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. 2020 Jan;9(2):783-796.
doi: 10.1002/cam4.2719. Epub 2019 Nov 26.

Antitumor activity of celastrol by inhibition of proliferation, invasion, and migration in cholangiocarcinoma via PTEN/PI3K/Akt pathway

Biqiang Zhu  1   2 , Yunwei Wei  1   2
Affiliations

Antitumor activity of celastrol by inhibition of proliferation, invasion, and migration in cholangiocarcinoma via PTEN/PI3K/Akt pathway

Biqiang Zhu et al. Cancer Med. 2020 Jan.

Abstract

Aim: Cholangiocarcinoma is a malignant tumor originating from bile duct epithelium. Currently, the treatment strategy is very limited and the prognosis is poor. Recent studies reported celastrol exhibits antigrowth and antimetastasis properties in many tumors. Our study aimed to assess the anti-CCA effects of cholangiocarcinoma (CCA) and the mechanisms involved in it.

Methods: In this study, the long-term and short-term antiproliferation effects was determined using colony formation and Cell Counting Kit-8 (CCK-8) assays, respectively. Flow cytometry was performed to quantify apoptosis. Furthermore, wound healing and transwell assays were performed to determine the cell migration and invasion capabilities, respectively. To further find the mechanism involved in the celastrol-induced biological functions, LY204002, a PI3K/Akt signaling inhibitor, and an Akt-1 overexpression plasmid were employed to find whether PI3K/Akt pathway was involved in the celastrol-induced CCA cell inhibition. Additionally, short interfering RNA (siRNA) was also used to investigate the mechanism involved in the celastrol-induced PI3K/Akt signaling inhibition. Western blotting and immunofluorescence assays were also performed to detect the degree of relative proteins. Moreover, we validated the antiproliferation and antimetastasis effects of celastrol in vivo by constructing subcutaneous and lung metastasis nude mice models.

Results: We discovered that celastrol effectively induced apoptotic cell death and inhibited the capacity of migration and invasion in CCA cells. Further mechanistic study identified that celastrol regulated the PI3K/Akt signaling pathway, and the antitumor efficacy was likely due to the upregulation of PTEN, a negative regulator of PI3K/Akt. Blockage of PTEN abolished the celastrol-induced PI3K/Akt signaling inhibition. Additionally, in vivo experiments conformed celastrol inhibited the tumor growth and lung metastasis with no serious side effects.

Conclusions: Overall, our study elucidated a mechanistic framework for the anti-CCA effects of celastrol via PTEN/PI3K/Akt pathway.

Keywords: Akt; apoptosis; celastrol; epithelial-to-mesenchymal transition; phosphatase and tensin homolog.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effects of celastrol on CCA cells viability. A, The chemical structure of celastrol. B and C, TFK‐1 and HuCCT‐1 cells were treated with celastrol (0, 5, 10, 20, or 40 μmol/L) for indicated time (24, 48, or 72 h). The cell viability was analyzed using CCK‐8 assay. D and E, The numbers of colonies were counted. *P < .05, **P < .01, or ***P < .001 vs control group
Figure 2
Figure 2
Celastrol‐induced CCA cell apoptosis. Cells were incubated with celastrol (0, 20, or 40 µmol/L) for 24 h. A, The apoptotic effect was analyzed via flow cytometry. B and C, Western blotting was performed to measure the degree of Bax, Bcl‐2, cleaved Caspase3, cleaved Caspase9, and Survivin. *P < .05, **P < .01 or ***P < .001 vs control group
Figure 3
Figure 3
Celastrol inhibits CCA cells migration and invasion. Cells were treated with certain concentrations of celastrol. A, Wound healing assays were performed to determine the wound closure rate after cell layer was scratched. The micrographs represent cell layers before and after scratches, respectively. (200 × magnification). B, The invasive ability was examined by transwell chamber assay. The micrographs represent invasive cells on the other side of the membrane. (200 × magnification). C, Represents the statistical results of wound healing and invasion assays. D, Shows the effect of celastrol on morphological changes. E, Shows the analysis of MMP‐2, MMP‐9, E‐Cadherin, and vimentin by western blot. *P < .05 vs control group
Figure 4
Figure 4
Celastrol‐induced PI3K/Akt signaling pathways blockage. A, Representative images of relative proteins expression in response to celastrol treatment in TFK‐1 cells by western blotting assay. B, p‐Akt levels were assessed using fluorescence microscopy. Original magnification: 400×. C, TFK‐1 cells were divided into Akt overexpression and low‐expression groups by cell transfection and LY294002 treatment at different celastrol concentrations. Cell viability in each group is on shown. D, Related proteins were assessed by western blotting in each group. *P < .05, **P < .01 or ***P < .001 vs control group. ###P < .001 vs control group. &&& P < .001 vs control group. ¢ P < .05 or ¢¢¢ P < .001 vs control group
Figure 5
Figure 5
Celastrol elevates PTEN expression in CCA cells. Following indicated concentrations of celastrol treatment, A, PTEN expression was evaluated via western blotting in TFK‐1 cells. B, PTEN levels were assessed using fluorescence microscopy in TFK‐1 cells. Original magnification: 400×. C and D, After specific siRNA‐PTEN transfection in the TFK‐1 cells, related proteins were evaluated via western blotting. *P < .05 or **P < .01 vs control group
Figure 6
Figure 6
Celastrol inhibited CCA cells growth and metastasis in vivo A, Tumors were extracted from the two groups and the photos are shown. B, Represents the changes of tumor volume. C, Represents tumor weight's statistical analysis. D, Incidence of lung metastasis and numbers of metastatic nodules per lung are shown. E, Representative photos of lung metastatic nodules (100 × magnification) are shown. F, Statistical analysis of body weight. G, Related proteins expression was analyzed. *P < .05 or **P < .01 vs control group
Figure 7
Figure 7
Immunohistochemical characterization of tumor tissues. A, Immunohistochemical analyses of Ki 67, cleaved caspase 3, vimentin, p‐AKT, and PTEN. Note the proliferation activity in tissue by Ki67‐positive tumor cells, apoptosis by cleaved caspase‐3, and EMT by vimentin (200 × magnification). B, The levels of relative proteins were analyzed. *P < .05 or **P < .01 vs control group
Figure 8
Figure 8
Diagram illustrating the proposed effects of celastrol on PI3K/Akt/mTOR and NF‐κB signaling pathways
See this image and copyright information in PMC

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