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Updated: Mar 26, 2026
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
Published on: October 10, 2011
[Reversal of stemness in multidrug-resistant hepatocellular carcinoma cells by SIS3]
Wei Yan1, Ting Wen1, Suqiong Lin1
1Department of General Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, China.
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.
Conclusion:
SIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.
Insights
SIS3, a Smad3 inhibitor, reverses stemness in multidrug-resistant hepatocellular carcinoma (HCC) cells. This finding offers potential new therapeutic strategies for treating drug-resistant HCC by targeting cancer stem cell properties.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Stem Cell Research
Background:
- Hepatocellular carcinoma (HCC) often develops multidrug resistance (MDR).
- Cancer stem cells (CSCs) contribute to MDR and tumor recurrence in HCC.
- The Smad3 signaling pathway plays a role in cancer progression.
Purpose of the Study:
- To investigate if SIS3, a Smad3 phosphorylation inhibitor, can reverse the stemness of MDR HCC cells.
- To evaluate the impact of Smad3 inhibition on CSC markers and tumorigenicity.
Main Methods:
- Developed multidrug-resistant HCC cell lines (Huh7.5.1/ADM) using Adriamycin (ADM).
- Assessed drug sensitivity (CCK-8 assay), CSC marker expression (CD133 via flow cytometry), and tumorigenicity (in vitro colony formation, in vivo xenografts).
- Analyzed protein expression changes (Western blotting) including CD133, Smad3, Bcl-2, Bax, and p-Smad3.
Main Results:
- Huh7.5.1/ADM cells exhibited stable MDR, increased CD133+ subpopulations (76.06% vs. 25.38%), enhanced tumorigenicity, and activated Smad3.
- SIS3 treatment significantly decreased the CD133+ subpopulation (48.49% vs. 76.06%) in Huh7.5.1/ADM cells.
- SIS3 also reduced MDR (e.g., ADM IC50 decreased from 0.215 to 0.112 µg/ml) and inhibited tumor formation and colony formation ability.
Conclusions:
- SIS3 effectively reverses stemness and multidrug resistance in HCC cells by inhibiting the Smad3 signaling pathway.
- Targeting Smad3 with inhibitors like SIS3 presents a promising therapeutic approach for overcoming MDR in hepatocellular carcinoma.
