Canine osteosarcoma (OS) stem cells are crucial factors for the formation, recurrence, and distant metastasis of osteosarcoma. Gambogenic acid (GNA), a broad-spectrum anti-tumor drug, has the advantage of low side effects. Whether GNA can intervene in the inhibition of stem cells to suppress the progression of canine OS still needs to be explored. This study targeted the stem cells regulatory factor - KLF transcription factor 5 (KLF5), to evaluate the functional impact of GNA on canine OS cells. In this study, the canine OS cell line McKinley was used to investigate the effects on proliferation, migration, and invasion, as well as stem cell transformation and stem cell characteristics after cancer stem cell sphere screening. The IC₅₀ of GNA on McKinley was 0.28 μM, and it exhibited dose-dependent inhibition of proliferation, migration, and invasion. GNA significantly inhibited the stemness transformation, stemness maintenance, and EMT of canine OS stem cells, while also inducing differentiation potential. KLF5 was highly expressed in canine OS cells and tissues. GNA significantly inhibited the expression and nucleation of KLF5. KLF5 over-expression promoted McKinley proliferation, migration, and invasion, enhancing the stemness transformation and stemness maintenance of stem cells, and reversed the inhibitory effect of GNA on the malignant biological behavior and stemness factors. This study found that KLF5 is a crucial factor in GNA against canine OS. This further improves the pharmacological mechanism of GNA anti-tumor and provides new insights and experimental basis for canine OS stem cell research.

To Cite This Article: Li S, Zhao Y, Zhang Y, Kang H, Yang T, Hou X, Zhang S, Wang W, Hou Q, Yang H, Sha J and Fan H, 2026. Gambogenic acid inhibited stemness and malignant biological behavior of canine osteosarcoma cells by regulating KLF5 expression. Pak Vet J, 46(6): 1473-1484. http://dx.doi.org/10.29261/pakvetj/2026.140