The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation

Abstract

Background and purpose: The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined.

Materials and methods: Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis.

Results: The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy.

Conclusions: Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines - one which is sensitive to the Akt-inhibitor ErPC3 and one which is more sensitive to the PI3K-inhibitor LY294002. Our findings underline the importance for the definition of predictive biomarkers that allow the selection patients that may benefit from the treatment with a specific signal transduction modifier.

Figure 1

Anti-neoplastic effects of ErPC3 and ionizing radiation on prostate cancer cells. The prostate cancer cell lines PC3, DU145, and LNCaP, were irradiated (RT) with 2, 5, or 10 Gy (A-C) or treated with 1-100 µM ErPC3, as indicated (D-F). 48 h after treatment a WST-1 Assay was performed. The absorption correlates with the number of viable cells and was normalized to that of untreated controls. PC3 (A) and DU145 (B) were not affected by ionising radiation whereas the number of viable LNCaP cells was reduced 48 h after irradiation (C). All cell lines responded to ErPC3-treatment in a concentration-dependent manner. The androgen-independent cell line PC3 was most sensitive to ErPC3 (D). 25 µM ErPC3 reduce the number of viable PC3 cells by approximately 50% whereas 50 µM ErPC3 were needed to affect the viability of DU145 (E) and PC3 cells (F).

Figure 2

Apoptosis induction in response to ErPC3 and ionizing radiation. PC3 and LNCaP cells were treated with 1-50 µM ErPC3 or irradiated with a single dose of 2 or 10 Gy. 48 h later, cells were stained with propidium iodide in a hypotonic citrate buffer containing Triton X-100 and subjected to flow cytometric analysis to estimate DNA fragmentation which occurs upon induction of apoptosis. 5 µM ErPC3 were sufficient to induce DNA fragmentation in PC3 cells (A), whereas 25 µM ErPC3 were required to trigger apoptotic DNA-fragmentation in LNCaP cells (B). Ionizing radiation up to 10 Gy did not induce DNA-fragmentation above a background level in PC3 (C) and LNCaP cells (D).

Figure 3

Activation of caspase-3 and regulation of Bcl-2 protein family members in response to ErPC3-treatment and irradiation. PC3 and LNCaP cells were treated with 0-25 µM ErPC3 or irradiated with 2 or 10 Gy. Cells lysates were generated 48 h after treatment, separated by electrophoresis, and protein expression was subsequently analyzed by western blotting. Both cells lines showed a concentration-dependent activation of caspase-3 in response to ErPC3-treatment (A, B). In PC3 cells, cleavage of the caspase-3 substrate PARP could already be detected after treatment with 12.5 µM ErPC3; PARP-cleavage was accompanied by a weak activation of caspase-3 detectable upon treatment with 12.5 µM ErPC3 (A). A weak cleavage of caspase-3 and PARP was also observed when LNCaP cells were treated with 12.5 µM ErPC3, but cleavage was clearly visible after treatment with 25 µM ErPC3 (B). No caspase-3 activation and PARP cleavage was observed in response to ionizing radiation. No change of protein levels of the pro-apoptotic Bak and Bax and the anti-apoptotic Bcl-xL was observed upon irradiation or in response to treatment with ErPC3 (C, D). A slight reduction in the levels of antiapoptotic Bcl-2 was observed upon irradiation in LNCaP and PC3 cells, whereas treatment with ErPC3 reduced the levels of the anti-apoptotic Mcl-1 in LNCaP cells. However, the changes of Mcl-1 expression levels did not correlate with the sensitivity of LNCaP cells to ErPC3.

Figure 4

Differential effects of ErPC3 and LY294002 on prostate cancer cell survival and p-Akt levels. (A, B, left panels) DU145, LNCaP, and PC3 cells were treated with solvent controls or 25-100 µM ErPC3 or 25-100 µM LY294002. 48 h later a WST-1 assay was performed to quantify the number of viable cells. PC3 cells were most sensitive to treatment with ErPC3 (A, left panel), whereas LNCaP cell were most susceptible to LY294002-treatment (B, left panel). Western blot analysis of lysates generated from PC3 cells 48 h after treatment with 0-25 µM ErPC3 showed a massive reduction of Akt-phosphorylation at serine 473 (p-Akt) whereas almost no reduction of p-Akt was found 48 h after irradiation with 2 or 10 Gy (A, right panel). ErPC3 also reduced p-Akt levels in LNCaP cells however with lower potency (A, right panel). Western blot analysis of lysates generated 0-48 h after treatment with 50 µM LY294002 showed a massive down-modulation of p-Akt-levels in LNCaP cells within 1 h after treatment; still, a considerable reduction in p-Akt levels could be detected 48 h after treatment. In contrast, LY294002 failed to reduce p-Akt levels in PC3 cells at any time point measured (B, right panel).

Figure 5

Anti-neoplastic effect of combined treatment with ErPC3 and ionizing radiation. PC3 cells (A), DU145 cells (B), and LNCaP cells (C) were treated with increasing concentrations of ErPC3 (1-100 µM) and ionizing radiation (2, 5, 10 Gy) alone or in combination as indicated. Cell viability was analyzed 48 h after treatment by using the WST-1 assay. PC3 and DU145 cell did not respond to irradiation alone but responded to single treatment with ErPC3 (A, B). The anti-neoplastic effects of the combination were mainly attributed to the effects of ErPC3 (A, B). In contrast, LNCaP cells were highly sensitive to treatment with radiation alone, as well as to ≥ 50 µM ErPC3 (C). When LNCaP cells were treated with subtoxic ErPC3-concentrations in combination with irradiation, the reduction in the number of viable cells was mainly due to ionizing radiation (C). However, the cell viability was further reduced when LNCaP cells were treated with toxic ErPC3-concentrations (≥ 50µM) in combination with irradiation.

Figure 6

Combined effects of ErPC3 and ionizing radiation on apoptosis induction in prostate cancer cell lines. PC3 and LNCaP cells were irradiated with 10 Gy, treated with 12.5 µM ErPC3 or both treatments were combined. DNA fragmentation (A, B) and caspase activation (C, D) were analyzed 48 h later. (A) Approximately 30% of PC3 cells showed DNA fragmentation after a single treatment with ErPC3, whereas radiation-induced apoptosis was below 10%. The amount of apoptotic cells significantly increased when cells were subjected to combined treatment (data show means ± SD; n = 3; ***: p < 0,001). (B) Although a single therapy with ionizing radiation or ErPC3 did not induce apoptotic DNA fragmentation in LNCaP cells, the combination of both treatments resulted in apoptosis levels comparable to that in PC3 cells. The results were confirmed by Western blotting analyzing caspase-3 and PARP cleavage (C, D). Cleavage of caspase-3 (PC3 and LNCaP cells) and PARP (LNCaP cells) was more effective when ErPC3 and ionizing radiation were combined (C, D).