OBJECTIVEto investigate the effects of antidiabetic drug metformin on proliferation and apoptosis in human hepatocellular carcinoma cell line Huh-7 cells.

METHODSHuh-7 cells were treated with metformin at different concentrations. Cell viability was determined by MTT assay. Cell apoptosis and CD133(+) expression rate were detected by flow cytometery (FCM). Expressions of PTEN, Akt, p-Akt, Bcl-2, Bax proteins in the cells were measured by Western blot. The effect of metformin on the hepatosphere formation was observed in the serum-free suspension culture. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the expression levels of stemness marker genes CD133, β-catenin, and ABCG2 mRNA in the hepatospheres.

RESULTSThe proliferation of Huh-7 cells was inhibited by metformin in a dose- and time-dependent manner. The early and late cell apoptosis rates induced by metformin at dose of 10 mmol/L for 48 hrs were (22.29 ± 0.8)% and (13.87 ± 1.2)%, respectively, and 25 mmol/L for 48 hrs (15.28 ± 2.1)% and (25.89 ± 2.3)%, respectively. Western blotting results revealed that the expression of CD133, phosphorylated Akt and the Bcl-2/Bax ratio were downregulated, and PTEN was upregulated in the Huh-7 cells after treated with 25 mmol/L metformin for 48 hrs. Metformin inhibited the formation of hepatospheres. Metformin also downregulated the expression of several cancer stem cells (CSCs)-related genes which are involved in the signaling pathways governing the self-renewal, proliferation and differentiation of CSCs in the hepatospheres.

CONCLUSIONSMetformin inhibits the proliferation of human hepatocellular carcinoma Huh-7 cells and enhances their apoptosis in vitro. It may be related to the downregulation of PI3K/Akt signal pathway and selectively targeting CD133(+) cells.

AC133 Antigen ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Antigens, CD ; genetics ; metabolism ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Glycoproteins ; genetics ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Metformin ; administration & dosage ; pharmacology ; Neoplasm Proteins ; genetics ; metabolism ; PTEN Phosphohydrolase ; metabolism ; Peptides ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; Time Factors ; bcl-2-Associated X Protein ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo investigate whether SIS3, a specific inhibitor of Smad3 phosphorylation, can reverse the stemness of multidrug-resistant(MDR) hepatocellular carcinoma cells.

METHODSMDR 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.

RESULTSADM 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.

CONCLUSIONSIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.

AC133 Antigen ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigens, CD ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; pathology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Glycoproteins ; metabolism ; Heterografts ; Humans ; Isoquinolines ; pharmacology ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; metabolism ; Neoplastic Stem Cells ; drug effects ; Peptides ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyridines ; pharmacology ; Pyrroles ; pharmacology ; Smad3 Protein ; antagonists & inhibitors ; metabolism ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism