Citations

Abstract Resistance to temozolomide (TMZ) in the majority of glioblastoma multiforme (GBM) is mediated by O6-methylguanine methyltransferase (MGMT). In this study, the efficacy of TMZ, a mono-functional methylating agent, was compared to a bi-functional TMZ analog (DP68) and a corresponding mono-functional TMZ analog (DP86) in established glioma lines (T98, U251), primary GBM xenograft lines (GBM12, GBM22, GBM6) and a TMZ-resistant model with high level MGMT expression (GBM12TMZ). Using a combination of CyQuant and neurosphere assays, the IC50 with TMZ treatment for MGMT expressing lines (U251, GBM12, GBM22) was much lower than non-expressing lines (T98, G6, G12TMZ), while all lines were sensitive to DP68 regardless of MGMT status (see Table). In the same assays, cells were less sensitive to DP86, but cytotoxicity was also independent of MGMT status. In both U251 and T98, DP68 resulted in a time-and dose-dependent induction of markers for DNA damage and checkpoint activation (P-H2AX, ATM, KAP1, Chk1 and Chk2) and a pronounced S-phase arrest within 24 hours of treatment. Based on these results and the hypothesis that DP68 induces DNA cross-links, siRNA knockdown was used to further dissect the importance of the ATM and ATR signaling pathways and the Fanconi Anemia (FA) cross-link repair pathway for DP68 cytotoxicity. Compared to no treatment, 1 microM DP68 resulted in 100% relative absorbance in siCONtrol transfected U251 cells, 41% for siATR, 35% for siFANCD2 and 90% for siATM. In contrast, only siATR and not siFAND2 nor siATM sensitized U251 cells to DP86. Similarly, inhibition of ATR (VE-821), but not ATM (KU-60019) potently sensitized cells to DP68 (see Table). Taken together, the inhibitory effects of DP68 are superior to TMZ in the GBM cell lines tested, regardless of MGMT expression. Furthermore, the siRNA and western blotting suggest that recovery from DP68 is dependent on ATR and the FA repair pathway. TMZ (IC50, microM) DP68 (IC50, microM)) DP86 (IC50, microM)) CyQuant Ctrl 1uM VE-821 1uM KU-60019 Ctrl 1uM VE-821 1uM KU-60019 Ctrl 1uM VE-821 1uM KU-60019 U251 25 20 20 2.5 0.8 2.1 70 40 65 T98 >320 200 260 9 2 7 115 60 115 Neurosphere Ctrl VE-821 KU-60019 Ctrl VE-821 KU-60019 Ctrl VE-821 KU-60019 G12 4 3.2 4.2 0.9 0.08 0.7 18 6 18 G22 40 NA NA 1 NA NA 40 NA NA G6 160 NA NA 0.5 NA NA 15 NA NA G12TMZ >300 NA NA 2 NA NA 22 NA NA Citation Format: Ann C. Mladek Tuma, Yulian Ramirez, Dimitrios Pletsas, Richard T. Wheelhouse, Roger M. Phillips, Ross H. Alonzo, Kaitlyn Knudson, Jann N. Sarkaria. Cytotoxicity of a novel bi-functional temozolomide analog, DP68, is independent of MGMT status in glioblastoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2013-4476

Steatosis, an early manifestation in alcoholic liver disease, is associated with the accumulation of hepatocellular lipid droplets (LDs). However, the role ethanol metabolism has in LD formation and turnover remains undefined. Here, we assessed LD dynamics following ethanol and oleic acid treatment to ethanol-metabolizing WIF-B cells (a hybrid of human fibroblasts (WI 38) and Fao rat hepatoma cells). An OA dose-dependent increase in triglyceride and stained lipids was identified which doubled ( P < 0.05 ) in the presence of ethanol. This effect was blunted with the inclusion of an alcohol metabolism inhibitor. The ethanol/ OA combination also induced adipophilin, LD coat protein involved in the attenuation of lipolysis. Additionally, ethanol treatment resulted in a significant reduction in lipid efflux. These data demonstrate that the metabolism of ethanol in hepatic cells is related to LD accumulation, impaired fat efflux, and enhancements in LD-associated proteins. These alterations in LD dynamics may contribute to ethanol-mediated defects in hepatocellular LD regulation and the formation of steatosis.