br Results br Bou reduces the viability of rectal cancer
3.1. Bou reduces the viability of rectal cancer cells without apoptosis
HCT-116 cells treated with Bou dose dependently decreased cell viability, with an IC50 value of 12.1 μM after 24 h of incubation
(Fig. 1A). In comparison, there was no detectable eﬀect of Bou on the cell viability of normal cells, such as HEK-293 cells (Fig. 1B). Next, we determined the inhibition eﬀect of Bou on proliferation activity in HCT-116 cells. Cells treated with Bou eﬃciently inhibited its proliferation activity after a 5-day incubation (Fig. C). This inhibition eﬀect did not involve in cytotoxicity (increased release of LDH into culture medium) (Fig. 1D). This inhibition eﬀect was further produced in SW480, SW620 and LOVO rectal cancer cell lines (Fig. 1E–G), and among the four rectal cancer cell lines, Bou treatment showed a stronger inhibition against HCT-116 cells. Consistent with the proliferation inhibition, HCT-116 cells treated with Bou inhibited clone sphere expansion after a 10-day cultured and arrested Nivolumab at both the G0/G1 and G2/M phases (Fig. 1H and I). Importantly, these inhibition eﬀects did not occur with cell apoptosis (Fig. 1G). r> 3.2. Bou reprograms glycolysis toward to aerobic oxidation
Incubation of HCT-116 cells with Bou dose dependently increased glucose uptake (Fig. 2A). Then the metabolic eﬀects of Bou were evaluated using an extracellular Flux Analyzer that allows the mea-surement of the ECAR and OCR in real time. As expected, Bou in-cubation showed a lower level of ECAR and an increase in OCR (Fig. 2B and C). This improved eﬀect was associated with an increase of mi-tochondrial copies (Fig. 2D and E) and oxygen consumption capacity in mitochondria of Bou-treated cells (Fig. 2F). Consistency, the activities of citrate synthase (CS), mitochondrial complexes I and II was also elevated in HCT-116 cells (Fig. 2G–I). In keeping with an increase in oxidative capacity, cells treated with Bou showed a decrease in the level of incomplete oxidation products of mitochondria, such as re-active oxygen species and malondialdehyde (MDA) (Fig. 2J).
3.3. Bou reprograms energy metabolism in HCT-116 cell is closely related to UCP2
Western blot assays revealed that Bou treatment induced an acti-vation of a range of key proteins indicative of the regulation of mi-tochondrial biogenesis and oxidative capacity in HCT-116 cells (Fig. 3A and B). The expression of the key mitochondrial biogenesis regulator PGC-1α was strongly increased with the treatment of Bou. The ex-pression of proteins indicative of mitochondrial oxidative capacity, such as carnitine palmityl transferase 1 beta (CPT-1β) and long-chain
acyl-CoA synthetase (ACSL), was also enhanced. Moreover, HCT-116 cells 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