Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents

Abstract

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

Figure 1. Genipin sensitizes drug-resistant MX2 cells to menadione toxicity.

A) The viability of HL-60 (filled diamond) and MX2 (filled square) following exposure to menadione. Cells were exposed for 24 h to different concentrations of menadione (0–100 µmol/L) followed by the assessment of the number of live cells. Viability was expressed as a percent of the control value. Kruskall-Wallis with a post-hoc Mann-Whitney test, n = 3, p<0.05. * indicates statistical significance for HL-60 cells compared to control and # indicates statistical significance for MX2 compared to control. B) Immunoblot analysis of anti-oxidant defense enzymes and UCP2. Mitochondria from MX2 (M) and HL-60 (H) cells were isolated and analyzed for UCP2, MnSOD, and GPx4. Cytosol was used for G6PDH determinations. NADP-ICDH served as the loading control for the cytosol and mitochondrial fractions. Spleen lysate (100 µg) was used as a UCP2 loading control. The molecular weight of UCP2 was confirmed using molecular mass markers. C) In situ analysis of the specificity of genipin for UCP2. Following the determination of basal oxygen consumption rates, the impact of genipin on OCR in CHO-UCP2 (white bars) cells was assessed. Cells were exposed to (0–50 µM) genipin for 15 min and then OCR was tested. Data was expressed as a % of CHO-EV OCR. INSET: immunoblot analysis of UCP2 in CHO-UCP2 and CHO-EV cells. SDH served as the loading control. Kruskall-Wallis with a post-hoc Mann-Whitney test, n = 3, p<0.05. * indicates statistical significance when compared to the control. D) Assessment of genipin toxicity and the response of drug-sensitive HL-60 and drug-resistant MX2 cells to simultaneous treatment with menadione and genipin. Cells were exposed to genipin (0–500 µmol/L) in the presence or absence of 20 µM menadione for 24 h. Following the exposure, cell viability was determined. ♦ and ▪ represent exposure to either genipin or genipin + menadione. Viability was expressed as a percent of the control. Kruskall-Wallis with a post-hoc Mann-Whitney test, n = 3, p<0.05. * corresponds to statistical significance when cells exposed to both menadione and genipin were compared to control. # corresponds to statistical significance when genipin-exposed cells were compared to the control cells. E) Treatment with genipin and menadione increases MX2 cell death. MX2 cells were exposed to 20 µmol/L menadione and genipin (0–500 µmol/L) for 24 h. The amount of cell death was determined using the PI assay. 1-way ANOVA with a post-hoc Tukey's test, n = 5, p<0.05. * represents significance when compared to 0 µM.

Figure 2. Genipin sensitizes MX2 cells to epirubicin and doxorubicin.

HL-60 and MX2 cells were grown to 70% confluency and then exposed to either epirubicin (0–0.5 µM) or doxorubicin (0–0.5 µM) in the absence or presence of 20 µM genipin. Amount of cell death was determined by PI assay. Results were expressed as a percent of the control. A) ♦ epirubicin only, ▪ epirubicin + genipin. 1-way ANOVA with a post-hoc Tukey's test, n = 5, * p<0.05. * corresponds to statistical significance when epirubicin and genipin treated cells were compared to control. # corresponds to statistical significance when epirubicin-treated cells were compared to control cells. B) ♦ doxorubicin only, ▪ doxorubicin + genipin. 1-way ANOVA with a post-hoc Tukey's test, n = 5, p<0.05. * corresponds to statistical significance when epirubicin and genipin treated cells were compared to control cells.

Figure 3. Genipin decreases cellular oxygen consumption in drug-resistant MX2 but not drug-sensitive HL-60 cells during oligomycin-induced state 4 respiration.

A) Oxygen consumption measurements in cells exposed to 20 µM genipin 24 h exposure. Oxygen consumption measurements were performed following a 30 min incubation in reaction medium in the absence (white bars) or presence (grey bars) of oligomycin. 1-way ANOVA with a post-hoc Tukey's test, n = 6, **p<0.01. Treated MX2 or HL-60 cells were only compared with their corresponding control mean values. B) Oxygen consumption measurements in cells exposed to genipin (20 µM) and/or menadione (20 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 4, *p<0.05,

Figure 4. Genipin treatment increases menadione-mediated ROS levels in drug-resistant cells.

ROS levels were assessed in intact HL-60 and MX2 cells using DCFH-DA. A) ROS levels in cells exposed to solely menadione (0–50 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, *p<0.05. B) ROS levels in cells exposed to 0 or 20 µM genipin and menadione (0–50 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, * p<0.05, **p<0.01.

Figure 5. Genipin treatment enhances the production of ROS by doxorubicin.

ROS levels were assessed in intact HL-60 and MX2 cells using DCFH-DA. A) ROS levels in HL-60 cells and MX2 cells exposed to doxorubicin (0–1 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, *p<0.05, **p<0.01. B) ROS levels in HL-60 cells and MX2 cells exposed to 20 µM genipin and doxorubicin (0–1 µM) for 24 h. 1-way ANOVA with a post-hoc Tukey's test, n = 5, *p<0.05, **p<0.01.