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

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.

Figure 1

Determination of optimal FA concentrations. Figure 1A-C illustrates the mean % live cells ± SEM of EHEB, JVM-2 and MEC-2 following 72 hour treatment with vehicle, or increasing concentrations of AA, EPA or DHA. Percent (%) live cells was determined after 72 hour treatments by trypan blue exclusion assay. Figure 1A illustrates the mean % live cells ± SEM of EHEB following treatment with vehicle, or increasing concentrations of AA, EPA or DHA. Significant reductions in % live cells were observed at 55 μM AA, 75 μM and 100 μM EPA, and 100 μM DHA as compared to vehicle. Although not statistically significant, concentrations of AA 35 μM and AA 45μM indicated viabilities of 57% and 54%. FA concentrations of AA 25 μM, EPA 50 μM and DHA 75 μM were chosen for the remainder of the study. Figure 1B illustrates the mean% live cells ± SEM of JVM-2 following treatment with vehicle, or increasing concentrations of AA, EPA or DHA. Significant reductions in % live cells were observed at 45 μM and 55 μM AA, and 75 μM and 100 μM EPA as compared to vehicle. Although not statistically significant, concentrations of DHA 75 μM and DHA 100 μM indicated cell viabilities of 65% and 55%. FA concentrations of AA 35 μM, EPA 50 μM and DHA 50 μM were chosen for the remainder of the study. Figure 1C illustrates the mean % live cells ± SEM of MEC-2 following treatment with vehicle, or increasing concentrations of AA, EPA or DHA. Significant reductions in % live cells were observed at 45 μM AA, 75 μM and 100 μM EPA and 75 μM and 100 μM DHA as compared to vehicle. FA concentrations of AA 25 μM, EPA 50 μM and DHA 50 μM were chosen for the remainder of the study. Statistical significant was determined by Multiple Comparison Test using Tukey’s correction. Abbreviations: AA- arachidonic acid, EPA- eicosapentaenoic acid, DHA- docosahexaenoic acid, α = 0.05, * <0.05, ** < 0.01, *** < 0.001.

Figure 2

In vitro sensitivity of EHEB following treatment with doxorubicin or fludarabine in the presence and absence vehicle, AA, EPA or DHA. Cell viability was determined by MTT assay. Percent (%) cell death was determined by Annexin-V/Propidium Iodide duel stain with flow cytometry. Figure 2A illustrates the % cell viability ± SEM of EHEB to doxorubicin (0–7.5 μM) in the presence or absence of vehicle, AA 25 μM, EPA 50 μM or DHA 75 μM. Cells pre-treated with either EPA or DHA had significantly greater decreases in cell viability as compared to vehicle when treatment with doxorubicin. Figure 2B illustrates the % cell viability ± SEM of EHEB to fludarabine (0-50 μM) in the presence or absence of vehicle, AA 25 μM, EPA 50 μM or DHA 75 μM. Pre-treatment of cells with EPA had significantly greater reductions in cell viability as compared to vehicle when treated with 30 μM and 40 μM fludarabine. Figure 2C illustrates the % dead cells ± SEM of EHEB following pre-treatment with vehicle, AA 25 μM, EPA 50μM or DHA 75 μM alone and following treatment with 1.5 μM doxorubicin or 40 μM fludarabine. Pre-treatment with DHA alone induced significantly greater cell death as compared to vehicle. Compared to vehicle, cells pre-treated with DHA had significantly higher cell death when treated with doxorubicin or fludarabine. Figure 2D provides 2D graphical representations of Annexin-V/PI Plots. ‘Early’ Apoptosis was defined as cells positive for Annexin-V-FITC only. ‘Late’ Apoptosis was defined as cells positive for Annexin-V-FITC and PI. ‘Necrotic’ was defined as cells positive for PI only. Statistical significance was determined by Multiple Comparison Test with Dunnet’s correction (Figure 2A and B) or Tukey’s correction (Figure 2C). α = 0.05, * <0.05, ** < 0.01, *** < 0.001.

Figure 3

In vitro sensitivity of JVM-2 following treatment with doxorubicin or vincristine in the presence and absence vehicle, AA, EPA or DHA. Cell viability was determined by MTT assay. Percent (%) cell death was determined by Annexin-V/Propidium Iodide duel stain with flow cytometry. Figure 3A illustrates the % cell viability ± SEM of JVM-2 to doxorubicin (0–7.5 μM) in the presence or absence of vehicle, AA 35 μM, EPA 50 μM or DHA 50 μM. Cells pre-treated with AA, EPA or DHA induced significantly greater decreases in cell viability as compared to vehicle when treated with doxorubicin. Figure 3B illustrates the % cell viability ± SEM of JVM-2 to vincristine (0-250 nM) in the presence or absence of vehicle, AA 35 μM, EPA 50 μM or DHA 50 μM. Compared to vehicle, only cells pre-treated with DHA had significantly greater reductions in cell viability when treatment with vincristine. Figure 3C illustrates the % dead cells ± SEM of JVM-2 following pre-treatment with vehicle, AA 35 μM, EPA 50 μM or DHA 50 μM alone and following treatment with 1.5 μM doxorubicin or 100 nM vincristine. Pre-treatment with DHA alone induced significantly greater cell death as compared to vehicle. Compared to vehicle, Cells pre-treated with DHA had significantly greater cell death when treated with doxorubicin or vincristine. Figure 3D provides 2D graphical representations of Annexin-V/PI Plots. ‘Early’ Apoptosis was defined as cells positive for Annexin-V-FITC only. ‘Late’ Apoptosis was defined as cells positive for Annexin-V-FITC and PI. ‘Necrotic’ was defined as cells positive for PI only.Statistical significance was determined by Multiple Comparison Test with Dunnet’s correction (Figure 3A and B) or Tukey’s correction (Figure 3C). α = 0.05, * <0.05, ** < 0.01, *** < 0.001.

Figure 4

In vitro sensitivity of MEC-2 following treatment with doxorubicin or vincristine in the presence and absence vehicle, AA, EPA or DHA. Cell viability was determined by MTT assay. Percent (%) cell death was determined by Annexin-V/Propidium Iodide duel stain with flow cytometry. Figure 4A illustrates the % cell viability ± SEM of MEC-2 to doxorubicin (0–7.5 μM) in the presence or absence of vehicle, AA 25 μM, EPA 50 μM or DHA 50 μM. Cells pre-treated with either EPA or DHA had significantly greater decreases in cell viability as compared to vehicle when treated with doxorubicin. Figure 4B illustrates the % cell viability ± SEM of MEC-2 to vincristine (0-250 nM) in the presence or absence of vehicle, AA 25 μM, EPA 50 μM or DHA 50 μM. Cells pre-treated with either EPA or DHA had significantly greater decreases in cell viability as compared to vehicle following treatment with vincristine. Figure 4C illustrates the % dead cells ± SEM of MEC-2 following pre-treatment with vehicle, AA 25 μM, EPA 50 μM or DHA 50 μM alone and following treatment with 1.5 μM doxorubicin or 100 nM vincristine. Compared to vehicle, pre-treatment DHA alone induced significantly greater cell death; whereas pre-treatment with either AA or EPA induced significantly lower cell death. Cells pre-treated with DHA had higher cell death, as compared to vehicle, when treated with doxorubicin or vincristine; however, this was only significant when compared against AA pre-treated cells. Figure 4D provides 2D graphical representations of Annexin-V/PI Plots. ‘Early’ Apoptosis was defined as cells positive for Annexin-V-FITC only. ‘Late’ Apoptosis was defined as cells positive for Annexin-V-FITC and PI. ‘Necrotic’ was defined as cells positive for PI only. Statistical significance was determined by Multiple Comparison Test with Dunnet’s correction (Figure 4A and B) or Tukey’s correction (Figure 4C). α = 0.05, * <0.05, ** < 0.01, *** < 0.001.

Figure 5

Chemo-sensitizing capability of DHA is mediated through generation of intracellular ROS and formation of lipid peroxides. Figure 5A illustrates mean relative fluorescence units ± SEM of MEC-2 over the course of 2 hours following 72 hour pre-treatment with vehicle, AA 25 μM, EPA 50 μM or DHA 50 μM. Linear regression analysis indicated a greater slope following treatment with EPA or DHA versus vehicle (0.483 and 0.683 versus 0.267) indicating an increased generation of ROS across time. Figure 5B illustrates mean ng MDA/μg protein ± SEM in the presence or absence of vehicle, AA 25 μM, EPA 50 μM or DHA 50 μM alone and following treatment with doxorubicin (1.5 μM) or vincristine (100 nM). Pre-treatment with DHA induced significantly higher levels of MDA as compared to vehicle. The addition of doxorubicin to DHA pre-treated cells induced significantly higher levels of MDA versus either alone. The addition of vincristine to either EPA or DHA pre-treated cells induced significantly lower levels of MDA as compared to n-3 or drug alone. Figure 5C illustrates the % cell viability ± SEM of MEC-2 following treatment with 1.5 μM doxorubicin and 50 μM vitamin-E alone and in combination in the presence or absence of vehicle, AA 25 μM, EPA 50 μM or DHA 50 μM. Compared to vehicle, cells pre-treated with DHA had significantly greater reductions in cell viability when treated with doxorubicin. Co-treatment of DHA pre-treated cells with doxorubicin and vitamin-E abrogated the enhanced sensitivity of MEC-2 to doxorubicin by DHA. Figure 5D illustrates mean ng MDA/μg protein ± SEM of MEC-2 following pre-treatment with DHA alone and after treatment with 1.5 μM doxorubicin alone and in combination with 50 μM vitamin-E. Co-treatment with doxorubicin and vitamin-E in DHA pre-treated cells indicated significantly lower levels of MDA versus doxorubicin alone. Statistical significance was determined by Multiple Comparison Test with Tukey’s correction. Abbreviations: MDA: malondialdehyde, α = 0.05, * <0.05, ** < 0.01, *** < 0.001.