The online version of this article (https://doi.org/10.1186/s13046-017-0661-7) contains supplementary material, which is available to authorized users.
Chemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells’ fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu’s cytotoxicity in vitro and in vivo.
Cell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice.
Curcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells.
Pre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.
Additional file 1: Figure S1. Viability of colon cancer HT29 cells treated with 5-Fu or curcumin, alone or in different combinations. A. Growth-inhibitory curves of HT29 cells exposing to gradient concentrations of 5-Fu or curcumin alone: (a) and (b) for 24 h, and (c) and (d) 48 h, respectively. B. Comparison of viabilities in HT29 cells treated for 48 h with different combinational protocols. Cur: curcumin alone, 5-Fu: 5-Fu alone, Pre-Cur: pretreated with curcumin for 24 h followed by 5-Fu for 24 h, Cur + 5-Fu: co-treated with 5-Fu and curcumin for 24 h, Pre-5-Fu: pretreated with 5-Fu for 24 h followed by curcumin for 24 h: (a) 10 μM curcumin/10 μM 5-Fu, (b) 10 μM curcumin/20 μM 5-Fu, (c) 20 μM curcumin/10 μM 5-Fu and (d) 20 μM curcumin and 20 μM 5-Fu. (PDF 148 kb)
Additional file 2: Figure S2. Images of colon carcinoma cells HCT116. (A) HCT116 cells were treated with 5-Fu for 24 h and 48 h, respectively. (B) HCT116 cells were treated with solvent for 48, pretreated with solvent for 24 h and then 20 μM 5-Fu for 24 h, pretreated with 20 μM Cur for 24 h and then 20 μM 5-Fu for 24 h, respectively. (PDF 441 kb)
Additional file 3: Figure S3. Western blot analysis of p62 and LC3 II/I in HT29 cells after exposing to varied concentrations of 5-Fu for 24 h. *, p < 0.05 and **, p < 0.01 compared to the vehicle (0 μM 5-Fu) cell group. (PDF 186 kb)
Additional file 4: Figure S4. Western blot analysis of Beclin-1, p62, LC3 II/I,P-AMPK and P-ULK1 in HT29 cells pretreated with varied concentrations of curcumin for 24 h and then 20 μM of 5-Fu for 24 h. *, p < 0.05 and **, p < 0.01 and ***, p < 0.001 compared to the placebo (0 μM curcumin) cell group. (PDF 218 kb)
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- Curcumin synergizes with 5-fluorouracil by impairing AMPK/ULK1-dependent autophagy, AKT activity and enhancing apoptosis in colon cancer cells with tumor growth inhibition in xenograft mice
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