Glutathione (GSH) and GSH-related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)-resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ-resistant cells than in sensitive cells. Furthermore, silencing GSR in drug-resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over-expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro-oxidant Lbuthionine -sulfoximine. The modulation of redox state by GSH or L-buthionine -sulfoximine regulated GSR-mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress-free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment.

Oxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-␣ were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during ␤-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-␣ and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury.

■ AbstractMany diabetic patients suffer from a cardiomyopathy that cannot be explained solely by poor coronary perfusion. This cardiomyopathy may be due to either organ-based damage like fibrosis, or to direct damage to cardiomyocytes. Mitochondrial-derived reactive oxygen species (ROS) have been proposed to contribute to this cardiomyopathy. To address these questions, we used the OVE26 mouse model of severe type 1 diabetes to measure contractility in isolated cardiomyocytes by edge detection and in vivo with echocardiography. We also assessed the source of ROS generation using both a general and a mitochondrial specific indicator. When contractility was assayed in freshly isolated myocytes, contraction was much stronger in control myocytes. However, contractility of normal myocytes became weaker during 24 hours of in vitro culture. In contrast, contractility of diabetic OVE26 myocytes remains stable during culture. Echocardiography revealed normal or hyperdynamic function in OVE26 hearts under basal conditions but with a sharply reduced response to isoproterenol, a β-adrenergic agonist. For ROS generation, we found that ROS production in diabetic myocytes was elevated after exposure to either high glucose or angiotensin II (AngII). Superoxide detection with the mitochondrial sensor MitoSOX Red confirmed that mitochondria are a major source of ROS generation in diabetic myocytes. These results show that contractile deficits in OVE26 diabetic hearts are due primarily to cardiomyocyte impairment and that ROS from mitochondria are a cause of that impairment.

Silkworm cocoon was recorded to cure carbuncle in the Compendium of Materia Medica. Previous studies have demonstrated that the supplemental silk protein sericin exhibits anticancer activity. In the present study, we investigated the effects of silk fibroin peptide (SFP) extracted from silkworm cocoons against human lung cancer cells in vitro and in vivo and its possible anticancer mechanisms. SFP that we prepared had high content of glycine (~ 30%) and showed a molecular weight of ~ 10 kDa. Intragastric administration of SFP (30 g/kg/d) for 14 days did not affect the weights, vital signs, routine blood indices, and blood biochemical parameters in mice. MTT assay showed that SFP dose-dependently inhibited the growth of human lung cancer A549 and H460 cells in vitro with IC values of 9.921 and 9.083 mg/mL, respectively. SFP also dose-dependently suppressed the clonogenic activity of the two cell lines. In lung cancer H460 xenograft mice, intraperitoneal injection of SFP (200 or 500 mg/kg/d) for 40 days significantly suppressed the tumor growth, but did not induce significant changes in the body weight. We further examined the effects of SFP on cell cycle and apoptosis in H460 cells using flow cytometry, which revealed that SFP-induced cell cycle arrest at the S phase, and then promoted cell apoptosis. We demonstrated that SFP (20-50 mg/mL) dose-dependently downregulates Bcl-2 protein expression and upregulates Bax protein in H460 cells during cell apoptosis. The results suggest that SFP should be studied further as a novel therapeutic agent for the treatment of lung cancer.

The novel pyrazoline derivative, BHX, has recently been shown to exhibit potent anti-tumour activity by blocking the Wnt/β-catenin signalling pathway. However, its effect on breast cancer growth and invasion are unknown. Our results show that BHX suppresses MDA-MB-231 cell viability and colony formation in a dose-dependent manner, and induces apoptosis and G0/G1 phase arrest. BHX-treated breast cancer cells showed morphological characteristics of cells undergoing apoptosis. Furthermore, BHX inhibited cell migration and invasion, which was associated with increased E-cadherin mRNA and protein expression, and down-regulation of SNAIL and vimentin. In addition, BHX induced the generation of intracellular ROS and decreased β-catenin protein and mRNA expression. We used a mouse xenograft model to investigate the effects of BHX in vivo, where the growth of MDA-MB-231 xenografted tumours was suppressed in nude mice treated continuously with BHX for 21 days. Finally, the rat plasma concentration of BHX was measured by ultra-performance liquid-chromatography tandem mass spectrometry and the pharmacokinetic parameters of BHX were processed by non-compartmental analysis. In conclusion, BHX merits further study as a novel therapeutic small molecule for the treatment of breast cancer.