Counteracting Colon Cancer by Inhibiting Mitochondrial Respiration and Glycolysis with a Selective PKCδ Activator

Abstract

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

Keywords: OXPHOS; PKCδ; Roy-Bz; anticancer agent; glycolysis.

Figure 1

Schematic representation of the molecular mechanism of action of Roy-Bz-induced apoptosis. Roy-Bz binds to the PKCδ-C1 domain, inducing its translocation to the perinuclear region. In the nucleus, PKCδ is cleaved by caspase-3, resulting in the generation of the PKCδ catalytic fragment (PKCδ-CF), which in turns phosphorylates nuclear substrates, such as histone H3 on Ser-10, that promotes apoptosis. Additionally, it triggers the transcription of apoptotic genes, namely TP53 and BAX, thus promoting a mitochondrial apoptotic pathway, with subsequent cytochrome c (cyt c) release, activation of caspase-3, PARP cleavage, and ultimately apoptosis induction.

Figure 2

Roy-Bz has cytotoxic effect on colon cancer cells by decreasing their metabolic activity. (A,B) HCT116 cells were treated with increasing concentrations (0.8 to 4.0 μM) of Roy-Bz or vehicle (DMSO), for 48 h. In (A), the dose-response curve for the growth of HCT116 cells was obtained from the SRB assay (IC₅₀ value of 1.65 ± 0.15 μM). Data are mean ± SEM of four independent experiments; growth obtained with vehicle was set as 100%. In (B), metabolic activity was determined by resorufin fluorescence measurement after 360 min incubation, using resazurin assay. Data are expressed as percentage of vehicle and are mean ± SEM of three independent experiments. Statistical analysis was carried out with a one-way ANOVA test followed by a Dunnett’s post hoc test; *** significantly different from control (cells treated with vehicle) with p < 0.001.

Figure 3

Roy-Bz inhibits cellular respiration in colon cancer cells. HCT116 cells were previously treated with 1.65 μM Roy-Bz or vehicle (control), except in (G) where they were treated with 1.65 and 3.30 μM Roy-Bz, for 48 h. (A) OCR were assessed with a Mito Stress Test using a Seahorse XF96 Analyzer, under basal conditions followed by sequential administration of 1 μM oligomycin, 0.5 μM FCCP, and 1 μM rotenone/1 μM antimycin A mix during the measurement. (B–F) Bioenergetics parameters, including (B) non-mitochondrial respiration (minimum rate measurement after rotenone/antimycin A injection), (C) proton leak associated OCR ((minimum rate measurement after oligomycin addition)-(non-mitochondrial respiration)), (D) ATP production associated OCR ((last rate measurement before oligomycin injection)-(minimum rate measurement after oligomycin injection)), (E) maximal respiration ((maximum rate measurement after FCCP injection)-(non-mitochondrial respiration)), (F) spare respiratory capacity ((maximal respiration)-(basal respiration)). Data are mean ± SEM of at least 4 independent experiments; values significantly different from control (* p < 0.05), unpaired Student’s t-test. (G,H) Expression levels of specific subunits of mitochondrial complexes I–V and mitochondrial-related proteins, respectively. Immunoblots represent one of the three independent experiments; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 4

Roy-Bz inhibits cellular respiration in tumor tissues of human colon xenograft mouse models. (A) Representative images of IHC of mitochondrial markers (COX4 and TOM20) detected in tumor tissues of HCT116 xenografts treated with 10 mg/kg Roy-Bz or vehicle (control) and collected at the end of the treatment (scale bar = 5 μm; magnification = ×200; hematoxylin and eosin (H&E)). (B) Quantification of IHC staining of HCT116 xenograft tumor tissues was assessed by evaluation of DAB intensity. Data are mean ± SEM; values significantly different from control (*** p < 0.001, **** p < 0.0001), unpaired Student’s t-test.

Figure 5

Roy-Bz inhibits the glycolytic pathway capacity in colon cancer cells. HCT116 cells were treated with 0.5 μM Roy-Bz or vehicle (DMSO), for 48 h. (A) ECAR curves were assessed with a Glycolysis Stress Test using a Seahorse XF24 Analyzer, under basal conditions followed by sequential injection of 10 mM glucose, 1 µM oligomycin, and 50 mM 2-DG during the measurement. (B–D) Bioenergetic parameters, including (B) glycolysis (maximal measurement after the addition of saturating amounts of glucose minus the measurement after adding 2-DG), (C) glycolytic reserve (maximal measurement following the addition of oligomycin minus maximal measurement before adding oligomycin), and (D) maximum glycolytic capacity (maximal measurement following the addition of oligomycin minus measurement after adding 2-DG). Data are mean ± SEM of at least 4 independent experiments; values significantly different from control (* p < 0.05, *** p < 0.001), unpaired Student’s t-test.

Figure 6

Roy-Bz reduces the levels of proteins involved in glycolysis in tumor tissues of human colon xenograft mouse models. (A) Representative images of IHC of glycolytic markers (GLUT1, HK2, MCT4, and TIGAR) detected in tumor tissues of HCT116 xenografts treated with 10 mg/kg Roy-Bz or vehicle (control) and collected at the end of the treatment (scale bar =5 μm; magnification = ×400; H&E). (B) Quantification of immunohistochemistry staining of HCT116 xenograft tumor tissues was assessed by evaluation of DAB intensity. Data are mean ± SEM; values significantly different from control (* p < 0.05, ** p < 0.01, *** p < 0.001), unpaired Student’s t-test.