Triptolide induces apoptotic cell death of human cholangiocarcinoma cells through inhibition of myeloid cell leukemia-1

Abstract

Background: Cholangiocarcinoma (CCA), a devastating neoplasm, is highly resistant to current chemotherapies. CCA cells frequently overexpress the antiapoptotic protein myeloid cell leukemia-1(Mcl-1), which is responsible for its extraordinary ability to evade cell death. Triptolide, a bioactive ingredient extracted from Chinese medicinal plant, has been shown to inhibit cell proliferation and induce apoptosis in several cancers.

Methods: CCK-8 assay was performed to detect cell survival rate in vitro. DAPI staining and Flow cytometry were used to analyze apoptosis. Western blot was performed to determine the expression levels of caspase-3, caspase-7, caspase-9, PARP, and Mcl-1. Quantitative real-time PCR and immunofluorescence were used to detect the expression levels of Mcl-1. The nude mice xenograft model was used to evaluate the antitumor effect of triptolide in vivo.

Results: Triptolide reduced cell viability in cholangiocarcinoma cell lines in a dose- and time-dependent manner, with IC50 values of 12.6 ± 0.6 nM, 20.5 ± 4.2 nM, and 18.5 ± 0.7 nM at 48 h for HuCCT1, QBC939, and FRH0201 respectively. Triptolide induced apoptosis in CCA cell lines in part through mitochondrial pathway. Using quantitative real-time PCR, western blot and immunofluorescence, we have shown that triptolide downregulates Mcl-1 mRNA and protein levels. Furthermore, triptolide inhibited the CCA growth in vivo.

Conclusions: Triptolide has profound antitumor effect on CCA, probably by inducing apoptosis through inhibition of Mcl-1. Triptolide would be a promising therapeutic agent for CCA.

Figure 1

Triptolide inhibited cell survival of CCA cells. (A-C) HuCCT1 (A), QBC939 (B), and FRH0201 (C) cells were incubated with triptolide (0–200 nM) for 24 and 48 h. Cell viability was evaluated by CCK-8 assay.

Figure 2

Triptolide induced apoptosis of CCA cells. (A) DAPI staining (original magnification, ×200) was used to determine the apoptosis. Arrows indicate cells with nuclear condensation and fragmentation. (B-D) HuCCT1 (B), QBC939 (C), and FRH0201 (D) cells were incubated with triptolide (0-100 nM) for 24 h and Annexin V levels were assessed by flow cytometry. *p < 0.05, ***p < 0.001, compared with control.

Figure 3

Triptolide induced caspase activation in CCA cells. (A-C) Caspase-3 activity in CCA cells were analyzed after treatment with indicated concentrations of triptolide for 12 h. Data are presented as fold increases as compared with control cells. (D) Protein expression of caspase-3 and caspase-7 were analyzed by western blot after treatment with triptolide (100 nM) for 24 h. (E) Protein expression of caspase-9 was analyzed by western blot after treatment with triptolide (100 nM) for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control.

Figure 4

Triptolide inhibited Mcl-1 mRNA expression in HuCCT1 and QBC939 cells. (A-B) Triptolide (25–100 nM) significantly reduced Mcl-1 mRNA expression (as assessed by real-time PCR) in both HuCCT1 (A) and QBC939 (B) cells. Expression of Mcl-1 was normalized against the housekeeping gene β-actin. ***p < 0.001, compared with control.

Figure 5

Triptolide time- and dose-dependently inhibited Mcl-1 protein expression and induced PARP cleavage in CCA cells. (A) Cells were treated with 100 nM triptolide for indicated times or (B) with indicated concentrations of triptolide for 12 h. Whole cell extracts were prepared and analyzed using indicated antibodies. β-actin served as a loading control. (C) HuCCT1 and QBC939 cells were treated with 100 nM triptolide for 24 h and then analyzed for the expression of Mcl-1 by immunofluorescence (original magnification, ×200).

Figure 6

Triptolide inhibited CCA cell growth in vivo. HuCCT1 cells were injected subcutaneously into the left flanks of athymic nude mice. When tumors reached a size of approximately 150 mm³, mice were i.p. with triptolide (0.2 mg/kg) or vehicle control every day for a total of 22 days. (A) Tumor volume was measured every three days using calipers and calculated as described in Materials and Methods (n = 5). (B) The mouse weight was measured every three days to monitor the drug effects. (C) At the end of the study, the excised tumors from each group were weighed. (D) Resected tumors from each group were photographed. (E) H & E and Ki67 staining in the tumor tissues of triptolide and vehicle treated mice (original magnification, ×200). Arrows show the Ki67 expression in tumor tissue. *p < 0.05, **p < 0.01, compared with control.