Synthesis and Anticancer Activity of A-Ring-Modified Derivatives of Dihydrobetulin

Area of Science:

• Medicinal Chemistry
• Oncology
• Molecular Biology

Background:

• Multidrug resistance (MDR) in cancer is a major clinical challenge, often mediated by ATP-binding cassette efflux transporters like P-glycoprotein (P-gp).
• Developing novel anticancer agents that overcome MDR is crucial for improving patient outcomes.

Purpose of the Study:

• To synthesize and evaluate novel 3,4-seco-lupane triterpenoids for anticancer activity, particularly against MDR cancer cells.
• To investigate the mechanism of action of the most potent derivative, methyl ketone 31 (MK), focusing on its interaction with P-gp and its cytotoxic effects.

Main Methods:

• Synthesis of semi-synthetic triterpenoids from dihydrobetulin.
• Cytotoxicity assays (MT-assay) against various human cancer cell lines, including P-gp overexpressing cells.
• In silico analysis for P-gp inhibition, in vitro Rhodamine 123 efflux tests, and combination studies with verapamil.
• Apoptosis assays (Annexin V-FITC), cell cycle analysis, mitochondrial function assessment, and Western blotting for caspase activation.

Main Results:

• Methyl ketone 31 (MK) demonstrated significant cytotoxicity (0.7-16.6 µM) against nine human cancer cell lines, including the P-gp overexpressing HBL-100/Dox subclone.
• In silico and in vitro studies indicated that MK is neither a P-gp inhibitor nor a substrate.
• MK induced apoptosis in HBL-100/Dox cells through ROS-mediated mitochondrial pathway activation, evidenced by cell cycle arrest, mitochondrial dysfunction, cytochrome c release, and caspase-9/-3 activation.

Conclusions:

• The novel triterpenoid derivative MK exhibits potent anticancer activity against MDR cancer cells.
• MK's cytotoxicity is mediated by the induction of apoptosis via the mitochondrial pathway, independent of P-gp inhibition.
• MK represents a promising lead compound for developing new therapeutic strategies against multidrug-resistant cancers.