Targeting PI3K/Akt represses Hypoxia inducible factor-1α activation and sensitizes Rhabdomyosarcoma and Ewing's sarcoma cells for apoptosis

Abstract

Background: Hypoxia inducible factor-1 α (HIF-1α) has been identified as an important novel target in apoptosis resistance of pediatric tumors such as Rhabdomyosarcoma (RMS) and Ewing's sarcoma (ES). Evidence suggests that PI3K/Akt signaling plays a role in regulation of HIF-1α activation as well as apoptosis resistance in various adult tumors. However the relevance of PI3K/Akt signaling in HIF-1bα activation and apoptosis resistance in childhood tumors has not been addressed yet. Thus, this study was to investigate whether PI3K/Akt signaling is involved in hypoxia induced activation of HIF-1α as well as in resistance to hypoxia-induced apoptosis in childhood tumors such as RMS and ES.

Methods: Constitutive activation of PI3K/Akt signaling was analyzed by Western blotting. Hypoxic activation of HIF-1α was determined by Western Blot analysis and electrophoretic mobility shift assay. Apoptosis was determined by flow cytometric analysis of the propidium iodine stained nuclei of cells treated with PI3K inhibitor LY294002 in combination with either TNF-related apoptosis-inducing ligand (TRAIL) or doxorubicin.

Results: This study demonstrated that PI3K/Akt signaling was constitutively activated in RMS and ES cell lines, A204 and A673, respectively. Targeting PI3K/Akt signaling by the inhibitor LY294002 (30 μM) significantly decreased the protein expression as well as DNA binding activity of HIF-1α and restored the apoptosis-inducing ability of cells in hypoxia Additionally, pretreatment with LY294002 sensitized A204 and A673 cells to TRAIL or doxorubicin induced apoptosis under hypoxia.

Conclusion: These results suggest that the constitutively active PI3K/Akt signaling contributes to hypoxic activation of HIF-1α as well as HIF1α-mediated apoptosis resistance in RMS and ES cells under hypoxia.

Keywords: Apoptosis; Ewing’s sarcoma; HIF-1α; Hypoxia; PI3K/Akt; Rhabdomyosarcoma.

Figure 1

PI3K/Akt signalling is constitutively activated in A204 and A673 cell lines. Protein levels and phosphorylation status of Akt and β-actin (loading control), were analyzed by Western blotting in A. 24 hours cell culture of A204 cells under normoxia, treated with 0, 10, 20, 30 μmol/L LY294002. B. 24 hours cell culture of A673 cells under normoxia, treated with 0, 10, 20, 30 μmol/L LY294002. C. 24 h culture of A204 cells under normoxia or hypoxia, medium containing either 0% (−) or 10% FCS (+; top) and/or treated with 30 μmol/L LY294002. D. 24 h culture of A673 cells under normoxia or hypoxia, medium containing either 0% (−) or 10% FCS (+; top) and/or treated with 30 μmol/L LY294002. E. Western blots obtained as indicated in C were densitometrically analyzed. Shown are means ± SEM of four independent experiments. Statistically significant differences between LY294002 treated and untreated (20% FCS) are indicated *, p < 0.05 and **, p < 0.01. Statistically significant differences between LY294002 treated and untreated (0% FCS) are indicated #, p < 0.05 and ##, p < 0.01. The difference between untreated hypoxic and normoxic condition was not significant (for 20% FCS, p = 0.1288; for 0% FCS, p = 0.8749). F. Western blots obtained as indicated in D were densitometrically analyzed. Shown are means ± SEM of four independent experiments. Statistically significant differences between LY294002 treated and untreated (20% FCS) are indicated *, p < 0.05 and **, p < 0.01. Statistically significant differences between LY294002 treated and untreated (0% FCS) are indicated #, p < 0.05 and ##, p < 0.01. The difference between untreated hypoxic and normoxic condition was not significant (for 20% FCS, p = 0.1529; for 0% FCS, p = 0.3275).

Figure 2

PI3K/Akt pathway is involved in activation of HIF-1α in A204 and A673 cell lines. Cells were pretreated with 0 or 30 μmol/L of LY294002 for 1 hour and then incubated for 24 hours or 48 hours either in normoxia (N) or hypoxia (H). A. Protein expression levels of HIF-1α and β-actin were analyzed by western blotting. B. DNA binding activity of Hif-1α in nuclear extracts was assessed by EMSA. Specific HIF-1 DNA binding was confirmed by using a radioactive labeled mutated (m) probe, in which the HIF-1α consensus binding site is inactivated, and by competition with unlabelled consensus (c) and mutant (c/m) DNA probes in 100 fold excess.

Figure 3

Sensitization of A204 and A673 cells to doxorubicin- and TRAIL-induced apoptosis by PI3K inhibition. A204 (A) and A673 (B) cells were pretreated or not with 30 μmol/L LY294002 for 1 hour, incubated with doxorubicin (0.1 μg/ml) or TRAIL (100 ng/ml) and cultured under normoxic or hypoxic conditions for up to 72 hours. 24 hours-white bars, 48 hours- black bars, 72 hours-hatched bars. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide–stained nuclei; the percentage of specific apoptosis is shown. Columns, mean of three independent experiments done in duplicate; bars, SD. For statistical analysis two-way ANOVA was performed comparing specific apoptosis of either TRAIL or doxorubicin-induced apoptosis under normoxia vs hypoxia (*p < 0.05, **p < 0.01, ***p < 0.001) and hypoxia induced apoptosis vs hypoxia + LY294002 (^##p < 0.01, ^###p < 0.001) as well as hypoxia + TRAIL or doxorubicin vs hypoxia + LY294002 + TRAIL or doxorubicin (^§§p < 0.01, ^§§§p < 0.001).

Figure 4

Sensitization of A204 and A673 cells for doxorubicin- and TRAIL- induced apoptosis is caspase dependent. A204 (A) and A673 (B) cells were pretreated with 0 or 30 μmol/L of LY294002 for 1 hour, and cultured under normoxic or hypoxic conditions with doxorubicin (0.5 μg/ml) or TRAIL (100 ng/ml) for up to 72 hours with or without or z-VAD-fmk (50 μmol/L) 24 hours-white bars, 48 hours-black bars, 72 hours-hatched bars Columns, mean of three independent experiments done in duplicate; bars, SD. For statistical analysis two-way ANOVA was performed comparing specific apoptosis of either LY294002 + TRAIL or doxorubicin--induced apoptosis without z-VAD-fmk vs with z-VAD-fmk under normoxia (*p < 0.05, **p < 0.01) or the same comparison of specific apoptosis under hypoxia (^#p < 0.05, ^##p < 0.01).