Abstract Cisplatin is one of the most commonly used drugs against human malignancies. Its cytotoxic effects are mainly mediated by formation of cisplatin-DNA adducts, which activate series of DNA damage-responsive proteins that ultimately trigger cell death. In spite of its potent activity, some malignancies treated with cisplatin may develop resistance after initial response while others may be intrinsically insensitive. Accordingly, cisplatin is frequently combined with other drugs to reduce resistance or to attain synergy. The presented work was conducted in EGFR-overexpressing cancer cells, DU-145 and MDA-MB-231, to examine the possible molecular effects of cisplatin alone or in combination with vorinostat, a histone deacetylase inhibitor. Proliferation assay, annexin V staining, western blotting and RT-PCR were used to study effects of those drugs on cell viability, apoptosis and molecular events in both cell lines. In the present study, incubation of DU-145 cells with cisplatin exerted dose-dependent antiproliferative and apoptosis-inducing effects. In addition, cisplatin significantly increased the apoptosis markers, cleaved PARP and cleaved caspase 7; depleted total EGFR and decreased ERK activation. This molecular response was also associated with dose-dependent increase in phosphorylated ATM (an early sensor of genotoxic stress), phosphorylated p38 and mRNA level of DDIT3 (a late sensor of DNA damage). On the other hand, in MDA-MB-231, cisplatin did not affect percent of apoptotic cells as compared with the untreated control, indicating a cisplatin-insensitive phenotype. The insensitive response towards cisplatin treatment was confirmed through absence of cleaved PARP and cleaved caspase 7 in total cell lysates. Although cisplatin increased the level of phosphorylated ATM and phosphorylated p38 in MDA-MB-231, it failed to affect DDIT3 mRNA level as compared with untreated cells. This may suggest that sensitivity towards cisplatin may be related to late DNA damage processing rather than early DNA damage response. Treating DU-145 and MDA-MB-231 cells with vorinostat elicited dose-dependent decrease in proliferation and increase in percentage of apoptotic cells. It also dose-dependently cleaved PARP and caspase 7, time-dependently degraded EGFR and decreased ERK activation in both cell lines. In spite of that potent activity, vorinostat synergized the effect of cisplatin only in DU-145 and failed to circumvent the insensitivity of MDA-MB-231 cells towards cisplatin. In conclusion, cisplatin decreased cell proliferation and induced apoptosis only in DU-145 cells but not MDA-MB-231 cells. Combination of vorinostat and cisplatin synergistically reduced percent cell viability and triggered apoptotic response in DU-145 cells when compared with the effects of individual drugs. However, in MDA-MB-231cells, addition of vorinostat to cisplatin did not significantly change any of the measured parameters. Citation Format: Asmaa Elsayed El-Kenawi, John Kenneth Cowell. Effects of cisplatin, vorinostat, and their combinations on EGFR-overexpressing cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4560. doi:10.1158/1538-7445.AM2014-4560