Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca2+ release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6ß, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017.

AKT serine/threonine protein kinase modulates bufalin-triggered intrinsic pathway of apoptosis in CAL 27 human oral cancer cells.

Bufalin has been reported to induce apoptosis in a variety of cancers but little is demonstrated in oral squamous cell carcinoma (OSCC) cells. The present study investigated the inhibition of proliferation, cell cycle arrest and apoptotic effects of bufalin in CAL 27 human oral cancer cells. Bufalin inhibited the growth of CAL 27 cells in a concentration-dependent manner and an IC50 value of bufalin was about 125 nM for 24 h treatment using the MTT assay. Moreover, the cell cycle distribution was arrested at the G0/G1 phase in CAL 27 cells after bufalin exposure. Upon bufalin stimulation, the expression of Bcl-2 was significantly decreased while that of cytochrome c, Apaf-1 and AIF was increased compared to the control group by western blot analysis. An increase in the expression of the active form of caspases was found in bufalin-treated cells, and the caspase activities were also elevated. Bufalin-triggered apoptosis was blocked by specific inhibitors of caspase-9 (z-LEHD-fmk) and caspase-3 (z-DEVD-fmk), respectively. In contrast, CAL 27 cells overexpressing constitutively active AKT (CAL 27/CA-AKT) were exposed to bufalin at different concentrations, and cell growth remained unchanged. Bufalin exhibited minimal apoptotic effects on CAL 27/CA-AKT cells. Taken together, bufalin induced G0/G1 phase arrest and provoked the intrinsic apoptotic pathway via AKT activation in CAL 27 cells. Our data suggest that bufalin could be potentially efficacious in the treatment of oral cancer in the future.