Mitochondria-dependent apoptosis in triptolide-induced hepatotoxicity is associated with the Drp1 activation.

How triptolide is associated with mitochondrial dysfunction and apoptosis in connection with its hepatotoxicity remains unclear. The objective of our study was to find out the link between mitochondrial dynamics and cell death in triptolide induced hepatotoxicity. We treated L02 cells with 25 nM concentration of triptolide. The results demonstrated that triptolide treatment caused an increase in apoptotic cell death, mitochondrial depolarization, ROS overproduction, a decrease in ATP production, and mitochondrial fragmentation which in turn is associated with the activation of Drp1 fission protein. Triptolide treatment led to the translocation of Drp1 from the cytosol into outer mitochondrial membrane where it started mitochondrial fission. This fission event is coupled with the mitochondrial release of cytochrome c into the cytosol and subsequently caspase-3 activation. TEM analysis of rat liver tissues revealed the distortion of mitochondrial morphology in triptolide-treated group. Western blot analysis explained that disruption in mitochondrial morphology was attached with the recruitment of Drp1 to mitochondria, cytochrome c release, and caspase-3 activation. However, Mdivi-1 co-treatment inhibited the activation of Drp1 and caspase-3 and blocked the release of cytochrome c into the cytosol. In short, inhibiting Drp1 protein activation may provide a new potential target for curing Drp1-associated apoptosis in triptolide-induced hepatotoxicity.

Drp1-associated mitochondrial dysfunction and mitochondrial autophagy: a novel mechanism in triptolide-induced hepatotoxicity.

Triptolide being an active ingredient of Chinese herbal plant Tripterygium wilfordii Hook f. has severe hepatotoxicity. Previous studies from our lab reported triptolide-induced mitochondrial toxicity in hepatocytes. However, biomolecular mechanisms involved in triptolide-induced mitochondrial dysfunction are not yet entirely clear. We explored the connection between mitochondrial fragmentation and mitophagy in triptolide-induced hepatotoxicity. Triptolide caused an increase in ROS production, a decrease in mitochondrial depolarization, a diminution of ATP generation, a decline in mitochondrial DNA copy number, mitochondrial fragmentation, and disturbance in mitochondrial dynamics in a concentration-dependent manner in L02 cells. Disturbance in mitochondrial dynamics was due to an increased expression of Drp1 fission protein in vitro and in vivo. L02 cells exhibited an increase in the colocalization of lysosomes with mitochondria and autophagosomes with mitochondria in triptolide treated group as compared to control group which was inhibited by Mdivi-1. Transmission electron micrographs of rat liver tissues treated with triptolide (400 µg/kg) revealed activation of mitophagy which was prevented by Mdivi-1 co-treatment. Taken together, our results showed that mitochondrial fission-associated mitophagy is a novel mechanism involved in triptolide-induced hepatotoxicity. For the alleviation of triptolide-induced hepatotoxicity, mitochondrial fission and mitochondrial autophagy signaling pathway can be targeted as a new therapeutic strategy. Graphical abstract ᅟ.

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library.

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both in vitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7 and 9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.