Moncpt Anti-tumor Effect And Mechanism Of Bladder Cancer Cells 5637

Bladder cancer is the most common urologic cancer in our country. Morethan 90% of bladder cancer is transitional cell carcinoma (TCC). About 70-80% ofcases are superficial at the time of diagnosis, of which 70% are stage Ta and 30%are stage T1. Low-grade non-invasive tumors may be treated with resection andfulguration. However, despite complete tumor resection, two thirds of patients willdevelop tumor recurrence in five years and by 15 years 88% of patients willdevelop a recurrence. Progression from superficial bladder cancer to deepmuscle invasion occurs in 15% of patients. The high rate of tumor recurrence andpotential progression provides an opportunity to institute chemoprevention orprophylactic therapy. Now intravesical therapy is the important modality forbladder cancer treatment.There are two main categories of intravesical therapy,that is, chemotherapy and immunotherapy. Camptothecin analogs is one of theimportant chemotherapeutic drugs for intravesical therapy.The structure-activity relationship (SAR) analysis of the CPTs hasdemonstrated that substitutions at the 7-, 9-, or 10-positions of most camptothecinanalogs can enhance their antitumor activity. Although the induction of hydroxylor alkoxy at position 10, such as, 10-methoxycamptothecin, has antitumor activity,its low therapy index limited further research. In contrast, the induction of a nitroat position 9, such as 9-nitrocamptothecin, has shown satisfactory activity with lowtoxicity. 9-nitrocamptothecin was difficultly prepared by nitration of camptothecinbecause the 12-nitrocamptothecin was the main product,and the 10-methoxy-9-nitrocamptothecin could easily be prepared from10-hydroxycamptothecin.The present study was conducted to estimate theanti-cancer activity of MONCPT against bladder cancer both in vitro and in vivo,and the molecular mechanisms of anti-tumor effect.Purposes: To determine the anticancer activity of MONCPT both in vitro and invivo against bladder cancer cell 5637; estimate the effect on cell cycle of bladdercancer cell 5637 and elucidate its mechanism.Methods:1) Cancer cell killing ability of MONCPT was measured by MTT method;2) Tumor growth inhibition activity of MONCPT was measured by murine bearinghuman xenografted tumor models in vivo;3) After MONCPT treating,cell cycle distribution was detected by flow cytometry;4) Protein (p53, CDK7, p21, AKT, Mat1, Wee1, Cyclin B1, CDK1, Cyclin H,Cdc25c) expression was detected by Western blotting;Results:1. Cytotoxicity AssayUsing the MTT method, we determined the cytotoxic activity of MONCPTagainst bladder cancer cell lines (5637 cells). All the cells exhibiteddose-dependent sensitivity to 48-hours exposure with MONCPT and the IC₅₀value was 1.31±0.16μM. 2. Effect of MONCPT on Tumor Growth in vivo in 5637 Xenografted Models.On the basis of the encouraging in vitro data, the antiproliferation activity ofMONCPT was evaluated in human tumor models xenografted in athymic mice.The study showed that MONCPT possessed of significant effect on tumorweight(P<0.01) compared with the negative control group , but not on athymicmice body weight (P > 0.05). The tumor weights were 0.63±0.12g (MONCPT5mg/kg), 0.24±0.06g (MONCPT10mg/kg) and 0.06±0.03g (MONCPT20 mg/kg).In the negative control group the tumor weight was 1.83±0.30g. Compared withthe control group, the tumor volumes were significantly inhibited from day 5 in thegroups treated with MONCPT (10.0 mg/kg) and Irinotican (10.0 mg/kg) (P<0.05-0.01) and from day 3 in the groups treated with MONCPT (20.0 mg/kg) (P<0.05) , from day 9 in the groups treated with MONCPT (5.0 mg/kg) (P <0.01).The relative tumor proliferation rates (T/C, %) of 5 mg/kg MONCPT were 103.2%,84.6%,95.7%,80.2%,62.0%,41.6% and 23.8% at treatment day 3, 5, 7, 9,11,13, and 15 respectively, while these of 10 mg/kg MONCPT were 95.3%,67.7%,61.4%,34.2%, 21.2%,11.1% and 6.2%. The T/Cs (%) of 20 mg/kg MONCPTwere 73.2%,51.9%,40.3%,20.9%,11.7%,6.9% and 3.3%, from day 5the T/Cs (%) < 60% . The T/Cs (%) of 10 mg/kg Irinotican were 88.1%,70.4%,50.5%,31.3%,20.8%,13.8% and 7.3%,from day 7 the T/Cs(%) <60%. 3. Cell cycle Assay of MONCPT against 5637.5637cells were respectively treated with 10,1, 0.1, 0.01μM MONCPT for 24hours. Collect the treated cells and the control cells, and then use the flowcytometry to detect the cell cycle distribution. It was showed that G2/M distributionwere 19.7±3.6%,46.9±7.3%,76.8±9.2%and 18.55±3.5%respectively,and 8.56±2.13% in control group . 0.1μM MONCPT has the greatest effectto arrest cell cycle in G2/M phase. 5637 cells were treated with 0.1μM MONCPTfor 12, 24, 36 and 48 hours, collectting the treated cells and the control cells, andthen used the flow cytometry to detect the cell cycle distribution. It was indicatedthat the increase of the cell cycle G2/M distribution was detected in 5637 cells.The percentages of cell cycle G2/M in 5637 cells induced by 0.1μM MONCPT for0, 12, 24, 36 and 48 hour were 11.45±2.3%, 22.4±3.85%(12h), 79.18±7.3%(24h), 50.15±6.25%(36h) and 29.7±3.85% (48h) respectively.4. Determination of the Expression of Cell cycle G2/M phase-relatedProteins.Incubate the 5637 cells with 0.1μM MONCPT for 12, 24, 36 and 48 hours,collectting the treated cells and the cells without exposure to MONCPT, followedby extracting proteins from these cells. It was showed that MAT1,CyclinB1 wereobviously down-regulated from 12 to 24h, and then up-regulated from 36 to 48hby MONCPT (0.1 pM). CDK1,CD7,Cdc25C,Cyclin H,AKT,Wee1 weredown-regulated within 48 hours by MONCPT (0.1μM). The high-level expression of p21 and p53 proteins at 24 h after treatment of MONCPT (0.1μM ) in 5637cells indicated that MONCPT exert its potency through affecting the expression ofproteins related with cell cycle.Discuss and Conclusion:MONCPT is a new synthetic derivative of Camptothecin. Our results showedthat MONCPT exerted potent antiproliferative action on human bladder cancercells in vitro and in vivo. We also identified that cell cycle G2/M phase waseffectively arrest by MONCPT in human bladder cancer 5637 cells, whichindicated that its mechanism of anti-bladder cancer effect was through cell cyclepathway.In previous study, it was reported that MONCPT induced cell cycle G2/Mphase arrest by several pathways simultaneously, including p53 pathway, theMAPK pathway and the activation of Akt. Cell cycle regulation and itsmodulation by various plant-derived agents are gaining widespread attention inrecent years. Mammalian cells respond to DNA-damaging agents by activatingcell cycle checkpoints, acting by delaying cell cycle progression until errors havebeen corrected. Usually these control mechanisms determine a temporaryarrest at a specific stage of the cell cycle to allow the cell to correct possibledefects. The arrest at G2/M prevented the cells from completing the cell cycleand proliferating. In contrast to the G1/S checkpoint, the mammalian G2/Mcheckpoint is poorly understood.This checkpoint prevents the improper segregation of chromosomes, which is likely to be important in humantumorigenesis. G2/M transition provides an effective checkpoint in cell cycleprogression that is regulated by the sequential activation and deactivation of Cdcfamily proteins and cyclin complexes. The Cdc2-cyclin B kinase is pivotal inregulating this transition. Cdc2 is inhibited simultaneously by four transcriptionaltargets of p53, Gadd45, p21, and 14-3-3σ. p53 is a transcription factor thatup-regulates a number of important cell cycle-modulating genes such as p21.Part of the mechanism by which p53 blocks cells at the G2 checkpoint involvesinhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Inmammalian cells, both cyclin binding and phosphorylation by CAK are required forthe activation of Cdc2-cyclin B complex, Thr161 on Cdc2 of the complex isphosphorylated by CAK, a complex containing cyclin H, MAT1 and CDK7. In theabsence of Cdc2 dephosphorylation by Cdc25C, and also the directphosphorylation of cyclin B by Plk1, the accumulation of cyclin B-Cdc2 in thenucleus is prevented and entry into mitosis is stalled. In addition, Plk1 also hasbeen shown to function via the Cdc25C and Cdc2/cyclin B1 positive feedbackloop at the onset of mitosis. Our results were showed that MAT1,CyclinB1 wereobviously down-regulated from 12 to 24h, and then up-regulated from 36 to 48hby MONCPT (0.1μM ). CDK1,CDK7,Cdc25C,Cyclin H,AKT,Wee1 weredown-regulated within 48 hours by MONCPT (0.1μM ) . The high-levelexpression of p21 and p53 proteins at 24 h after treatment of MONCPT (0.1μM)in 5637 cells indicates that MONCPT exert its potency through affecting theexpression of proteins related with cell cycle. In conclusion, MONCPT exhibited high anti-proliferation activity in humanbladder cancer 5637 cell line both in vivo and in wfro.lt was shown that MONCPTcould induce cell cycle G2/M phase arrest in human bladder cancer 5637 cellsand its mechanism may be associated with cyclinB1-CDK1 complex, p53, CDK7and Cdc25c.