New combination chemotherapy of cisplatin with an electron-donating compound for treatment of multiple cancers

Abstract

Cisplatin is the first and most widely used platinum-based chemotherapy drug and is the cornerstone agent in treating a broad spectrum of cancers. However, its clinical application is often limited by severe toxic side effects and drug resistance. Based on the discovered dissociative electron transfer mechanism of cisplatin, a novel combination of cisplatin with [9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride (basic violet 10, BV10) is proposed to potentiate the chemotherapeutic effect of cisplatin. Here, we show that this combination enhances the anti-cancer effect of cisplatin in both in vitro cell lines and in vivo xenograft mouse models of cisplatin-sensitive and -resistant lung, ovarian and cervical cancers while introducing minimal additional toxic side effects. Furthermore, femtosecond time-resolved laser spectroscopic measurements demonstrate that cisplatin reacts with BV10 via an electron transfer mechanism. These results indicate that the combination of cisplatin with BV10 is promising for improving the chemotherapy of cancers with various extents of cisplatin resistance.

Figure 1

DNA damage measurements using agarose gel electrophoresis. (A) Gel images of plasmid DNA treated with various concentrations of CDDP with/without the presence of 10 μM RDM-B (BV10), where the three separate bands from top to bottom represent DNA with single-strand break (SSB), double-strand break (DSB, red dashed square), and intact supercoiled DNA (SC, blue dashed square). The yields of DSB DNA and the amount of SC DNA are shown in (B) and (C), respectively.

Figure 2

Cellular DNA DSB measurements in different cancer cell lines using γH2AX labeling. Representative pictures of γH2AX labeling are shown in (A), (C) and (E), and those of dead green staining are shown in (B), (D) and (F) in HeLa, A549 and NIH:OVACR-3 cells, respectively, with the treatments indicated.

Figure 3

In vitro cell viability tests on various human cancer cell lines and a normal cell line. All viabilities are represented as percentages with respect to the control (untreated cells, taken as 100% survival). The MTT assay was performed to obtain RDM-B (BV10) cytotoxicity and toxicity profiles (A), cell-killing efficacies of CDDP and its combination with BV10 in cervical cancer HeLa cells (B) and ME-180 cells (C), lung cancer A549 cells (D), ovarian cancer NIH:OVCAR-3 cells (E), and normal cells (F), in which error bars represent the standard deviation of data obtained in each group. All MTT experiments were performed at 24 h post-treatment. The clonogenic assay was performed in A549 (G), ME-180 (H), NIH:OVCAR-3 (I), and HeLa (J) cells for 2 h treatment with CDDP and its combination with BV10, in which error bars represent the standard error of the mean (s.e.m.) of data obtained in each group. The p values reported on the graph were obtained from unpaired two-tail student t tests: ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., nonsignificant (p ≥ 0.05).

Figure 4

In vitro apoptosis detection in cervical cancer HeLa cells. (A) Representative pictures (black arrows indicating morphological changes) of HeLa cells treated with 10 μM and 30 μM CDDP with/without the combination of 10 μM RDM-B (BV10) for 12 h and then incubated with the CellEvent Caspase-3/7 Green Detection Reagent and analyzed using fluorescence microscopy. (B) Percentages of activated caspases 3/7. (C)–(H) Early/late apoptosis measurements of HeLa cells treated with 20 μM and 40 μM CDDP with/without the combination of 20 μM BV10 for 18 h using an Annexin V-FITC Apoptosis Detection Kit, where the cells were double stained with Annexin-V-FITC and PI. Quantitative analyses of the cell images in A and flow cytometry data in (C)–(H) were performed using an ImageJ software (https://imagej.nih.gov/ij/) and a FlowJo software (https://www.flowjo.com/solutions/flowjo), respectively.

Figure 5

Mouse xenograft models of human lung (A549) cancer, human cervical (ME-180) cancer, and human ovarian (NIH:OVCAR-3) cancer treated by CDDP or RDM-B (BV10) alone and their combination. (A–C) Tumor growth curves for the A549, NIH:OVCAR-3, and ME-180 models, respectively, where error bars represent the standard error of the mean (s.e.m.). (D) Representative pictures of mice bearing ovarian tumors. The statistical analysis results indicated in (A)–(C) were obtained from either two-way ANOVA (multiple groups in A and C) or unpaired two-tail student t tests (two groups in B): ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., nonsignificant (p ≥ 0.05). Statistical analyses were performed with GraphPad Prism 9.0.0 (https://www.graphpad.com/scientific-software/prism/) and Microsoft Excel.

Figure 6

Femtosecond time-resolved (pump-probe) laser spectroscopic observations of the dissociative electron transfer (DET) reaction between cisplatin (CDDP) and Rhodamine-B (BV10) in aqueous solutions. (A) Transient absorption measurements of BV10 cation radical (RDM-B^+•) in 10 μM BV10 only, 3 mM CDDP only, and the mixtures of 10 μM BV10 and 1.5 mM or 3 mM CDDP. (B,C) Transient absorption measurements of prehydrated electrons (e_pre⁻) in pure water, 60 μM and 120 μM BV10 only solutions, and in pure water, 3 mM CDDP only and the mixtures of 3 mM CDDP with 60 μM or 120 μM BV10.