Wang Hua, Liu Zhuo, Wang Zong-ping, Li Fang-yin, Zhao Yang, Chen Gui-ping, Li De-chuan
Department of Urology, Zhejiang Cancer Hospital, Hangzhou 310022, China (Email:
Zhonghua Zhong Liu Za Zhi. 2013 Jun;35(6):412-7.
To investigate the therapeutic efficacy of double-mutated oncolytic adenovirus AxdAdB-3 in combination with gemcitabine for treating bladder cancer in an orthotopic nude mouse model.
The susceptibility to the adenovirus was evaluated in bladder cancer cell lines YTS-1, T24, 5637 and KK47, and normal cell lines HCV29 and WI38. The cells were infected with AxCAlacZ and stained with 5-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal). Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells. Flow cytometry was used to determine the YTS-1 cells in S phase of cell cycle after adenovirus infection. Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay. Orthotopic bladder cancer model was established in nude mice, and the inhibitory efficacy of intravesical instillation therapy with AxdAdB-3 or/and gemcitabine was assessed.
Gene transduction efficiency was different among the cell lines, and correlated with expression of CAR. 5637 and KK47 cells with high expression of CAR were more susceptible to the adenovirus, whereas YTS-1 and T24 cells with little CAR expression were resistant to adenoviral infection. Immunostaining showed that the expression levels of hexon protein varied among the cell lines. Normal cells infected with AxdAdB-3 expressed little hexon protein. The proportion of S-phase cells was (39 ± 3) % and (49 ± 5) % in the AxCAlacZ- and AxdAdB-3-infected bladder cancer cells, respectively. AxdAdB-3 effectively induced S-phase entry of cell cycle (P < 0.05). AxdAdB-3 combined with gemcitabine significantly inhibited the growth of bladder cancer cell lines. In vivo, the mean weight of the bladder tumors in mice treated with intravesical instillation of AxCAlacZ, gemcitabine, AxdAdB-3, and AxdAdB-3 + gemcitabine were 400.6, 126.4, 82. 0, 40.4 mg, respectively. Either AxdAdB-3 (P < 0.0001) and gemcitabine (P < 0.0001) suppressed the tumor growth in nude mice, and the combination therapy reduced tumors more effectively than either AxdAdB-3 (P < 0.0001) or gemcitabine (P < 0.0001) alone.
Intravesical instillation therapy with AxdAdB-3 in combination with gemcitabine can effectively inhibit the orthotopic bladder cancer in nude mouse, and further relevant clinical studies are guaranteed.
在原位裸鼠模型中研究双突变溶瘤腺病毒AxdAdB-3联合吉西他滨治疗膀胱癌的疗效。
评估腺病毒在膀胱癌细胞系YTS-1、T24、5637和KK47以及正常细胞系HCV29和WI38中的易感性。细胞用AxCAlacZ感染并用5-溴-4-氯-3-吲哚-β-半乳糖苷（X-Gal）染色。进行针对腺病毒六邻体蛋白的免疫染色以确定AxdAdB-3在癌细胞中的选择性复制。采用流式细胞术测定腺病毒感染后YTS-1细胞处于细胞周期S期的比例。通过CCK-8法测定AxdAdB-3和/或吉西他滨处理后的细胞活力。在裸鼠中建立原位膀胱癌模型，并评估膀胱内灌注AxdAdB-3或/和吉西他滨的抑制疗效。
各细胞系间基因转导效率不同，且与柯萨奇病毒和腺病毒受体（CAR）的表达相关。CAR高表达的5637和KK47细胞对腺病毒更敏感，而CAR表达少的YTS-1和T24细胞对腺病毒感染有抗性。免疫染色显示各细胞系中六邻体蛋白的表达水平不同。感染AxdAdB-3的正常细胞六邻体蛋白表达少。在感染AxCAlacZ和AxdAdB-3的膀胱癌细胞中，S期细胞比例分别为（39±3）%和（49±5）%。AxdAdB-3有效诱导细胞周期进入S期（P<0.05）。AxdAdB-3联合吉西他滨显著抑制膀胱癌细胞系的生长。在体内，膀胱内灌注AxCAlacZ、吉西他滨、AxdAdB-3和AxdAdB-3+吉西他滨处理的小鼠膀胱肿瘤平均重量分别为400.6、126.4、82.0、40.4mg。AxdAdB-3（P<0.0001）和吉西他滨（P<0.0001）均抑制裸鼠肿瘤生长，联合治疗比单独使用AxdAdB-3（P<0.0001）或吉西他滨（P<0.0001）更有效地缩小肿瘤。
膀胱内灌注AxdAdB-3联合吉西他滨可有效抑制裸鼠原位膀胱癌，值得进一步开展相关临床研究。
