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Comparative Study
. 2006 Oct 2:6:232.
doi: 10.1186/1471-2407-6-232.

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

Henning Schulze-Bergkamen  1 , Binje Fleischer, Marcus Schuchmann, Achim Weber, Arndt Weinmann, Peter H Krammer, Peter R Galle
Affiliations
Comparative Study

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

Henning Schulze-Bergkamen et al. BMC Cancer. .

Abstract

Background: Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro.

Methods: RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively.

Results: Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition.

Conclusion: Our data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies.

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Figures

Figure 1
Figure 1
Sensitivity of Huh7 cells towards chemotherapeutic drug-induced apoptosis. Huh7 cells were treated with 5-FU, mitomycin C, epirubicin, or cisplatin for 24 h and 48 h, respectively (concentration as indicated). Cells were then harvested and analyzed for apoptosis induction by flow cytometry according to the method of Nicoletti et al. [29]. Assays were performed in triplicates and are representative for three independent experiments. Values are means + SD.
Figure 2
Figure 2
Blocking of different protein kinases and its effect on Mcl-1 expression and chemotherapeutic drug-induced apoptosis. (A) Huh7 cells were treated with the kinase inhibitors LY294002 (25 μM, blocking PI3 kinase), PD98059 (50 μM, blocking MEK1), SP600125 (1–100 μM, blocking JNK1), AG1478 (0.5–5 μM, blocking EGF receptor tyrosine kinase), rapamycin (50 pM, blocking mTOR kinase) and Raf I-kinase inhibitor (0.1–1 μM) for the time indicated. Whole cell lysates were prepared, separated, and immunoblotted with antibodies against Mcl-1 and alpha-tubulin. Blots shown are representative for at least two independent experiments. (B) Huh7 cells were treated with LY294002 (25 μM), PD98059 (50 μM), rapamycin (50 pM). After 1 h of pre-incubation, cells were additionally treated with epirubicin, 5-FU or left untreated for 24 h (concentrations as indicated). Cells were then harvested and analyzed by flow cytometry for apoptosis induction according to Nicoletti et al. [29]. Assays were performed in triplicates and are representative for three independent experiments. Values are means + SD. *p < 0.01, **p < 0.001.
Figure 3
Figure 3
Silencing of Mcl-1 expression in HCC cells by RNA interference results in sensitization towards chemotherapy. (A) Huh7 cells were transfected with siRNA specific for Mcl-1 (40 nM) or transfected with siRNA specific for green fluorescent protein (GFP) as control. Cells were lysed 24, 48 and 72 h after transfection and analyzed for Mcl-1 expression by Western Blot (upper panel). Tubulin expression was used to control equal loading. siRNA concentration was titrated as indicated and expression of Mcl-1 and tubulin was analyzed by Western Blot 24 h after transfection (middle panel). 24 h after transfection with siRNA (40 nM), total RNA was extracted and analyzed for Mcl-1 mRNA expression by quantitative real-time PCR (lower panel). Expression of Mcl-1 was normalized to actin in each sample. Relative Mcl-1 expression was calculated as described in the materials and methods section. (B) Huh7 cells were transfected with siRNA specific for Mcl-1 (40 nM, "Mcl-1 siRNA +") or transfected with siRNA specific for GFP as control ("Mcl-1 siRNA -"). 24 h after transfection cells were treated with different chemotherapeutic drugs for 24 h (mitomycin C, 10 μM, 5-FU, 150 μg/ml (with or without valproic acid, VA, 100 μg/ml) and epirubicin, 1 μg/ml). Some cultures were irradiated with UV (7.5 and 15 mJ/cm2, respectively). 24 h later, cells were harvested and analyzed by flow cytometry for apoptosis induction according to the method by Nicoletti [29]. Assays were performed in triplicates and are representative for at least three independent experiments. Values are means + SD. *p < 0.01, **p < 0.001. (C) Cells were incubated for 24 h with epirubicin (1 μg/ml). Caspase-3 (upper panel) and caspase-9 (lower panel) activity were measured by fluorometric assays as described in the materials and methods section. Bars = SD.
Figure 4
Figure 4
Apoptosis sensitivity of HCC cells towards different kinase inhibitors and TRAIL after downregulation of Mcl-1. Huh7 cells were treated for 24 h with different kinase inhibitors (LY294002, 25 μM; PD98059, 50 μM; PP2, 10 μM; rapamycin, 50 pM; SU5614, 15 μM; and TRAIL, 100 ng/ml). Cells were transfected 24 h before with siRNA specific for Mcl-1 (40 nM, "Mcl-1 siRNA +") or with siRNA specific for green fluorescent protein as a control (40 nM, "Mcl-1 siRNA -"). Apoptosis was determined by FACS analysis according to Nicoletti [29]. Values are means + SD. *p < 0.01, **p < 0.001.
Figure 5
Figure 5
Mcl-1 interference sensitizes HCC cells towards combined treatment modalities. (A) Huh7 cells were transfected with siRNA specific for Mcl-1 (grey bars) or transfected with siRNA specific for GFP as control (light bars). After 24 h, cells were treated with chemotherapeutic drugs for another 24 h: cisplatin, 1 μg/ml, epirubicin, 200 ng/ml, 5-FU, 150 μg/ml (+ valproic acid (VA), 100 μg/ml), and mitomycin C, 5 μM. Some cultures were pre-incubated for 1 h with the kinase inhibitors LY294002 (25 μM, blocking PI3K), PD98059 (50 μM, blocking MEK1), AG1478 (5 μM, blocking EGF receptor tyrosine kinase) and a Raf I-kinase inhibitor (100 nM). Apoptosis was assessed by FACS analysis. *p < 0.05, **p < 0.001. n.s., not significant. (B) and (C) Huh7 cells were transfected with siRNA (40 nM) as described above and treated 24 h thereafter. In (B), cells were treated with 5-FU (150 μg/ml) and VA (100 μg/ml) for 8 or 24 h, as indicated. Cells were harvested and caspase-3 (upper panel) or caspase-9 activities (lower panel) were measured by applying caspase-specific fluorogenic substrates as described in the materials and methods section. *p < 0.01, **p < 0.001. (C) Cells were treated with 5-FU (150 μg/ml) for 48 h with or without simultaneous treatment with TRAIL as indicated. Apoptosis was measured using flow cytometrical analysis. Values are means + SD. *p < 0.05, **p < 0.001, n.s., not significant.
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