Hung Sheng Shang

Deguelin Inhibits the Migration and Invasion of U-2 OS Human Osteosarcoma Cells via the Inhibition of Matrix Metalloproteinase-2/-9 in Vitro

Osteosarcoma is the most common malignant primary bone tumor in children and young adults and lung metastasis is the main cause of death in those patients. Deguelin, a naturally occurring rotenoid, is known to be an Akt inhibitor and to exhibit cytotoxic effects, including antiproliferative and anticarcinogenic activities, in several cancers. In the present study, we determined if deguelin would inhibit migration and invasion in U-2 OS human osteosarcoma cells. Deguelin significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells which was associated with a reduction of activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). Furthermore, results from western blotting indicated that deguelin decreased the cell proliferation and cell growth-associated protein levels, such as SOS1, PKC, Ras, PI3K, p-AKT(Ser473), IRE-1α, MEKK3, iNOS, COX2, p-ERK1/2, p-JNK1/2, p-p38; the cell motility and focal adhesion-associated protein levels, such as Rho A, FAK, ROCK-1; the invasion-associated protein levels, such as TIMP1, uPA, MMP-2. MMP-9, MMP-13, MMP-1 and VEGF in U-2 OS cells. Confocal microscopy revealed that deguelin reduced NF-κB p65, Rho A and ROCK-1 protein levels in cytosol. MMP-7, MMP-9 and Rho A mRNA levels were suppressed by deguelin. These in vitro results provide evidence that deguelin may have potential as a novel anti-cancer agent for the treatment of osteosarcoma and provides the rationale for in vivo studies in animal models.

Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI-3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo

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.

Sulforaphane‐induced apoptosis in human leukemia HL‐60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN‐induced cytotoxic effects and whether or not they went through cell‐cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL‐60 cells. Cell viability, cell‐cycle distribution, sub‐G1 (apoptosis), reactive oxygen species (ROS) and Ca2+ production, levels of mitochondrial membrane potential (ΔΨm), and caspase‐3, −8, and −9 activities were assayed by flow cytometry. Apoptosis‐associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub‐G1 phase development. Furthermore, SFN increased ROS and Ca2+ production and decreased the levels of ΔΨm and activated caspase‐3, −8, and −9 activities in HL‐60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas‐L, caspase‐8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl‐x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase −8, −3, −4, −6, and −7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL‐60 cells via Fas‐ and mitochondria‐dependent pathways.

Casticin impairs cell migration and invasion of mouse melanoma B16F10 cells via PI3K/AKT and NF‐κB signaling pathways

Casticin, a polymethoxyflavone, is one of the major active components obtained from Fructus viticis, which have been shown to have anticancer activities including induce cell apoptosis in human cancer cells. The aim of this study was to investigate the molecular mechanisms by which casticin inhibits cell migration and invasion of mouse melanoma B16F10 cells. Cell viability was examined by MTT assay and the results indicated that casticin decreased the total percentages of viable cells in dose‐dependent manners. Casticin affected cell migration and invasion in B16F10 cells were examined by wound healing mobility assay and Boyden chamber migration and invasion assay and results indicated that casticin inhibited cell migration and invasion in dose‐dependent manners. Western blotting was used to examine the protein expression of B16F10 cells after exposed to casticin and the results showed that casticin decreased the expressions of MMP‐9, MMP‐2, MMP‐1, FAK, 14‐3‐3, GRB2, Akt, NF‐κB p65, SOS‐1, p‐EGFR, p‐JNK 1/2, uPA, and Rho A in B16F10 cells. Furthermore, cDNA microarray assay was used to show that casticin affected associated gene expression of cell migration and invasion and the results indicated that casticin affected some of the gene expression such as increased SCN1B (cell adhesion molecule 1) and TIMP2 (TIMP metallopeptidase inhibitor 2) and decreased NDUFS4 (NADH dehydrogenase (ubiquinone) Fe‐S protein4), VEGFA (vascular endothelial growth factor A), and DDIT3 (DNA‐damage‐inducible transcript 3) which associated cell migration and invasion in B16F10 cells. Based on those observations, we suggest that casticin could be used as a novel anticancer metastasis of melanoma cancer in the future.

Benzyl isothiocyanate (BITC) induces apoptosis of GBM 8401 human brain glioblastoma multiforms cells via activation of caspase-8/Bid and the reactive oxygen species-dependent mitochondrial pathway

Benzyl isothiocyanate (BITC) is one of member of the isothiocyanate family which has been shown to induce cancer cell apoptosis in many human cancer cells. In the present study, we investigated the effects of BITC on the growth of GBM 8401 human brain glioblastoma multiforms cells. Results indicated that BITC‐induced cell morphological changes decreased in the percentage of viable GBM8401 cells and these effects are dose‐dependent manners. Results from flow cytometric assay indicated that BITC induced sub‐G1 phase and induction of apoptosis of GBM 8401 cells. Furthermore, results also showed that BITC promoted the production of reactive oxygen species (ROS) and Ca2+ release, but decreased the mitochondrial membrane potential (ΔΨm) and promoted caspase‐8, ‐9, and ‐3 activates. After cells were pretreated with Z‐IETD‐FMK, Z‐LEHD‐FMK, and Z‐DEVD‐FMK (caspase‐8, ‐9, and ‐3 inhibitors, respectively) led to decrease in the activities of caspase‐8, ‐9, and ‐3 and increased the percentage of viable GBM 8401 cells that indicated which BITC induced cell apoptosis through caspase‐dependent pathways. Western blotting indicated that BITC induced Fas, Fas‐L, FADD, caspase‐8, caspase ‐3, and pro‐apoptotic protein (Bax, Bid, and Bak), but inhibited the ant‐apoptotic proteins (Bcl‐2 and Bcl‐x) in GBM 8401 cells. Furthermore, BITC increased the release of cytochrome c, AIF, and Endo G from mitochondria that led to cell apoptosis. Results also showed that BITC increased GADD153, GRP 78, XBP‐1, and ATF‐6β, IRE‐1α, IRE‐1β, Calpain 1 and 2 in GBM 8401 cells, which is associated with ER stress. Based on these observations, we may suggest that BITC‐induced apoptosis might be through Fas receptor, ROS induced ER stress, caspase‐3, and mitochondrial signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC‐caused growth inhibition and induced apoptotic cell death of GBM 8401 cells.

Chitosan oligosaccharides in combination with Agaricus blazei Murill extract reduces hepatoma formation in mice with severe combined immunodeficiency

Chitosan and Agaricus blazei Murill (ABM) extracts possess antitumor activities. The aim of the present study was to investigate whether chitosan, ABM extract or the two in combination were effective against tumors in tumor-bearing mice. The mice were subcutaneously injected with SK-Hep 1 cells and were then were divided into the following six groups: Group 1, control group; group 2, chitosan 5 mg/kg/day; group 3, chitosan 20 mg/kg/day; group 4, ABM (246 mg/kg/day) and chitosan (5 mg/kg/day) combined; group 5, ABM (984 mg/kg/day) and chitosan (20 mg/kg/day) combined; and group 6, ABM (984 mg/kg/day). The mice were treated with the different concentrations of chitosan, ABM or combinations of the two for 6 weeks. The levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and vascular endothelial growth factor (VEGF), and tissue histopathological features were examined in the surviving animals. Based on the results of the investigation, the treatments performed in groups 2, 3 and 4 were identified as being capable of reducing the weights of the tumors, however, group 4, which was treated with chitosan (5 mg/kg/day) in combination with ABM (246 mg/kg/day) was able to reduce the levels of GOT and VEGF. As a result, treatment with chitosan in combination with ABM may offer potential in cancer therapy and requires further investigation.