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

Fahrmann and Hardman Lipids in Health and Disease 2013, 12:36 http://www.lipidworld.com/content/12/1/36 RESEARCH Open Access 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 and W Elaine Hardman* Abstract 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. Keywords: Omega 3, Chemo-sensitization, Chronic lymphocytic leukemia * Correspondence: Department of Biochemistry and Microbiology, Marshall University School of Medicine, Huntington, WV, USA © 2013 Fahrmann and Hardman; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fahrmann and Hardman Lipids in Health and Disease 2013, 12:36 http://www.lipidworld.com/content/12/1/36 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). Page 2 of 13 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 followi (...truncated)