engleski

Sensitisation of malignant glioma cells to temozolomide-induced cell death via integrin silencing

Integrin signalling regulates numerous cellular processes including proliferation, migration, metastasis, cancer-induced angiogenesis and cell death. Previously it was shown that the patients having MGMT negative tumors can benefit from the cilengitide therapy. This is due to the fact that temozolomide (TMZ) can achieve a maximal cytotoxic effect in those tumors, since TMZ-induced primary DNA damage (O6-Methylguanine) cannot be repaired. The aim of this study was to investigate the role of integrins αvβ3, αvβ5, α3β1 and α4β1 in a putative resistance to TMZ, a monoalkylating agent used in the first-line therapy of malignant gliomas. For this we determined the integrin status (together with MGMT and p53 status) in a set of human adult malignant glioma cell lines, from which four MGMT negative cell lines (LN229, LN308, U87MG and U138MG) were chosen for further experiments. Decreased expression of all examines integrins αvβ3, αvβ5, α3β1 and α4β1 achieved by β3, β5, αv, α3 or α4-specific siRNA transfection sensitized the high-grade glioma cell lines to TMZ irrespective of the p53 status, as determined by MTT, colony formation and sub-G1 (PI) flow cytometric assay. Also, treatment of the cell lines with a cilengitide-similarRGD integrin inhibitir sensitized them to TMZ. Silencing of integrins αvβ3, αvβ5 and α4β1 expressed on LN229 and of αvβ3, αvβ5, α4β1 and α3β1 expressed on U138MG cells led to abrogation of the FAK/ILK/Akt pathway, leading to more pronounced induction of TMZ-induced γH2AX foci (DNA double-strand breaks), reduction in Ead51 protein level and apoptosis, as determined by caspase-3 cleavage and decreased Bcl-xL and survivin expression, in comparison to TMZ alone. Also, subcutaneous U87MG xenografts in mice treated i.p. with the integrin inhibitor and TMZ achieved stronger tumor regression compared to those treated with TMZ or with the integrin antagonist alone. Protein extracts isolated from the ablated tumor xenografts that have been exposed to TMZ and integrin antagonist showed the strongest level of H2AX phosphorilation and caspase-3 cleavage, pointing to increased DNA damage and cell death, again suggesting that integrin silencing impairs DNA repair.

glioma; integrin; sensitisation; temozolomid

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