Zhijia Fang

Genotoxicity of tri- and hexavalent chromium compounds in vivo and their modes of action on DNA damage in vitro.

Chromium occurs mostly in tri- and hexavalent states in the environment. Hexavalent chromium [Cr(VI)] compounds are extensively used in diverse industries, and trivalent chromium [Cr(III)] salts are used as micronutrients and dietary supplements. In the present work, we report that they both induce genetic mutations in yeast cells. They both also cause DNA damage in both yeast and Jurkat cells and the effect of Cr(III) is greater than that of Cr(VI). We further show that Cr(III) and Cr(VI) cause DNA damage through different mechanisms. Cr(VI) intercalates DNA and Cr(III) interferes base pair stacking. Based on our results, we conclude that Cr(III) can directly cause genotoxicity in vivo.

Inhibition of type i insulin-like growth factor receptor tyrosine kinase by picropodophyllin induces apoptosis and cell cycle arrest in T lymphoblastic leukemia/lymphoma

Abstract It has been recently shown that the type I insulin-like growth factor receptor (IGF-IR) contributes significantly to the survival of T lymphoblastic leukemia/lymphoma (T-LBL) cells, and it was therefore suggested that IGF-IR could represent a legitimate therapeutic target in this aggressive disease. Picropodophyllin (PPP) is a potent, selective inhibitor of IGF-IR that is currently used with notable success in clinical trials that include patients with aggressive types of epithelial tumors. In the present study, we tested the effects of PPP on Jurkat and Molt-3 cells; two prototype T-LBL cell lines. Our results demonstrate that PPP efficiently induced apoptotic cell death and cell cycle arrest of these two cells. These effects were attributable to alterations of downstream target proteins. By using proteomic analysis, seven different proteins were found to be affected by PPP treatment of Jurkat cells. These proteins are involved in various aspects of cellular metabolism, cytoskeleton organization and signal transduction pathways. The results suggest that PPP affects multiple signaling molecules and inhibits fundamental pathways that control cell growth and survival. Our study also provides novel evidence that PPP could be potentially utilized for the treatment of aggressive T-LBL.

Effect of Oleic Acid on the Levels of Eight Metal Ions in Human Hepatoma SMMC-7721 Cells

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Its incidence is rising worldwide. However, no specific therapy has been shown to be effective in its treatment. In the present study, the in vitro NAFLD model was established in human SMMC-7721 cells by using oleic acid (OA). Then, content changes of eight cations, including sodium, magnesium, potassium, calcium, iron, copper, zinc, and manganese, were investigated in the experimental model. The results showed that OA induced a decrease in magnesium level, while an increase in iron one. Additionally, the supplementation of magnesium in the cell culture model was studied. It showed that magnesium ameliorated lipid accumulation induced by OA. Our results suggest that magnesium could decrease the risk of NAFLD and be used as a promising candidate for the treatment of NAFLD.

Trivalent chromium alleviates oleic acid induced steatosis in SMMC-7721 cells by decreasing fatty acid uptake and triglyceride synthesis.

Trivalent chromium [Cr(III)] has been shown as an essential trace element for human health. Previous studies depict that Cr(III) plays important roles in maintaining normal glucose and lipid metabolism, whereas its effect on the hepatic lipid metabolism is still unknown. In the present study, we investigated the effects and underlying mechanisms of Cr on hepatic steatosis induced by oleic acid (OA) in human hepatoma SMMC-7721 cells. Hepatic steatosis model was co-administered with Cr. Indexes of lipid accumulation were determined and associated genes expression were analyzed. The data showed that OA could induce lipid accumulation and triglyceride (TG) content in SMMC-7721 cells, and significantly increase the expression of cluster of differentiation 36 (CD36) and diacylglycerol acyltransferase 2 (DGAT2). This steatosis effect of OA was ameliorated by Cr. The TG accumulation and up-regulation of CD36 and DGAT2 genes followed steatosis induction were inhibited by Cr. After the treatment of Cr, excessive intracellular OA content was also attenuated. Furthermore, Cr still performed inhibitory effect of DGAT2 expression at the presence of DGAT2 agonist or inhibitor, which indicated that the inhibitory effect of Cr on lipogenesis is associated with the downregulation of DGAT2 expression. These findings demonstrate that Cr alleviates hepatic steatosis via suppressing CD36 expression to prevent fatty acid uptake, as well as suppressing DGAT2 expression to inhibit TG synthesis. It suggests that CD36 and DGAT2 might become the novel drug targets for their properties in hepatic steatosis. Most importantly, Cr may be a potential anti-steatosis candidate to offer protective effects against liver damage.

Phosphatidate phosphatase-1 is functionally conserved in lipid synthesis and storage from human to yeast.

Phosphatidate phosphatase-1 (PAP1) enzymes (yeast Pah1p/Smp2p, mammalian lipin1-3) have a key role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate (PA) and its product diacylglycerol (DAG). Recent investigation shows that mammalian lipin-1 complements phenotypes exhibited by yeast pah1Δ mutant cells, which indicates the functions of PAP1 enzymes are evolutionarily conserved. The observation was confirmed after transformation of human LPIN1 into PAH1-defective yeast, which resulted in human LPIN1-induced accumulation of triacylglycerol (TAG )and lipid droplet formation. In double mutants lacking Tgl3p and Tgl4p, overexpression of PAH1 or LPIN1 induced TAG accumulation and excessive obesity. Furthermore, the obese yeast was used as a model to study the anti-obesity effects of PAP1 activity inhibitors, including propranolol and clenbuterol. The data showed that the inhibitors significantly suppressed TAG accumulation and lipid droplets formation. These findings demonstrate that LPIN1 plays a functional role in lipid synthesis and storage, a role which is highly conserved from human to yeast. Inhibition of TAG synthesis will become an efficacious treatment strategy for obesity and our excessive obesity model will provide a very useful tool for discovery of new anti-obesity drugs in the future.

Toona Sinensis and Moschus Decoction Induced Cell Cycle Arrest in Human Cervical Carcinoma HeLa Cells.

Toona sinensis and Moschus are two herb materials used in traditional Chinese medicine, most commonly for their various biological activities. In this study, we investigated the inhibitory effect of three decoctions from Toona sinensis, Moschus, and Toona sinensis and Moschus in combination on cell growth in several normal and cancer cell lines by cell viability assay. The results showed that the combined decoction exhibited the strongest anticancer effects, compared to two single decoctions. The observations indicated that the combined decoction did not induce cell apoptosis and autophagy in HeLa cells by fluorescence microscopy. Flow cytometry analysis revealed that the combined decoction arrested HeLa cell cycle progression in S-phase. After the decoction incubation, among 41 cell cycle related genes, eight were reduced, while five were increased in mRNA levels by real-time PCR assay. Western blotting showed that there were no apparent changes of protein levels of Cyclin E1, while P27 expression significantly declined and the levels of CDC7 and CDK7 obviously increased. The data suggest that the RB pathway is partially responsible for the decoction-induced S-phase cell cycle arrest in HeLa cells. Therefore, the combined decoction may have therapeutic potential as an anticancer formula for certain cancers.

Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae

ABSTRACT The heavy metal cadmium is widely used and released into the environment, posing a severe threat to crops and humans. Saccharomyces cerevisiae is one of the most commonly used organisms in the investigation of environmental metal toxicity. We investigated cadmium stress and the adaptive mechanisms of yeast by screening a genome-wide essential gene overexpression library. A candidate gene, OLE1, encodes a delta-9 desaturase and was associated with high anti-cadmium-stress activity. The results demonstrated that the expression of OLE1 was positively correlated with cadmium stress tolerance and induction was independent of Mga2p and Spt23p (important regulatory factors for OLE1). Moreover, in response to cadmium stress, cellular levels of monounsaturated fatty acids were increased. The addition of exogenous unsaturated fatty acids simulated overexpression of OLE1, leading to cadmium resistance. Such regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. IMPORTANCE A S. cerevisiae gene encoding a delta-9 desaturase, OLE1, was associated with high anti-cadmium-stress activity. The data suggest that the regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. The discovery of OLE1 involvement in membrane stability may indicate a novel defense strategy against cadmium stress.

Effects of cadmium on intracellular cation homoeostasis in the yeast Saccharomyces cerevisiae

Previous studies have demonstrated that cadmium can induce biochemical and physiological changes in yeast Saccharomyces cerevisiae. However, studies on the influence of cadmium on the ion balance in the cell and the interaction between cadmium and other ions are still relatively few in number. By using inductively coupled plasma-atomic emission spectrometry, the contents of some cations, including Zn2+, Ca2+, Fe3+, Cu2+, Mg2+, K+, and Na+ were measured. The data showed that the levels of Zn2+ and Fe3+ were increased, while those of Cu2+, K+, and Na+ were decreased after cadmium treatment. Afterwards, using the drop test assay, the interactions between cadmium and the selected ions were investigated. The results suggested that the cytotoxicity of cadmium could be attributable to the interference of cadmium with the intracellular cation homoeostasis. Calcium channel transporter Cch1 participates in the intracellular uptake of cadmium. Additionally, Zn2+, Ca2+, Fe3+, Mg2+, and K+ can rescue the toxic effect of cadmium in yeast.

A novel HAC1-based dual-luciferase reporter vector for detecting endoplasmic reticulum stress and unfolded protein response in yeast Saccharomyces cerevisiae.

Unfolded protein response (UPR) is an important cellular phenomenon induced by over-accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen. ER stress and UPR are implicated in human diseases such as diabetes, atherosclerosis and neurodegenerative diseases. Current methods for measuring ER stress levels and UPR activation usually include cells lysis and other complicated procedures such as reverse transcription-PCR (RT-PCR). These methods typically have low sensitivity and are not suitable for live detection. In this study, we developed a dual-luciferase gene reporter system to monitor UPR activation in live cells of the yeast Saccharomyces cerevisiae by taking advantage of the HAC1 intron and its unconventional splicing-regulation mechanism. We showed that this reporter can be used to monitor UPR in live cells with high sensitivity.

OLE1 reduces cadmium-induced oxidative damage in Saccharomyces cerevisiae

Cadmium has been shown to be an important environmental pollutant. Our previous studies have shown that the regulation of OLE1 in the synthesis of unsaturated fatty acids may act as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage induced by cadmium. However, the involvement of OLE1 in cadmium-induced oxidative stress is still unclear. In this study, we explored the effects of OLE1 on cadmium-induced oxidative stress in yeast. Different from ascorbic acid, the overexpression of OLE1 did not affect the activities of superoxide dismutase, catalase and glutathione peroxidase. Although OLE1 overexpression induced an increase in GSH levels, the anti-oxidative mechanism of OLE1 was GSH1 independent. On the other hand, similar to ascorbic acid, OLE1 overexpression significantly reduced the levels of superoxide radical, carbonyl protein and lipid peroxidation. Additionally, overexpression of OLE1 effectively prevented cell membrane damage induced by cadmium. OLE1 could also reduce the cytotoxicities of other heavy metals stress, including copper, tri- and hexavalent chromium. Thus, our results indicate that overexpression of OLE1 reduces oxidative stress induced by cadmium possibly through the inhibition of lipid peroxidation and protection of the cytoplasmic membrane from damage in yeast cells. Furthermore, we determined that the anti-oxidative effect of OLE1 is independent of antioxidant enzymes and GSH1.