br acyl CoA synthetase ACSL
acyl-CoA synthetase (ACSL), was also enhanced. Moreover, HCT-116 Doxorubicin treated with Bou dose and time dependently activated the SIRT1/AMPK axis as indicated by increases in the phosphorylation level of AMPKα (Thr-172) and its downstream target acetyl-CoA carboxylase (ACC) (Ser-89). The SIRT1 activity increased, resulting in a decrease in the total acetylation level in cells (Fig. 3A–C). Interestingly, Bou treatment increased the expression of UCP2 in a dose- and time-de-pendent manner.
Studies have revealed that activation of UCP2 induced a metabolic reprogramming in cancer cells (Esteves et al., 2014). To test the hy-pothesis that UCP2 plays an essential role in Bou-induced metabolic reprogramming and its anti-rectal cancer eﬀect, we transfected the UCP2 expression plasmid into HCT-116 cells. Overexpression of UCP2 resulted in the activation of the SIRT1/AMPK axis that was associated with SIRT1 activity enhanced (Fig. 3D and E). Meanwhile, over-expression of UCP2 in cells resulted in a decrease of ECAR and an in-crease of OCR (Fig. 3F and G). Notably, in these metabolic eﬀects, no major change was observed in the combination group of UCP2 trans-fection and Bou treatment compared with UCP2 transfection alone, suggesting that Bou induced metabolic reprogramming in HCT-116 cells was related to UCP2.
Apart from this, overexpression of UCP2 in HCT-116 cells sig-nificantly inhibited clone sphere formation after a 10 days incubation and also no major diﬀerence was observed in the combination group in comparison with UCP2 transfection alone (Fig. 3H).
3.4. Bou up-regulates UCP2 level through PGC-1α enrichment via de-acetylation
PGC-1α is a critical transcriptional coactivator, and studies have revealed that PGC-1α induces UCP1 expression because the UCP1 promoter contains a well-defined PGC-1α binding site that may un-derlie the induction of UCP1 (Pan et al., 2009). To explore whether Bou-activated UCP2 was associated with PGC-1α, we treated HCT-116 cells with Bou in the presence or absence of wild-type PGC-1α transfection. Both Bou treatment and PGC-1α transfection activated the expression of PGC-1α and UCP2, and an additional eﬀect of activating PGC-1α and UCP2 was observed in the presence of the PGC-1α plasmid (Fig. 4A and B). In addition, we used chromatin immunoprecipitation to study whether PGC-1α can be recruited to the UCP2 promoter region under the treatment of Bou. As expected, the signal robustly increased
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, cells 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-