Cucurbitacin E Glucoside as an Apoptosis Inducer in Melanoma Cancer Cells by
Modulating AMPK/PGK1/PKM2 Pathway
Authors: Hussein Mohammed Abdalla , Sallam Aya Sayed , Mohamed Shaza Ahmed , Mahmoud Abdel-Rady Amera , Maghrabe Adam Mostafa , Soltan Abdelrahman Wahdan , Abdelhamid Hanan Mohamed , Eldesoky Gaber E. , Alam Seikh Mafiz , Islam Mohammad Shahidul 
Published: January 2025

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.