Triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro.

Abstract

Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood-brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour-specific markers. To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro. We evaluated the sensitising effect of CCNU alone and in combination with TMZ-irradiation in canine glioma J3T-BG cells and long-term drug-exposed subclones by using clonogenic survival and proliferation assays. Bisulphite-SEQ and Western Blot were used to investigate molecular alterations. TMZ (200μM) or CCNU alone (5μM) reduced the irradiated survival fraction (4Gy) from 60% to 38% (p= 0.0074) and 26% (p= 0.0002), respectively. The double-drug combination reduced the irradiated survival fraction (4Gy) more potently to 12% (p< 0.0001). After long-term drug exposure, both subclones show higher IC50 values against CCNU and TMZ. For CCNU-resistant cells, both, single-drug CCNU (p= 0.0006) and TMZ (p= 0.0326) treatment combined with irradiation (4Gy) remained effective. The double-drug-irradiation combination reduced the cell survival by 86% (p< 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ-resistant cells, only the double-drug combination with irradiation (4Gy) reduced the cell survival by 88% (p= 0.0057) while single-drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P-gp expression while MGMT-methylation profile analysis showed a general high methylation level in the parental and long-term treated cell lines. Our findings indicate that combining CCNU with TMZ-irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.