br Fig Bou reprograms glycolysis
Fig. 2. Bou reprograms glycolysis toward aerobic oxidation in HCT-116 cells. (A) Glucose uptake assay. (B–C) ECAR and OCR level. (D) mtDNA/nDNA. (E) Representative image of TEM, Original magnification, 5000 × . (F) Oxygen consumption in mitochondria isolated from HCT-116 cells. (G) Citrate synthase (CS) activity. (H–I) Mitochondrial complexes (I and II) activity. (J) MDA and reactive oxygen species level. *P < 0.05, **P < 0.01, vs control (Ctrl) group. Data were expressed as Mean ± S.E.M. from 4 independent experiments.
with Bou treatment. Bou stimulation enriched PGC-1α concentration at the UCP2 promoter region by more than two fold when compared with the control group (Fig. 4C).
SIRT1 is a key energy sensor involved in maintaining energy homeostasis, which regulates PGC-1α level via de-acetylating PGC-1α (Nemoto et al., 2005). As reported, immunoprecipitation of PGC-1α from cell lysate revealed that Bou treatment increased the level of PGC-1α as well as the coimmunoprecipitation (CoIP) between proteins SIRT1 and PGC-1α. Importantly, BMS-936558 treated with Bou had decreased total acetylation and PGC-1α acetylation levels in parallel with a
weakened interaction between the proteins SIRT1 and PGC-1α (Fig. 4D). Moreover, we transfected wild-type SIRT1 and SIRT1H355A mutant plasmids (without enzymatic activity) into HCT-116 cells in the presence of PGC-1α. As noted in Fig. 4E–H, expression of wild-type SIRT1 but not the SIRT1H355A mutant reduced PGC-1α acetylation le-vels in association with an increase in SIRT1 de-acetylation activity and a corresponding increase in PGC-1α and UCP2 transcriptional activity, while an opposite eﬀect was observed in SIRT1H355A cells. Interestingly, cells co-treated with Bou and wild-type SIRT1 showed more potent increases in PGC-1α transcriptional activity as well as SIRT1 de-
acetylation activity. Conversely, SIRT1H355A mutant induction abol-ished the stimulation eﬀect of Bou on PGC-1α and UCP2.
Moreover, the SIRT1 inhibitor nicotinamide (NAM) treatment markedly decreased PGC-1α transcriptional activity and thoroughly abolished the stimulation eﬀect of Bou (Fig. 4I and J). Western blot analysis revealed that NAM treatment increased the acetylated level of PGC-1α and down-regulated PGC-1α level in HCT-116 cells. These ef-fects were also observed in Bou-treated cells. NAM treatment thor-oughly abolished the stimulation of Bou to a similar level to that of NAM treatment alone (Fig. 4K), suggesting that Bou treatment medi-ated PGC-1α activation depends on de-acetylation.
3.5. Bou inhibits rectal tumor growth in a xenograft mouse model without toxicity
To examine the eﬀects of Bou on rectal cancer in vivo, HCT-116 cells
were implanted subcutaneously into the dorsal flank of BALB/nude mice followed by administration of Bou (50 mg/kg, i.p., every other day) for 16 days. The administration of Bou significantly inhibited rectal tumor growth (by 31%) and reduced tumor weight (by 37%) with a slight decrease in body weight (Fig. 5A-B), this may due to its body weight-decreasing eﬀect as our previous study . Be noted, the rectal cancer-induced liver injury was attenuated in Bou-treated mice as measured by H&E staining (Fig. 5C).
To understand the mechanism underlying the therapeutic eﬀects of Bou on tumorigenesis in vivo, we conducted immunohistochemistry in the tumor tissue to examine the level of Ki-67 as a biomarker of cell proliferation. As expected, the expression level of Ki-67 was sig-nificantly decreased in Bou-treated mice compared with control mice (Fig. 5C). Moreover, mice treated with Bou increased mitochondria content and oxidation capacity of mitochondria as indicated by en-hanced mitochondrial complex I activity and increased oxygen
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Fig. 4. Bou upregulates UCP2 level through enrichment PGC-1α via de-acetylating. HCT-116 cells were transfected with a PGC-1α luciferase promoter construct (PGC-1α-Luc) and an UCP1 construct (UCP2-Luc) followed by stimulation with Bou (25 μM) for 24 h. (A) PGC-1α luciferase activity. (B) UCP2 luciferase activity determination under the stimulation of PGC-1α or Bou (25 μM) treatment. Normalized luciferase activities are shown as fold change. (C) PGC-1α recruitment to the UCP2 promoter region in Bou-treated HCT-116 cells analyzed by ChIP. Fold enrichment are given. N = 5 per condition. (D) SIRT1 directly interacted with protein PGC-1α as analyzed by CoIP. (E–G) Cells were transfected with wild type SIRT1 or SIRT1 mutant (SIRT1H355A) under the treatment of Bou (25 μM) for 24 h. (E) SIRT 1 activity. (F–G) PGC-1α and UCP2 luciferase activity assay. Normalized luciferase activities are shown as fold change. (H) SIRT1 directly interacted with protein PGC-1α as analyzed by CoIP. (I–K) HCT-116 cells were treated with NAM (10 μM) and Bou (25 μM) alone or together for 24 h. (I) SIRT 1 activity. (J) PGC-1αluciferase activity assay. Normalized luciferase activities are shown as fold change. (K) Expression of PGC-1α, UCP2 and acetylated PGC-1α. *P < 0.05, **P < 0.01, compared with the control (Ctrl) group. #P < 0.05, compared with the Bou-treated group. Data were expressed as Mean ± S.E.M. from 4 independent experiments.