title: [Reversal of stemness in multidrug-resistant hepatocellular carcinoma cells by SIS3].
abstract: OBJECTIVE
  To investigate whether SIS3, a specific inhibitor of Smad3 phosphorylation, can reverse the stemness of multidrug-resistant(MDR) hepatocellular carcinoma cells.
  
  
  METHODS
  MDR HCC Huh7.5.1/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. CCK-8 assay was used to determine the cellular sensitivity of various anticancer drugs. Flow cytometry (FCM) was used to analyze the expression level of cancer stem cell marker CD133. Clone formation assay and mouse subcutaneous xenograft tumors were used to investigate the tumorigenicity in vitro and in vivo. Western blotting (WB) was used to analyze the changes of expressions of CD133, Smad3, Bcl-2, Bax and p-Smad3 in different conditions.
  
  
  RESULTS
  ADM treatment of HCC cells in vitro resulted in a development of subline, Huh7.5.1/ADM cells, with CSC phenotypes: stable MDR phenotype (besides ADMc Huh7.5.1/ADM cells were also more resistant to some other anticancer drugs including VCR, MMC and CTX ) (IC50: 0.215 ± 0.018 vs. 0.123 ± 0.004, 0.145 ± 0.009 vs. 0.014 ± 0.002, 1.021 ± 0.119 vs. 0.071 ± 0.006, 27.007 ± 1.606 vs. 1.919 ± 0.032) (unit: µg/ml) (P<0.05). Huh7.5.1/ADM cells enriched the cancer stem-like cell fraction (CD133-positive subpopulation) (76.06 ± 2.948% vs. 25.38 ± 4.349%) (P<0.05), had stronger tumorigenicity in vivo and colony formation ability, and activated the Smad3 activity. Inhibition of Smad3 activity by SIS3 decreased stemness of the Huh7.5.1/ADM cells: CD133-positive subpopulation (48.49 ± 2.304% vs. 76.06 ± 2.948%) (P<0.05); ADM IC50: (0.112 ± 0.019 vs. 0.215 ± 0.018), VCR IC50 (0.065 ± 0.013 vs. 0.145±0.009), MMC IC₅₀ (0.749 ± 0.121 vs. 1.021 ± 0.119), CTX IC50 (10.576 ± 1.248 vs. 27.007 ± 1.606) (unit: µg/ml) (P<0.05), and decreased tumorigenicity and colony formation ability.
  
  
  CONCLUSIONS
  SIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.
authors.affiliation: Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yan
authors.affiliation: Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Wen
authors.affiliation: Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
authors.forename: Suqiong
authors.initials: S
authors.lastname: Lin
authors.affiliation: Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
authors.forename: Zhongcai
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China; Email: wuguoyang_mail@aliyun.com.
authors.forename: Guoyang
authors.initials: G
authors.lastname: Wu
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006023:Glycoproteins / Q000378:metabolism; D064593:Heterografts; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D010455:Peptides / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D014410:Tumor Stem Cell Assay; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C508504:6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride; D000071916:AC133 Antigen; D000903:Antibiotics, Antineoplastic; D015703:Antigens, CD; C494826:BAX protein, human; D006023:Glycoproteins; D007546:Isoquinolines; D009363:Neoplasm Proteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; D011758:Pyrroles; D051900:Smad3 Protein; D051028:bcl-2-Associated X Protein; D004317:Doxorubicin