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. 2022 Jan 20;14(2):238.
doi: 10.3390/pharmaceutics14020238.

The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation

Dina Baier  1   2   3 , Beatrix Schoenhacker-Alte  1   2   3 , Mate Rusz  1   4 , Christine Pirker  2   3 , Thomas Mohr  2 , Theresa Mendrina  1   2   3 , Dominik Kirchhofer  2 , Samuel M Meier-Menches  4 , Katharina Hohenwallner  1   4 , Martin Schaier  1   4 , Evelyn Rampler  1   4 , Gunda Koellensperger  4 , Petra Heffeter  2   3 , Bernhard Keppler  1   3 , Walter Berger  2   3
Affiliations

V体育安卓版 - The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation

Dina Baier et al. Pharmaceutics. .

Abstract

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG) VSports手机版. In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity. .

Keywords: 2-deoxy-d-glucose; BOLD-100/KP1339; ER stress; autophagy; chemotherapy resistance; colon cancer; glycolysis; lysosome; pancreatic cancer; ruthenium. V体育安卓版.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Whole genome gene expression profiling identifies transcriptional deregulations of cellular carbohydrate metabolism as a response to BOLD-100 and in acquired resistance against BOLD-100 in HCT116 and Capan-1 cells. (a) Cell viability of acquired BOLD100-resistant HCTR and CapanR cells as compared to their respective parental HCT116 and Capan-1 cells upon 72 h of treatment with increasing BOLD-100 concentrations, as determined by MTT assay. The statistical significance of the differences was determined using a two-way ANOVA with a Bonferroni post-test: * p < 0.05, *** p < 0.001. Asterisks are given above BOLD-100-resistant cells at the indicated concentrations and indicate the level of significance of a difference in comparison to parental cells. (b) ICP-MS analysis of whole cell lysates of indicated cells after 24 h of treatment with 100 µM BOLD-100. Statistical significance of differences was determined using a two-tailed unpaired Student´s t-test: no significant (ns) difference was detected. (c) GSEA of HCTR vs. HCT116 cells identifies “GALACTOSE_METABOLISM” (nominal p-value < E−7; FDR 0.010) and “FRUCTOSE_AND_MANNOSE_METABOLISM” (nominal p-value < E−7; FDR 0.084) as the third and fourth most significantly enriched gene sets in the KEGG database. Heat maps display the differentially regulated genes of the respective gene sets. (d) GSEA of CapanR vs. Capan-1 cells identifies “KEGG_PYRUVATE_METABOLISM” (nominal p-value 0.147; FDR 0.596) as the eighth and “KEGG_N_GLYCAN_BIOSYNTHESIS” (nominal p-value 0.193; FDR 0.703) as the tenth most significantly upregulated gene set. The heat map displays differentially regulated genes of the “KEGG_PYRUVATE_METABOLISM” gene sets. (e) mRNA regulation of SLC2A4, SLC2A9, and SLC2A12 of HCTR and CapanR as compared to respective parental counterparts. The bar on the right indicates fold-change (FC) values.
Figure 2
Figure 2
Acquired BOLD-100-resistant HCTR cells are characterized by enhanced glucose uptake and glycolytic activity, leading to increased intracellular pyruvate and decreased lactate levels. Flow cytometry analysis of fluorescence intensity of glucose-starved HCT116 and HCTR cells after 1 h of incubation with 25 µM 2-NBDG following treatment with solvent control (a) or 100 µM BOLD-100 (b) for 24 h. The results are the means of triplicates ± SD of two independent experiments. In (b) data are normalized to the respective control (dashed line). The statistical significance of differences was calculated with a two-tailed unpaired Student’s t-test (a) or a two-way ANOVA with Tukey’s multiple comparisons test (b): * p < 0.05, *** p < 0.001, **** p < 0.0001. (c) Gene-metabolite network of whole genome gene expression data computed together with metabolomics data of HCTR vs. HCT116 cells. Dots and hexagons indicate regulated genes or metabolites, respectively. Differences in the color intensity correspond to the strength of gene expression change and the green fringe indicates significant regulation. (d) Metabolomics of HCTR vs. HCT116 cells with 24 h of treatment with DMSO or 100 μM BOLD-100; all metabolites in pmol/μg protein, n = 6 biological replicates.
Figure 3
Figure 3
2-DG targets enhanced glycolytic activity in HCTR cells and synergizes with BOLD-100. (a) Cell viability assay of HCT116 and HCTR cells after 72 h of treatment with 2-DG relative to their respective controls (dashed line). Respective significance levels relative to the controls are indicated above the dashed line. The statistical significance of differences was determined using a two-way ANOVA with Tukey´s multiple comparisons test: * p < 0.05, **** p < 0.0001. The significance between respective treatment groups is given between the cell lines. (b) Cell viability assay of HCT116 vs. HCTR cells after 72 h of treatment with the glutaminase inhibitor BPTES relative to their respective controls (dashed line). The statistical significance of differences was determined using a two-way ANOVA with Tukey´s multiple comparisons test: * p < 0.05, ** p < 0.01; ns: non-significant. (c) Cell viability of indicated cells upon 72 h of treatment with BOLD-100 in combination with 2-DG at the indicated concentrations, as determined by MTT assay. One representative of three independent experiments is shown. (d) Combination indices (CI) based on the cell viability data from HCT116 and HCTR cells treated with BOLD-100 in combination with 2-DG at the indicated concentrations for 72 h. CI < 0.9, synergism; CI = 0.9–1.2, additive effects; or CI > 1.2, antagonism. (e) Crystal violet-stained clone formation assay showing clonogenic cell growth of HCT116 and HCTR cells treated with 50 µM BOLD-100, 0.5 mM 2-DG, or the combination of both compounds for seven days. For the solvent control, cells were treated with the amount of DMSO equivalent to the respective BOLD-100 samples. Quantification is shown in the lower panel. Results are normalized to the respective control (dashed line). The statistical significance of differences was determined with a two-way ANOVA with Tukey’s multiple comparisons test: * p < 0.05, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
Glucose deprivation by 2-DG differentially regulates ER stress and leads to apoptotic cell death upon combination with BOLD-100. (a) Representative composite images show morphological changes of HCT116 or HCTR cells detected with dual staining of Hoechst 33342/PI. Cells were treated for 72 h with DMSO (control, equivalent to BOLD-100), 2.5 mM 2-DG, 100 µM BOLD-100 or their combination and imaged by fluorescence microscopy (magnification 20×). Grey arrows indicate examples of mitotic nuclei. Green arrows indicate live cells with apoptotic nuclei. Red arrows indicate dead cells with late apoptotic nuclei. (b) Quantification of treatment-respective early and late apoptotic nuclei depicted in (a). Statistical significance of differences was calculated using a two-way ANOVA with Tukey´s multiple comparisons test: * p < 0.05, **** p < 0.0001; ns: non-significant. (c) Expression levels of GRP78, peIF2A (Ser51), eIF2A, HSP70, and PARP after 24 h of treatment of HCT116 and HCTR cells with the indicated concentrations of BOLD-100, 2-DG, or their combination, analyzed by Western blotting. Two different control states were included, i.e., medium control without DMSO for 2-DG and with DMSO as a solvent control for BOLD-100. β-actin served as the loading control. The numbers below indicate quantified Western blot signal intensities normalized to their respective controls: blue, medium; black, DMSO; and red, HCTR vs. HCT116 cells.
Figure 5
Figure 5
Survival and death from glucose deprivation by 2-DG is associated with differential regulation of the lysosomal compartment. (a) GSEA of HCTR vs. HCT116 cells identifies the “LYSOSOME” (nominal p-value < E−7; FDR 0.010) as the fifth most significantly enriched gene set in the KEGG database. The heat map displays differentially regulated genes of the respective gene set. (b) Flow cytometry analysis of fluorescence intensity of HCT116 and HCTR cells after 72 h or 24 h of treatment with DMSO, 100 µM BOLD-100, 10 mM 2-DG, or their combination, stained for 30 min with 0.5 µM Lysotracker red. The statistical significance of differences was calculated with a two-way ANOVA with Tukey´s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: non-significant. (c) Representative spinning-disc live cell fluorescence images (magnification 192×) of Lysotracker (red) and Hoechst 33342 (blue)-stained HCT116 and HCTR cells after 72 h or (d) 24 h of treatment with 100 µM BOLD-100, 10 mM 2-DG, or their combination.
Figure 6
Figure 6
2-DG differentially regulates autophagy depending on BOLD-100 sensitivity or resistance, and the combination synergistically disturbs the autophagic flux. (a) Expression levels of beclin-1, p62, LC3B, and LC3B I/II in HCT116 and HCTR cells treated with the indicated concentrations of BOLD-100, 2-DG, or their combination for 24 h, analyzed by Western blotting. Two different control states were included, i.e., without DMSO for 2-DG and with DMSO as a solvent control for BOLD-100. β-actin served as the loading control. The numbers below indicate the quantified Western blot signal intensities normalized to their respective controls: blue, medium; black, DMSO; and red, HCTR vs. HCT116 cells. (b) Representative spinning-disc live cell fluorescence images (magnification 192×) of Lysotracker (red) and Hoechst 33342 (blue)-stained HCT116 and HCTR cells after 24 h of treatment with 100 µM BOLD-100, 10 mM 2-DG, or their combination. (c) Quantification of LC3B signal of two independent Western blot experiments. (d) Calculation of the treatment-respective LC3B II/I ratio from protein expression detected in (a). (e) Cell viability of HCT116 and HCTR cells upon 72 h of treatment with CQ in combination with 2-DG at the indicated concentrations, determined by MTT assay. The statistical significance of differences was calculated with a two-way ANOVA with Tukey´s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: non-significant.
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