Yung Luen Shih

Kaempferol Induces DNA Damage and Inhibits DNA Repair Associated Protein Expressions in Human Promyelocytic Leukemia HL-60 Cells

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60 cells, which may be the factors for kaempferol induced cell death in vitro.

Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways

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.

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 promotes immune responses, ameliorates glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, but enhances lactate dehydrogenase levels in normal mice in vivo

Chitosan, a naturally derived polymer, has been shown to possess antimicrobial and anti-inflammatory properties; however, little is known about the effect of chitosan on the immune responses and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) activities in normal mice. The aim of the present study was to investigate whether chitosan has an effect on the immune responses and GOT, GPT and LDH activities in mice in vivo. BALB/c mice were divided into four groups. The negative control group was treated with a normal diet; the positive control group was treated with a normal diet plus orally administered acetic acid and two treatment groups were treated with a normal diet plus orally administered chitosan in acetic acid at doses of 5 and 20 mg/kg, respectively, every other day for 24 days. Mice were weighed during the treatment, and following the treatment, blood was collected, and liver and spleen samples were isolated and weighted. The blood samples were used for measurement of white blood cell markers, and the spleen samples were used for analysis of phagocytosis, natural killer (NK) cell activity and cell proliferation using flow cytometry. The results indicated that chitosan did not markedly affect the body, liver and spleen weights at either dose. Chitosan increased the percentages of CD3 (T-cell marker), CD19 (B-cell marker), CD11b (monocytes) and Mac-3 (macrophages) when compared with the control group. However, chitosan did not affect the phagocytic activity of macrophages in peripheral blood mononuclear cells, although it decreased it in the peritoneal cavity. Treatment with 20 mg/kg chitosan led to a reduction in the cytotoxic activity of NK cells at an effector to target ratio of 25:1. Chitosan did not significantly promote B-cell proliferation in lipopolysaccharide-pretreated cells, but significantly decreased T-cell proliferation in concanavalin A-pretreated cells, and decreased the activity of GOT and GPT compared with that in the acetic acid-treated group,. In addition, it significantly increased LDH activity, to a level similar to that in normal mice, indicating that chitosan can protect against liver injury.

Chitosan promotes immune responses, ameliorating total mature white blood cell numbers, but increases glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, and ameliorates lactate dehydrogenase levels in leukemia mice in vivo

The aim of the present study was to investigate the effect of chitosan (a naturally derived polymer) on the immune responses and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) levels in WEHI-3 cell-generated leukemia mice. Mice were divided into control, WEHI-3 control, acetic acid (vehicle)-treated, and 5 and 20 mg/kg chitosan-treated groups. Mice were subsequently weighed, blood was collected, and liver and spleen samples were isolated and weighed. Blood samples were measured for cell markers, the spleen underwent phagocytosis and natural killer (NK) cell activity examination, and cell proliferation was analyzed by flow cytometry. Chitosan did not significantly affect the weights of body, liver and spleen at 5 and 20 mg/kg treatment. Chitosan increased the percentage of CD3 (T cells marker), decreased the levels of CD19 (B-cell marker) and CD11b at 5 mg/kg treatment, and decreased the levels of Mac-3 at 5 and 20 mg/kg treatment. Chitosan significantly increased macrophage phagocytosis of PBMCs, but did not significantly affect macrophage phagocytosis in the peritoneal cavity. Chitosan treatment did not significantly affect the cytotoxic activity of NK cells, and also did not affect T- and B-cell proliferation. Chitosan significantly increased total white blood cell numbers, and GOT and GPT activities were both significantly increased. However, chitosan did not significantly affect LDH activity in leukemia mice. Chitosan may aid in future studies on improving immune responses in the treatment of leukemia.

Ouabain impairs cell migration, and invasion and alters gene expression of human osteosarcoma U-2 OS cells

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.