Doxorubicin-transferrin conjugate alters mitochondrial homeostasis and energy metabolism in human breast cancer cells

Abstract

Doxorubicin (DOX) is considered one of the most powerful chemotherapeutic agents but its clinical use has several limitations, including cardiomyopathy and cellular resistance to the drug. By using transferrin (Tf) as a drug carrier, however, the adverse effects of doxorubicin as well as drug resistance can be reduced. The main objective of this study was to determine the exact nature and extent to which mitochondrial function is influenced by DOX-Tf conjugate treatment, specifically in human breast adenocarcinoma cells. We assessed the potential of DOX-Tf conjugate as a drug delivery system, monitoring its cytotoxicity using the MTT assay and ATP measurements. Moreover, we measured the alterations of mitochondrial function and oxidative stress markers. The effect of DOX-Tf was the most pronounced in MDA-MB-231, triple-negative breast cancer cells, whereas non-cancer endothelial HUVEC-ST cells were more resistant to DOX-Tf conjugate than to free DOX treatment. A different sensitivity of two investigate breast cancer cell lines corresponded to the functionality of their cellular antioxidant systems and expression of estrogen receptors. Our data also revealed that conjugate treatment mediated free radical generation and altered the mitochondrial bioenergetics in breast cancer cells.

Figure 1

Principle of the mitochondrial stress assay. (A) Proposed definitions for cellular bioenergetics based on oxygen consumption rate (OCR). (B) Targets of each compound in the electron transport chain. (C) OCR levels refer to breast cancer and normal cells (left and right y axis, respectively) versus time (x axis). Injection of the three compounds oligomycin, FCCP, and antimycin A/rotenone delimited four time intervals within each of which OCR was roughly constant. Oligomycin is a specific inhibitor of the ATPase complex V and prevents protons from crossing the membrane through this complex to phosphorylate ADP. Oligomycin was used here to inhibit cellular respiration completely. FCCP is a mitochondrial uncoupler that allows protons to leak freely across the mitochondrial membrane and uncouples oxygen consumption from phosphorylation of ADP. Antimycin A is a specific inhibitor of Complex III, and rotenone is a specific inhibitor of Complex I. They were used together to block all mitochondrial respiration even in the presence of an uncoupler such as FCCP.

Figure 2

Comparisons of cytotoxicity of free DOX and DOX–Tf conjugate in breast cancer and endothelial cell lines. (A) Viability of MCF-7, MDA-MB-231 and HUVEC-ST cells measured by MTT assay after incubation of the cells for 72 h with increasing concentrations of DOX and DOX–Tf. The values are mean ± SD of five independent experiments with six replicates in each experiment. (B) IC₅₀ values (calculated on basis of cell viability curves) for free DOX and DOX–Tf conjugate in MCF-7, MDA-MB 231 and HUVEC-ST cells. (C) Cellular ATP level in MCF-7, MDA-MB-231, and HUVEC-ST cells treated with IC₅₀ concentrations of DOX alone or DOX–Tf for 4, 24 and 72 h. All values were normalized to untreated, control cells, taken as 100%. Data are expressed as mean ± SD (n = 6). Asterisks refer to the level of significance @@(**p < 0.01, ***p < 0.001).

Figure 3

Oxidative stress markers in MCF-7, MDA-MB-231 and HUVEC-ST cell lines treated with DOX–Tf conjugate or DOX alone for 4, 24 and 72 h. (A) Cellular ROS level assessed by H₂DCFDA oxidation after treatment of MCF-7, MDA-MB-231 and HUVEC-ST cells with DOX–Tf conjugate or free DOX (IC₅₀ dose) for 4, 24 and 72 h in the absence or presence of ROS scavenger NAC (5 mM). (B) Activity of NADH oxidase in MCF-7, MDA-MB-231 and HUVEC-ST cells was measured after 4, 24 and 72 h exposure of cells to free DOX and DOX–Tf conjugate. Data represent mean ± SD of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 denote a statistically significant difference compared with control cells; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 denote a significant difference observed between cells incubated with DOX in comparison to DOX–Tf conjugate; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001; n = 6: difference between expression in the cells treated with DOX or DOX–Tf.

Figure 4

Changes in MMP of MCF-7, MDA-MB-231 and HUVEC-ST cells. (A) Effect of DOX–Tf conjugate and free DOX on MMP in human breast cancer cells and endothelial cells. MMP of cells treated with IC₅₀ concentrations of DOX–Tf or DOX alone (after 4, 24 and 72 h) was estimated with JC-1. Each result represents mean ± SD of four independent experiments, related to fluorescence of the control sample assumed as 100%. *p < 0.05 significantly different compared to the respective untreated cells; ⁺p < 0.05, significant differences between samples incubated with DOX or DOX–Tf. (B) Collapse of MMP in human breast cancer cells and HUVEC-ST endothelial cell line incubated with FCCP for 4, 24 and 72 h. Fluorescence ratio of JC-1 dimers/JC-1 monomers in control cells was assumed as 100%. Results are presented as mean ± SD of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 denote statistically significant changes in comparison with the control cells (not treated with FCCP) taken as 100%.

Figure 5

Fluorescent microscopy images of control cells incubated with PBS, and cells treated with IC₅₀ concentrations of DOX or DOX–Tf for 24 h. Red fluorescence of JC-1 dimers was present in the cell areas with high MMP, while green fluorescence of JC-1 monomers was prevalent in the areas with diminished MMP. JC-1-stained cells were visualized with an inverted fluorescence microscope (Olympus IX70, Japan); 400 × magnification.

Figure 6

Mitochondrial respiration of MCF-7, MDA-MB 123 and HUVEC-ST cells after incubation with DOX–Tf. Data are expressed as pmol O₂/s/mg protein in permeabilized cells measured after DOX and DOX–Tf treatment with IC₅₀ concentrations. The following mitochondrial states were evaluated: routine respiration (A–C) State D (D–F) and State ETS (G–I) after 4, 24 and 72 h incubation with DOX or DOX–Tf. Data were expressed as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 denote statistically significant changes in comparison with the control untreated cells; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 significant differences between samples incubated with DOX or DOX–Tf.

Figure 7

Selected parameters of mitochondrial bioenergetics in human breast cancer cell lines (MCF-7 and MDA-MB-231) and noncancer HUVEC-ST cells. The following mitochondrial parameters were evaluated: RCR (A–C) L/E, (D–F) and P/E (G–I). Measurement was performed after 4, 24 and 72 h incubation with DOX alone or DOX–Tf conjugate (IC₅₀ doses). Data are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 denote statistically significant changes in comparison with the control untreated cells; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 significant differences between samples incubated with DOX or DOX–Tf.

Figure 8

Expression of Bax, Bcl2 and Cytochrome c gene transcripts (relative to HPRT1 housekeeping gene) in MCF-7, MDA-MB-231 and HUVEC-ST cells exposed to IC₅₀ concentrations of DOX or DOX–Tf. Asterisks refer to the level of significant (⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001; n = 6) difference between expression in the cells treated with DOX or DOX–Tf.