Deficiency in Cardiolipin Reduces Doxorubicin-Induced Oxidative Stress and Mitochondrial Damage in Human B-Lymphocytes

<div><p>Cardiolipin (CL) is an inner mitochondrial membrane phospholipid which plays an important role in mitochondrial function. Perturbation in CL biosynthesis alters mitochondrial bioenergetics causing a severe genetic disorder commonly known as Barth syndrome. Barth syndrome patients are known to have a reduced concentration and altered composition of CL. Cardiolipin is also known to have a high affinity for the chemotherapeutic agent doxorubicin (Dox), resulting in an extensive mitochondrial accumulation of the drug. Our results indicate that B-lymphocytes from healthy individuals are more sensitive to Dox-induced oxidative stress and cellular toxicity compared to the B-lymphocytes from Barth syndrome as indicated by greater cell death and greater level of cleaved caspase-3 following Dox treatment. Barth lymphocytes, when compared to healthy lymphocytes, showed a greater basal level of mitochondrial reactive oxygen species (mito-ROS), yet exhibited a lower level of induced mito-ROS production in response to Dox. Significantly less ATP content and slightly greater OXPHOS protein levels were detected in healthy cells compared to Barth cells after Dox treatment. Consistent with greater mitochondrial ROS, treatment with Dox induced a higher level of lipid peroxidation and protein carbonylation in healthy lymphocytes compared to Barth lymphocytes. The final remodeling of CL during CL synthesis is catalyzed by the tafazzin protein. Knockdown of <i>tafazzin</i> gene in H9c2 cardiomyocytes using siRNA showed decreased oxidant-induced damage, as observed in Barth lymphocytes. Our findings demonstrate that a deficiency in CL might provide a therapeutic advantage in favor of oxidant-induced anticancer activities.</p></div

Dox-induced cell death in Barth and healthy lymphocytes.

<p>(A) Annexin V and propidium iodide staining was used to measure induction of apoptosis by 0.1, 0.5, and 1 μM of Dox following 24 h treatment. VP16 (10 μM) was used as a positive control. (B) Representative western blot showing the level of cleaved caspase-3 in healthy and Barth lymphocytes after 0.5 and 1 μM Dox treatment for 24 h. (C) Quantitative analysis of cleaved caspase-3 from (B) normalized by α-tubulin. Cleaved caspase-3 levels for control (untreated) healthy and Barth lymphocytes were normalized to 100 and Dox-induced changes in cleaved caspase-3 were normalized against corresponding control. (D) Representative western blot showing the level of autophagy induction determined by LC3-II levels in healthy and Barth lymphocytes after 0.5 and 1 μM Dox treatment for 24 h with or without 5 nM bafilomycin A1 for final 2 h of Dox treatment. (E) Quantitative analysis of LC3-II from (D) normalized by α-tubulin. LC3-II levels for control (untreated) healthy and Barth lymphocytes were normalized to 100 and Dox-induced changes in LC3-II level in the presence or absence of bafilomycin A1 were normalized against corresponding control. *p<0.05; **p<0.001; H = healthy B-lymphocytes, B = Barth B-lymphocytes, Baf = bafilomycin A1.</p

Determination of Dox-induced OXPHOS protein levels in healthy and Barth lymphocytes.

<p>(A) Representative western blot for the detection of OXPHOS proteins in healthy and Barth lymphocytes. Healthy and Barth lymphocytes were treated with 0.1, 0.5, 1, and 5 μM of Dox for 24 h and OXPHOS protein levels were determined by western blot using an antibody cocktail containing five mouse mAbs against mitochondrial complex subunits I (NDUF8-20 kDa)), II (SDHB-30 kDa), III (UQCRC2-48 kDa), IV (MTCO1-40 kDa) and V (ATP5a-55 kDa). Actin was used as a loading control to determine the relative levels of each subunit. (B-F) Quantitative determination of mitochondrial complex subunits from (A) normalized by β-actin. In each panel, basal level of mitochondrial complex subunits for control (untreated) healthy and Barth lymphocytes were normalized to 100; and Dox-induced changes in the band intensities of each subunit in the western blot were normalized against corresponding control.</p

Determination of specific carbonylated proteins in MCF-12A, MDA-MB-231, and MDA-MB-468 cell lines by two-color western.

<p>Representative two-color western blots to detect carbonylation in (A) filamin A, (B) EPRS, and (C) HSP90β proteins using anti-filamin A, anti-EPRS and anti-HSP90β antibodies respectively in combination with anti-DNP antibody. Protein carbonyls were detected using anti-goat 800CW (green), and specific protein bands were detected using donkey anti-rabbit or anti-mouse 680LT (red) as the secondary antibodies. 12A = MCF-12A, 231 = MDA-MB-231, 468 = MDA-MB-468.</p

Mitochondrial accumulation of Dox in healthy and Barth lymphocytes.

<p>(A) Representative western blot showing the purity of the mitochondrial fractions. Protein lysates from cell fractionation were loaded in duplicate for healthy and Barth lymphocytes. (B) Quantitative determination of mitochondrial Dox by fluorescence method (λ_ex = 478 nm λ_em = 594 nm) in healthy and Barth cells. Cells were incubated with 1μM Dox for 24 h, washed with PBS, then incubated in fresh media without Dox for 2 h, then cells were harvested and subjected to isolate the mitochondrial fractionation for Dox measurement. (C) Standard curve generated from the known concentrations of Dox solutions that was used to quantify mitochondrial Dox.</p

Identification of carbonylated proteins in human breast cancer and adjacent healthy tissue by two-color western.

<p>Representative two-color western blots to detect carbonylation in (A) filamin A, (B) HSP90β, and (C) EPRS proteins. Arrow in each panel indicates the overlapping bands between corresponding protein (red) and carbonyl (green). Figure panels (D), (E), and (F) represent the two-color western for immunoprecipitated samples using anti-filamin A, anti-HSP90β and anti-EPRS antibodies respectively in combination with anti-DNP antibody. Carbonylated protein bands were detected using anti-goat 800CW (green) and specific protein bands were detected using donkey anti-rabbit or anti-mouse 680LT (red) as the secondary antibodies. N = adjacent healthy tissue, T = breast tumor tissue.</p

Determination of lipid peroxidation and total protein carbonylation in healthy and Barth lymphocytes.

<p>Quantitative determination of lipid peroxidation product in (A) healthy and Barth lymphocytes at basal level and (B) Dox-induced lipid peroxidation in healthy and Barth lymphocytes normalized to control. MDA-TBA adduct was measured at 532 nm and normalized to mg of protein in the cell lysate. To determine the Dox-induced changes in lipid peroxidation between healthy and Barth lymphocytes, basal levels of MDA for control (untreated) healthy and Barth lymphocytes were normalized to 100; and Dox-induced changes in MDA level were normalized against corresponding control. (C) Total protein carbonylation in healthy and Barth lymphocytes with and without Dox (1μM) treatment was determined by ELISA. *p<0.05; **p<0.001.</p

<i>Tafazzin</i> knockdown and Dox-sensitivity in H9c2 cardiomyocytes.

<p>(A) knockdown of <i>tafazzin</i> was confirmed in H9c2 cells after transfection with 20 nM <i>tafazzin</i> siRNA for 48 h by western blot analysis. (B) Representative western blot showing the level of cleaved caspase-3. After transfection with <i>tafazzin</i> and NTP siRNA for 24 h, cells were treated with 0.5 and 1 μM Dox for 24 h and cleaved caspase-3 levels were quantified using tubulin as a loading control. (C) Quantitative analysis of cleaved caspase-3 from (B) normalized by α-tubulin. (D) Mitochondrial superoxide levels were measured using MitoSOX Red dye in <i>tafazzin</i> and NTP transfected control H9c2 cells at basal levels and (E) after 0.5 and 1μM Dox treatment for 24 h. The changes in mean fluorescence intensity of mitochondrial superoxide sensitive dye MitoSOX Red in healthy and Barth lymphocytes was normalized by the corresponding control sample to compare the Dox-induced effect in both cell types. Mitochondrial complex III inhibitor Antimycin A (20 μM) was used as a positive control. (F) Measurement of mitochondrial membrane potential using JC-1 dye for NTP and Taz siRNA transfected H9c2 after treatment with Dox for 2 h. Y-axis represents the relative fluorescence ratio of 590/540 nm normalized to control without Dox treatment. Basal level of mitochondrial membrane potential in Taz siRNA transfected cells was slightly lower than NTP siRNA transfected cells (1.03 for NTP siRNA transfected cells and 0.9 for Taz siRNA transfected cells). CCCP (10 μM), a commonly used mitochondrial membrane depolarization agent was used as a positive control. To determine the Dox-induced effect between NTP and Taz siRNA transfected H9c2 cells, basal level of ROS and mitochondrial membrane potential of untreated (control) NTP and Taz siRNA transfected H9c2 cells were normalized to 100; and Dox-induced changes in the ROS and mitochondrial membrane potential were normalized against corresponding control. *p<0.05; **p<0.001.</p

Determination of Dox-induced ATP levels in healthy and Barth lymphocytes.

<p>Healthy and Barth lymphocytes were treated with vehicle (control) or 0.5, 1, and 5 μM of Dox for 8 and 24 h and cellular ATP content was determined (A) at basal levels without Dox treatment and (B-C) in control and Dox-treated lymphocytes using a kit from Molecular Probes. Amount of ATP was calculated as pmol of ATP per 1x10⁶ cells. The y-axis shows the amount of ATP per 1x10⁶ cells. To determine the Dox-induced changes in the ATP level between healthy and Barth lymphocytes, basal level of ATP for control (untreated) healthy and Barth lymphocytes were normalized to 100; and Dox-induced changes in ATP level were normalized against corresponding control. **p<0.001.</p

Determination of Dox-induced mitochondrial superoxide production and mitochondrial membrane potential in healthy and Barth lymphocytes.

<p>Quantitative analysis of changes in mean fluorescence intensity of mitochondrial superoxide sensitive dye MitoSOX Red (A) in healthy and Barth lymphocytes at basal level without Dox treatment and (B) after 0.5 and 1 μM Dox treatment for 24 h. Mitochondrial complex III inhibitor Antimycin A (20 μM) was used as a positive control. All values were normalized to mean fluorescence intensity of control. Quantitative representation of mitochondrial membrane potential of healthy and Barth lymphocytes at basal levels without Dox treatment (C), and after treatment with 1 and 5 μM Dox (D). CCCP (10 μM), a commonly used mitochondrial membrane depolarization agent was used as a positive control. To determine the Dox-induced oxidative stress between healthy and Barth lymphocytes, basal levels of ROS for control (untreated) healthy and Barth lymphocytes were normalized to 100; and Dox-induced changes in the ROS were normalized against corresponding control. Mitochondrial membrane potential was analyzed in a similar way. *p<0.05; **p<0.001; AntA = Antimycin A.</p