Pyracrenic acid induces oxidative stress-mediated mitochondrial dysfunction in non-small cell lung cancer (NSCLC) by modulating the PI3K/AKT/GSK3Β pathway leading to caspase-dependent and -independent cellular death

Abstract

Lung cancer remains one of the most prevalent and lethal malignancies worldwide, with non small cell lung cancer (NSCLC) accounting for the majority of cases. Platinum based chemotherapies—most notably cisplatin—remain central to treating advanced NSCLC; however, their usefulness is limited by off target toxicity, acquired resistance, and suboptimal efficacy. These shortcomings highlight the urgent need for more selective and less toxic therapies. Natural products have long served as a source for drug discovery, either directly as sources of pure drugs, indirectly as raw materials for semisynthetic derivatives, or as structural templates for designing lead molecules. In this context, pyracrenic acid (PA), a pentacyclic triterpenoid, was isolated from Melaleuca quinquenervia fruit by column chromatography and identified via NMR. Despite being an ester, PA showed robust stability toward cellular esterases, as verified by HPLC analysis. Functionally, PA exhibited potent cytotoxicity against the human NSCLC cell line A549 (IC₅₀ = 2.8 ± 0.02 µM; p < 0.001), outperforming its potential breakdown products, betulinic acid (IC₅₀ = 73.4 ± 1.9 µM) and caffeic acid (IC₅₀ > 100 µM). Its activity was also superior to that of cisplatin (IC₅₀ = 3.3 ± 0.03 µM; p > 0.05). Selectivity indices further indicated greater toxicity toward A549 cells than toward several non malignant cell lines, Vero E6 (SI = 2.5), NIH/3T3 (SI = 2.1), and MRC 5 (SI = 3.0).Mechanistic studies revealed that PA triggers apoptosis, evidenced by dose dependent increase in caspase 3/7 activity and enhanced RealTime Glo™ Annexin V signals, alongside elevated levels of cleaved caspase 3, caspase 9, and PARP. PA also modulated cell cycle regulators—upregulating p53 and p21 while downregulating CDK2 and cyclin D1—consistent with induction of cell cycle arrest. PA induced a dose-dependent increase in oxidative stress and mitochondrial dysfunction, as measured by the ROS-Glo™ H₂O₂ assay (Promega) and JC-1 dye, respectively. This was accompanied by upregulation of pro-apoptotic proteins Bax, Bad, and cytochrome c, along with downregulation of the anti-apoptotic protein Bcl-2. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, effectively reduced both oxidative stress and mitochondrial membrane depolarization. In contrast, cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (mPTP), did not alleviate oxidative stress, suggesting that oxidative stress precedes and causes mitochondrial dysfunction, rather than resulting from it. Notably, co-treatment with either NAC or CsA significantly (p < 0.01) improved cell viability following 24 hours of exposure to PA (10 µM), compared to PA treatment alone. Although PA elevated the levels of cleaved caspase 3 and caspase 9, co treatment with the pan caspase inhibitor Z VAD FMK did not significantly (p > 0.05) restore A549 cell viability, implying that PA also induces caspase independent cytotoxicity. To explore whether autophagy was involved, we assessed autophagic markers and found that PA provoked a dose dependent increase in monodansylcadaverine (MDC) staining alongside higher LC3 II and Beclin 1 levels. Notably, blocking autophagy with 3 methyladenine (3 MA) failed to significantly (p > 0.05) reverse PA induced cytotoxicity and instead further lowered cell viability, indicating that autophagy serves a protective role rather than acting as an alternative cell death pathway. The effect of PA on the PI3K/Akt/GSK3β signaling pathway was thoroughly examined. PA was found to inhibit this pathway by promoting the dephosphorylation of PI3K and Akt, which subsequently led to the dephosphorylation and activation of GSK3β. Co-treatment with lithium chloride (LiCl), a known GSK3β inhibitor, significantly improved the viability of A549 cells exposed to PA, highlighting the critical role of GSK3β activation in PA-induced cytotoxicity. Furthermore, PA demonstrated strong anti-proliferative activity against a cisplatin-resistant A549 cell line, where it similarly inhibited the PI3K/Akt signaling pathway, underscoring its potential to overcome drug resistance in NSCLC. Collectively, these findings position PA as a promising, naturally derived lead compound for the development of more effective NSCLC therapies.