Huey-Wen Shyu

Nickel(II)-Induced Oxidative Stress, Apoptosis, G2/M Arrest, and Genotoxicity in Normal Rat Kidney Cells

In order to elucidate the effects of nickel (Ni) on oxidative stress, apoptosis, and genotoxicity in renal cells, the levels of intracellular oxidants, lipid peroxidation, apoptotic proteins, and DNA damage were measured in normal rat kidney (NRK) cells after nickel chloride (NiCl2) treatment. NiCl2 appeared to increase the formation of the fluorescent oxidized compound (dichlorofluorescein, DCF) and levels of thiobarbituric acid-reactive substances (TBARS). In flow cytometric analysis, a rise in cell proportion in sub-G1 phase occurred in a concentration-dependent manner. After Ni treatment, there was reduced expression of Bcl-2 and Bcl-xL proteins, while induced Bad and Bax proteins expression was higher. Single-strand DNA breakage induced by Ni in NRK cells was determined by comet assay. Significant increase DNA damage score (arbitrary units) was noted in a concentration-related manner after treatment with Ni. Induction of intracellular oxidants by Ni was accompanied by an increasing frequency of DNA strand breakage. Our data indicate that Ni-induced oxidative stress and genotoxicity in NRK cells may involve reactive oxygen intermediates, and that Bcl family-mediated signaling pathway may be involved in positive regulation of Ni‐induced renal cytotoxicity.

Combined effects of terazosin and genistein on a metastatic, hormone-independent human prostate cancer cell line

Metastatic prostate cancer progresses from androgen-dependent to androgen-independent. Terazosin, a long-acting selective alpha1-adrenoreceptor antagonist, induces apoptosis of prostate cancer cells in an alpha1-adrenoreceptor-independent manner, while genistein, a major soy isoflavone, inhibits the growth of several types of cancer cells. The present study was designed to test the therapeutic potential of a combination of terazosin and genistein using a metastatic, hormone-independent prostatic cancer cell line, DU-145. Terazosin or genistein treatment inhibited the growth of DU-145 cells in a dose-dependent manner, whereas had no effect on normal prostate epithelial cells. Addition of 1 microg/ml of terazosin, which was inactive alone, augmented the growth inhibitory effect of 5 microg/ml of genistein. Co-treatment with terazosin resulted in the genistein-induced arrest of DU-145 cells in G2/M phase being overridden and an increase in apoptotic cells, as evidenced by procaspase-3 activation and PARP cleavage. The combination also caused a greater decrease in the levels of the apoptosis-regulating protein, Bcl-XL, and of VEGF165 and VEGF121 than genistein alone. In conclusion, the terazosin/genistein combination was more effective in inhibiting cell growth and VEGF expression as well as inducing apoptosis of the metastatic, androgen-independent prostate cancer cell line, DU-145, than either alone. The doses used in this study are in lower and nontoxic anticancer dosage range, suggesting this combination has potential for therapeutic use.

Phenethyl isothiocyanate induces DNA damage-associated G2/M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations.

Phenethyl isothiocyanate (PEITC) is a naturally occurring cruciferous vegetable-derived compound that inhibits cell growth and induces apoptosis in oral cancer cells. However, the exact mechanism of PEITC action has not been fully elucidated. This study investigated the molecular mechanism and anticancer potential of PEITC in oral squamous cell carcinoma (OSCC) cells with various p53 statuses. PEITC inhibited the growth of OC2, SCC4, and SCC25 cells (functional p53 mutants) in a dose-dependent manner with low toxicity to normal cells. Treatment with PEITC induced reactive oxygen species production, nitric oxide generation, and GSH depletion and triggered DNA damage response as evidenced by flow cytometry, 8-OHdG formation, and comet assay. Furthermore, the subsequent activation of ATM, Chk2, and p53 as well as the increased expression of downstream proteins p21 and Bax resulted in a G2/M phase arrest by inhibiting Cdc25C, Cdc2, and cyclin B1. The PEITC-induced apoptotic cell death, following a diminished mitochondrial transmembrane potential, reduced the expression of Bcl-2 and Mcl-1, released mitochondrial cytochrome c, and activated caspase 3 and PARP cleavage. The p53 inhibitor pifithrin-α and the antioxidants N-acetylcysteine and glutathione (GSH) protected the cells from PEITC-mediated apoptosis. However, mito-TEMPO, catalase, apocynin, and L-NAME did not prevent PEITC-induced cell death, suggesting that PEITC induced G2/M phase arrest and apoptosis in oral cancer cells via a GSH redox stress and oxidative DNA damage-induced ATM-Chk2-p53-related pathway. These results provide new insights into the critical roles of both GSH redox stress and p53 in the regulation of PEITC-induced G2/M cell cycle arrest and apoptosis in OSCCs.

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

Caffeine inhibits adipogenic differentiation of primary adipose-derived stem cells and bone marrow stromal cells.

Caffeine consumption has been related to loss of body weight and modulates lipid metabolism. However, impacts of caffeine on adipogenic differentiation have not been well determined yet. The present study evaluated the effects of caffeine on adipogenesis using primary rat adipose-derived stem cells (ADSCs) and a mouse bone marrow stromal cell line (M2-10B4) in vitro. ADSCs and M2-10B4 were continuously exposed to caffeine (0.1-1mM) during adipogenic differentiation for 7 and 12 days, respectively. Oil red O and Nile red staining showed that caffeine reduced lipid droplet and adipocyte levels in both cell types. In addition, Nile red staining and FACScan flow cytometry showed that caffeine dose-dependently decreased adipocyte differentiation from 20% to 50% of the control ADSCs and M2-10B4 cells. Caffeine decreased the expression of adipogenesis-related genes including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, adipocyte lipid binding protein, lipoprotein lipase, leptin, and TNFα in a dose-dependent manner. Rather, low concentration of caffeine (0.1mM) significantly increased IL-6 expression, but unexpectedly inhibited that at a concentration more than 0.3mM. Taken together, caffeine was able to effectively inhibit adipogenic differentiation of ADSCs and M2-10B4 cells partly through its inhibition of adipogenesis-related factors.

Caffeine regulates osteogenic differentiation and mineralization of primary adipose-derived stem cells and a bone marrow stromal cell line

Caffeine consumption reportedly influences bone mineral density and body weight. However, the effects of caffeine on bone metabolism are still controversial, and whether the dosage of caffeine influences osteogenic differentiation is yet to be clarified. In the present study, we cultured primary adipose-derived stem cells (ADSCs) and a bone marrow stromal cell line (M2-10B4) in osteogenic differentiation media containing varying concentrations of caffeine. Caffeine had biphasic effects: 0.1 mM caffeine significantly enhanced mineralization and alkaline phosphatase (ALP) activity. Consistent with these observations, a caffeine concentration of 0.1 mM upregulated the osteogenic differentiation marker genes ALP and osteocalcin (OCN), and elevated osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2) and Sirtuin 1 (SIRT1) levels. However, a concentration of caffeine greater than 0.3 mM suppressed the differentiation of both the cell types. These findings indicate that caffeine has a beneficial effect on ADSCs and bone marrow stromal cells, enhancing differentiation to osteoblasts; this effect, which is mediated via RUNX2 activation at low doses is significantly suppressed at high doses.

The Preventive Effect of Biochanin A on Bone Loss in Ovariectomized Rats: Involvement in Regulation of Growth and Activity of Osteoblasts and Osteoclasts

Biochanin A (BCA) is a major isoflavone abundant in red clover (Trifolium pretense). The protective effect of BCA on bone loss in an ovariectomized (OVX) animal model has never been clarified. The objective of this study was to investigate the biological effects of BCA on bone loss in OVX rats in vivo and on the development of osteoblasts and osteoclasts in vitro. Ovariectomy resulted in a marked increase in body weight and a decrease in femoral bone mineral density and trabecular bone volume that was prevented by BCA or 17β-estradiol (E2) treatment. However, an increase in uterine weight was observed in E2-treated OVX rats, but not in response to BCA treatment. Treatment with BCA increased the mRNA expression of osterix, collagen type I, alkaline phosphatase (ALP), and osteocalcin and decreased the mRNA expression of tartrate-resistant acid phosphatase (TRAP) and the receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio in the femur of OVX rats. Treatment with BCA or E2 prevented the OVX-induced increase in urinary deoxypyridinoline (DPD) and serum tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). In vitro, BCA induced preosteoblasts to differentiate into osteoblasts and increased osteoblast mineralization. BCA inhibited preosteoclasts and osteoclast proliferation and decreased osteoclast bone resorption. These findings suggest that BCA treatment can effectively prevent the OVX-induced increase in bone loss and bone turnover possibly by increasing osteoblastic activities and decreasing osteoclastic activities.

Biochanin A Promotes Osteogenic but Inhibits Adipogenic Differentiation: Evidence with Primary Adipose-Derived Stem Cells

Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1–1 μM) were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

Human herpesvirus 8 viral FLICE-inhibitory protein retards cell proliferation via downregulation of Id2 and Id3 expression

Death receptor-mediated apoptosis is potently inhibited by viral FLIP (FLICE/caspase 8 inhibitory protein) through reduced activation of procaspase 8. In this study, we show that the human herpesvirus 8-encoded vFLIP retards cell proliferation. Overexpression of vFLIP caused cell cycle arrest, with an apparent decrease of cells in the S phase. The Id (inhibitor of DNA binding) proteins are considered as dominant negative regulators of differentiation pathways, but positive regulators of cellular proliferation. The mechanisms by which Id proteins promote the cell cycle are diverse, but appear to involve affecting the expression of cell cycle regulators. RT-PCR results demonstrated that the expression of vFLIP decreased the expression levels of Id2 and Id3 as well as cyclin E and cyclin A compared with the vFLIP-null cells. These indicate that vFLIP affects cell proliferation by decreasing the expression levels of cell cycle regulatory proteins.

Epigallocatechin-3-Gallate Suppresses Human Herpesvirus 8 Replication and Induces ROS Leading to Apoptosis and Autophagy in Primary Effusion Lymphoma Cells

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has been shown to induce cell death in cancer cells. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by human herpesvirus 8 (HHV8). In this study, we examined the role of EGCG on PEL cells in cell death and HHV8 replication. We performed trypan blue exclusion assay to assess the cell viability of PEL cells, flow cytometry analysis to examine the cell cycle distribution and reactive oxygen species (ROS) generation, caspase-3 activity to assay apoptosis, acridine orange staining to determine autophagy, and immunoblotting to detect the protein levels involved in apoptosis and autophagy as well as mitogen activated protein kinases (MAPKs) activation upon EGCG treatment. The expression of the HHV8 lytic gene was determined by luciferase reporter assay and reverse transcription-PCR, and viral progeny production was determined by PCR. Results revealed that EGCG induced cell death and ROS generation in PEL cells in a dose-dependent manner. N-acetylcysteine (NAC) inhibited the EGCG-induced ROS and rescued the cell from EGCG-induced cell death. Even though EGCG induced ROS generation in PEL cells, it reduced the production of progeny virus from PEL cells without causing HHV8 reactivation. These results suggest that EGCG may represent a novel strategy for the treatment of HHV8 infection and HHV8-associated lymphomas.