Cytotoxicity of ungeremine towards multi-factorial drug resistant cancer cells and induction of apoptosis, ferroptosis, necroptosis and autophagy.

Background: Successful cancer chemotherapy is hampered by resistance of cancer cells to established anticancer drugs. Numerous natural products reveal cytotoxicity towards tumor cells.

Purpose: The present study was aimed to determine the cytotoxicity of a betaine-type alkaloid, ungeremine, towards 9 cancer cell lines including various sensitive and drug-resistant phenotypes. The mode of action of this compound was further investigated.

Methods: The cytotoxicity, ferroptotic and necroptotic cell death were determined by the resazurin reduction assay. Caspase activation was evaluated using the caspase-Glo assay. Flow cytometry was applied for the analysis of cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (HDCFH-DA). Apoptotic, necroptotic and autophagic markers were determined by Western blotting. CCRF-CEM leukemia cells were used for all mechanistic studies.

Results: Ungeremine displayed cytotoxic activity towards the 9 cancer cell lines tested, including drug-sensitive and MDR phenotypes. The ICvalues obtained varied from 3.67 µM (in MDA-MB-231-BCRP breast carcinoma cells) to 75.24 µM (against in CEM/ADR5000 leukemia cells) for ungeremine and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin (control drug). Ungeremine induced ferroptosis, necroptosis, autophagy as well as apoptosis mediated by caspase activation, MMP alteration and increase ROS production.

Conclusion: The present investigation showed that ungeremine is a promising cytotoxic compoundthat could be further explored in the future to develop new anticancer drugs to fight sensitive and resistant phenotypes.