Peiminine Induces G0/G1-Phase Arrest, Apoptosis, and Autophagy via the ROS/JNK Signaling Pathway in Human Osteosarcoma Cells in Vitro and in Vivo

Aims: Peiminine has been reported to have various pharmacological properties, including anticancer activity. In this study, we investigated the effect of this alkaloid on osteosarcoma and explored the underlying mechanisms.Methods: To evaluate the antiosteosarcoma effects of peiminine in vitro, cell viability was assessed by CCK-8 and live/dead assays; the effects of the drug on apoptosis and the cell cycle were examined by flow cytometry; the effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively, while its effects on autophagy were observed by transmission electron microscopy and an LC3 fluorescent puncta formation assay. The role of autophagy in the peiminine-mediated effects in osteosarcoma cells was evaluated by CCK-8 assay and western blotting after the application of the autophagy inhibitor chloroquine. The effect of peiminine on reactive oxygen species (ROS) production was analyzed using fluorescence confocal microscopy and spectrophotometry. Additionally, peiminine-treated osteosarcoma cells were exposed to SP600125, a JNK inhibitor, and N-acetylcysteine, a ROS scavenger, after which the contribution of the ROS/JNK signaling pathway to osteosarcoma was assessed using cell viability and LC3 fluorescent puncta formation assays, flow cytometry, and western blotting. A xenograft mouse model of osteosarcoma was generated to determine the antitumor effects of peiminine in vivo.Results: Peiminine suppressed proliferation and metastasis and induced cell cycle arrest, apoptosis, and autophagy in osteosarcoma cells. These anticancer effects of peiminine were found to be dependent on intracellular ROS generation and activation of the JNK pathway. In line with these results, peiminine significantly inhibited xenograft tumor growth in vivo.Conclusions: Peiminine induced G0/G1-phase arrest, apoptosis, and autophagy in human osteosarcoma cells via the ROS/JNK signaling pathway both in vitro and in vivo. Our study may provide an experimental basis for the evaluation of peiminine as an alternative drug for the treatment of osteosarcoma.

Induction of Apoptosis by Isoalantolactone in Human Hepatocellular Carcinoma Hep3B Cells through Activation of the ROS-Dependent JNK Signaling Pathway

Isoalantolactone (IALT) is one of the isomeric sesquiterpene lactones isolated from the roots of Inula helenium L. IALT is known to possess various biological and pharmacological activities, but its anti-cancer mechanisms are not well understood. The aim of the present study was to investigate the anti-proliferative effects of IALT in human hepatocellular carcinoma (HCC) cells and to evaluate the potential anti-cancer mechanisms. Our results demonstrated that IALT treatment concentration-dependently suppressed the cell survival of HCC Hep3B cells, which was associated with the induction of apoptosis. IALT increased the expression of death-receptor-related proteins, activated caspases, and induced Bid truncation, subsequently leading to cleavage of poly (ADP-ribose) polymerase. In addition, IALT contributed to the cytosolic release of cytochrome c by destroying mitochondrial integrity, following an increase in the Bax/Bcl-2 expression ratio. However, IALT-mediated growth inhibition and apoptosis were significantly attenuated in the presence of a pan-caspase inhibitor, suggesting that IALT induced caspase-dependent apoptosis in Hep3B cells. Moreover, IALT activated the mitogen-activated protein kinases signaling pathway, and the anti-cancer effect of IALT was significantly diminished in the presence of a potent c-Jun N-terminal kinase (JNK) inhibitor. IALT also improved the generation of intracellular reactive oxygen species (ROS), whereas the ROS inhibitor significantly abrogated IALT-induced growth reduction, apoptosis, and JNK activation. Furthermore, ROS-dependent apoptosis was revealed as a mechanism involved in the anti-cancer activity of IALT in a 3D multicellular tumor spheroid model of Hep3B cells. Taken together, our findings indicate that IALT exhibited anti-cancer activity in HCC Hep3B cells by inducing ROS-dependent activation of the JNK signaling pathway.