Identification of a Noncanonical Necrotic Cell Death Triggered via Enhanced Proteolysis by a Novel Sapogenol Derivative

Abstract

Small molecules which activate distinct cell death pathways have promising high potential for anticancer drug research. Especially, regulated necrosis draws attention as an alternative cell death mechanism to overcome the drug resistance. Here, we report that a new semisynthetic saponin analogue (AG-08) triggers necrotic cell death with unprecedented pathways. AG-08-mediated necrosis depends on enhanced global proteolysis involving calpains, cathepsins, and caspases. Moreover, AG-08 generates several alterations in lysosomal function and physiology including membrane permeabilization, redistribution toward the perinuclear area, and lastly excessive tubulation. As a consequence of lysosomal impairment, the autophagic process was abolished via AG-08 treatment. Collectively, in addition to its ability to induce necrotic cell death, which makes AG-08 a promising candidate to cope with drug resistance, its unique activity mechanisms including autophagy/lysosome impairment and enhancement of proteolysis leading a strong death capacity emphasizes its potential for anticancer drug research.

Sapogenin based self-assembly structures activating a non-apoptotic cell death via multiple pathways

“…Interestingly, CHOP protein level was enhanced by 8 µM AG-08 treatment but surprisingly decreased by 16 µM (Figure 6B). Since similar results was observed in the p62 protein levels in the previous study [1], we suggested that the CHOP levels were induced due to ER stress, but the enhancement of proteolysis by the prolonged or higher dose treatments of AG-08 might lead to degradation of full-length CHOP protein.…”

“…After the discovery of regulated necrosis, the induction of necrotic cell death has attracted huge attention and numerous studies state that necrotic death inducers possess high potential for cancer treatment. In a recent study, we reported a new semi-synthetic saponin analog (AG-08) triggering necrotic cell death along with unprecedented pathways including enhancement of global proteolysis and several alterations in lysosomal function and physiology [1]. The subsequent studies revealed an unexpected property of AG-08: After one freeze thaw cycle of its solution, AG-08 lost its cytotoxicity without any change in its chemical structure.…”

“…Next, the Lactate Dehydrogenase (LDH) release assay, which was previously used to demonstrate necrotic cell death for AG-08[1] was performed, and as shown in Fig 8A, all the tested compounds caused substantial release of LDH in a dose dependent manner. After LDH…”

“…The reaction mixture was stirred at room temperature for 12 h. Reaction mixture quenched with water and extracted three times with 100 mL EtOAc. Crude product was purified by silica gel column chromatography (cyclohexane:EtOAc; 8:2) to give AG-05 (13.9 mg lyophilized white-off powder) 1. H NMR (400 MHz, CDCl3): δ =7.77 (dd, J=8.2, 1.7 Hz, 2H), 7.32 (dd, J=8.2, 1.7 Hz, 2H), 5.evaporator.…”

The role of Cycloastragenol at the intersection of Nrf-2/ARE, telomerase, and proteasome activity

“…HEKn cells were seeded on 6 well plate with coverslips for fluorescence microscopy (Uner et al, 2020). The next day, cells were treated with the indicated concentration of CA for 24 h. Then, cells on coverslips were washed twice with cold 1X PBS and fixed with 4% paraformaldehyde in PBS for 30 min at 4°C.…”