Differential induction of apoptosis and autophagy by pyrrolizidine alkaloid clivorine in human hepatoma Huh-7.5 cells and its toxic implication

Abstract

Growing evidence suggests that the pyrrolizidine alkaloids (PAs)-induced hepatotoxicity is mediated by multiple cell death/defence modalities. However, the detailed mechanisms are still lacking. In this study, the hepatotoxic effects of four PAs including three retronecine-type ones (senecionine, seneciphylline and monocrotaline) and one otonecine-type (clivorine) on the proliferation of Huh-7.5 cells and the possible mechanisms were investigated. The results showed that all the PAs could inhibit cell proliferation and induce apoptosis in a concentration-dependent manner. Among them clivorine was the most significant one. In addition to its effect on apoptosis, clivorine treatment could promote autophagy in Huh-7.5 cells, as evidenced by the accumulation of autophagosomes, the enhancement of LC3B expression at the concentrations close to its IC0 value, and the increased conversion of LC3B-I to LC3B-II in the presence of lysosomal inhibitor (chloroquine) and decreased formation of green fluorescent protein (GFP)-LC3 positive puncta in the presence of autophagic sequestration inhibitor (3-methyladenine). Among the other tested PAs, senecionine and seneciphylline also activated autophagy at the same concentrations used for clivorine but monocrotaline did not. Furthermore, our study demonstrated that suppression or enhancement of autophagy resulted in the remarkable enhancement or suppression of senecionine, seneciphylline and clivorine-induced apoptosis at the concentration close to the IC10 for clivorine, respectively, indicating a protective role of autophagy against the PA-induced apoptosis at the low level of exposure. Collectively, our data suggest that PAs in different structures may exert different toxic disturbances on the liver cells. Apoptosis may be one of the most common models of the PA-induced cytotoxicity, while autophagy may be a structure-dependent defence model in the early stage of PA intoxication. Differential induction of apoptosis and autophagy probably depending on the concentration is essential for the cytotoxic potency of clivorine.

CHOP Regulates Endoplasmic Reticulum Stress-Mediated Hepatoxicity Induced by Monocrotaline

“…Sustained apoptosis causes cell death and ultimately leads to cell dysfunction ( D’arcy, 2019 ). Previous studies have shown that some PAs can induce apoptosis in primary mouse hepatocytes ( Yang et al, 2017b ) or cell lines, including human live L-02 cells ( Ji et al, 2008 ), human hepatoma cells HepG2 ( Ebmeyer et al, 2019 ), and Huh-7 ( Liu et al, 2017 ). In this study, we tested the toxic effects of MCT with different concentrations and time on primary rat hepatocytes.…”

Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

“…Software-based analyses of the regulated genes indicated that, among others, cellular mechanisms such as cell death, apoptosis and necrosis could be impaired [ 27 ]. Other studies have also indicated connections between PA exposure and the induction of apoptosis in the liver for the PAs monocrotaline, clivorine, retrorsine and senecionine [ 28 , 29 , 30 , 31 , 32 ]. Based on these data, we focused on structural differences in the induction of cytotoxicity and especially apoptosis by 22 different PAs.…”

“…The activation of the initiator caspases 8 and 9 was less pronounced. PA-induced apoptosis seems to be more likely to occur via the intrinsic signaling pathway (caspase 9-mediated) [ 28 , 30 , 32 ]. Waizenegger et al (2018) and Ebmeyer et al (2019) could nevertheless show that the extrinsic signaling pathway (mediated by Fas via caspase 8) may also be involved.…”

Seneciphylline, a main pyrrolizidine alkaloid in Gynura japonica, induces hepatotoxicity in mice and primary hepatocytes via activating mitochondria‐mediated apoptosis

“…PAs are mainly divided into three types: retronecine (RET)‐type, otonecine (OTO)‐type and platyphylline (PLA)‐type (Figure 1A). The former two bear an unsaturated 1, 2‐double bond in their structure and are highly toxic (Li, Kan, Li, & Lin, 2013; Liu et al, 2017). PAs can be metabolized by cytochrome P450 enzymes (CYP450s) to form dehydropyrrolizidine alkaloids (DHPAs).…”