Omega 3 fatty acids increase the chemo-sensitivity of B-CLL-derived cell lines EHEB and MEC-2 and of B-PLL-derived cell line JVM-2 to anti-cancer drugs doxorubicin, vincristine and fludarabine

Johannes F Fahrmann 0 W Elaine Hardman 0 0 Department of Biochemistry and Microbiology, Marshall University School of Medicine , Huntington, WV , USA Background: B-Cell chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the United States. Clinical treatment of CLL is often limited due to drug resistance and severe therapy-induced toxicities. We hypothesized that the omega 3 (n-3) fatty acids, eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), would increase the sensitivity of malignant B-lymphocytes to anti-cancer drugs doxorubicin, vincristine and/or fludarabine in vitro and that increased sensitivity is achieved by alterations in cell-cycle progression leading to growth inhibition and/or enhanced cell death. We further postulate that enhanced sensitivity is dependent on the formation of lipid peroxides and to the generation of reactive oxygen species (ROS). Methods: In the present study, B-CLL-derived leukemic cell lines EHEB and MEC-2 and the B-Prolymphocytic leukemic-derived (PLL) cell line JVM-2 were tested for in vitro sensitivity against doxorubicin, vincristine or fludarabine in the presence or absence of vehicle, arachidonic acid (omega 6), EPA or DHA. Cell cycle analysis and Annexin-V assays were performed to determine cell cycle progression and % apoptotic cells, respectively. Assays for malondialdehyde, a measure of lipid peroxidation, and DCF fluorescence assays, a measure of intracellular ROS, were performed to determine if enhanced sensitivity of cells to the drugs by n-3 was dependent on the formation of ROS. Results: Our results indicated that: 1) EPA and DHA differentially sensitized B-leukemic cell lines EHEB, JVM-2 and MEC-2 to doxorubicin, vincristine and fludarabine in vitro; 2) n-3 alone and with drug treatment increased cell death and induced G2/M arrest in a cell-type specific manner; 3) lipid peroxidation increased in the presence of n-3; 4) there was higher lipid peroxidation in MEC-2 cells in presence of DHA and doxorubicin than with either alone; 5) n-3 increased generation of ROS in MEC-2, and 6) the addition of vitamin-E abrogated the increase in ROS generation and chemo-sensitivity of MEC-2 to doxorubicin by DHA. Conclusion: N-3's are promising chemo-sensitizing agents for the treatment of CLL. Selective enhancement of chemo-sensitivity of EHEB, JVM-2 and MEC-2 to drugs by n-3 that is not dependent on increased lipid peroxidation and ROS generation indicates alternative mechanisms by which n-3 enhances chemo-sensitivity. - Introduction B-Cell chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the United States [1]. CLL is a disease of the elderly with two thirds of patients being over 65 years of age at time of diagnosis [1]. CLL remains largely incurable outside of allogeneic transplantation [1]. Despite the success of current treatments such as fludarabine, many patients develop drug resistance and disease relapse [2]. As such, clinical treatment of CLL is often hindered by drug resistance and the non-selectivity of most drugs [3]. Additionally, treatment options for CLL patients who require aggressive treatment are limited due to significant side-effect profiles which are often too toxic for the elderly or those with comorbidities [1]. Given the age group of patients diagnosed with CLL, a therapeutic intervention that can increase the sensitivity of CLL cells to chemotherapy without causing additional adverse effects would be clinically beneficial. Omega 3 and omega 6 polyunsaturated fatty acids (PUFAs) are essential fatty acids (FAs) which must be obtained from diet. Long chain omega 3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) are primarily found in fish oils [4]. The omega 6 fatty acid, arachidonic acid (AA), is primarily found in the meat of animals that consumed corn or soybeans. The ratio of omega 3 FAs to omega 6 FAs in the average western diet is heavily weighted in favor of omega 6 [5,6]. Omega 3 fatty acids have consistently been shown to enhance sensitivity of various solid tumor cells to chemotherapy in vitro [7,8] and in vivo [9-11]. However, it has not been shown whether n-3 can enhance the sensitivity of CLL to anti-cancer drugs. Previous studies performed by our group have shown that consumption of an omega 3 supplement, predominantly composed of EPA and DHA, increased the sensitivity of malignant B lymphocytes isolated from patients with early CLL (RAI stages 0, 1) to doxorubicin in an in vitro assay [12]. These findings prompted us to further evaluate the potential use of omega 3 as a chemo-sensitizing agent for the treatment of CLL. The primary objective of this study was to determine whether EPA and/or DHA could increase the sensitivity of malignant B-lymphocytes to doxorubicin, vincristine and/or fludarabine in vitro. Secondary objectives were to elucidate potential mechanism (s) by which n-3 enhance chemo-sensitivity. We hypothesized that EPA and/or DHA would increase the sensitivity of malignant B-lymphocytes to doxorubicin, vincristine and fludarabine in vitro and that enhanced sensitivity is mediated by alterations in cell cycle progression leading to enhanced growth inhibition and/or enhanced cell death. We further postulate that increased chemo-sensitivity is dependent, in part, on the formation of lipid peroxides, and the generation of reactive oxygen species (ROS). In this study we assayed for: 1) fatty acid lipid composition, 2) in vitro sensitivity of B-CLL-derived cell lines EHEB, and MEC-2 and B-Prolymphocytic-derived (PLL) cell line JVM-2 against doxorubicin, vincristine and fludarabine in the presence of vehicle (no added FA), AA, EPA or DHA, 3) % of apoptotic cells, 4) cell cycle distribution, 5) generation of intracellular reactive oxygen species (ROS), and 6) levels of lipid peroxidation. Results N-3 and N-6 fatty acids induce cell death Figures 1A-C illustrates the % alive cells SEM of EHEB, JVM-2 and MEC-2 following treatment with vehicle, or increasing concentrations of AA, EPA and DHA. Cell viability was assessed by Trypan Blue Exclusion assay following treatment for 72 hours. Treatment with AA, EPA or DHA induced dose-responsive reductions in cell viability as compared to vehicle in all three cell lines. We wanted to determine the chemo-sensitizing effects of FA following treatment with concentrations of FA that alone did not induce significant cytotoxicity. Thus, we chose to use concentrations of AA at 25 M, 35 M and 25 M, EPA at 50 M (all cell lines) and DHA at 75 M, 50 M and 50 M for EHEB, JVM-2 and MEC-2, respectively. The chosen FA concentrations used in this study are clinically achievable [12]. Gas chromatography post 72 hour of FA treatment validated FA incorporation in all cells (Supplementary Data-1). EPA and DHA differentially sensitize malignant B-lymphocytes to doxorubicin, vincristine and fludarabine in vitro In our study, we wanted to determine whether FA pretreatment would syne (...truncated)