ABSTRACT
Objective To research the monoclonal antibody KMP1 inhibited bladder cancer EJ cell lines growth and metastasis in vivo by bioluminescence imaging. Methods Immunohistochemistry was used to determine the KMP1 binding to EJ and EJ-GFP cell lines. The xenograft tumor cell growth and distribution were measured by vernier calipers and dynamic in vivo fluorescence imaging. Immunohistochemistry and H&E counterstaining researched the feature of the xenograft tumor. Results Cell growth curves of EJ and EJ-GFP cells were similar. EJ-GFP had a green fluorescence. In EJ-GFP nude mouse tumor model, the addition of KMP1 significantly inhibited tumor growth and extended the average life span of nude mice. Both EJ and EJ-GFP cells can bind to KMP1,and the weight of transplanted tumors in the KMP1 treatment group was significantly lower than that of the mIgG control group (P＜0.001).Conclusion KMP1 has a promising antitumor effect in vivo. It might be valuable for development as a promising targeted agent for bladder cancer.
ABSTRACT
AbstractFucoidan, a sulfated polysaccharide found in marine algae and brown seaweeds, has been shown to inhibit the in vitro growth of human cancer cells. This study was conducted in cultured human bladder cancer EJ cells to elucidate the possible mechanisms by which fucoidan exerts its anti-proliferative activity, which until now has remained poorly understood. Fucoidan treatment of EJ cells resulted in dose-dependent inhibition of cell growth and induced apoptotic cell death. Flow cytometric analysis revealed that fucoidan led to G1 arrest in cell cycle progression. It was associated with down-regulation of cyclin D1, cyclin E, and cyclin-dependent-kinases (Cdks) in a concentration-dependent manner, without any change in Cdk inhibitors, such as p21 and p27. Furthermore, dephosphorylation of retinoblastoma protein (pRB) by this compound was associated with enhanced binding of pRB with the transcription factors E2F-1 and E2F-4. Overall, our results demonstrate that fucoidan possesses anticancer activity potential against bladder cancer cells by inhibiting pRB phosphorylation.
ABSTRACT
Objective: To investigate the effects of p21/Waf1 gene silencing on replicative potential of bladder cancer-specific oncolytic adenovirus and the proliferation inhibition of EJ cells. Methods: The shRNA targeting p21/Waf1 gene (p21/Waf1-shNA) was designed and synthesized, then the annealing oligonucleotide fragments were subcloned into pMagic 7.1 vector containing coding gene of green fluorescent protein (GFP) to construct recombinant lentiviral plasmid pLVT1051, which was confirmed by PCR and DNA sequencing. The 293T cells were co-transfected with three plasmids including pLVT1051, pCMV-VSV-G and pCMV-dR8.91 to produce the recombinant lentivirus. The EJ cells were infected with recombinant lentivirus carrying p21/Waf1-shRNA, and the puromycin was used to screen out the stably infected EJ cells. The expression levels of p21/Waf1 mRNA and protein in EJ cells after infection with recombinant lentivirus carrying p21/Waf1-shRNA were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. The p21/Waf1 gene-silenced EJ cells were infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, then the expression levels of virus replication-related proteins E1A and Hexon were detected by Western blotting, and the cytotoxic effect on EJ cells was examined by MTT method. Results: Recombinant lentiviral vector carrying p21/Waf1-shRNA targeting p21/Waf1 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus was harvested. The stably infected EJ cells were successfully screened out by using puromycin forr two weeks. The expression levels of p21/Waf1 mRNA and protein in EJ cells infected with recombinant lentivirus carrying p21/Waf1-shRNA were significantly reduced (both P < 0.05). The expression levels of E1A and Hexon proteins in p21/Waf1 gene-silenced EJ cells infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A were up-regulated, and the proliferation inhibition of EJ cells was enhanced (P < 0.01). Conclusion: The p21/Waf1 gene-silenced EJ cells are successfully constructed, and p21/Waf1 gene silencing can enhance the replicative ability of bladder cancer-specific oncolytic adenovirus and significantly inhibit the proliferation of EJ cells.