Longikaurin A, a natural ent-kaurane, suppresses proliferation, invasion and tumorigenicity in oral squamous cell carcinoma cell by via inhibiting PI3K/Akt pathway in vitro and in vivo
Background: Longikaurin A (LK-A), a naturally occurring ent-kaurane diterpenoid, has emerged as a promising candidate in cancer therapy. This study aims to explore the anti-tumor effects of LK-A on oral squamous cell carcinoma (OSCC) cells and investigate its underlying molecular mechanisms.
Methods: In vitro assays, including CCK-8 and EdU assays, were used to evaluate cell viability and proliferation. The migratory and invasive capabilities of OSCC cells were assessed by Transwell migration and invasion assays. Apoptosis was analyzed using Annexin V-FITC/PI staining and TUNEL assays. Western blotting was performed to assess protein expression related to cell cycle regulation, apoptosis, and the PI3K/Akt signaling pathway. In vivo, mouse xenograft models were treated with LK-A, and tumor growth, as well as signaling pathway activity, were evaluated using immunohistochemistry and Western blotting.
Results: LK-A significantly inhibited OSCC cell viability and proliferation in a dose-dependent manner, with IC50 values of 4.36 μM and 4.93 μM at 24 hours, and 1.98 μM and 2.89 μM at 48 hours for CAL27 and TCA-8113 cells, respectively. EdU assays showed a marked reduction in cell proliferation, while cell cycle analysis indicated G2/M phase arrest. Western blotting confirmed a decrease in CyclinB1 and Cdc2 expression. LK-A also suppressed cell migration and invasion, as evidenced by reduced MMP-2 and MMP-9 levels. Apoptosis was significantly enhanced, with increased expression of Bax and cleaved caspase-3, and MMP-9-IN-1 decreased Bcl-2 expression. Additionally, LK-A inhibited the PI3K/Akt/mTOR signaling pathway, as indicated by a decrease in the phosphorylation of PI3K, Akt, and mTOR. The pro-apoptotic and antiproliferative effects of LK-A were reversed by the AKT activator SC79. In vivo, LK-A markedly inhibited tumor growth in mouse xenograft models, resulting in smaller tumor weights and volumes, with no significant reduction in body weight. Immunohistochemistry and Western blotting further confirmed the suppression of p-Akt and Ki-67 expression.
Conclusion: These findings suggest that LK-A exerts potent antiproliferative, anti-migratory, and pro-apoptotic effects on OSCC cells, primarily through the inhibition of the PI3K/Akt signaling pathway. This supports its potential as a therapeutic agent for OSCC.