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Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

Dzieran J, Fabian J, Feng T, Coulouarn C, Ilkavets I, Kyselova A, Breuhahn K, Dooley S, Meindl-Beinker NM - PLoS ONE (2013)

Bottom Line: TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling.Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients.RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7.

View Article: PubMed Central - PubMed

Affiliation: Molecular Hepatology - Alcohol Associated Diseases, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

ABSTRACT
Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

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TGF-β induces cell death and/or inhibits proliferation in PLC, HepG2, HuH6, Hep3B and HuH7.(A) Left side: HCC cell lines were treated with 5 ng/ml TGF-β for indicated time points. Changes in c-MYC and P21 expression were detected using Western blot analysis with GAPDH as loading control. As HCC-T cells show a delayed TGF-β response, c-MMYC was induced after 72 h of TGF-β treatment. Right side: After TGF-β treatment for 0 (grey line), 2 (white bars) or 6 days (black bars) cell proliferation was evaluated by MTT assays. Treated samples were normalized to the corresponding control. (B) Left side: Western blot analysis of PARP and Caspase 3 cleavage using GAPDH expression as loading control after 0 and 48 h TGF-β treatment. In the case of HuH7 cells, the control sample was treated with TGF-β for 3 h. Right side: Cell death induced by 5 ng/ml TGF-β over 72 h (filled bars) was quantified by detecting LDH release normalized to total amount of LDH. Untreated samples were defined as 0 (grey line). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
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pone-0072252-g001: TGF-β induces cell death and/or inhibits proliferation in PLC, HepG2, HuH6, Hep3B and HuH7.(A) Left side: HCC cell lines were treated with 5 ng/ml TGF-β for indicated time points. Changes in c-MYC and P21 expression were detected using Western blot analysis with GAPDH as loading control. As HCC-T cells show a delayed TGF-β response, c-MMYC was induced after 72 h of TGF-β treatment. Right side: After TGF-β treatment for 0 (grey line), 2 (white bars) or 6 days (black bars) cell proliferation was evaluated by MTT assays. Treated samples were normalized to the corresponding control. (B) Left side: Western blot analysis of PARP and Caspase 3 cleavage using GAPDH expression as loading control after 0 and 48 h TGF-β treatment. In the case of HuH7 cells, the control sample was treated with TGF-β for 3 h. Right side: Cell death induced by 5 ng/ml TGF-β over 72 h (filled bars) was quantified by detecting LDH release normalized to total amount of LDH. Untreated samples were defined as 0 (grey line). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

Mentions: Sensitivity to TGF-β induced cytostasis was evaluated in 10 HCC cell lines. 2 days TGF-β treatment significantly inhibited cell proliferation in Hep3B, HuH7 and PLC, as determined by MTT-assay (Figure 1A, right panel), HepG2 and HuH6 displayed no or a weak response after 2 days, respectively. However, they show a strong response 6 days upon TGF-β addition. Proliferation of HCC-M, HCC-T, HLE, FLC-4 and HLF was not reduced. HCC-T even displayed an increased proliferation after 6 days treatment. In line with MTT-assay data, expression of proliferation associated marker P21 showed the strongest induction comparing before and after TGF-β treatment in those cell lines with TGF-β dependent proliferation inhibition. Consistently, expression of proliferation facilitating protein c-MYC was specifically down regulated in cell lines sensitive to TGF-β induced proliferation control, whereas it was even induced late in HCC-T cells (Figure 1A, left panel, Figure S2). Western Blot analysis was not sensitive enough to confirm weak cell death (+7%) of HepG2 upon TGF-β treatment on protein level.


Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

Dzieran J, Fabian J, Feng T, Coulouarn C, Ilkavets I, Kyselova A, Breuhahn K, Dooley S, Meindl-Beinker NM - PLoS ONE (2013)

TGF-β induces cell death and/or inhibits proliferation in PLC, HepG2, HuH6, Hep3B and HuH7.(A) Left side: HCC cell lines were treated with 5 ng/ml TGF-β for indicated time points. Changes in c-MYC and P21 expression were detected using Western blot analysis with GAPDH as loading control. As HCC-T cells show a delayed TGF-β response, c-MMYC was induced after 72 h of TGF-β treatment. Right side: After TGF-β treatment for 0 (grey line), 2 (white bars) or 6 days (black bars) cell proliferation was evaluated by MTT assays. Treated samples were normalized to the corresponding control. (B) Left side: Western blot analysis of PARP and Caspase 3 cleavage using GAPDH expression as loading control after 0 and 48 h TGF-β treatment. In the case of HuH7 cells, the control sample was treated with TGF-β for 3 h. Right side: Cell death induced by 5 ng/ml TGF-β over 72 h (filled bars) was quantified by detecting LDH release normalized to total amount of LDH. Untreated samples were defined as 0 (grey line). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
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pone-0072252-g001: TGF-β induces cell death and/or inhibits proliferation in PLC, HepG2, HuH6, Hep3B and HuH7.(A) Left side: HCC cell lines were treated with 5 ng/ml TGF-β for indicated time points. Changes in c-MYC and P21 expression were detected using Western blot analysis with GAPDH as loading control. As HCC-T cells show a delayed TGF-β response, c-MMYC was induced after 72 h of TGF-β treatment. Right side: After TGF-β treatment for 0 (grey line), 2 (white bars) or 6 days (black bars) cell proliferation was evaluated by MTT assays. Treated samples were normalized to the corresponding control. (B) Left side: Western blot analysis of PARP and Caspase 3 cleavage using GAPDH expression as loading control after 0 and 48 h TGF-β treatment. In the case of HuH7 cells, the control sample was treated with TGF-β for 3 h. Right side: Cell death induced by 5 ng/ml TGF-β over 72 h (filled bars) was quantified by detecting LDH release normalized to total amount of LDH. Untreated samples were defined as 0 (grey line). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).
Mentions: Sensitivity to TGF-β induced cytostasis was evaluated in 10 HCC cell lines. 2 days TGF-β treatment significantly inhibited cell proliferation in Hep3B, HuH7 and PLC, as determined by MTT-assay (Figure 1A, right panel), HepG2 and HuH6 displayed no or a weak response after 2 days, respectively. However, they show a strong response 6 days upon TGF-β addition. Proliferation of HCC-M, HCC-T, HLE, FLC-4 and HLF was not reduced. HCC-T even displayed an increased proliferation after 6 days treatment. In line with MTT-assay data, expression of proliferation associated marker P21 showed the strongest induction comparing before and after TGF-β treatment in those cell lines with TGF-β dependent proliferation inhibition. Consistently, expression of proliferation facilitating protein c-MYC was specifically down regulated in cell lines sensitive to TGF-β induced proliferation control, whereas it was even induced late in HCC-T cells (Figure 1A, left panel, Figure S2). Western Blot analysis was not sensitive enough to confirm weak cell death (+7%) of HepG2 upon TGF-β treatment on protein level.

Bottom Line: TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling.Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients.RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7.

View Article: PubMed Central - PubMed

Affiliation: Molecular Hepatology - Alcohol Associated Diseases, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

ABSTRACT
Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

Show MeSH
Related in: MedlinePlus