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. 2022 Sep 11;18(15):5698-5712.
doi: 10.7150/ijbs.78345. eCollection 2022.

Nobiletin suppresses cholangiocarcinoma proliferation via inhibiting GSK3β

Liping You  1   2 , Jiacheng Lin  2 , Zhuo Yu  1 , Yihan Qian  2 , Yuting Bi  1   2 , Fang Wang  2 , Lei Zhang  1 , Chao Zheng  1 , Jinghao Zhang  1 , Wenxuan Li  1   2 , Yaxuan Cai  3 , Yueqiu Gao  1 , Xiaoni Kong  2 , Xuehua Sun  1
Affiliations

Nobiletin suppresses cholangiocarcinoma proliferation via inhibiting GSK3β

Liping You et al. Int J Biol Sci. .

Abstract

Background: Cholangiocarcinoma (CCA) is a type of hepatobiliary cancer characterized by uncontrolled cell proliferation, with a poor prognosis and high mortality. Nobiletin (NBT) is a promising anti-tumor compound derived from the peels of oranges and other citrus plants, citrus plant. But the effect of NBT on CCA remains unknown. Results: Our data showed that NBT suppressed CCA cell proliferation in vitro and in vivo. Colony formation and Edu assay indicated that NBT inhibited cell proliferation. Cell cycle analysis showed that NBT arrested the cell cycle in G0/G1 phase. Target prediction showed that GSK3β was a direct target. Western blot and immunofluorescence confirmed that NBT reduced the phosphorylation of GSK3β. The antiproliferative effect of NBT was intercepted in GSK3β knockdown CCA cells. The cellular thermal shift assay (CETSA) showed NBT directly bound to GSK3β. Finally, NBT showed an anti-proliferative effect in tumor-bearing mice with no hepatotoxicity. Conclusion: NBT could inhibit CCA proliferation, and the pharmacological activity of NBT in CCA was attributed to its direct binding to GSK3β. We suggested that NBT might be a potential natural medicine in CCA treatment.

Keywords: Cholangiocarcinoma; GSK3β; Nobiletin; natural medicine; β-catenin.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
NBT inhibited CCA cells proliferation. (a) The chemical structure of NBT. (b) The viability of primary hepatocyte was assessed after treatment with different doses of NBT for 48h. (c) The viability of TFK1 cells and RBE cells was assessed after treatment with different doses of NBT and at different times. (d) Representative cell morphological changes. (e) Representative results of annexin V/FITC/PI staining and quantitative analysis, *p< 0.05, **p< 0.01. (f) Monolayer culture; quantitative analyses of colony numbers are shown, *p< 0.05, **p< 0.01. (g) Representative results of EdU staining and quantitative analyses, *p< 0.05, **p< 0.01.
Figure 2
Figure 2
NBT induced CCA cells cycle arrest in G0/S1. (a) Representative results of cell cycle and quantitative analyses after 48 hours of NBT treatment. (b, c), *p< 0.05, **p< 0.01. (D) The expression of G0/G1 cell cycle signal regulators, Cyclin D1 and CDK4 were examined by western blot after NBT treatment for 24 hours and 48 hours.
Figure 3
Figure 3
Prediction and validation of NBT suppression CCA cell growth. (a) The intersection of target proteins of NBT and CCA. (b) Representative results of p-GSK3β, GSK3β, active β-catenin, β-catenin, p-JNK1, and JNK1 protein levels by Western blot analysis after TFK1 and RBE cells were treated with NBT (0, 50, 100 µM) for 24 hours and 48 hours. (c) Protein levels of ESR1 by Western blot analysis.
Figure 4
Figure 4
The biological function of tumor cells was significantly inhibited after GSK3β knockdown. (a) GSK3β mRNA levels of tumor cells after GSK3β knockdown by siRNA. (b) GSK3β protein levels of tumor cells after GSK3β knockdown by siRNA. (c) The cell viability of tumor cells after GSK3β knockdown by CCK-8 assay, *p< 0.05, **p< 0.01, # p< 0.05, ## p< 0.01. (d) The proliferation level of tumor cells after GSK3β knockdown by EdU staining, *p< 0.05, **p< 0.01. (e) Cell cycle analysis after GSK3β knockdown, *p< 0.05, **p< 0.01.
Figure 5
Figure 5
The biological function of tumor cells remained after JNK1 knockdown. (a) JNK1 mRNA levels of tumor cells after JNK1 knockdown by siRNA. (b) JNK1 protein levels of tumor cells after JNK1 knockdown by siRNA. (c) The cell viability of tumor cells after JNK1 knockdown by CCK-8 assay. (d) The proliferation level of tumor cells after JNK1 knockdown by EdU staining, *p< 0.05, **p< 0.01. (e) Cell cycle analysis after JNK1 knockdown, *p< 0.05, **p< 0.01.
Figure 6
Figure 6
NBT reduced the nuclear shift of β-catenin by targeting GSK3β. (a, b) Molecular docking showed the NBT has a high affinity on GSK3β and the binding pose located on the active pocket which could affect the phosphorylation of GSK3β. (c) The illustration of the CETSA following quantification of the Western blots. (d) Localization of active β-catenin in TFK1 and RBE cells treated with NBT 50 µm for 24 hours or vehicle detected by immunofluorescence staining. (e) Protein expression of β-catenin in TFK1 and RBE cells treated with NBT or vehicle determined in nuclear fractions by Western blot. Lamin B1 was used as nuclear protein control.
Figure 7
Figure 7
Anti-tumor function of NBT on CCA in vivo. (a) Scheme of CCA inoculation and systemic injection. (b) Representative image of TFK1 xenograft tumors in each group. (c) Tumor volume in each group. Data were expressed as the means ± standard deviations (SDs). (d) Tumor weight in each group. Data were expressed as the means ± standard deviations (SDs). *p< 0.05, **p< 0.01. (e) Immunohistochemical staining of Ki67and TUNEL assay in tumors from the vehicle or NBT-H treated mice and Ki67 quantitative analysis, *p< 0.05, **p< 0.01. (f) The expression of p-GSK3β, GSK3β, Cyclin D1 and active β-catenin were detected by western blot.
Figure 8
Figure 8
Bodyweight, ALT, and H&E staining of xenograft tumor sections. (a) Bodyweight changes between each group. (b) ALT changes between control and NBT-H treated mice. (c) H&E staining of major organs.
Figure 9
Figure 9
Schematic diagram showing the mechanism of NBT inhibiting proliferation in CCA.
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