Mitochondrial relocation of a common synthetic antibiotic: A non-genotoxic approach to cancer therapy

Abstract

Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major issue in cancer treatment. Currently, only a few examples of potentially non-genotoxic drugs have been reported. Mitochondrial re-localization of ciprofloxacin, one of the most commonly prescribed synthetic antibiotics, is reported here as a new approach. Conjugating ciprofloxacin to a triphenyl phosphonium group (giving lead Mt-CFX), is used to enhance the concentration of ciprofloxacin in the mitochondria of cancer cells. The localization of Mt-CFX to the mitochondria induces oxidative damage to proteins, mtDNA, and lipids. A large bias in favor of mtDNA damage over nDNA was seen with Mt-CFX, contrary to classic cancer chemotherapeutics. Mt-CFX was found to reduce cancer growth in a xenograft mouse model and proved to be well tolerated. Mitochondrial relocalization of antibiotics could emerge as a useful approach to generating anticancer leads that promote cell death via the selective induction of mitochondrially-mediated oxidative damage.

Keywords: Ciprofloxacin; DNA damage; Mitochondria; Non-genotoxic cancer therapy; Prodrug; Reactive oxygen species; Targeted therapeutics.

Figure 1.. Mt-CFX induces cell death in a mitochondrial membrane potential (MMP)-dependent manner

(A) Chemical structure of Mt-CFX. (B) Concentration-dependent cell-viability of MDA-MB-231 cells incubated with Mt-CFX, CFX, and TPP, as determined by a Cyto-Tox96 Assay (72h). (C) Concentration-dependent cell-viability of MCF10A cells incubated with Mt-CFX, CFX, and TPP, as determined by a CytoTox96 Assay (72h). (D) Proportion of JC-1 monomer fluorescence in a flow cytometry assay of MDA-MB-231 pretreated with 30 μM Mt-CFX with incubation times as indicated in the figure.* (E) Time depended Annexin V-FITC flow cytometry analysis of MDA-MB-231 pretreated with 30 μM Mt-CFX per the incubation times indicated in the figure. (F) Concentration-dependent cell-viability of different cell lines incubated with Mt-CFX, as indicated in the figure, as determined by a CytoTox96 Assay (72h).* (G) Different proportions of JC-1 monomer fluorescence in a flow cytometry assay of untreated cells. (H) Summarized JC-1 monomer fractions from panel G and IC₅₀ values of Mt-CFX-treated cell lines from panel F.* All cytotoxicity experiments were carried out three times and in triplicate wells. Data are represented as mean ± SEM. Statistical significance was determined using a one-way ANOVA test with post-hoc Bonferroni test. *Different letters (e.g., a–d) signify data which are statistically different (p < 0.05).

Figure 2.. Bo-Mt-CFX, a ROS-triggered DDS system incorporating Mt-CFX

(A) Peroxide-induced self-immolative activation mechanism of Bo-Mt-CFX (B) Localization of resorufin release from 10 μM Bo-Mt-CFX in MDA-MB-231 cells, compared with MitoTracker Green FM, LysoTracker Green DND-26, and ER-Tracker Green. (C) Western blotting of apoptosis-related proteins in MDA-MB-231 cells treated with 10 μM Bo-Mt-CFX or a control. (D) Western blotting of cytochrome c release from mitochondria to the cytosol seen in MDA-MB-231 cells treated with 3 μM or 10 μM Bo-Mt-CFX, as well as a control.

Figure 3.. Mt-CFX induces mitochondrial ROS production with minimal nuclear DNA damage

(A) Confocal microscopic fluorescence intensity of MDA-MB-231 cells treated with Mt-CFX (30 μM) or a control co-incubated with 400 μM Amplex Red® for 30 min (n = 6). (B) Confocal microscopic fluorescence intensity of Mt-CFX (30 μM) or control-treated MDA-MB-231 cells incubated with 5 μM Mito-Sox for 15 min (n = 5). (C) Confocal microscopic fluorescence intensity of Mt-CFX (30 μM) or control treated MDA-MB-231 cells incubated with 10 μM CM-H2DCFDA for 30 min (n = 15). (D) ELISA DNA oxidation and protein carbonylation assay results of MDA-MB-231 cells treated with 30 μM Mt-CFX, CFX as well as control samples. Colorimetric malondialdehyde (MDA) assay results, as a measure of lipid peroxidation, of similarly treated MDA-MB-231 cells. (E) q-PCR analysis of DNA repair genes in MDA-MB-231 cells treated with 30 μM Mt-CFX, CFX, DOX, and control samples. (F) q-PCR analysis of housekeeping genes in MDA-MB-231 cells treated with 30 μM Mt-CFX, CFX, DOX, and control samples. (G) Taq1 restriction site mutation assay using MDA-MB-231 cells treated with 30 μM Mt-CFX, CFX, DOX, and control samples. (H) Long-range q-PCR DNA lesion assay using MDA-MB-231 cells treated with Mt-CFX (30 μM), temozolomide (TMZ) (1 μM), camptothecin (CPT) (250 nM), cis-platin (CDDP) (3 μM), chlorambucil (CLB) (50 μM), and fluorouracil (5-FU) (30 μM), respectively. Concentrations were selected on the basis of their respective IC₅₀ values. Data are represented as mean ± SEM. (n = 3, unless specified otherwise). Statistical significance was determined using a one-way ANOVA test with post-hoc Bonferroni test. Different letters (e.g., a–d) signify data which are statistically different (p < 0.05).

Figure 4.. Identification of cellular processes affected by Mt-CFX

(A) Heatmap representing relative expression levels of the DEGs (differentially expressed genes) to control versus 10 μM CFX and Mt-CFX. (B) KEGG pathway enrichment analysis of the 197 DEGs. (C-F) q-PCR gene expression levels of selected genes in cells MDA-MB-231 cells treated with 10 μM Mt-CFX, CFX and a control sample. (C) Cellular metabolism.* (D) Mitochondrial detoxification.* (E) Cellular repair and inflammasome induction.* (F) Autophagy and mitophagy.* Data are represented as mean ± SEM. (n = 3, in panels C-F). Statistical significance in panels c-f was determined using a one-way ANOVA test with post-hoc Bonferroni test. *Different letters (e.g., a–d) signify data which are statistically different (p < 0.05).

Figure 5.. In vivo tumor growth reduction by Mt-CFX and Bo-Mt-CFX.

(A) In vivo images of MDA-MB-231 xenograft mouse 1 h, 24 h, and 48 h post intravenous tail vein injection of a single dose of 0.5 μmol kg⁻¹ Bo-Mt-CFX. Excitation at 550 nm. Emission at 650 nm. (B) Ex vivo imaging of excised tumors and organs 48 h post injection of a single dose of 0.5 μmol kg⁻¹ Bo-Mt-CFX or vehicle only. Excitation at 550 nm. Emission at 650 nm. (C) In vivo tumor volume determination (1/2 × length × width²) of mice treated with 0.5 μmol kg⁻¹ CFX, Mt-CFX, Bo-Mt-CFX, or vehicle alone, once a week for 3 weeks.* (D) Body weight of mice during the treatment regime. (E) Excised tumors at the treatment endpoint. (F) Excised tumor weight per treatment group.* (G-H) Blood serum AST and ALT activity levels, as determined using a colorimetric assay.* (I) Immunohistochemistry (cleaved caspase 3 and Ki-67) of representative tumor tissue slices of the different treatment groups. (J) H&E staining of representative tissue slices of the different treatment groups. Data are represented as mean ± SEM. Panels C, D, F: n = 4 mice and n =8 tumors per group, Panels G, H: n = 4. Statistical significance was determined using a one-way ANOVA test with post-hoc Bonferroni test. *Different letters (e.g., a–d) signify data sets that are statistically different (p < 0.05).