Experimental Study On Treatment Of Bladder Cancer With A Combination Of TRAIL And MMC

PartⅠExperimental study of a combination of TRAIL and MMC on treatment of bladder cancer cell strains in vitroObjective:To explore the effect of TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand) on inhibition and apoptosis of bladder cancer cell strains T24 and 5637 in vitro,and to explore the anti-tumor synergistic effects of TRAIL and MMC on bladder cancer cell strains.Methods:T24 and 5637 bladder cancer cell strains were employed in our study, 100μl of target cell suspension were added to each well of 96-well plates,by adding a concentration of 1,10,100μg/L TRAIL,0.1,1.0,10.0 mg/L mitomycin C(MMC) respectively.The bladder cancer cells were incubated for 24 hours with TRAIL,MMC and TRAIL combined with MMC at different concentration.MTT working solution was added to each culture well and calculated the survival rates of bladder cancer cells and compared the deference.Bladder cancer cell suspension was added to 12-well plates and incubated for 24h,and then incubated for 12 hours with TRAIL,MMC and TRAIL combined with MMC at different concentration.The rates of apoptosis and death of bladder cancer cells were detected by flow cytometry,and compared the deference within TRAIL group,MMC group and the group of TRAIL combined with MMC.Results:TRAIL inhibited efficiently the growth of human bladder cancer cell strains T24,5637 significantly.①MTT experiment results:the growth inhibition rate(GIR) of 1,10,100μg/L TRAIL on bladder cancer T24 cell strain were 26.4%,29.3%and 37.7%, there was a significant difference(P＜0.01) to the non-drug group.The GIR of three concentrations of TRAIL on bladder tumor 5637 cell strain were 13.7%,21.9%and 59.1%, respectively.Compare to the group of non-drug,there was no significant difference to the group of 1μg/L TRAIL(P＞0.05),whereas there was a significant difference to the group 10,100μg/L TRAIL(P＜0.01).10.0mg/L MMC on bladder cancer T24,5637 cell strains, the GIR were 36.0%,26.7%,whereas 100μg/L TRAIL combined with 10.0mg/L MMC on the bladder tumor cell strains T24,5637,the GIR reached 58.4%,90.7%,respectively, There was significant difference to 100μg/L TRAIL and 10.0mg/L MMC,both have obvious synergy(P＜0.05).②the results of detecting by flow cytometry:100μg/L TRAIL induced bladder tumor cell strains T24,5637 apoptosis,apoptosis rates were 20.1%,47.5%, and there was a significant differences(P＜0.01) to the apoptosis rate 1.9%,5.1%of the group of non-drug;The apoptosis rates were 13.1%,18.1%,respectively.when use individually 10.0mg/L MMC on bladder cancer cell strains T24,5637;whereas 100μg/L TRAIL combined with 10.0mg/L MMC on the bladder tumor cell strains T24,5637,the apoptosis rate reached 41.5%,61.5%,respectively.There was significant difference to 100μg/L TRAIL and 10.0mg/L MMC,both have obvious synergy in two cell strains (p＜0.05).Conclusions:In vitro,①TRAIL can effectively inhibit the growth of bladder cancer cell strains T24,5637,and it also can induce apoptosis in human bladder tumor cell strains T24,5637.②TRAIL can enhance the effect of MMC on inhibiting the growth of human bladder cancer cell strains T24,5637,and they also have the synergistic effect on induction of human bladder cancer cell strains apoptosis.PartⅡThe effect of Ad-TRAIL and MMC on bladder tumors in vivoObjective:To study the therapeutic effects of eukaryotic expression vector coding human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) combined with Mitomycin C(MMC) on bladder carcinoma in vivo.Methods:Methods:①T24 human bladder cancer cell strain cultivate in vitro.Five 6-week-old immune-deficient mice(BALB/c-nu),the roots of the right forelimb dorsal skin with 75%alcohol disinfection,then inoculated subcutaneously with 0.2ml concentration of 1×10~7/ml of the T24 human bladder cancer cell line suspension.Up to the tumor size of 1cm in diameter,the mice were sacrificed and tumors excised,check the texture uniformity of department organizations,and cut into about 2 mm~3,10～15mg size, 40 mice of 6-week-old BALB/c-nu were transplanted to subcutaneous dorsal roots of the right forelimb.Transplantation methods:using 75%alcohol disinfection of transplanted skin,cut the size of 0.4cm skin incision,separation of subcutaneous tissue,the subcutaneous tissue implants,0~# suture the skin incision line.Continue to conventional feeding mice and to observe changes in the injection site.②These 40 BALB/c-nu mice, tumor grow to 0.4～0.6cm were randomly divided into four groups,namely group MMC(A group):intraperitoneal injection of 0.1g/L MMC(10μl/g body weight) keep on 7d; intratumoral injection of 5×10~8 pfu/ml Ad empty vector 0.1ml×3,the tumors were injected in 4,8,12 points,1/2d,a total of 3 times;Group Ad-TRAIL(B group):abdominal injection of PB S(10μl/g)×7d,intratumoral injection of 5×10~8 pfu/ml Ad-TRAIL 0.1ml×3, 1/2d,a total of 3 times;Group MMC and Ad-TRAIL(C group):abdominal injection of MMC as the group A,intratumoral injection of Ad-TRAIL as the group B;group control (group D):intraperitoneal injection of PBS(10μl/g)×7d,intratumoral injection of 5×10~8 pfu/ml Ad empty vector 0.1ml×3 sites,1/2d,a total of 3 times.Two weeks after drug delivery,to measure mouse body weight,diameter and weight of tumors,observe the tumor cells under the microscope(×400),and measure the TRAIL mRNA and TRAIL protein expression within tumor tissue by using RT-PCR and western blot.Results:①the first batch of five mice,has three tumor-bearing model of success,to cells after injection of 50d,3 tumor-beating mice diameter reach 0.6～1.0cm,and confirmed by histopathology;the second batch of 40 mice tumor model for all of the success of tumor rate was 100%,4 weeks after transplantation all mice tumor diameter are 0.4～0.6cm.②pre-delivery weight and the load average diameter of the tumor was no significant difference in four groups of mice(P＞0.05);Tumor diameter and weight of group A,B,C were significantly smaller than group D(P＜0.001)2 weeks after delivery, while the group C was significantly smaller than group A and B(P＜0.01),group B was significantly smaller than group A(P＜0.001);Weight of group A and C were significantly less than group D(P＜0.01),while there was no significant difference between group B and group D(P＞0.05);Under the microscope,the necrosis of tumor cells, group C＞B＞A＞D;TRAIL mRNA and TRAIL protein expressed in tumor cell within Group B and C,and the difference of expression of the relative volume of Group B and C was not statistically significance(P＞0.05),while group A and D did not express the TRAIL mRNA and TRAIL protein in tumor cells.Conclusion:①Constructed successfully in nude mice of human bladder cancer cell strain T24 nude model;②Experiments in vivo,Ad-TRAIL was successful transfected into T24 bladder cancer and expressed TRAIL protein;③MMC and Ad-TRAIL have obvious inhibitory effect to T24 human bladder cancer in nude mice transplanted tumor,and both has synergistic effect with combination.