| Renal cell carcinoma (RCC) is one of the most common malignant tumors ofurinary system, accounting for80%to90%of renal malignant tumors. Clear cellcarcinoma is the common pathological type of RCC. Distant metastasis is the leadingcause of death for RCC patients. Activation in the signaling pathways ofRAF-MEK-ERK and PI3K-Akt, and up-regulation of gene expression in Mcl-1andTrkB play important roles in anoikis resistance, which is closely related to survivaland metastasis for tumor cell and has been suggested to be the first prerequisite forcancer cells to metastasize. So, whether survival and metastasis of RCC are associatedwith anoikis resistance or not? How they work and are they also relative to the abovemolecular events? Do the biological characteristics in the cancer cell of RCC changealong with the above molecular events? At present, few studies have been found onthe molecular mechanisms of anoikis resistance in human renal cell carcinoma. Basedon these backgrounds, we designed this experiment.In this research, the model of anoikis resistance in ACHN cells of RCC, whichwas regarded as the object of study, was initially established. This study, targetingRAF-MEK-ERK, PI3K-Akt, Mcl-1and TrkB, serving Sorafenib and K252a as thetreatment factors and using siRNA to silence TrkB gene, was designed to explore thebiological characteristics and possible molecular mechanisms in anoikis-resistantACHN cells of RCC in vitro, consequently, to acquire new ideas and new ways forthe prevention and treatment of RCC metastasis. The full text is divided into as thefollowing parts: Objective: To establish a stable and reliable model of anoikis resistance in ACHNcells of RCC, and to observe their changes in morphology and biologicalcharacteristics.Methods: Suspension culture in ultra-low-adhesion plate for10days and subsequentre-adherent culture was used to induce parental ACHN cells of human RCC intoanoikis-resistant ones. The morphological changes in anoikis-resistant ACHN cellswere observed by inverted phase contrast microscope; Apoptosis of ACHN cells withsuspension culture and adherent culture was observed by AO (acridine orange)/EB(ethidium bromide) staining; MTT test, clonogenic assay, AnnexinV-FITC/PI test onflow cytometry, scarification test and Transwell test were applied to investigate thechanges in biological characteristics of anoikis-resistant ACHN cells of RCC.Results: ACHN cells of RCC with suspension culture were aggregated to grow. Whilere-adherent culure, these ACHN cells turned more processes, irregular polygon anddisorderly arrangement from regular fusiform and orderly arrangement in morphology.Under a fluorescence microscope after AO/EB staining, ACHN cells with adherentculture for72hours were seen to grow well, however, great apoptosis occurred inACHN cells with suspension culture for the same periods. The proliferation,clonogenic capacity, anoikis resistance, abilities of mobility and invasion inanoikis-resistant ACHN cells were significantly higher than those in parental ones(P＜0.05).Conclusion: A method of suspension culture in ultra-low-adhesion plate for10daysand subsequent re-adherent culture could be applied to establish a stable and reliablemodel of anoikis resistance in ACHN cells of RCC; Objective: To study the molecular mechanisms in anoikis-resistant ACHN cells ofRCC in vitro.Methods: In this study, Sorafenib and K252a were served as treatment factors. MTTtest, clonogenic assay, AnnexinV-FITC/PI test on flow cytometry, scarification testand Transwell test were used to investigate the effects of different treatment factors onbiological characteristics in anoikis-resistant ACHN cells of RCC. Real-time PCRanalysis was applied to detect mRNA of genes relative to cancer cells survival andmetastasis in parental ACHN cells and anoikis-resistant ones. Western blot assay wasused to explore the effects of different treatment factors on signaling pathways andexpression of genes associated with tumor cells survival and metastasis in parentalACHN cells and anoikis-resistant ones.Results:1.Anoikis-resistant ACHN cells were treated by different factors for72h. Theproliferation, clonogenic abilities, apoptotic resistance, mobility capacity andinvasiveness in Sorafenib group, K252a group and Sorafenib+K252a group weresignificantly decreased as compared to negative control group(P＜0.05). Comparingwith Sorafenib group and K252a group, the above biological characteristics inSorafenib+K252a group also significantly declined(P＜0.05). However, there is nodifference between Sorafenib group and K252a group (P＞0.05).2. The relative amounts mRNA of genes associated with cancer cells survival andmetastasis in anoikis-resistant ACHN cells were significantly higher than those inparental ones(P＜0.05).3.The expression of proteins in signaling pathways such as p-ERK and p-AKT, and the expression of proteins associated with tumor cells survival and metastasis such asMCL-1,TrkB,VEGF and MMP9in negative control group of anoikis-resistant ACHNcells, were significantly up-regulated as compared to negative control group ofparental ACHN cells (P＜0.05). After anoikis-resistant ACHN cells treated bydifferent factors for72hours, expression of the above proteins in Sorafenib group,K252a group and Sorafenib+K252a group appeared to be significantlydown-regulated comparing with the negative control group of anoikis-resistant ACHNcells(P＜0.05). The expression of the above proteins in Sorafenib+K252a group wereremarkably lower than those in Sorafenib group and K252a group(P＜0.05). However,there is no difference between Sorafenib group and K252a group (P＞0.05).Conclusion:1. Sorafenib and K252a dramatically affect the biological characteristics inanoikis-resistant ACHN cells of RCC. Moreover, both of them may have additive orsynergistic effects.2. Anoikis-resistant ACHN cells with higher proliferation and higher metastaticpotential, which were induced from parental ACHN cells of RCC, is closelyassociated with activation of PI3K-AKT and RAF-MEK-ERK, and with up-regulationof some proteins relative to tumor cells survival and metastasis such asMCL-1,TrkB,VEGF and MMP9. Objective: To further confirm the relationship between TrkB gene and ACHN ofRCC with anoikis resistance subsequently caused to survive and metastasize.Methods: Three chemical synthesis candidate Oligos of siRNA targeting human TrkB-mRNA were designed to be transfected into anoikis-resistant ACHN cells ofRCC. At48hours after transfection, real-time PCR analysis and Western blot assaywere applied to detect the relative amounts of TrkB-mRNA and expression of itsprotein respectively, so as to screen the most effective TrkB(NTRK2)-siRNA forsubsequent study; MTT test, AnnexinV-FITC/PI test on flow cytometry, scarificationtest and Transwell test were used to explore the changes in biological characteristicsand proteins associated with tumor cells survival and metastasis such as p-AKT,MCL-1, VEGF and MMP9in anoikis-resistant ACHN cells, whose TrkB weresilenced with the most effective NTRK2-siRNA; After TrkB was silenced withNTRK2-siRNA3for48hours, anoikis-resistant ACHN cells were treated with thefinal concentration at1.0μmol/L(less than the threshold concentration). MTT test,AnnexinV-FITC/PI test on flow cytometry and Western blot assay were applied todetect the proliferation, apoptosis rate and the expression of MCL-1in the aboveanoikis-resistant ACHN.Results:1. The relative amounts of TrkB-mRNA and expression of its proteins inNTRK2-siRNA3group appeared to be significantly lower than those in the remaininggroups(P＜0.05).2. After TrkB in anoikis-resistant ACHN cells of RCC was silenced withNTRK2-siRNA3for48hours, the proliferation, apoptotic resistance, mobilitycapacity and invasiveness were notably weaken(P＜0.05). The expression levels ofproteins such as p-AKT, MCL-1, VEGF and MMP9in NTRK2-siRNA3group weresignificantly down-regulated as compared to blank control group(P＜0.05).3. There is no difference in proliferation, apoptosis rate and the expression of MCL-1between Sorafenib(1.0μmol/L) group and blank control group (P＞0.05). Among theSorafenib（1.0μmol/L）group, NTRK2-siRNA3group and NTRK2-siRNA3+Sorafenib（1.0μmol/L）group, there were significant differences in proliferation,apoptosis rate and the expression of MCL-1between every two groups(P＜0.05). Conclusion:1. NTRK2-siRNA3could effectively interfere TrkB in anoikis-resistant ACHN cellsof RCC, therefore, is used to the subsequent study.2.Silencing TrkB in anoikis-resistant ACHN cells of RCC with NTRK2-siRNA3could inhibit the activation of PI3K-Akt, and down-regulate the expression of genesassociated with cancer cells survival and metastasis such as MCL-1, VEGF andMMP9, consequently, could affect their biological characteristics.3.Silencing TrkB with NTRK2-siRNA3could improve the sensitivity ofanoikis-resistant ACHN cells of RCC treated with Sorafenib, which has a certainrelationship with higher sensitivity in inhibition of MCL-1with Sorafenib. |
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