Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells

Figures

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  Figure 1. Cytotoxicity of free doxorubicin and doxorubicin–transferrin conjugate in human leukemia cell lines and peripheral blood mononuclear cells. (A): Viability of CCRF-CEM, K562, and PBMC cells after 48 h incubation with increasing concentrations of doxorubicin–transferrin (DOX–Tf) conjugate (violet symbols) or free doxorubicin (DOX) (blue symbols). Cell viability was measured by XTT assay. Values represent the means ± SD, (n = 5) * p < 0.05, ** p < 0.01 in comparison to untreated, control cells, (+) statistically significant differences noted between the probes incubated with free DOX compared to the conjugate, + p < 0.05. (B): Comparison of the cytotoxicity of free DOX and transferrin-bound DOX in CCRF-CEM and K562 cell lines or PBMCs. ## p < 0.05, ### p < 0.01 in comparison to normal, non-cancer cells, (++, +++) statistically significant differences noted between the probes incubated with free DOX compared to the conjugate, ++ p < 0.01, +++ p < 0.001. The values are the IC50 mean [nM] ± SD of five independent experiments with a 95% confidence interval. (C): Morphological changes observed with microscopy. Inverted phase contrast microscopy images were obtained following treatment of…

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  Figure 2. Doxorubicin–transferrin conjugate induced modifications of histone H2AX in human leukemia cells (A):The ratio of phosphorylation of histone H2AX (γH2AX) in comparison to total cellular content of this protein after treatment of CCRF-CEM and K562 cells or PBMCs with IC50 concentrations of doxorubicin (DOX) alone and doxorubicin–transferrin (DOX–Tf) conjugate for 6, 24, or 48 h. All values were normalized to untreated control cells, taken as 1. Data are expressed as the means ± SD, (n = 3). * p < 0.05 indicates statistically significant difference compared with control cells; and ++ p < 0.01 shows a difference of γH2AX level between cells treated with DOX or DOX–Tf. (B): The level of mRNA transcripts for the histone H2AX gene in the examined human leukemia cell lines as well as PBMCs exposed to IC50 concentrations of free DOX or DOX–Tf for 24 h. Data are expressed as the means ± SD, (n = 3). Asterisks refer to the level of significant difference (** p < 0.01) in mRNA level in the conjugate-treated cells compared to untreated control cells.

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  Figure 3. DNA damage is correlated with doxorubicin–transferrin-induced apoptosis in CCRF-CEM and K562 cell lines and in PBMCs (A) The influence free doxorubicin (DOX) and transferrin (Tf)-bound DOX (IC50 concentrations, respectively) on the induction of apoptosis in human leukemia cells as well as normal PBMCs was determined by transferase dUTP nick end labeling (TUNEL) assay. Quantitative results, calculated as the level of TUNEL-positive cells, are presented as mean ± SD (≥3). * p < 0.05, ** p < 0.01 relative to the control (untreated cells); + p < 0.05 indicates statistically significant difference between cells treated with DOX or DOX–Tf. (B) Typical cytometric dot-blots obtained after 24 h of incubation of human cancer and normal cells with IC50 concentrations of DOX alone or DOX–Tf conjugate. Lower left down corner, TUNEL-negative cells, propidium iodide (PI)-negative; lower right corner, TUNEL-positive cells, PI-negative; left upper corner, TUNEL-negative, PI-positive cells; upper right corner, TUNEL-positive, PI-positive cells.

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  Figure 4. Doxorubicin -transferrin conjugate alters TRAIL ligand expression in human leukemia cells. (A) The production of TRAIL-ligand (TRAIL-L) in cancer and normal cells incubated with IC50 concentrations of doxorubicin (DOX) and doxorubicin–transferrin (DOX–Tf) conjugate for 6, 24, or 48 h. The results represent mean ± SD of three independent experiments. * p < 0.05 in comparison to respective control cells taken as 100%, (+) statistically significant difference observed between cells incubated with DOX in comparison to DOX–Tf, + p < 0.05. (B) TRAIL-L transcript levels (relative to Hydroxymethylbilane synthase (HPRT1) and hydroxymethylbilane synthase (HMBS) housekeeping genes) in CCRF-CEM and K562 cell lines and PBMCs exposed to either form of DOX for 24 h at IC50 concentrations, n = 3. Asterisks refer to significant differences (* p < 0.05) in the transcription levels in cells treated with free drug or DOX–Tf compared to the untreated cells. + indicates statistically significant changes between samples incubated with DOX alone or with DOX–Tf (+ p < 0.05).

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  Figure 5. Changes in TNF-α expression in K562 and CCRF-CEM cell lines, and in PBMCs treated with transferrin-bound doxorubicin or free drug (A) The effect of IC50 concentrations of doxorubicin (DOX) and doxorubicin–transferrin (DOX–Tf) conjugate on TNF-α expression. The data represent mean ± SD of three independent experiments. * p < 0.05 in comparison to untreated, control cells, (+) statistically significant differences noted between the probes incubated with free DOX compared to the conjugate, + p < 0.05. (B) Expression of TNF-α gene transcript (relative to HPRT1 and HMBS housekeeping genes) in investigated human leukemia cell lines and PBMCs exposed to IC50 concentrations of DOX alone and DOX–Tf conjugate. Asterisks refer to the level of significance (* p < 0.05, n = 3) whereas symbol “+” indicates a difference between expression in the cells treated with DOX or DOX–Tf, + p < 0.05.

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  Figure 6. The evaluation of pro-inflammatory cytokines level after leukemia cells treatment with DOX and DOX–Tf conjugate. (A,B): The concentrations of interleukin 4 (IL-4) and interleukin 6 (IL-6) secreted in the supernatants of CCRF-CEM, K562, and PBMC cultures treated with IC50 concentrations of doxorubicin–transferrin (DOX–Tf) conjugate and DOX alone for 6, 24, or 48 h. The results show mean ± SD of three independent experiments. * p < 0.05,in comparison to untreated, control cells, (+) statistically significant differences observed between the probes incubated with free DOX in comparison to the conjugate, + p < 0.05. (C,D): Level of mRNA transcripts for IL-6 and IL-4 genes in CCRF-CEM, K562, and PBMCs exposed to IC50 concentrations of DOX–Tf conjugate and free doxorubicin for 48 h. Data (ΔΔCt values) were transformed into relative copy number values (the number of mRNA copies of the examined genes per housekeeping gene index, calculated as the average Ct value of the HPRT1 and HMBS housekeeping genes) and standardized to the level of mRNA transcripts in untreated cells, taken as 1. Results are presented as the means ± SD, (n = 6). Asterisks refer to the level of significant difference…

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  Figure 7. A simplified model of the differential effects of the doxorubicin–transferrin (DOX–Tf) conjugate on the pro-inflammatory response and induction of apoptosis via the extrinsic pathway. DOX–Tf conjugate stimulates the production of TNF-α and TRAIL ligand, the cell signaling proteins. They are also known as two major extrinsic mediators of apoptosis. Acute lymphoblastic cells and chronic myeloid malignancies have different isoreceptors for these cytokines. Binding of TRAIL or TNF-α to their receptors, results in death receptors aggregation (DR4, DR5) and Fas-associated death domain-containing protein (FADD). The death domain of FADD binds to an analogous domain of caspase-8 to form the death-inducing signaling complex (DISC). This process activates downstream caspases and leads to apoptosis. In parallel, in the presence of typical hallmarks of apoptosis, the DOX–Tf conjugate affects cellular genome and mediates the production of pro-inflammatory response. The transcription of IL-4 and IL-6 genes mediates the secretion of these cytokines to the extracellular matrix.