Research On Relationships Between PI3K/Akt Pathway,Tumor Recurrence And Camptothecin Chemosensitization In Non-muscle-invasive Bladder Cancer

BackgroundBladder cancer is the9th most common cancer worldwide. Over the last60to70years, the incidence rate of bladder cancer has been rising, especially in undeveloped countries and in our contrary too. Over90%of bladder tumors are urothelial carcinoma, within which about70-80%of tumors are non-muscle-invasive. Transurethral resection of bladder cancer (TURBt) plus intravesical adjunctive therapy is the initial and standard treatment for non-muscle-invasive bladder cancer (NMIBC). Tumor recurrence is still a major issue of NMIBC, although intravesical chemotherapy could reduce recurrence rate significantly. Chemoresistance plays an important role in tumor recurrence.Mutation of FGFR3(Fibroblast growth factor receptor-3) is another feature of NMIBC of which activating mutations of FGFR3is found in up to70%. PI3K/Akt pathway is a major downstream of FGFR3and it plays an important role in cell survival, cell growth, cell proliferation, angiogenesis, cellular metabolism, cell migration and invasion, which confers chemoresistance. Akt plays the central role in PI3K/Akt pathway. Activating mutation of FGFR3could enhance the PI3K/Akt pathway which results in proliferation promotions and resistance to cytotoxic stimulations. There are few studies investigating the relationship between PI3K/Akt pathway and NMIBC, so it becomes more interesting and important to study PI3K/Akt pathway and Akt status in NMIBC.Part1. The relationship between pAkt expression and recurrent free survival of NMIBC patients1.1. ObjectsStudy of Akt status in NMIBC and the relationship with recurrent free survival of patients.1.2. MethodsWith the methods of immunohistochemistry, we tested tumor samples from89patients of primary NMIBC. We analyzed the relationship between pAkt and clinical characteristics (age, sex, grade, stage, tumor number, tumor size). Kaplan-Meier method was used to explore the relationship between these factors and RFS (recurrent free survival) of patients. COX regression was applied to test the independence of these risk factors.1.3. ResultsThe positive rate of pAkt in NMIBC was73.0%and the overexpression rate was22.5%, significant higher (p<0.01) than that in normal bladder tissues20.0%and0%. The levels of pAkt had no correlation with clinical characteristics (age, sex, grade, stage, tumor number, tumor size). Kaplan-Meier analyze showed that high pAkt expression, tumor size bigger than3cm and high grade tumor were all correlated with reduced RFS. But COX regression analyze showed that only pAkt was independent risk factor, relative risk (RR) value was0.28. 1.4. ConclusionThe positive staining rates of pAkt were high in NMIBC and the level of pAkt is an independent risk factor of predicting RFS of NMIBC.Part2. Regulation of Akt expression and activation by CPT in NMIBC2.1. ObjectsStudy of the influence of a common intravesical chemotherapy reagent camptothecin (CPT) on the regulation of Akt and pAkt levels in NMIBC.2.2. MethodsCPT sensitive cell line RT4and CPT insensitive cell line5637were used. And we also established CPT resistant RT4cell line, RT4/CPT. CCK-8was applied to test the CPT chemosensitivity. Thereafter cells were treated with high-dose HCPT (Water soluble CPT) to mimic intravesical instillation. The changes of Akt and pAkt levels were tested by western blot. We also investigated pAkt signaling in paired primary and recurrent samples from18patients of NMIBC who had CPT intravesical chemotherapy and early tumor relapse.2.3. ResultsChemosensitivity to CPT in RT4/CPT,5637and RT4cells was tested by CCK-8, RT4cells were the most sensitive to CPT cytotoxic effect, reaching half-cell viability at12h after CPT treatment;5637cells were less sensitive, reaching half-cell viability at48h; RT4/CPT cells were least sensitive, remaining75%viability at48h. Western blot showed that Akt and pAkt levels of RT4/CPT cells were higher than that in RT4cells and pAkt level of5637was higher than that in RT4cells too, which indicated that pAkt was an indicator of chemosensitivity of CPT to bladder cancer cells. Then we mimicked in vivo intravesical instillation therapy. After HCPT incubation, the levels of pAkt in RT4, RT4/CPT and5637cells were all up-regulated and reached peak at2h,8h and10h respectively. The expression levels of Akt and pAkt were both up-regulated in recurrent tumor samples compared with corresponding primary ones.2.4. ConclusionHigh levels of pAkt correlate with insensitivity to CPT in bladder cancer cells. CPT could up-regulates Akt and pAkt levels in bladder cancer cells and tissues.Part3. In Vitro study of chemosensitivity of PI3K inhibitor and DNA-PK inhibitor in NMIBC3.1. ObjectsStudy of the chemosensitization after blocking of Akt activation and DNA repair (by blocking DNA-PK). PI3K inhibitor wortmannin and DNA-PK inhibitor Nu7441were applied to block Akt activation.3.2. MethodsThe apoptosis rates were tested by flow cytometry in RT4/CPT and5637cells. Normal transitional cells SV-HUC-1were also tested as normal control for drug safety. As the cytotoxic effects caused by CPT is mainly due to DNA damage, which requires DNA-PK, so we also studied the chemosensitivity of DNA-PK inhibitor Nu7441in RT4/CPT,5637and SV-HUC-1cells.3.3. ResultsFlow cytometry results showed in RT4/CPT cells, CPT, wortmannin and Nu7441alone could not induce apoptosis, but could increase apoptosis rate to37.8%(CPT40μM) when pretreated with wortmannin while pretreatment with Nu7441could not reach this effect; in5637cells, CPT alone had a dose-dependent cytotoxic effect, inducing apoptosis rate of15%,24%and27%at the dose of10μM、20μM and40μM; wortmannin could also sensitize5637to CPT (40μM) induced apoptosis rate of51.3%, while Nu7441still could not. In SV-HUC-1cells, treatment with CPT, wortmanin or Nu7441alone could not induce apoptosis and wortmannin could increase apoptosis rate of CPT (40μM) to14.6%, while Nu7441still could not. 3.4. ConclusionThe response of NMIBC cells to cytotoxic stimulates of CPT is mainly through PI3K/Akt activation to promote cell survival but not DNA-PK dependent DNA damage repair pathway. PI3K inhibitor has a synergistic effect with CPT in NMIBC cells, and this effect is relatively safe to normal transitional cells.