Characterization of cyclotide Vdif A from Viola diffusa inhibits non-small cell lung cancer cells via regulation of CKS2.

[BACKGROUND] Cyclotides are plant-derived cyclic peptides with remarkable structural stability and broad-spectrum bioactivities, including anticancer potential. However, the mechanisms of action of these agents against tumors remain largely unclear. Viola diffusa, a traditional medicinal plant native to southern China, contains abundant cyclotides, whose therapeutic potential against non-small cell lung cancer (NSCLC) remains to be thoroughly investigated.

[PURPOSE] This study aimed to isolate and characterize cyclotides from V. diffusa, evaluate their anticancer activity against NSCLC, and explore the underlying molecular mechanisms, focusing on their regulation of key proteins involved in cancer cell proliferation and survival.

[METHODS] Cyclotides were identified through transcriptomic sequencing and mass spectrometry. Purified peptides were tested for cytotoxicity against A549 NSCLC cells and other cancer lines. The effects of the lead cyclotide, Vdif A, on proliferation, cell cycle, apoptosis, and DNA damage were assessed by colony formation assays, flow cytometry, Hoechst staining, siRNA Transfection, and Western blotting. Quantitative proteomic profiling and bioinformatics analyses identified differentially expressed proteins. The antitumor efficacy and safety of Vdif A were validated in a Lewis lung carcinoma (LLC) syngeneic allograft model.

[RESULTS] Thirty cyclotides were identified from V. diffusa, with Vdif A exhibiting the most potent cytotoxicity against A549 cells (IC = 0.455 μM). Vdif A dose-dependently suppressed proliferation (0.25-1 μM), induced S-phase arrest, triggered apoptosis, and caused DNA damage via activation of the mitochondrial pathway. Proteomic analysis revealed significant downregulation of CKS2, a key cell cycle regulator, in Vdif A-treated cells. CKS2 functional validation experiments confirmed that CKS2 knockdown potentiates the anticancer effects of Vdif A in A549 Cells. In vivo, Vdif A (2-4 mg/kg) inhibited tumor growth by 46.58% with limited systemic toxicity at higher doses. Immunohistochemistry results confirmed reduced cell proliferation and increased apoptosis in Vdif A-treated tumor tissues.

[CONCLUSION] Unlike previous studies that mainly describe cytotoxic screening of cyclotides without exploring their underlying mechanisms, this study identifies Vdif A as a potent anticancer cyclotide from V. diffusa that inhibits NSCLC growth by regulating CKS2 and disrupting cell-cycle and survival pathways. These findings highlight Vdif A as a promising lead compound for cyclotide-based NSCLC therapy.