Precision Design in Cancer Treatment: Cucurbitacin E Modulates AMPK, PGK1, and PKM2 to Optimize Radiation Efficacy in Melanoma

INTRODUCTION: Cucurbitacin E (CE), a naturally occurring triterpenoid derived from Cucurbitaceae plants, exhibits potent anticancer activity, particularly against melanoma. Previous studies indicate that CE suppresses melanoma progression by activating AMP-activated protein kinase (AMPK), thereby disrupting metabolic pathways and inducing apoptosis. Despite these findings, the potential synergistic interaction between CE and γ-irradiation in melanoma cell death remains unexplored. OBJECTIVE: This study investigates the combined effects of CE and γ-irradiation on apoptosis in A375 melanoma cells, aiming to uncover the underlying molecular mechanisms. The goal is to develop a novel radiosensitization approach to enhance melanoma treatment efficacy. METHODS: A375 cells were categorized into four groups: untreated control (Group I), CE-only (47.146 μg/mL; Group II), γ-irradiation-only (6 Gy/well; Group III), and combined CE + γ-irradiation (Group IV). Synergistic efficacy was assessed using the Chou-Talalay method. Cell viability and apoptosis were evaluated via MTT assays and Annexin V/PI staining. Gene expression (AMPK, PGK1, PKM2) was analyzed using qPCR, while oxidative stress markers (GSH, GR, SOD, GPx, CAT, MDA), cell cycle regulators (P53, P21, cyclin D1, cyclin E2, cdk2, cdk4), and apoptosis-related proteins (Bax, caspase-3, Bcl-2) were quantified via ELISA. Flow cytometry assessed cell cycle arrest and apoptosis. Molecular docking studies analyzed CE's binding affinity with AMPK, PGK1, and PKM2. RESULTS: CE demonstrated dose-dependent cytotoxicity (IC₅₀ = 47.146 μg/mL). The combination of CE (100 μg/mL) and γ-irradiation (6 Gy/well) significantly reduced A375 cell viability, confirming CE's radiosensitizing role. Combination Index analysis confirmed a synergistic interaction between CE and γ-irradiation (CI = 0.68), enhancing melanoma cell radiosensitivity. Flow cytometry revealed CE-induced G2/M arrest and amplified apoptosis when paired with radiation. Mechanistically, CE upregulated AMPK while suppressing PGK1 and PKM2, inhibiting glycolysis. Oxidative stress markers (MDA, P21) increased, whereas antioxidant enzymes (GSH, GR, SOD, GPx, CAT) and cell cycle proteins (cyclin D1, cyclin E2, cdk2) declined. Apoptosis- related analysis showed elevated Bax and caspase-3 levels, alongside reduced Bcl-2 levels, indicating activation of the intrinsic apoptotic pathway. Molecular docking identified strong binding between CE and AMPK (ΔG = -8.1 kcal/mol), PGK1 (ΔG = -7.7 kcal/mol), and PKM2 (ΔG = -7.9 kcal/mol), validating its direct interaction with metabolic targets. DISCUSSION: CE synergizes with γ-irradiation by disrupting glycolysis, inducing oxidative stress, activating intrinsic apoptosis, and arresting the cell cycle at G2/M. Its dual role as a metabolic inhibitor and radiosensitizer overcomes melanoma's resistance mechanisms. These findings align with prior studies on cucurbitacins but are the first to demonstrate CE's radiosensitizing effects in A375 cells. Limitations include the need for invivo validation. CONCLUSION: CE acts as a potent radiosensitizer, augmenting γ-irradiation-induced apoptosis in A375 cells through metabolic disruption, oxidative stress modulation, intrinsic apoptotic activation, and cell cycle arrest. These results highlight its potential for improving melanoma radiotherapy. Future research should optimize dosing regimens and explore clinical translation to advance precision oncology strategies.