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

To assess the role of PI3K pathway in HIF-1α induction the phosphorylation status of Akt, which was used as surrogate for PI3K activity, was examined by Western blot analysis. As shown in Figure 1A and B, A204 and A673 cells had high levels of Akt phosphorylation on Ser473 in normoxia. Next we used LY294002, the pharmacologic inhibitor of PI3K to interfere with phosphorylation of Akt, and here we show that upon treatment with LY294002 the level of p-Akt was decreased in a dose dependent manner in both A204 and A673 cells in normoxia (Figure 1A, B). To address whether PI3K/Akt signaling was sustained in hypoxia, phosphorylation of Akt was examined in the presence or absence of LY294002 in both normoxia and hypoxia. In addition, due to the growth factors present in serum, which can induce Akt phosphorylation, we also tested serum-deprived cells. Accordingly, pretreatment of A204 and A673 cells by 30 μM LY294002 decreased phosphorylation of Akt in both conditions whereas protein levels of total Akt were not altered (Figure 1C, D). As seen in Figure 1C and D, levels of p-Akt-Ser473 were similar in A204 and A673 cells either in normoxia or hypoxia and did not change by serum deprivation but suppressed by LY294002 addition. Densitometry analysis also confirmed these data (Figure 1E and F) suggesting in A204 and A673 cells in normoxia p-Akt levels when normalized to Akt levels, is significantly decreased in the presence of LY294002 whether or not FCS is withdrawn. In contrast, no significant differences were detected in p-Akt levels between hypoxia and normoxia in both cells. In hypoxia in A673 cells p-Akt levels again did not change by serum deprivation whereas it seemed to be increased in A204 cells though it was not significant. Moreover, addition of LY294002 was able to suppress p-Akt levels significantly either in the presence or absence of FCS also in hypoxia in both cells. Whereas pretreatment of A204 and A673 cells by 30 μM LY294002 did not alter the protein levels of total Akt. Hence, we conclude that levels of p-Akt were sustained in both cells under hypoxia and did not change by serum deprivation either in normoxia or hypoxia.

Thus, these data demonstrated that activation of PI3K/Akt signaling is constitutive in both cell lines in normoxia and hypoxia, as evidenced by high levels of phosphorylated p-Akt-Ser473, the downstream effector of PI3K (Figure 1C, D).

In order to examine whether constitutive activation of PI3K/Akt signaling is involved in hypoxic induction of HIF-1α protein, either pretreated with 30 μM of LY294002, or left untreated (control) A204 and A673 cells were incubated under hypoxic conditions and subsequently subjected to Western blot analysis for stabilization of HIF-1α protein. As shown in Figure 2A, HIF-1α protein was stabilized 24 h after exposure to hypoxia and remained its levels up to 48 h post exposure in both cell lines. Remarkably, pre-treatment with LY294002 decreased the expression of HIF-1α suggesting that induction of HIF-1α by hypoxia requires activation of PI3K pathway. Further, the effect of PI3K inhibitor on hypoxia-induced DNA-binding activity of HIF-1α was investigated by EMSA utilizing a 3^′ HRE-derived oligonucleotide probe. Using a mutant probe, performing competition assays (Figure 2B) confirmed the identity of the HIF-1α band. Under hypoxic conditions HIF-1α showed increased DNA-binding activity at 24 h and the level of activity was still high at 48 h in both A204 and A673 cells (Figure 2B). Pretreatment with LY294002 reduced hypoxia-induced DNA binding activity of HIF-1α in both cell lines, although this effect was more pronounced in A204 cells after 24 h compared to A673 cells.

Next, we investigated whether or not decreased stabilization and DNA binding activity of HIF-1α by LY294002, can sensitize A204 and A673 cells to apoptosis under hypoxia. In order to examine long lasting effects of LY294002, cells were cultured for up to 72 h under hypoxic conditions in the presence of 30 μM LY294002 and apoptosis was assessed every 24 hours. As seen in Figure 3A while hypoxia alone did not trigger apoptosis in both cell lines, pretreatment with LY294002 induced 15,5 (±1,7)% and 16,0 (±1,1)% apoptosis in A204 and A673 cells, respectively, in a time dependent manner (Figure 3A). These data suggest that decreased protein level and DNA binding activity of HIF-1α by LY294002 treatment restores apoptosis sensitivity in A204 RMS and A673 ES cells.

In our previous work, we showed that hypoxia protects against death receptor- (e.g. TRAIL) and cytotoxic drug- (e.g. Doxorubicin) induced apoptosis in A204 and A673 cells. Therefore cells were pretreated with LY294002 and cultured for up to 72 h in the presence or absence of TRAIL in both normoxia and hypoxia. Apoptosis was assessed every 24 hours, and as seen in Figure 3A, without LY294002 pretreatment, after 72 h TRAIL - induced apoptosis in normoxia was at least 10% higher than to that of in hypoxia, underlining the protective role of hypoxia in both cell lines. Interestingly, pretreatment with LY294002 significantly sensitized cells for TRAIL-induced apoptosis and rendered the protective effect of hypoxia (Figure 3A).

Next, the effect of HIF-1α inhibition by LY294002 treatment in combination with doxorubicin, typically triggering apoptosis via the mitochondrial pathway, was also tested. In contrast to TRAIL, doxorubicin-induced apoptosis was substantial in A204 and A673 cells under either normoxia or hypoxia, though a slight protective effect of hypoxia was still present. Pretreatment of cells with LY294002 greatly enhanced doxorubicin-induced apoptosis. When pretreated with LY294002 the rate of apoptosis was at least 20% higher in both A204 and A673 cells after 72 h exposure to hypoxia (Figure 3B).

Moreover, the broad range caspase inhibitor z-VAD-fmk was used to test requirement for caspases during TRAIL- or doxorubicin-induced apoptosis under hypoxia. Apoptosis induced by combined treatments of LY294002 and TRAIL, or doxorubicin was significantly blocked in the presence of z-VAD-fmk under both normoxia and hypoxia in both cell lines in a time dependent manner (Figure 4A and B). These results indicated that apoptosis induced by combined treatments with LY294002 and either TRAIL, or doxorubicin was mediated by caspases.

Human Rhabdomyosarcoma (A204) and Ewing’s sarcoma (A673) cell lines were obtained from American Type Culture Collection (Manassas, VA) and were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Eggenstein, Germany) containing 10% heat inactivated fetal calf serum (Biochrom, Berlin, Germany), 100 IU/ml penicillin, 100 μg/ml streptomycin (Biochrom), 10 mM glutamine (Biochrom) in a humidified atmosphere at 37 °C with 5% CO₂ unless otherwise specified. Hypoxic conditions (0.5% O₂) were achieved by incubation in a humidified internal incubator of a hypoxia glove box (Coy Laboratory Products, Inc.). After an initial exposure to low oxygen, all subsequent treatments were given within the glove box to prevent cellular damage due to reoxygenation.

Apoptosis was assessed by fluorescence-activated cell-sorting (FACScan, Becton Dickinson, Heidelberg, Germany) analysis of DNA fragmentation of propidium-iodide stained nuclei as described previously [2]. The percentage of specific apoptosis was calculated as follows: 100 × [experimental apoptosis (%) − spontaneous apoptosis (%)] [100% − spontaneous apoptosis (%)].

Total cell extracts were prepared from cells grown in 6-well plates at 90% confluence. Cells were exposed to 20% O₂ or 0.5% O₂ for the indicated time points and lysed in lysis buffer (20 mM Tris, pH 7.5 (Sigma), 150 mM KCl (Sigma), 1 mM EDTA, 1% Triton X-100 (Sigma) supplemented with protease inhibitor mixture (Complete®; Roche Applied Science, Mannheim, Germany). 0.2 mM phenylmethylsulfonyl fluoride (PMSF); 0.5 mM dithiothreitol (DTT) and 1 mM sodium-ortho-vanadate before use. Western blot analysis was done as described previously using primary antibodies, mouse anti-Hif-1α monoclonal (1:250; BD Biosciences; Heidelberg, Germany), rabbit anti-phospho Akt (Ser 473) (D9E) and rabbit anti-Akt (Cell Signaling, Beverly, MA), followed by goat-anti-mouse IgG or goat-anti-rabbit IgG conjugated to horseradish peroxidase (1:5,000; Santa Cruz Biotechnology) [2]. Mouse anti-β-actin monoclonal antibody (1: 5000; Sigma), was used as a loading control. All proteins were visualized using enhanced chemiluminescence (Amersham Biosciences).

Nuclear extracts were prepared essentially as described [2]. Oligonucleotide DNA probes containing the HIF-1α binding sequence 5^′-TCTGTACGTGACCACACTCACCTC [43] and a mutant probe 5^′-TCTGTAAAAGACCACACTCACCTC, labelled with with γ-32P-ATP (Amersham, Freiburg, Germany) and annealed with complementary oligonucleotides, were used for EMSA [43]. EMSA was done as described previously [2]. In general, unless otherwise stated, all qualitative analyses were repeated at least three times.