Ascorbic acid (vitamin C) synergistically enhances the therapeutic effect of targeted therapy in chronic lymphocytic leukemia

Abstract

Background: Novel, less toxic, cost-effective and safe therapeutic strategies are needed to improve treatment of chronic lymphocytic leukemia (CLL). Ascorbic acid (AA, vitamin C) has shown a potential anti-cancer therapeutic activity in several cancers. However, the anti-cancer effects of ascorbic acid on CLL B-cells have not been extensively studied. We aimed in this study to evaluate the in vitro therapeutic activity using clinically relevant conditions.

Methods: Primary CLL B-cells and two CLL cell lines were exposed to a dose that is clinically achievable by AA oral administration (250 μM), and cell death and potential mechanisms were assessed. The role of the protective CLL microenvironment was studied. Synergistic interaction between AA and CLL approved drugs (Ibrutinib, Idelalisib and Venetoclax) was also evaluated.

Results: Ascorbic acid is cytotoxic for CLL B-cells at low dose (250 μM) but spares healthy B-cells. Ascorbic-acid-induced cytotoxicity involved pro-oxidant damage through the generation of reactive oxygen species in the extracellular media and in CLL cells, and induced caspase-dependent apoptosis. We also found that AA treatment overcame the supportive survival effect provided by microenvironment including bone marrow mesenchymal stem cells, T-cell cues (CD40L + IL-4), cytokines and hypoxia. Our data suggest that resistance to AA could be mediated by the expression of the enzyme catalase in some CLL samples and by the glucose metabolite pyruvate. We also demonstrated that AA synergistically potentiates the cytotoxicity of targeted therapies used in or being developed for CLL.

Conclusion: These preclinical results point to AA as an adjuvant therapy with potential to further improve CLL treatments in combination with targeted therapies.

Keywords: Ascorbic acid; Chronic lymphocytic leukemia; Cytotoxicity; Drug combination; Vitamin C.

Fig. 1

Ascorbic acid selectively kills CLL B-cells and has low toxicity toward B-cells from healthy donors (HD B-cells). Ascorbic acid’s effects are due to H₂O₂ generation and are reversed by catalase, sodium pyruvate (SP 1 mM) and the iron chelator deferoxamine (DFX). a: Viability of primary HD B-cells and CLL B-cells after 24 h of treatment with various concentrations of AA (***: p < 0.001 vs. Ctrl, CLL n = 16, HD B-cells n = 4). b: Extracellular H₂O₂ levels in the culture medium (RPMI) after 4 h of treatment with different concentrations of AA in the presence or absence of catalase (600 U/ml) or SP (1 mM) (n = 6). c: Primary CLL B-cell viability after 24 h of treatment with AA, dehydroascorbic acid (DHA) or AA 2-phosphate (Asc-2P); (n = 3) (**: p < 0.01, ***: p < 0.001 vs. Ctrl). d, e and f: Viability of CLL B-cells after 24 h of treatment with 250 μM AA in the presence or absence of DFX (100 μM) (*: p < 0.05; n = 5) (d), catalase (**: p < 0.01; n = 10) (e) and SP (1 mM) (***: p < 0.001; n = 20) (f). Data are presented as mean ± SEM

Fig. 2

The JVM3 CLL cell line is less-sensitive to AA’s effects than the OSU-CLL cell line. a: Viability of OSU-CLL and JVM3 cells, assessed in a flow cytometry assay using annexin-V-APC and 7-AAD staining after treatment with vehicle or 250 μM AA. b: Viability of JVM3 and OSU-CLL cell lines after treatment with increased concentrations of AA (**: p < 0.01, ***: p < 0.001 vs. OSU-CLL at the same concentrations; n = 5). c, d: Mitochondrial ROS levels, as recorded in a MitoSox flow cytometry assay in OSU-CLL (c) and JVM3 cell lines (d) treated for 6 h with 250 μM AA and expressed as the fold change vs. vehicle (*: p < 0.05 vs. vehicle; n = 7). Data are presented as the fold change in mean ± SEM fluorescence intensity (MFI) for the MitoSox dye. e, f: The GSH/GSSG ratio in OSU-CLL (e) and JVM3 (f) cell lines was assessed after 2 h of treatment with 250 μM AA or vehicle, *: p < 0.05, **: p < 0.01 vs. vehicle (n = 3)

Fig. 3

CLL B-cells’ sensitivity to AA is altered by catalase expression. a: catalase protein expression in OSU-CLL and JVM3 cell lines. b: Viability of OSU-CLL cells after AA treatment for 24 h in the presence or absence of catalase (600 U/ml) (**: p < 0.01; n = 6). c: Viability of primary CLL B-cells after treatment with AA for 24 h (***: p < 0.001; n = 40). d: Relative mRNA expression of catalase vs. GAPDH in CLL B-cells from AA-sensitive patients (S-CLL B-cells) or AA-non-sensitive patients (NS-CLL B-cells) and B-cells from healthy donors (HD B-cells). e: Catalase protein expression (normalized against β-actin) in HD B-cells and CLL B-cells (*: p < 0.05, ***: p < 0.001). f: Upper panel: Western blot and quantification of catalase protein levels following treatment of JVM3 cells for 48 h and 72 h with a control siRNA (siCtrl) or siRNA against catalase (siCAT). Relative catalase protein levels were quantified using ImageJ software. Lower panel: At 72 h, siRNA-transfected cells were treated by AA and cell viability was assessed after 24 h using the CellTiter-Glo Luminescent Cell Viability Assay Kit (n = 2 in duplicate). *: p < 0.05, **: p < 0.01. Data are presented as the mean ± SEM

Fig. 4

AA-induced apoptosis is caspase-dependent. a: Western blot of PARP cleavage, cleaved caspase-3, cleaved caspase-8, and β-actin in OSU-CLL cells treated with 250 μM AA for 6 h in the presence or absence of sodium pyruvate (SP, 1 mM). b, c, and d: Quantification of cleaved PARP (n = 5) (B), cleaved caspase-3 (n = 5) (c) and cleaved caspase-8 (n = 3) (d), normalized against β-actin. Data are presented as the fold-change vs. the control (mean ± SEM) (*: p < 0.05, **: p < 0.01 vs. Ctrl)

Fig. 5

Effects of the microenvironment on AA-induced apoptosis in CLL B-cells. a: CLL cells were co-cultured for 6 h with MSCs prior to AA treatment (250 μM). After 24 h of AA treatment, CLL cells were collected and analyzed for cell viability by an annexin V/7AAD staining (**: p < 0.01, ***: p < 0.001; n = 12). b: Effects of AA on cell viability in the presence of CD40L + IL-4, CpG or anti-IgM (*: p < 0.05, **: p < 0.01, ***: p < 0.001 vs. Ctrl; n = 7). c: CLL B-cell viability in the presence or in absence of a combination of cytokines after 24 h of treatment with 250 μM AA (*: p < 0.05, **: p < 0.01, ***: p < 0.001 vs. vehicle; n = 6). d: Effects of 250 μM AA on the viability of CLL B-cells cultured in the presence of autologous patient serum (10%) or 10% FBS (Ctrl) (*: p < 0.05; ***: p < 0.001 vs. Ctrl; n = 10). e: Effects of increased concentrations of AA on the viability of CLL B-cells cultured in the presence of 10% autologous serum (*: p < 0.05; **: p < 0.01; ***: p < 0.001 vs. Ctrl; n = 5). f: OSU-CLL cells were pre-treated for 24 h with CoCl₂ (100 μM) then incubated with different concentrations of AA. Upper panel: Western blot analysis showing HIF-1α levels under normoxia and CoCl₂-induced hypoxia conditions. Lower Panel: Effect of AA on cell viability was assessed by the CellTiter-Glo cell viability assay (**: p < 0.01; ***: p < 0.001; n = 3 in duplicate). Data are presented as the mean ± SEM

Fig. 6

Ascorbic acid synergistically increases the effects of targeted therapies on CLL B-cells. a: Viability of CLL B-cells treated with 250 μM AA alone or in combination with the approved drugs fludarabine (35 μM) and cyclophosphamide (100 μM) (n = 6), ibrutinib (15 μM) (n = 11), idelalisib (50 μM) (n = 12), and venetoclax (10 nM) (n = 16)) (*: p < 0.05; **: p < 0.01, ***: p < 0.001). Cell viability was determined by an annexin V/7AAD staining. b: Synergistic efficacy of combination of AA and CLL targeted therapies in primary CLL B-cells. Left panels: Primary CLL cells (n = 6) were treated with ascorbic acid (AA) and ibrutinib or idelalisib or venetoclax for 24 h. CellTiter-Glo cell viability assay was performed to detect cell kill synergy. The curves show the dose-effect of single drugs and that of drugs combination. Each data point is the mean of six samples. Right panels: Tables show the combination index (CI) of each combination for each patient (P). The CI values were calculated using the Chou-Talalay method by the software Compusyn. Fa: fraction affected (fraction of cells affected by a particular drug dose)

Fig. 7

Ascorbic acid synergistically potentiates the effects of mitochondrial metabolism targeting therapies on CLL B-cells. a: Effects of metabolic enzyme inhibitors oligomycin A (5 μM), CPI-613 (100 μM) and metformin (1 mM) on the viability of CLL B-cells after treatment alone or in combination with 250 μM AA for 24 h (*: p < 0.05, **: p < 0.01, ***: p < 0.001; n = 5). Cell viability was determined by an annexinV/7AAD staining. Data are presented as the mean ± SEM. b: Table show the coefficient of drug interaction (CDI) for drug combination with AA showed in (a). CDI values < 0.7 were considered as synergistic