Bone cancer is one of the cancer-related diseases, and there are increased numbers of patients with bone cancer worldwide. Therefore the efficacy of treatment of bone cancer is considered extremely vital. Bufalin has been showed to have biological activities including anticancer activities in vitro and in vivo. However, the exact associated mechanisms for bufalin induced apoptosis in human bone cancer cells are still unclear. In the present study, we investigated the effect of bufalin on the cytotoxic effects in U-2 OS human osteosarcoma cells. For examining apoptotic cell deaths, we used flow cytometry assay, Annexin V/PI double staining, and TUNNEL assay. Reactive oxygen species (ROS), Ca2+, mitochondrial membrane potential (ΔΨm), and caspase-8, -9 and -3 activities were measured by flow cytometry assay. Furthermore, western blotting and a confocal laser microscopy examination were used for measuring the alterations of apoptotic associated protein expression and translocation, respectively. The results indicated that bufalin induced cell morphological changes, decreased the viable cell number, induced apoptotic cell death, and increased the apoptotic cell number, and affected apoptotic associated protein expression in U-2 OS cells. Bufalin increased apoptotic proteins such as Bak, and decreased anti-apoptotic proteins such as Bcl-2 and Bcl-x in U-2 OS cells. Furthermore, bufalin increased the protein levels of cytochrome c (Cyto c), AIF (Apoptosis inducing factor) and Endo G (Endonuclease G) in cytoplasm that were also confirmed by confocal microscopy examination. Based on those findings, bufalin induced apoptotic cell death in U-2 OS cells may be via endoplasmic reticulum (ER) stress, caspase-, and mitochondria-dependent pathways; thus, we may suggest that bufalin could be used as an anti-cancer agent for the treatment of osteosarcoma in the future, and further in vivo studies are needed.

Laminarin did not affect animal appearances, but increased the body weight at all doses. It reduced the weight of liver at 2.5 and 5 mg/ml and of spleen at 5 mg/ml. Laminarin increased CD3 (2.5 mg/ml) and CD19 (1 and 5 mg/ml) cell populations but reduced CD11b (5 mg/ml) cell populations, however, these did not affect Mac-3 marker level. Laminarin at 1 mg/ml increased phagocytosis by macrophages from peripheral blood mononuclear cell, but did not affect those from the peritoneal cavity. Laminarin increased NK cell cytotoxic activity at all doses and at a target ratio of 25:1 and 50:1. Laminarin did not affect B-cell proliferation, but at 5 mg/ml significantly reduced T-cell proliferation. Laminarin restored glutamate oxaloacetate transaminase (2.5 and 5 mg/ml) and glutamate pyruvate transaminase (2.5 mg/ml) levels. Based on these results, we suggest that laminarin can promote immune responses and protect against liver injury.

Abstract. Background Western blotting was used to examine the levels of apoptotic-associated protein and results indicated that fisetin increased expression of pro-apoptotic proteins such as B-cell lymphoma 2 (BCL2) antagonist/killer (BAK) and BCL2-associated X (BAX) but reduced that of anti-apoptotic protein such as BCL2 and BCL-x, and increased the cleaved forms of caspase-3, -8 and -9, and cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (ENDO G) in HSC3 cells. Confocal microscopy showed that fisetin increased the release of cytochrome c, AIF and ENDO G from mitochondria into the cytoplasm. Conclusion: Based on these observations, we suggest that fisetin induces apoptotic cell death through endoplasmic reticulum stress-and mitochondria-dependent pathways.Oral cancer is a subtype of head and neck squamous cell carcinoma. Oral squamous cell carcinoma (OSCC) comprises of a large proportion of head and neck SCC (1). In men, oral cancer constitutes approximately 3% of all malignant 1103

Cardamonin, the chalcone class, is one of the natural components from the spicy herbaceous plant (Alpinia conchigera Griff) and has anticancer activities in many human cancer cell lines. There is, however, no information to show that cardamonin induces cell apoptosis and alters apoptosis associated gene expressions in mouse leukemia cells. Thus, we investigated the effects of cardamonin on the apoptotic cell death and associated gene expression in mouse leukemia WEHI-3 cells in vitro. Results indicated that cardamonin decreased total viable cell number via induced cell morphological changes and apoptotic cell death in WEHI-3 cells that were assay by contrast-phase microscopy and flow cytometry examinations, respectively. The flow cytometry assay indicated that cardamonin increased reactive oxygen species (ROS) and Ca[Formula: see text] production, decreased the levels of mitochondrial membrane potential ([Formula: see text] and increased caspase-3, -8 and -9 activities in WEHI-3 cells. Western blotting was performed to analyze expression of relevant pro- and anti-apoptotic proteins and results showed that cardamonin decreased anti-apoptotic protein of Bcl-2 but increased pro-apoptotic protein of Bax in WEHI-3 cells. Furthermore, cardamonin increased cytochrome c, AIF and Endo G release, increased GRP78, caspase-12 that were associated with ER stress and increased Fas, Fas-Ligand and FADD expression. Furthermore, cardamonin increased the gene expressions of DAP (death-associated protein), TMBIM4 transmembrane (BAX inhibitor motif containing 4), ATG5 (autophagy related 5) but decreased the gene expression of DDIT3 (DNA-damage inducible transcript 3), DDIT4 (DNA-damage-inducible transcript 4), BAG6 (BCL2-associated athanogene 6), BCL2L13 [BCL2-like 13 (apoptosis facilitator)] and BRAT1 (BRCA1-associated ATM activator 1) that are associated with apoptosis pathways. Based on those findings, we may suggest cardamonin induced apoptotic cell death through Fas and Fas-Ligand-, caspase- and mitochondria-dependently pathways and also affects the apoptotic gene expression in WEHI-3 cells in vitro.

Ouabain, the specific Na /K -ATPase blocker, has biological activity including anti-proliferative and anti-metastasis effects in cancer cell. There is no study to show ouabain inhibiting cell migration and invasion in human osteosarcoma U-2 OS cells. Thus, we investigated the effect of ouabain on the cell migration and invasion of human osteosarcoma U-2 OS cells. Results indicated that ouabain significantly decreased the percentage of viable cells at 2.5-5.0 μM, thus, we selected 0.25-1.0 μM for inhibiting studies. Ouabain inhibited cell migration, invasion and the enzymatic activities of MMP-2, and also affected the expression of metastasis-associated protein in U-2 OS cells. The cDNA microarray assay indicated that CDH1, TGFBR3, SHC3 and MAP2K6 metastasis-related genes were increased, but CCND1, JUN, CDKN1A, TGFB1, 2 and 3, SMAD4, MMP13, MMP2 and FN1 genes were decreased. These findings provide more information regarding ouabain inhibited cell migration and invasion and associated gene expressions in U-2 OS cells after exposed to ouabain.