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Chemo-sensitisation of HeLa cells to etoposide by a benzoxazine in the absence of DNA-PK inhibition.

ABSTRACT: The benzoaxines have been developed from structurally similar chromones as specific inhibitors of the PI3K family to sensitize cancer cells to the effects of chemotherapeutic agents; most have been shown to do this through specific inhibition of DNA-PK and DNA repair mechanisms. In this study we examined the benzoxazine, 2-((3-methoxybut-3en-2-yl)amino)-8methyl-4H-benzo[1,3]oxazin-4one (LTUSI54). This compound had no DNA-PK or PI3K inhibitory activity but still sensitized HeLa cells to the effects of Etoposide. LTUSI54 works synergistically with Etoposide to inhibit growth of HeLa cells and sub G1 analysis indicates that this is not due to an increase in apoptosis. LTUSI54 neither enhances DSB formation due to Etoposide nor does it delay the repair of such damage. Cell cycle analysis shows a clear G2 block with Etoposide alone while, in combination with LTUSI54 there is an additional S phase arrest. Phospho-kinase analysis indicated that LTUSI54 engages key regulators of cell cycle progression, specifically p38?, p53 and ERK 1/2. From our results we hypothesize that LTUSI54 is promoting the cell cycle arrest through activation of p38? pathways, independent of p53 mechanisms. This results in a decrease in p53 phosphorylation and hence, restricted apoptosis. Changes in cell number appear to be the result of p38? pathways disrupting cell cycle progression, at the S and G2 checkpoints. Further investigation into the finer mechanisms by which LTUSI54 effects cell cycle progression would be of great interest in assessing this compound as a chemosensitising agent.

SUBMITTER: Fitzgibbon C

PROVIDER: S-EPMC3825418 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Chemo-sensitisation of HeLa cells to etoposide by a benzoxazine in the absence of DNA-PK inhibition.

Fitzgibbon Cheree C Ihmaid Saleh S Al-Rawi Jasim J Meehan-Andrews Terri T Bradley Christopher C

Investigational new drugs 20130922 6

The benzoaxines have been developed from structurally similar chromones as specific inhibitors of the PI3K family to sensitize cancer cells to the effects of chemotherapeutic agents; most have been shown to do this through specific inhibition of DNA-PK and DNA repair mechanisms. In this study we examined the benzoxazine, 2-((3-methoxybut-3en-2-yl)amino)-8methyl-4H-benzo[1,3]oxazin-4one (LTUSI54). This compound had no DNA-PK or PI3K inhibitory activity but still sensitized HeLa cells to the effec ...[more]

PMID: 24057508

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Project description:Non?small cell lung cancer (NSCLC) is radioresistant to X?rays due to powerful cellular DNA damage repair mechanisms. DNA?dependent protein kinase (DNA?PK) is a key enzyme involved in DNA damage repair and the phenomenon and molecular mechanism of NSCLC radionsensitivity were investigated following inhibition of DNA?PK activity. In the present study A549 cells were treated with the DNA?PK inhibitor NU7026 and/or siRNA directed against ataxia telangiectasia mutated (ATM), followed by exposure to 4 Gy X?ray irradiation. Radiosensitivity, DNA damage, apoptosis and protein expression were measured by colony formation assay, ?H2AX foci immunofluorescence, Annexin V/PI staining and western blotting, respectively. A Balb/c?nu/nu xenograft mouse model was established by subcutaneous injection of A549 cells and was used to examine the effect of administering NU7026 via intraperitoneal injection prior to 4 Gy X?ray exposure. The xenograft tumors were weighed and observed by hematoxylin and eosin staining after irradiation. NU7026 treatment followed by X?ray irradiation significantly decreased the colony formation ratio of A549 cells, and increased ?H2AX foci and cell apoptosis. Furthermore, the combined treatment of NU7026 and X?rays resulted in growth inhibition and cell apoptosis in A549 xenograft tumors. Consequently, apoptosis regulators full?length transactivating (TA) p73 and an N?terminally truncated (DN) p73 were upregulated and downregulated respectively, leading to activation of glucosyltransferases and Rab?like GTPase activators and myotubularins domain?containing 4 (GRAMD4) protein to reduce the Bcl?2/Bax protein ratio. In addition, ATM siRNA efficiently prevented ?H2AX foci formation, and enhanced NU7026?induced inhibition of survival and promoted apoptosis. In conclusion, inhibition of DNA?PK activity increased the radiosensitivity of A549 cells to X?ray irradiation. NU7026 treatment activated the ATM?dependent DNA damage response and induced p73 apoptosis pathway. DNA?PK inhibitor may be an effective constituent of radiosensitization products. DNA damage repair pathway could be a potential target for radiosensitization.

Dataset Information

Chemo-sensitisation of HeLa cells to etoposide by a benzoxazine in the absence of DNA-PK inhibition.

Publications

Chemo-sensitisation of HeLa cells to etoposide by a benzoxazine in the absence of DNA-PK inhibition.

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