Abstract 1247: Reduction of oxidative stress promotes daunorubicin resistance in acute lymphoblastic leukemia cells

Abstract

Abstract The regulation of oxidative stress is fundamental to cell survival and function. Oxidative stress is a result of accumulation of reactive oxygen species (ROS), which are produced by cell metabolism and in response to certain stressors. Cancer cells have higher than normal ROS levels and elevated antioxidant defense mechanisms. Daunorubicin (DNR), a widely used chemotherapy, is a topoisomerase II inhibitor that induces cell death through DNA double strand breaks and increased ROS. We have previously shown that adipocytes protect acute lymphoblastic leukemia (ALL) cells against DNR in vitro and that media cultured by both adipocytes and ALL cells together (ALCM) can protect ALL cells from DNR. We hypothesize that this protection is in part mediated by adipocytes alleviating oxidative stress in ALL cells. Murine pre-B ALL cells isolated from a BCR/ABL transgenic mouse (8093) and human leukemia cell lines (BV173, Nalm6, REH, SEM, RS4;11, HL60, and Molt4) were used. The ALL cells were co-cultured with 3T3-L1 adipocytes using a transwell system. Cells were collected for gene expression analysis using qPCR. Intracellular ROS was measured by flow cytometry with 2′-7′-dichlorodihydrofluorescein (DCFH-DA). Four of the six human leukemia cell lines tested were protected by adipocytes against DNR treatment. Western blots showed that DNR induced a significant increase in the cleaved to procaspase-3 ratio, which was reversed by ALCM (No drug: 0.24±0.2, DNR: 0.83±0.14, ALCM+DNR: 0.24±0.19; p = 0.004 for DNR vs. ALCM+DNR, n = 4). Expression of the oxidative stress response genes gclc, gclm and p21 was upregulated in 8093 cells upon DNR treatment (fold change over baseline: 1.82±0.37, 2.35±0.73, 5.55±1.50; p<0.05, n = 3), while the presence of adipocytes prevented this upregulation (fold change over baseline: 0.83±0.11, 0.88±0.02, 1.75±0.57; p<0.05, n = 3). To investigate the oxidative stress induced by DNR, Nalm6 cells were treated with five doses of DNR for 6 hours alone or in the presence of pre-adipocytes or adipocytes. There was a dose-dependent increase in%ROShigh cells regardless of feeder type. At 200nM DNR, there were significantly fewer%ROShigh cells when co-cultured with adipocytes compared to no feeder or pre-adipocytes (p<0.05, n = 3). To test whether alleviating oxidative stress alone would cause DNR resistance, BV173, Nalm6 and REH cells were treated with DNR (100, 200 and 300nM, respectively) or a combination of DNR and the anti-oxidant, N-acetyl cysteine (NAC; 20mM). The addition of NAC significantly protected the cells from DNR (p<0.05, n = 3). These findings demonstrate that adipocytes contribute to drug resistance by alleviating oxidative stress in ALL cells. Further research is required to identify the soluble factors responsible for these protective effects. Citation Format: Xia Sheng, Jonathan Tucci, Steven D. Mittelman. Reduction of oxidative stress promotes daunorubicin resistance in acute lymphoblastic leukemia cells. [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 1247. doi:10.1158/1538-7445.AM2015-1247