Effects of Clinacanthus nutans Extracts on Cell Proliferation and Apoptosis in Triple-Negative Breast Cancer: Mechanistic Insights

Jiafang Xu,1,* Jincha Long,2,* Zhen ying Li,3,* Chaoqun Wang,4 Yonggang Zhang,5 Huifang He,2 Qingjie Hu,2 Siqi Yin,2 Hai Li,2 Naizhen Wang,2 Qiang Gao,2 Shuaining Tang,2 Yongkang Zhu,6 Peng Wang,3 Renjun Feng,3 Yu Liu2,6 1Reproductive Medicine Center, The First Afffliated Hospital of Hainan Medical University, Haikou, People’s Republic of China; 2Department of Breast Surgery, The First Afffliated Hospital of Hainan Medical University, Haikou, People’s Republic of China; 3Key Laboratory of Tropical Translational Medicine of Ministry of Education & Key Laboratory of Brain Science Research Transformation in Tropical Environment of Hainan Province, School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou, People’s Republic of China; 4Department of Nuclear Medicine, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, People’s Republic of China; 5Department of General Surgery, People’s Hospital of Rizhao, Rizhao, People’s Republic of China; 6Department of General Surgery, Nanjing University of Chinese Medicine Affiliated Hospital, Nanjing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yu Liu, Department of Breast Surgery, The First Afffliated Hospital of Hainan Medical University, Haikou, People’s Republic of China, Email [email protected] Renjun Feng, Department of General Surgery, Nanjing University of Chinese Medicine Affiliated Hospital, Nanjing, People’s Republic of China, Email [email protected]: To explore the effects of Clinacanthus nutans extract (CnE) on triple-negative breast cancer (TNBC) and mechanism of action.Methods: In vitro, the human TNBC cell lines were treated with the extract at various concentrations. Cell viability was assessed using the CCK8 assay. In vivo, establishing a subcutaneous xenograft tumor model of TNBC, Hematoxylin-eosin staining and TUNEL assay were used to evaluate the effect of CnE on tumor proliferation. Tumor proteins were extracted, Quantitative proteomics and subsequently analyzed using bioinformatics approaches. Finally, immunohistochemistry evaluates the protein expression differences of ATP2A3, PLA2G4A, and ITPK1.Results: In vitro, CnE inhibited TNBC cell proliferation in a concentration-dependent manner, with IC50 values of 420 ± 35 μg/mL (MDA-MB-231) and 380 ± 28 μg/mL (MDA-MB-468), showing maximal 68.5% inhibition at 800 μg/mL (p < 0.001). The TNBC xenograft model was successfully established, and tumours in the extract-treated group were markedly smaller than those in the saline group. On day 28, the tumour inhibition rate was 28.66%, significantly higher than that in the saline group (P < 0.05). Haematoxylin–eosin staining staining and TUNEL assay showed increased tumor necrosis and apoptosis induction.(P < 0.001). Proteomic analysis showed that among the 4,908 identified proteins, 80 were upregulated, and 7 were downregulated. Bioinformatics analysis indicated involvement in the extracellular matrix, fatty acid metabolism, cell apoptosis, ferroptosis, immune response, choline metabolism, and amino acid metabolism. Immunohistochemistry revealed increased expression of ATP2A3 (1.3-fold, p < 0.05), PLA2G4A (1.6-fold, p < 0.05) and ITPK1 (3.2-fold, p < 0.01) proteins in the extract group compared to the control group.Conclusion: CnE inhibits TNBC cell proliferation, suppresses tumor growth, The mechanism likely involves multiple biological processes and pathways, Key pathways included apoptosis, ferroptosis, and necroptosis signaling.Keywords: Clinacanthus nutans, triple-negative breast cancer, proteomics, ATP2A3, PLA2G4A, ITPK1