Cucurbitacin E Glucoside as an Apoptosis Inducer in Melanoma Cancer Cells by Modulating AMPK/PGK1/PKM2 Pathway.

Abstract

Background: Cucurbitacin E glucoside (CEG), a prominent constituent of Cucurbitaceae plants, exhibits notable effects on cancer cell behavior, including inhibition of invasion and migration, achieved through mechanisms such as apoptosis induction, autophagy, cell cycle arrest, and disruption of the actin cytoskeleton.

Objective: Melanoma, the fastest-growing malignancy among young individuals in the United States and the predominant cancer among young adults aged 25 to 29, poses a significant health threat. This study aims to elucidate the apoptotic mechanism of CEG against the melanoma cancer cell line (A375).

Methods: The study estimated the IC50 of CEG against the A375 cell line and assessed cell viability, apoptosis, and necrosis upon CEG treatment. Additionally, IC50 values of CEG against Phosphoglycerate kinase1 (PGK1) and Pyruvate Kinase M2 (PKM2) were determined at various levels of concentrations. The impact of CEG on intracellular glutathione (GSH) levels and the activity of key enzymes (GR, SOD, GPx, CAT), as well as markers of apoptosis (P53), and cell cycle regulation (cyclin D1, cyclin E2, cdk2, cdk4), were estimated. Finally, the level of AMP-activated protein kinase (AMPK), PGK1, and PKM2 gene expression levels in A375 cells were also evaluated.

Results: The IC50 value of CEG against A375 cells was determined to be 41.87 ± 2.47 µg/mL. A375 cells treated with CEG showed a significant increase in the G0/G1 phase and a decrease in the S and G2/M phases, indicating cell cycle arrest and reduced proliferation. Additionally, there was an increase in the sub-G1 peak, suggesting enhanced apoptosis. Additionally, the pharmacological analysis revealed potent inhibitory activity of CEG against both PGK1 and PKM2 gene expression, with IC50 values 27.89, 11.70, 7.43 and 2.74 µg/mL after incubation periods intervals of 30, 60, 90 and 120 minutes, respectively. In In-Silico study, computational simulations showed a strong binding affinity of CEG towards AMPK, PGK1, and PKM2 activities, with estimated binding energy (∆G) values of -6.5, -7.9, and -8.3 kcal/mol, respectively. Furthermore, incubation of A375 cells with CEG (at concentrations of 20.9, 41.87, and 83.74 µg/mL) led to a significant decrease in GSH levels and the activity of GR, SOD, GPx, CAT, cyclin D1, cyclin E2, cdk2, and cdk4. Notably, CEG treatment upregulated AMPK levels while downregulating PGK1 and PKM2 gene expression significantly.

Conclusion: CEG induces apoptosis in melanoma cancer cells (A375) through various mechanisms, including enhanced production of P53 and MDA, inhibition of key enzymes (GR, SOD, GPx, CAT) involved in oxidative stress defense and production of cell cycle regulating enzymes (cyclin D1, cyclin E2, cdk2, cdk4, and upregulation of AMPK and downregulation PGK1, and PKM2 in A375 tumor cells pathways. The downregulation of PKM2 in CEG-treated A375 cells inhibits ATP generation via aerobic glycolysis, a metabolic preference of cancer cells.