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Hepatocyte growth factor enhances death receptor-induced apoptosis by up-regulating DR5.

Li Y, Fan X, Goodwin CR, Laterra J, Xia S - BMC Cancer (2008)

Bottom Line: Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported.No cell death was associated with HGF alone.Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hugo W, Moser Research Institute at Kennedy Krieger, Baltimore, MD, USA. yli44@jhu.edu

ABSTRACT

Background: Hepatocyte growth factor (HGF) and its receptor c-MET are commonly expressed in malignant gliomas and embryonic neuroectodermal tumors including medulloblastoma and appear to play an important role in the growth and dissemination of these malignancies. Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported.

Methods: Human medulloblastoma cells were treated with HGF for 24-72 hours followed by death receptor ligand TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) for 24 hours. Cell death was measured by MTT and Annexin-V/PI flow cytometric analysis. Changes in expression levels of targets of interest were measured by Northern blot analysis, quantitative reverse transcription-PCR, Western blot analysis as well as immunoprecipitation.

Results: In this study, we show that HGF promotes medulloblastoma cell death induced by TRAIL. TRAIL alone triggered apoptosis in DAOY cells and death was enhanced by pre-treating the cells with HGF for 24-72 h prior to the addition of TRAIL. HGF (100 ng/ml) enhanced TRAIL (10 ng/ml) induced cell death by 36% (P<0.001). No cell death was associated with HGF alone. Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase. Cell death induced by TRAIL+HGF was predominately apoptotic involving both extrinsic and intrinsic pathways as evidenced by the increased activation of caspase-3, 8, 9. Promotion of apoptosis by HGF occurred via the increased expression of the death receptor DR5 and enhanced formation of death-inducing signal complexes (DISC).

Conclusion: Taken together, these and previous findings indicate that HGF:c-Met pathway either promotes or inhibits medulloblastoma cell death via pathway and context specific mechanisms.

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Modulation of pro- and anti-apoptotic proteins by HGF. A. Western blot analysis of FADD, FLIP, Bax, Bcl-xl and Bim protein levels in response to HGF, TRAIL and HGF + TRAIL. Cells were treated with HGF for 72 h before incubation with TRAIL for another 24 h. FADD and Bim were slightly down regulated by HGF + TRAIL. B. Northern blot analysis of DR5 mRNA levels in response to HGF. DAOY cells were treated with HGF for the indicated times. Total cell RNA was hybridized with probes for DR5 and 28S RNA (control) as described in Materials and Methods. DR5 isoform 1 mRNA levels were increased between 1–72 h as shown (* P < 0.001). C. DAOY cells were pre-incubated with inhibitors for 30 min, and then treated with HGF for 24 h followed by Northern blot analysis for DR5 expression. The PI3K inhibitor LY294002 (30 μM) or wortmannin (1 μM) did not affect the DR5 up-regulation induced by HGF. The MAPK inhibitor PD98059 (30 μM) reversed up-regulation of DR5 by HGF (# P < 0.05). Data represent mean ± SE (N = 6).
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Figure 4: Modulation of pro- and anti-apoptotic proteins by HGF. A. Western blot analysis of FADD, FLIP, Bax, Bcl-xl and Bim protein levels in response to HGF, TRAIL and HGF + TRAIL. Cells were treated with HGF for 72 h before incubation with TRAIL for another 24 h. FADD and Bim were slightly down regulated by HGF + TRAIL. B. Northern blot analysis of DR5 mRNA levels in response to HGF. DAOY cells were treated with HGF for the indicated times. Total cell RNA was hybridized with probes for DR5 and 28S RNA (control) as described in Materials and Methods. DR5 isoform 1 mRNA levels were increased between 1–72 h as shown (* P < 0.001). C. DAOY cells were pre-incubated with inhibitors for 30 min, and then treated with HGF for 24 h followed by Northern blot analysis for DR5 expression. The PI3K inhibitor LY294002 (30 μM) or wortmannin (1 μM) did not affect the DR5 up-regulation induced by HGF. The MAPK inhibitor PD98059 (30 μM) reversed up-regulation of DR5 by HGF (# P < 0.05). Data represent mean ± SE (N = 6).

Mentions: The effects of HGF on key regulatory components of the extrinsic and intrinsic apoptotic pathways were examined by Western blot analysis. To summarize, total cell levels of the pro-apoptotic protein FADD were diminished slightly by TRAIL in both control and HGF-treated cells (0.9 and 0.85-fold, respectively). Levels of pro-apoptotic Bim were decreased by TRAIL in HGF-treated cells only (0.55-fold). The anti-apoptotic protein Bcl-xl was found to be substantially up-regulated by TRAIL in HGF-treated cells (1.62-fold). There was no significant change in Bax or FLIP protein levels (Fig. 4A). Bcl-2 and Bad were not detected in DAOY cells (data not shown). Thus, HGF does not appear to sensitize cells to TRAIL by affecting levels of these specific anti- or pro-apoptotic proteins.


Hepatocyte growth factor enhances death receptor-induced apoptosis by up-regulating DR5.

Li Y, Fan X, Goodwin CR, Laterra J, Xia S - BMC Cancer (2008)

Modulation of pro- and anti-apoptotic proteins by HGF. A. Western blot analysis of FADD, FLIP, Bax, Bcl-xl and Bim protein levels in response to HGF, TRAIL and HGF + TRAIL. Cells were treated with HGF for 72 h before incubation with TRAIL for another 24 h. FADD and Bim were slightly down regulated by HGF + TRAIL. B. Northern blot analysis of DR5 mRNA levels in response to HGF. DAOY cells were treated with HGF for the indicated times. Total cell RNA was hybridized with probes for DR5 and 28S RNA (control) as described in Materials and Methods. DR5 isoform 1 mRNA levels were increased between 1–72 h as shown (* P < 0.001). C. DAOY cells were pre-incubated with inhibitors for 30 min, and then treated with HGF for 24 h followed by Northern blot analysis for DR5 expression. The PI3K inhibitor LY294002 (30 μM) or wortmannin (1 μM) did not affect the DR5 up-regulation induced by HGF. The MAPK inhibitor PD98059 (30 μM) reversed up-regulation of DR5 by HGF (# P < 0.05). Data represent mean ± SE (N = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2590617&req=5

Figure 4: Modulation of pro- and anti-apoptotic proteins by HGF. A. Western blot analysis of FADD, FLIP, Bax, Bcl-xl and Bim protein levels in response to HGF, TRAIL and HGF + TRAIL. Cells were treated with HGF for 72 h before incubation with TRAIL for another 24 h. FADD and Bim were slightly down regulated by HGF + TRAIL. B. Northern blot analysis of DR5 mRNA levels in response to HGF. DAOY cells were treated with HGF for the indicated times. Total cell RNA was hybridized with probes for DR5 and 28S RNA (control) as described in Materials and Methods. DR5 isoform 1 mRNA levels were increased between 1–72 h as shown (* P < 0.001). C. DAOY cells were pre-incubated with inhibitors for 30 min, and then treated with HGF for 24 h followed by Northern blot analysis for DR5 expression. The PI3K inhibitor LY294002 (30 μM) or wortmannin (1 μM) did not affect the DR5 up-regulation induced by HGF. The MAPK inhibitor PD98059 (30 μM) reversed up-regulation of DR5 by HGF (# P < 0.05). Data represent mean ± SE (N = 6).
Mentions: The effects of HGF on key regulatory components of the extrinsic and intrinsic apoptotic pathways were examined by Western blot analysis. To summarize, total cell levels of the pro-apoptotic protein FADD were diminished slightly by TRAIL in both control and HGF-treated cells (0.9 and 0.85-fold, respectively). Levels of pro-apoptotic Bim were decreased by TRAIL in HGF-treated cells only (0.55-fold). The anti-apoptotic protein Bcl-xl was found to be substantially up-regulated by TRAIL in HGF-treated cells (1.62-fold). There was no significant change in Bax or FLIP protein levels (Fig. 4A). Bcl-2 and Bad were not detected in DAOY cells (data not shown). Thus, HGF does not appear to sensitize cells to TRAIL by affecting levels of these specific anti- or pro-apoptotic proteins.

Bottom Line: Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported.No cell death was associated with HGF alone.Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hugo W, Moser Research Institute at Kennedy Krieger, Baltimore, MD, USA. yli44@jhu.edu

ABSTRACT

Background: Hepatocyte growth factor (HGF) and its receptor c-MET are commonly expressed in malignant gliomas and embryonic neuroectodermal tumors including medulloblastoma and appear to play an important role in the growth and dissemination of these malignancies. Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported.

Methods: Human medulloblastoma cells were treated with HGF for 24-72 hours followed by death receptor ligand TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) for 24 hours. Cell death was measured by MTT and Annexin-V/PI flow cytometric analysis. Changes in expression levels of targets of interest were measured by Northern blot analysis, quantitative reverse transcription-PCR, Western blot analysis as well as immunoprecipitation.

Results: In this study, we show that HGF promotes medulloblastoma cell death induced by TRAIL. TRAIL alone triggered apoptosis in DAOY cells and death was enhanced by pre-treating the cells with HGF for 24-72 h prior to the addition of TRAIL. HGF (100 ng/ml) enhanced TRAIL (10 ng/ml) induced cell death by 36% (P<0.001). No cell death was associated with HGF alone. Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase. Cell death induced by TRAIL+HGF was predominately apoptotic involving both extrinsic and intrinsic pathways as evidenced by the increased activation of caspase-3, 8, 9. Promotion of apoptosis by HGF occurred via the increased expression of the death receptor DR5 and enhanced formation of death-inducing signal complexes (DISC).

Conclusion: Taken together, these and previous findings indicate that HGF:c-Met pathway either promotes or inhibits medulloblastoma cell death via pathway and context specific mechanisms.

Show MeSH
Related in: MedlinePlus