce epithelial-mesenchymal transformation. The mechanism may be that MK actived ERK1/2-NF-κB (P65) signaling pathways and maked NF-kappaB (P65) transfer to the cell nucleus to combine with the target gene, promoting the transcription and synthesis of target genes, mediating colon cancer cell epithelial-mesenchymal transition to promote the upregulation of Bcl-2and P-gp expression, eventually leaded colorectal cancer cells to acquire resistance.