Lanatoside C decelerates proliferation and induces apoptosis through inhibition of STAT3 and ROS-mediated mitochondrial membrane potential transformation in cholangiocarcinoma

Chao Zhang; Hong-Ying Yang; Long Gao; Ming-Zhen Bai; Wen-Kang Fu; Chong-Fei Huang; Ning-Ning Mi; Hai-Dong Ma; Ya-Wen Lu; Ning-Zu Jiang; Liang Tian; Teng Cai; Yan-Yan Lin; Xing-Xing Zheng; Kun Gao; Jian-Jun Chen; Wen-Bo Meng

doi:10.3389/fphar.2023.1098915

Lanatoside C decelerates proliferation and induces apoptosis through inhibition of STAT3 and ROS-mediated mitochondrial membrane potential transformation in cholangiocarcinoma

Front Pharmacol. 2023 Jun 15:14:1098915. doi: 10.3389/fphar.2023.1098915. eCollection 2023.

Authors

Chao Zhang^{1

2}, Hong-Ying Yang³, Long Gao^{1

4}, Ming-Zhen Bai^{1

4}, Wen-Kang Fu^{1

4}, Chong-Fei Huang^{1

4}, Ning-Ning Mi^{1

4}, Hai-Dong Ma^{1

4}, Ya-Wen Lu^{1

4}, Ning-Zu Jiang^{1

4}, Liang Tian^{1

4}, Teng Cai^{1

4}, Yan-Yan Lin^{1

4}, Xing-Xing Zheng⁵, Kun Gao³, Jian-Jun Chen³, Wen-Bo Meng^{1

4}

Affiliations

¹ The First Clinical Medical College, Lanzhou University, Lanzhou, Gansu, China.
² Department of Orthopedics, The First Hospital of Lanzhou University, Lanzhou, Gansu, China.
³ State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, China.
⁴ Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, Gansu, China.
⁵ Department of Ophthalmology, The Second Hospital of Lanzhou University, Lanzhou, Gansu, China.

Abstract

Introduction: The incidence of cholangiocarcinoma (CCA) has increased worldwide in recent years. Given the poor prognosis associated with the current management approach of CCA, new therapeutic agents are warranted to improve the prognosis of this patient population. Methods: In this study, we extracted five cardiac glycosides (CGs) from natural plants: digoxin, lanatoside A, lanatoside C, lanatoside B, and gitoxin. Follow-up experiments were performed to assess the effect of these five extracts on cholangiocarcinoma cells and compounds with the best efficacy were selected. Lanatoside C (Lan C) was selected as the most potent natural extract for subsequent experiments. We explored the potential mechanism underlying the anticancer activity of Lan C on cholangiocarcinoma cells by flow cytometry, western blot, immunofluorescence, transcriptomics sequencing, network pharmacology and in vivo experiments. Results: We found that Lan C time-dependently inhibited the growth and induced apoptosis of HuCCT-1 and TFK-1 cholangiocarcinoma cells. Besides Lan C increased the reactive oxygen species (ROS) content in cholangiocarcinoma cells, decreased the mitochondrial membrane potential (MMP) and resulted in apoptosis. Besides, Lan C downregulated the protein expression of STAT3, leading to decreased expression of Bcl-2 and Bcl-xl, increased expression of Bax, activation of caspase-3, and initiation of apoptosis. N-acetyl-L-cysteine (NAC) pretreatment reversed the effect of Lan C. In vivo, we found that Lan C inhibited the growth of cholangiocarcinoma xenografts without toxic effects on normal cells. Tumor immunohistochemistry showed that nude mice transplanted with human cholangiocarcinoma cells treated with Lan C exhibited decreased STAT3 expression and increased caspase-9 and caspase-3 expression in tumors, consistent with the in vitro results. Conclusion: In summary, our results substantiates that cardiac glycosides have strong anti-CCA effects. Interestingly the biological activity of Lan C provides a new anticancer candidate for the treatment of cholangiocarcinoma.

Keywords: Lanatoside C; apoptosis; cholangiocarcinoma; mitochondrial membrane potential; reactive oxygen species; stat3.

Grants and funding

This work was supported by Grant(s) from the National Natural Science Foundation of China (82060551 to W-BM, 81973193 to J-JC, 22177044 to KG), Lanzhou Science and Technology Development Guiding Plan Project (2020-ZD-79 to CZ), Foundation of Lanzhou University: Izuyxcx-2022-157, The Health Industry Scientific Research Program of Gansu Province (GSWSKY 2020-11 to Y-YL).