Capsaicin Enhances the Drug Sensitivity of Cholangiocarcinoma through the Inhibition of Chemotherapeutic-Induced Autophagy

Abstract

Cholangiocarcinoma (CCA), a devastating cancer with a poor prognosis, is resistant to the currently available chemotherapeutic agents. Capsaicin, the major pungent ingredient found in hot red chili peppers of the genus Capsicum, suppresses the growth of several malignant cell lines. Our aims were to investigate the role and mechanism of capsaicin with respect to the sensitivity of CCA cells to chemotherapeutic agents. The effect of capsaicin on CCA tumor sensitivity to 5-fluorouracil (5-FU) was assessed in vitro in CCA cells and in vivo in a xenograft model. The drug sensitivity of QBC939 to 5-FU was significantly enhanced by capsaicin compared with either agent alone. In addition, the combination of capsaicin with 5-FU was synergistic, with a combination index (CI) < 1, and the combined treatment also suppressed tumor growth in the CCA xenograft to a greater extent than 5-FU alone. Further investigation revealed that the autophagy induced by 5-FU was inhibited by capsaicin. Moreover, the decrease in AKT and S6 phosphorylation induced by 5-FU was effectively reversed by capsaicin, indicating that capsaicin inhibits 5-FU-induced autophagy by activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in CCA cells. Taken together, these results demonstrate that capsaicin may be a useful adjunct therapy to improve chemosensitivity in CCA. This effect likely occurs via PI3K/AKT/mTOR pathway activation, suggesting a promising strategy for the development of combination drugs for CCA.

Fig 1. The susceptibility of CCA cells to chemotherapeutics and capsaicin.

(A) Cytotoxic effects of common chemotherapeutics, including capsaicin, on three CCA cell lines. Cell viability was measured by MTT assay after treatment for 48 h. (B) Three CCA cell lines were treated with different concentrations of capsaicin for 24 h, 48 h, and 72 h. Cell viability was measured by MTT assay.

Fig 2. The effects of capsaicin in combination with 5-FU on QBC939 cells.

(A) Co-treatment with capsaicin and the autophagy inhibitor 3-MA enhanced the cytotoxic effect of 5-FU on QBC939 cells. Cell viability was measured by MTT assay after 48 h. Three drug combinations (RARA, 3-MA, and capsaicin) were compared to 5-FU at each treatment time, respectively. (B) Co-treatment with capsaicin increased 5-FU-induced apoptosis. Apoptosis was detected by Annexin V-FITC/PI staining after 48 h. 5-FU (40 μM), RARA (2 mM), 3-MA (1 mM), capsaicin (60 μM). (Capsaicin + 5-FU) vs. 5-FU. * P<0.05, ** P<0.01.

Fig 3. The cytotoxic effect of 5-FU in combination with capsaicin in vivo.

(A) Combination treatment of 5-FU and capsaicin significantly reduced the xenografted CCA tumor volume. (B) Combination treatment with 5-FU and capsaicin effectively suppressed CCA tumor growth. (C) Capsaicin increased 5-FU-induced apoptosis in vivo. Apoptosis was detected by TUNEL assay. * P<0.05, ** P<0.01.

Fig 4. 5-FU-induced autophagy was inhibited by capsaicin.

(A) Acridine orange staining showing lysosomal (red or orange) staining in the cells in the 5-FU treatment group. (B and D) The expression of beclin1 and atg5 mRNA was detected by real-time polymerase chain reaction (PCR) in vitro (B) and in vivo (D). (C and E) Western blot analysis of LC I/II protein expression exposed to different treatments in vitro (C) and in vivo (E). (F) Western blot analysis of the AKT/mTOR pathway in QBC939 cells exposed to different treatments after 24 h. GAPDH was used as a loading control. 5-FU (40 μM), capsaicin (40 μM), control/vehicle (PBS). * P<0.05, ** P<0.01.