Pharmacological Activity of Matrine in Inhibiting Colon Cancer Cells VM Formation, Proliferation, and Invasion by Downregulating Claudin-9 Mediated EMT Process and MAPK Signaling Pathway

Qiu Du; Yingda Lin; Changping Ding; Ling Wu; Yuan Xu; Qingling Feng

doi:10.2147/DDDT.S417077

Pharmacological Activity of Matrine in Inhibiting Colon Cancer Cells VM Formation, Proliferation, and Invasion by Downregulating Claudin-9 Mediated EMT Process and MAPK Signaling Pathway

Drug Des Devel Ther. 2023 Sep 11:17:2787-2804. doi: 10.2147/DDDT.S417077. eCollection 2023.

Authors

Qiu Du^#^{1

2}, Yingda Lin^#^{3

4}, Changping Ding^#⁵, Ling Wu³, Yuan Xu³, Qingling Feng⁶

Affiliations

¹ Department of Neurosurgery, the Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225012, People's Republic of China.
² Department of Central Laboratory, the Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225012, People's Republic of China.
³ Department of Pharmacy, the Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225012, People's Republic of China.
⁴ Department of Pharmacy, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, People's Republic of China.
⁵ Department of Medical Laboratory, the Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225012, People's Republic of China.
⁶ Department of Emergency Intensive Care Unit, the Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225012, People's Republic of China.

^# Contributed equally.

Abstract

Purpose: Matrine (Mat), the main active ingredient of traditional Chinese herbal plant Sophora flavescens Ait, has significant antitumor effects, but its pharmacological mechanism on colon cancer (CC) remains unclear. This study aimed to investigate the therapeutic effect of Mat on CC as well as the potential mechanism.

Methods: The vasculogenic mimicry (VM) of CC cells was observed by three-dimensional (3D) Matrigel cell culture. Cell proliferation, apoptosis, migration, invasion, and actin filament integrity were detected by CCK8, flow cytometry, wound healing, Transwell and Phalloidin staining assays. qRT-PCR and Western blotting were applied to detect the expression of EMT factors. RNA-sequencing was conducted to screen differentially expressed genes (DEGs), and the GO and KEGG pathway enrichment analyses were performed. Then, the expression of the key MAPK pathway genes and the target gene Claudin-9 (Cldn9) were analyzed. RNA interference was used to silence Cldn9 expression, and the effects of Cldn9 silencing and simultaneous treatment with Mat on VM formation, proliferation, apoptosis, invasion, and migration were investigated. Finally, the expression of EMT factors and MAPK pathway key genes was detected.

Results: CT26 cells formed the most typical VM structure. Mat disrupted the VM of CT26 cells, significantly suppressed their proliferation, migration, invasion, actin filament integrity, induced apoptosis, and inhibited EMT process. RNA-sequencing revealed 163 upregulated genes and 333 downregulated genes in Mat-treated CT26 cells, and the DEGs were significantly enriched in cell adhesion molecules and MAPK signaling pathways. Further confirmed that Mat significantly inhibited the phosphorylation levels of JNK and ERK, and the target gene Cldn9 was significantly upregulated in human CC tissues. Silencing Cldn9 markedly inhibited the VM, proliferative activity, invasiveness, and actin filament integrity of CT26 cells, blocked the EMT process, and downregulated the phosphorylation of JNK and ERK, whereas Mat intervention further strengthened the above trends.

Conclusion: This study indicated that Mat may synergistically inhibit the EMT process and MAPK signaling pathway through downregulation Cldn9, thereby exerting pharmacological effects on inhibiting VM formation, proliferation, and invasion of CC cells.

Keywords: Cldn9; EMT; MAPK signal pathway; colon cancer; matrine.

MeSH terms

Cell Proliferation
Claudins* / genetics
Colonic Neoplasms* / drug therapy
Epithelial-Mesenchymal Transition*
Humans
MAP Kinase Signaling System
Matrines*

Substances

Claudins
Matrines
CLDN9 protein, human

Grants and funding

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20200936), the Natural Science Research Project of the Higher Educational Institutions of Jiangsu Province (20KJB320007), the Science and Technology Planning Project of Yangzhou City (YZ2021082), and the Entrepreneurship and Innovation Program of Jiangsu Province (2020-30200).