Inhibition of glucose-6-phosphate dehydrogenase sensitizes cisplatin-resistant cells to death

Abstract

The mechanisms of cisplatin resistance, one of the major limitations of current chemotherapy, has only partially been described. We previously demonstrated that cisplatin-resistant ovarian cancer cells (C13), are characterized by reduced mitochondrial activity and higher glucose-dependency when compared to the cisplatin-sensitive counterpart (2008). In this work we further characterized the role of metabolic transformation in cisplatin resistance. By using transmitochondrial hybrids we show that metabolic reprogramming of cisplatin-resistant cell is not caused by inherent mtDNA mutations. We also found that C13 cells not only present an increased glucose-uptake and consumption, but also exhibit increased expression and enzymatic activity of the Pentose Phosphate pathway (PPP) enzyme Glucose-6-Phosphate Dehydrogenase (G6PDH). Moreover, we show that cisplatin-resistant cells are more sensitive to G6PDH inhibition. Even if the metabolomic fingerprint of ovarian cancer cells remains to be further elucidated, these findings indicate that PPP offers innovative potential targets to overcome cisplatin resistance.

Keywords: PPP; cancer metabolism; cisplatin; drug resistance; transmitochondrial hybrids.

Figure 1. Mitochondrial function in cancer cisplatin-resistant cells and their derived cybrids

A. Oxygen consumption of ovarian cancer cells (2008-C13) and their derived cybrids (H2008-HC13). B. Representative plots of the mitochondrial potential (ΔΨm) measured by flow cytometry. C. Effect of 5 mM galactose and D. 0.1 μM rotenone on cell viability after 24 hours of treatment. Data are expressed as percentage of cell number compared to the relative control. The data were obtained from at least 3–5 independent cultures. ***p < 0.001, **p < 0.01, *p < 0.05; C13 vs 2008. +++p < 0.001, ++p < 0.01, +p < 0.05; treatment vs control.

Figure 2. Cisplatin-resistant cells present a reduced mitochondrial mass

A. Representative confocal images of Mitotracker Green (MTG) staining. B. Representative plots of Acridine Orange 10-Nonyl bromide (NAO) mean fluorescence intensity measured by flow cytometry.C. MTG mean fluorescence intensity, calculated after a volumetric reconstruction of the mitochondrial network. D. Expression levels of genes involved in mitochondrial biogenesis or F. in mitophagy as tested by qRT-PCR. All genes were normalized to β-actin as endogenous control. E. LC3 protein expression measured from western blotting. The data were obtained from at least 3–5 independent cultures. ***p < 0.001, **p < 0.01, *p < 0.05; C13 vs 2008.

Figure 3. Cisplatin-resistant cells show an increased dependency to glucose

A. Glucose uptake measured after incubation with the glucose analogue 6-NBDG. Data are normalized to cisplatin-sensitivecells. B. Expression levels of glycolytic genes measured by qRT-PCR. All genes were normalized to β-actin. C–D. Cell viability after 24 hours of glucose deprivation with (D) or without (C) 1 mM 2-DG. Data are expressed as percentage of cell number compared to control. The data were obtained from at least 3–4 independent cultures. ***p < 0.001, **p < 0.01, *p < 0.05; C13 vs 2008. +++p < 0.001; treatment vs control.

Figure 4. Cisplatin-resistant cells present an increased dependency on glutamine for TCA intermediates biosynthesis

A. Abundance of TCA intermediates measured using LC-MS normalised to total ion current. B. Incorporation of 13C-labelled carbons into glutamate, succinate, fumarate and malate after growing cells for 24 hours in the presence of [U-¹³C]glutamine. C–D. Effect of glutamine deprivation on 2008 (C) and C13 (D) cell viability measured by trypan blue exclusion assay. The data were obtained from at least three independent cultures. ***p < 0.001, *p < 0.05; C13 vs 2008. +++p < 0.001, ++p < 0.01; treatment vs control.

Figure 5. Redox homeostasis is required for survival of C13 cisplatin-resistant cells

A–B. Cellular GSH and GSSG content measured by enzymatic assay (A) and GSH abundance (normalised to total ion count) measured by LC-MS (B). C. Intracellular levels of ¹³C₅-GSH after growing cells for 24 hours in the presence of [U-¹³C]glutamine. Data are normalized on the total pool of intracellular GSH (B) D. G6PD mRNA levels quantified by qRT-PCR, E. protein expression measured by western blotting and F. G6PDH enzymatic activity of 2008 and C13 cells. G–H. Isobologram of cisplatin-resistant cells showing additive effect of 6-AN or DHEA in association with cisplatin treatment. Data are expressed as percentage of cell number compared to control. The graph was obtained using iso-effective drug concentrations causing 25% of cytotoxic effect. Straight line indicates the theoretical additivity line. The data were obtained from at least 3–5 independent cultures. ***p < 0.001, **p < 0.01, *p < 0.05; C13 vs 2008. +++p < 0.001; treatment vs control.