Experimental Study Of The Effect Of Antitumor In Bladder Cancer And The Molecular Mechanism Of Honokiol

Objective To study the effect of antitumor in bladder cancer and the molecular mechanism of honokiol in vitro and in vivo.Methods Cell viability assay was used to study the effect of honokiol on T24and5637human bladder cancer cell lines. Colony-formation assay was used to examine bladder cancer cell growth with honokiol treatment. Selecting T24and5637cell treated in24hours by honokiol to study cell growth cycle and the percentage of apoptotic cell on the different drug concentration by Flow CytoMeter.T24and5637cells were stained with Hoechst33342, and the nuclear morphology was examined using fluorescence microscope with an ultraviolet filter. Wound healing assay was used to determine the effect of honokiol on the migration of T24and5637bladder cancer cell. Invasion assay was used to determine the effect of honokiol on the invasion of T24and5637cell.Extrcting the protein of T24and5637cell treated by different honokiol drug concentration, western blot assay was used to determine the protein level of proCaspase3,PARP,Bcl2,Bax,CyclinDl,p21,p27,MMP9,CD44,Notch-1,Sox-2,EZH2and its target protein H3K27me3.Athymic mouse tumor model of T24cell was made and observed the effect of honokiol on tumor growth and the toxicity and side effects on mice.The tissue and cell morphologic change were observed by HE stain after treatment with honokiol. Immunohistochemistry was used to detect the protein expression of CD44and Ki67. Extrcting the protein of T24cell xenograft tumor treated by honokiol, western blot was used to determine the protein level of MMP9,CD44,Notch-1,Sox-2,EZH2and H3K27me3.Results T24and5637bladder cancer cells were treated with0,4.8,7.2,9.6,14.4or19.2ug/ml honokiol and cell viability was assayed at24,48and72hours using cell viability assays. Treatment with honokiol resulted in inhibition in clonogenicity formation, whereas higher concentrations (4.8μg/ml and9.6μg/ml HNK) were more inhibitory.These findings demonstrated that honokiol inhibited cell viability in a dose dependent manner. Wound healing assay showed that the cellular motility was evidently inhibited in dose dependent manner. The results of the in vitro invasion assay displayed that honokiol can inhibit invasion ablilty of bladder cancer cell dose dependently. Flow cytometric analysis was used to detect cell cycle changing and cell apoptosis after honokiol treated T24and5637cell for24hours.The percentage of cell in the G1phase of cell cycle in T24cell on different honokiol concentration were48.2%,48.4%,77.5%and62.4%;Sub G1phase were1.2%,2%,3%and14.1%;G1phase cell in5637cell were44.9%,41%,50.3%and62%;Sub G1phase were0.8%,0.9%,1.2%and3.1%.T24and5637cell were stained with Hoechst33342and observed under fluorescence microscopy.The results showed that the honokiol treatment induced nuclear condensation and fragmentation obviously at24hours comparing with control cells.Western blot analysis indicated that treatment of T24and5637bladder cancer cell with honokiol resulted in a decreased expression of proapoptosis protein (proCaspase3and PARP), anti-apoptosis protein Bcl2and cell cycle protein CyclinD1, and increased expression of the cleaved poly (ADP-ribose) polymerase(PARP),cell cycle inhibitors p21, p27and BAX. Western blot analysis indicated that treatment of T24and5637bladder cancer cell with honokiol resulted in a dose-dependent decreased expression of MMP9,CD44,Notch-1,Sox-2,EZH2and its target protein H3K27me3.In T24xenograft tumor mice models in vivo,compared with control group,the tumor size of the groups treated with honokiol were significantly reduced, volumes of the three groups were1240.95±2.396mm3,529.875±3.402mm3and244.663±1.243mm3respectively; and the tumor weight of groups treated with honokiol were also significantly reduced,weight of the three groups were0.885±0.232g,0.471±0.383g and0.255±0.174g; but the liver,spleen and renal weight of the three groups were no significantly altered by honokiol treatment.It can be observed by light microscopy in HE staining sections that tumor cells in tumor tissue is not uniform in shape and size.Heteromorphism of cell nucleus was obvious,pathologic karyokinesis can be seen.CD44and Ki67were expressed largely in the tumor cells, compared with the control group,the expression of CD44and Ki67in the groups treated with honokiol was significantly reduced. These data showed that honokiol could reduce the expression of CD44and Ki67in bladder cancer tissue.Western blot analysis indicated that honokiol could reduce the expression of MMP9,CD44,Notch-1,Sox-2,EZH2and H3K27me3in T24xenograft tumor.Conclusion Honokiol can inhibit the growth and migration of human bladder cancer, and causes cell cycle arrest and induce cell apoptosis, which may be associated with decrease in cancer stem cell functions.