ku-55933 and Prostatic-Neoplasms

External beam radiation therapy, alone or combined with androgen deprivation, is a well‑established treatment for prostate cancer (PCa). However, not all patients benefit from radiotherapy due to congenital or acquired radioresistance. The preliminary results of the present study indicated that the loss of disabled homolog 2 interactive protein (DAB2IP) expression in PCa and normal prostate epithelia results in the resistance to γ‑rays. To further explore the association between DAB2IP and ionizing radiation (IR), PCa cells were fractionally irradiated 12 times with 2 Gy of γ‑rays and the change in DAB2IP mRNA expression was monitored. Notably, along with a continuous reduction of DAB2IP expression levels, increased expression levels of ataxia‑telangiectasia mutated (ATM) was observed in IR‑treated cells. In order to improve the sensitivity of DAB2IP‑deficient cells to IR, α‑particles, a type of high linear energy transfer radiation and KU55933, an ATM inhibitor, were used in the current study. It was determined that α‑particle irradiations were more effective than γ‑rays on cells expressing expected and decreased levels of DAB2IP. However, cells with a dysfunctional DAB2IP gene were resistant to α‑particle irradiation. Treatment with KU55933 did not enhance cell sensitivity to α‑irradiation. Therefore, this suggested that DAB2IP downregulation induced by radiotherapy may be associated with acquired radioresistance in patients with PCa.

Topics: Alpha Particles; Cell Line, Tumor; Cell Survival; Discoidin Domain Receptors; Gamma Rays; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Morpholines; Prostatic Neoplasms; Pyrones; Radiation Tolerance; ras GTPase-Activating Proteins; RNA, Messenger; Signal Transduction

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.

Topics: Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Chromones; Comet Assay; DNA Breaks, Double-Stranded; DNA Repair; DNA-Activated Protein Kinase; DNA-Binding Proteins; Flow Cytometry; Humans; Male; Morpholines; Nuclear Proteins; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Pyrones; Tumor Suppressor Proteins

Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells.. Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis.. IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK alpha subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21(waf/cip) as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further.. We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21(waf/cip) and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21(waf/cip) pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

Topics: AMP-Activated Protein Kinases; Ataxia Telangiectasia Mutated Proteins; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Enzyme Activation; Female; G2 Phase; Humans; Lung Neoplasms; Male; Metformin; Morpholines; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; Pyrones; Radiation Tolerance; RNA, Small Interfering; Tumor Suppressor Protein p53; Tumor Suppressor Proteins