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. 2013 Jan 24;4(1):e471.
doi: 10.1038/cddis.2012.200.

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms

N Charette  1 , C De Saeger, Y Horsmans, I Leclercq, P Stärkel
Affiliations

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms

N Charette et al. Cell Death Dis. .

Abstract

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.

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Figures

Figure 1
Figure 1
Salirasib sensitizes HCC cells to TRAIL-induced cell death. (a) HepG2 (left column), Huh7 (central column) and Hep3B (right column) cells were seeded in 96-wells plates and incubated for 24 h with DMSO or the indicated concentration of salirasib and the indicated concentration of human recombinant TRAIL (n=8–16 for each group) for 24 h. Cell viability was assessed by a WST-1 assay. (b) HepG2, Huh7 and Hep3B cells were seeded in 96-wells plates and incubated with DMSO or the indicated concentration of salirasib for 24 h. The culture medium was then replaced by a medium containing the same concentration of salirasib and the indicated concentration of human recombinant TRAIL (n=12–16 per group) for further 24 h and cell viability was assessed by a WST-1 assay. (c) Cells were seeded in 96-wells plates and incubated with DMSO or 150 μM salirasib for 24 h. TRAIL (50 ng/ml) was added for further 24 h. LDH release was then assessed. Results are expressed as percentage of the maximum LDH release obtained with a lysis buffer. Data are presented as mean±S.E.M. **P<0.001, ***P<0.0001 in treated groups versus control group
Figure 2
Figure 2
Apoptosis induced by treatment with salirasib and TRAIL involves both the DR and the mitochondrial apoptotic pathways. (a) HepG2, Huh7 and Hep3B cells were seeded in 6-wells plates and incubated for 24 h with DMSO or 150 μM salirasib. Culture medium was then replaced by a medium containing the same concentration of salirasib with or without 50 ng/ml TRAIL (n=6 per group) for 24 h. Cell cycle distribution was then assessed by flow cytometry and the sub-G0 population was quantified. (b–c). Cells were seeded in 96-wells plates and treated as in a. Caspase-3 (b), −8 (c) and −9 (d) were then assessed using a commercially available kit (n=8). Data are presented as mean±S.E.M. *P<0.05, **P<0.001, ***P<0.0001 in treated groups versus control group
Figure 3
Figure 3
Salirasib-induced sensitization to TRAIL-induced apoptosis parallels survivin down-regulation. (a) Quantification of survivin mRNA by quantitative PCR after 1 or 2 days of treatment with 75 or 150 μM of salirasib (n=6 per group). (b) Representative western blot after 1 or 2 days of treatment with 75 or 150 μM of salirasib. (c) Quantification of survivin protein expression by densitometric analysis (n=5–6 per group). Data are expressed as relative change versus control set arbitrarily at 1 and are presented as mean±S.E.M. *P<0.05, **P<0.001, ***P<0.0001 in treated groups versus control group
Figure 4
Figure 4
Survivin inhibition sensitizes HCC cells to TRAIL-induced apoptosis. (a) HepG2, Huh7 and Hep3B cells were plated in 96-wells plates and incubated with various doses of YM155 for 3 days (n=8 for each dose). Cell viability was assessed by a WST-1 assay and IC50 was determined using non linear regression analysis. Data are presented as mean±S.E.M. (b) Representative western blot after 2 days of treatment with 150 nM of YM155. (c) Quantification of survivin protein expression by densitometric analysis (n=5–6 per group). Data are expressed as relative change versus control set arbitrarily at 1 and are presented as mean±S.E.M. (d) HepG2, Huh7 and Hep3B cells were seeded in 6-wells plates and incubated for 24 h with DMSO or 150 nM YM155. Culture medium was then replaced by a medium containing the same concentration of YM155 with or without 50 ng/ml TRAIL (n=6 per group) for 24 h. Cell cycle distribution was then assessed by flow cytometry and the sub-G0 population was quantified. *P<0.05, **P<0.001, ***P<0.0001 in treated groups versus control group
Figure 5
Figure 5
TRAIL-induced apoptosis in salirasib pretreated cells is mediated by DR5. (a) Quantification of DR4 and DR5 mRNA by quantitative PCR after 24 h of treatment with 150 μM of salirasib. (b) HepG2, Huh7 and Hep3B cells were seeded in 96-wells plates and incubated for 24 h with DMSO or 150 μM salirasib with or without anti-DR4 and/or anti-DR5 antibodies. Cell viability was then assessed by a WST-1 assay. (c) Cells were seeded in 96-wells plates and cultured with DMSO or the indicated concentration salirasib for 24 h. They were then further incubated for 24 h in a medium containing DMSO or salirasib supplemented with the indicated concentration of agonistic anti-DR5 antibody. Data are presented as relative change compared with untreated controls (mean±S.E.M.). *P<0.05, **P<0.001, ***P<0.0001 in treated groups versus control group (salirasib+TRAIL group in b), #P<0.05, ##P<0.001, ###P<0.0001 compared to the salirasib alone group in b
Figure 6
Figure 6
Treatment with salirasib and TRAIL is not toxic for human hepatocytes. Primary human hepatocytes seeded in collagen-coated 96-wells plates were incubated in a culture medium containing DMSO or 150 μM salirasib for 24 h. The culture medium was then replaced by a medium containing the same concentration of salirasib with or without 100 ng/ml TRAIL (n=8 per group) for 24 h. Cell viability was then assessed using a WST-1 assay (a) and cell death was assessed by a LDH release assay (b). Data are presented as relative change compared with untreated controls (mean±S.E.M.)
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