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

<p>Abstract</p> <p>Background and Purpose</p> <p>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.</p> <p>Materials and methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner

Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca2+ signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0–10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca2+-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca2+-overload-induced mitochondrial superoxide anion formation

Radiation-induced apoptosis mechanisms and relevance for the clonogenic cell death

Bei der Behandlung von Tumoren spielt die Strahlentherapie eine wesentliche Rolle. Es ist jedoch wenig über die molekularen Mechanismen bekannt, die die Strahlensensitivität beeinflussen und zur Tumorregression führen. Ziel dieser Arbeit war deshalb die Aufklärung der apoptotischen Signalwege nach Bestrahlung. Hier wurde gezeigt, dass Caspasen, die Effektoren der apoptotischen Kaskade, nach Bestrahlung sekundär zu mitochondrialen Schäden aktiviert werden. Caspase-8 fungiert in diesem Fall nicht wie bisher angenommen als Initiatorcaspase sondern wird durch andere Caspasen aktiviert. Weiterhin ist Caspase-8 zwar nicht essentiell für strahleninduzierte Apoptose, für Apoptose nach Stimulierung des Zelltodrezeptors CD95 jedoch unerläßlich. Außerdem konnte erstmalig nachgewiesen werden, dass das pro-apoptotische Molekül Bid, ein Substrat von Caspase-8, ebenso durch die DEVD-spezifischen Proteasen, vermutlich Caspase-3 oder Caspase-7, unabhängig von Caspase-8 aktiviert werden kann. Die PT-Pore, ein unspezifischer mitochondrialer Kanal mit essentieller Funktion in einigen apoptotischen Signalwegen, scheint nach Bestrahlung dagegen keine wesentliche Rolle zu spielen, da ihre Blockierung mit Cyclosporin A keine Wirkung zeigte.Radiation therapy plays an important role in cancer treatment. However, little is kown about the molecular mechanisms that affect the radiation sensitivity and lead to tumor regression. Therefore the aim of this study was to enlighten of the apoptotic signal transduction pathways in response to irradiation. These data revealed that caspases, the major players in the apoptotic cascade, were activated secondarily to mitochondrial damage after irradiation. In contrast to previous assumptions, Caspase-8 does not act as initiation caspase but is activated by other caspases. Further on, caspase-8 is not essential for radiation-induced apoptosis but is necessary for apoptosis induced by death receptors. In addition, the pro-apoptotic molecule Bid, known as a substrate for caspase-8, may also be cleaved by DEVD-specific proteases, presumable by caspase-3 or caspase-7. The permeability transition pore, an unspecific mitochondrial channel with relevant function in some apoptotic pathways, seems to be dispensable in radiation-induced apoptosis since blocking this pore with cyclosporine A had no effect. Using Bcl-2 constructs that allow a specific expression either at the ER or the mitochondria it has been shown that endoplasmic and mitochondrial as well as wildtype Bcl-2 protect against radiation-induced apoptosis. Deletion of the Bcl-2 anchorage domain results in a loss of protection. In contrast, none of these constructs were able to block death receptor-induced apoptosis. Finally, using clonogenic tests it has been shown that the anti-apoptotic function of Bcl-2 does not correlate inevitably with an improved survival after irradiation. The radiation sensitivity rather depends on the expression of other factors, that varies between the cell systems

Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1

Myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, is often overexpressed in tumor cells limiting the therapeutic success. Mcl-1 differs from other Bcl-2 members by its high turnover rate. Its expression level is tightly regulated by ubiquitylating and deubiquitylating enzymes. Interaction of Mcl-1 with certain Bcl-2 homology domain 3 (BH3)-only members of the Bcl-2 family can limit the access to Mcl-1 ubiquitin ligase E3 and stabilizes the antiapoptotic protein. In addition, the overexpression of the deubiquitinase ubiquitin-specific protease 9x (USP9x) can result in the accumulation of Mcl-1 by removing poly-ubiquitin chains from Mcl-1 preventing its proteasomal degradation. Analyzing radiation-induced apoptosis in Jurkat cells, we found that Mcl-1 was downregulated more efficiently in sensitive parental cells than in a resistant subclone. The decline of Mcl-1 correlated with cell death induction and clonogenic survival. Knockdown of BH3-only proteins Bim, Puma, and Noxa did not affect Mcl-1 level or radiation-induced apoptosis. However, ionizing radiation resulted in activation of USP9x and enhanced deubiquitination of Mcl-1 in the radioresistant cells preventing fast Mcl-1 degradation. USP9x knockdown enhanced radiation-induced decrease of Mcl-1 and sensitized the radioresistant cells to apoptosis induction, whereas USP9x knockdown alone did not change Mcl-1 level in unirradiated cells. Together, our results indicate that radiation-induced activation of USP9x inhibits Mcl-1 degradation and apoptosis resulting in increased radioresistance

Protein Levels of Anti-Apoptotic Mcl-1 and the Deubiquitinase USP9x Are Cooperatively Upregulated during Prostate Cancer Progression and Limit Response of Prostate Cancer Cells to Radiotherapy

Background: Radiotherapy constitutes an important therapeutic option for prostate cancer. However, prostate cancer cells often acquire resistance during cancer progression, limiting the cytotoxic effects of radiotherapy. Among factors regulating sensitivity to radiotherapy are members of the Bcl-2 protein family, known to regulate apoptosis at the mitochondrial level. Here, we analyzed the role of anti-apoptotic Mcl-1 and USP9x, a deubiquitinase stabilizing Mcl-1 protein levels, in prostate cancer progression and response to radiotherapy. Methods: Changes in Mcl-1 and USP9x levels during prostate cancer progression were determined by immunohistochemistry. Neutralization of Mcl-1 and USP9x was achieved by siRNA-mediated knockdown. We analyzed Mcl-1 stability after translational inhibition by cycloheximide. Cell death was determined by flow cytometry using an exclusion assay of mitochondrial membrane potential-sensitive dye. Changes in the clonogenic potential were examined by colony formation assay. Results: Protein levels of Mcl-1 and USP9x increased during prostate cancer progression, and high protein levels correlated with advanced prostate cancer stages. The stability of Mcl-1 reflected Mcl-1 protein levels in LNCaP and PC3 prostate cancer cells. Moreover, radiotherapy itself affected Mcl-1 protein turnover in prostate cancer cells. Particularly in LNCaP cells, the knockdown of USP9x expression reduced Mcl-1 protein levels and increased sensitivity to radiotherapy. Conclusion: Posttranslational regulation of protein stability was often responsible for high protein levels of Mcl-1. Moreover, we demonstrated that deubiquitinase USP9x as a factor regulating Mcl-1 levels in prostate cancer cells, thus limiting cytotoxic response to radiotherapy