title: The SMAD2/3 pathway is involved in hepaCAM-induced apoptosis by inhibiting the nuclear translocation of SMAD2/3 in bladder cancer cells.
abstract: 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.
authors.affiliation: The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: E
authors.initials: E
authors.lastname: Chen
authors.affiliation: The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Tang
authors.affiliation: The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Xue
authors.initials: X
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Quan
authors.affiliation: Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Xiaohou
authors.initials: X
authors.lastname: Wu
authors.affiliation: The Key Laboratory of Diagnostics Medicine, Ministry of Education, Chongqing Medical University, Chongqing, 400016, People's Republic of China. luochunli79@126.com.
authors.forename: Chunli
authors.initials: C
authors.lastname: Luo
mesh_terms: D000368:Aged; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D002295:Carcinoma, Transitional Cell / Q000378:metabolism* / Q000473:pathology; D020169:Caspases / Q000096:biosynthesis / Q000235:genetics; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000502:physiology*; D009367:Neoplasm Staging; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000235:genetics; D021381:Protein Transport; D011506:Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051899:Smad2 Protein / Q000502:physiology*; D051900:Smad3 Protein / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D001749:Urinary Bladder Neoplasms / Q000378:metabolism* / Q000473:pathology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D018797:Cell Cycle Proteins; C487620:HEPACAM protein, human; D009363:Neoplasm Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases
keywords: Bladder cancer; Caspase protein; Cell apoptosis; HepaCAM; p-SMAD2/3