Wang Xiaorong, Chen E, Tang Min, Yang Xue, Wang Yin, Quan Zhan, Wu Xiaohou, Luo Chunli
The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China.
Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
Tumour Biol. 2016 Aug;37(8):10731-43. doi: 10.1007/s13277-016-4821-8. Epub 2016 Feb 12.
The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-β1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-β1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-β1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.
本研究旨在探讨肝细胞黏附分子（hepaCAM）与膀胱癌中SMAD家族成员2/3（SMAD2/3）之间的相关性，以及SMAD2/3信号通路在hepaCAM诱导的肿瘤细胞凋亡中的作用。采用免疫组织化学法检测膀胱癌组织中hepaCAM和p-SMAD2/3蛋白水平。运用流式细胞术和Hoechst染色研究hepaCAM对细胞凋亡的影响。分别使用hepaCAM过表达腺病毒、半胱天冬酶抑制剂（Z-VAD-FMK）和SMAD2/3激活剂（转化生长因子（TGF）-β1），通过蛋白质印迹法检测hepaCAM以及SMAD2/3/半胱天冬酶信号通路分子的表达。通过免疫荧光和蛋白质印迹法检测p-SMAD2/3的转位。与癌旁组织相比，膀胱癌组织中hepaCAM蛋白显著降低（P < 0.05），而p-SMAD2/3蛋白显著升高（P < 0.05）。然而，hepaCAM低表达和p-SMAD2/3高表达与患者的临床病理特征无统计学关联。Pearson分析显示hepaCAM与p-SMAD2/3呈负线性相关（r = -0.712/-0.724，P = 0.008/0.011）。hepaCAM过表达激活了半胱天冬酶3/8/9，下调了聚ADP核糖聚合酶（PARP）和p-SMAD2/3。与单独使用Z-VAD-FMK相比，用Z-VAD-FMK + hepaCAM处理膀胱癌细胞可显著下调前半胱天冬酶3/8/9和PARP，并诱导细胞凋亡。同样，与单独使用TGF-β1相比，TGF-β1 + hepaCAM联合处理可显著下调p-SMAD2/3、前半胱天冬酶3/8/9和PARP，并诱导膀胱癌细胞凋亡。hepaCAM过表达可阻止膀胱癌细胞BIU-87和T24中p-SMAD2/3从细胞质转位至细胞核。我们的研究结果揭示了p-SMAD2/3信号通路在hepaCAM诱导的癌细胞凋亡中起关键作用，并为当前及未来的Ad-hepaCAM和p-SMAD2/3临床试验提供了有价值的见解。
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