Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, have been shown to exhibit antineoplastic ability against many human cancer cells. In this study, we found that exposure of human osteogenic sarcoma U-2 OS cells to BITC and PEITC led to induce morphological changes and to decrease the percentage of viable cells in a time-and dose-dependent manner. BITC and PEITC induced cell cycle arrest at G2/M phase at 48 h treatment and inhibited the levels of cell cycle regulatory proteins such as cyclin A and B1 in U-2 OS cells but promoted the level of Chk1 and p53 that led to G2/M arrest. BITC and PEITC induced a marked increase in apoptosis (DNA fragmentation) and poly(ADP-ribose)polymerase (PARP) cleavage, which was associated with mitochondrial dysfunction and the activation of caspase-9 and -3. BITC and PEITC also promoted the ROS production in U-2 OS cells and the N-acetylcysteine (NAC, an antoxidant agent) was pretreated and then treated with both compounds which led to decrease the levels of ROS and increase the cell viability. Interestingly, BITC and PEITC promoted the levels of NO production and increased the iNOS enzyme. Confocal laser microscope also demonstrated that BITC and PEITC promoted the release of cytochrome c and AIF, suggesting that both compounds induced apoptosis through ROS, caspase-3 and mitochondrial, and NO signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC and PEITC-caused growth inhibition, G2/M arrest, and apoptotic death of U-2 OS cells. ß
Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI-3 cells in vitro and used WEHI-3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI-3 cells through the G0/G1 phase arrest and induction of caspase-3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI-3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac-3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI-3 cells. DATS also promotes immune responses in leukemia and normal 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, Ca release and mitochondrial membrane potential (ΔΨ ), 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, ATF6β, 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.
Bufalin has been shown to be effective against a variety of cancer cells, but its role in lung cancer has never been studied in an animal model. In this study, we evaluated bufalin effects in a human lung cancer cell line NCI-H460 both in vitro and in vivo. Bufalin caused significant cytotoxicity in NCI-H460 cells at a concentration as low as 1 μM. DNA condensation was observed in bufalin-treated cells in a dose-dependent manner. Mitochondrial membrane potential (ΔΨ ) was reduced and reactive oxygen species (ROS) were increased in bufalin-treated NCI-H460 cells. Levels of several proapoptotic proteins such as Fas, Fas-ligand, cytochrome c, apoptosis protease activating factor-1, endonuclease G, caspase-3 and caspase-9 were increased after bufalin treatment. At the same time, anti-apoptotic B-cell lymphoma 2 protein levels were reduced. Bufalin decreased glucose regulated protein-78 gene expression but increased growth arrest- and DNA damage-inducible 153 gene expression. Bufalin injected intraperitoneally in a dose-dependent manner reduced tumor size in BALB/C nu/nu mice implanted with NCI-H460 cells. Bufalin injection did not produce significant drug-related toxicity in experimental animals except at a high dose (0.4 mg kg ). In conclusion, low concentrations of bufalin can induce apoptosis in the human lung cancer cell line NCI-H460 in vitro. Bufalin also reduced tumor size in mice injected with NCI-H460 cells without significant drug-related toxicity. These results indicate that bufalin may have potential to be developed as an agent for treating human non-small cell lung cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1305-1317, 2017.
Abstract:Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells' adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future.
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