Abstract 1044: A combination of cisplatin and 2-deoxy-d-glucose results in synergistic cell death in both normoxia and hypoxia by the attenuation of autophagy
Authors: Jalota Akansha , Das Bhudev C. , Yadav Ajay K. , Chosdol Kunzang , Sinha Subrata 
Published: August 2015

Background and objectives

Hypoxia contributes markedly to the drug resistance of glial tumors which in themselves are highly refractory to therapy. 2-Deoxy-D-glucose (2-DG) has been tried in several tumors including glioma, and is known to induce autophagy as a protective response. Cisplatin (CP) has been earlier used as a second line drug and is a prototype DNA damaging agent. Since these drugs are well studied as single agents and are known to have a limited value, we studied the efficacy and mechanism of action of their combination in glial cell lines (p53 wild type A172 and p53 mutant type LN229) under both normoxia and hypoxia.

Methods

The cell lines A172 and LN229 (which vary in p53 function) were used in the study. Cell viability was determined by MTT assay for the combination of 2-DG and CP and combination index was determined. Caspase assay as well as Propidium Iodide (PI) staining was performed to determine the efficacy of the synergistic combination. Autophagic and apoptotic markers were analyzed by Real time PCR and confirmed through western blotting. Effect on cellular metabolism was confirmed through ATP assay. Cell survival after exposure to the combination of 2-DG and AKT inhibitor (LY294002) which is known to reverse autophagy was also studied.

Results

The combination of 2-DG with CP was synergistic under both normoxia and hypoxia in A172 and LN229 cell lines with combination index less than 1. Reduction of autophagy induced by 2-DG and induction of apoptosis by the combination in both normoxia and hypoxia was further confirmed by caspase activity and PI staining. Autophaghic markers (Beclin1 and conversion of LC3 I to II form) were reduced and apoptotic markers (Bax, caspase 3 and cytochrome c) were upregulated in the combination of CP and 2-DG in both normoxic and hypoxic conditions. ATP activity was further reduced in the combination of CP and 2-DG as compared to single agent. Combination of LY294002 (which is known to inhibit autophagy) with 2-DG also reduced cell survival in both normoxia and hypoxia in both the cell lines. The critical role of autophagy was confirmed by the analysis of appropriate markers.

Interpretation and Conclusions

This in vitro study has been able to identify a combination of known drugs which is more effective than either single agent alone in cell lines of different genetic backgrounds under varying degrees of tissue oxygenation. The mechanism of a reduction in autophagy resulting from this combination could be further generalized to identify other rational drug combinations.

Citation Format: Akansha Jalota, Bhudev C. Das, Ajay K. Yadav, Kunzang Chosdol, Subrata Sinha. A combination of cisplatin and 2-deoxy-d-glucose results in synergistic cell death in both normoxia and hypoxia by the attenuation of autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1044. doi:10.1158/1538-7445.AM2015-1044