Osthole Attenuates Bleomycin-Induced Pulmonary Fibrosis by Modulating NADPH Oxidase 4-Derived Oxidative Stress in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the extensive accumulation of myofibroblasts and collagens. However, the exact mechanism that underlies this condition is unclear. Growing evidence suggests that NADPH oxidases (NOXs), especially NOX4-derived oxidative stress, play an important role in the development of lung fibrosis. Bleomycin (BLM) is a tumor chemotherapeutic agent, which has been widely employed to establish IPF animal models. Osthole (OST) is an active constituent of the fruit of Cnidium ninidium. Here, we used an in vivo mouse model and found that OST suppressed BLM-induced body weight loss, lung injury, pulmonary index increase, fibroblast differentiation, and pulmonary fibrosis. OST also significantly downregulated BLM-induced NOX4 expression and oxidative stress in the lungs. In vitro, OST could inhibit TGF-ß1-induced Smad3 phosphorylation, differentiation, proliferation, collagen synthesis, NOX4 expression, and ROS generation in human lung fibroblasts in a concentration-dependent manner. Moreover, NOX4 overexpression could prevent the above effects of OST. We came to the conclusion that OST could significantly attenuate BLM-induced pulmonary fibrosis in mice, via the mechanism that involved downregulating TGF-ß1/NOX4-mediated oxidative stress in lung fibroblasts.

Acrylamide induces HepG2 cell proliferation through upregulation of miR-21 expression.

Acrylamide, a potential carcinogen, exists in carbohydrate-rich foods cooked at a high temperature. It has been reported that acrylamide can cause DNA damage and cytotoxicity. The present study aimed to investigate the potential mechanism of human hepatocarcinoma HepG2 cell proliferation induced by acrylamide and to explore the antagonistic effects of a natural polyphenol curcumin against acrylamide via miR-21. The results indicated that acrylamide (≤100 µmol/L) significantly increased HepG2 cell proliferation and miR-21 expression. In addition, acrylamide reduced the PTEN expression in protein level, while induced the expressions of p-AKT, EGFR and cyclin D1. The PI3K/AKT inhibitor decreased p-AKT protein expression and inhibited the proliferation of HepG2 cells. In addition, curcumin effectively reduced acrylamide-induced HepG2 cell proliferation and induced apoptosis through the expression of miR-21. In conclusion, the results showed that acrylamide increased HepG2 cell proliferation via upregulating miR-21 expression, which may be a new target for the treatment and prevention of cancer.

Tyrosol Attenuates High Fat Diet-Induced Hepatic Oxidative Stress: Potential Involvement of Cystathionine ß-Synthase and Cystathionine γ-Lyase.

The Mediterranean diet is known for its cardioprotective effects. Recently, its protective qualities have also been reported in patients with non-alcoholic fatty liver disease (NAFLD). Oxidative stress is one of the important factors responsible for the development and progression of NAFLD. Hydrogen sulfide (H2S), a multifaceted gasotransmitter, has emerged as a potential therapeutic target in NAFLD. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are major enzymes responsible for endogenous H2S synthesis. Since oxidative stress contributes to NAFLD pathogenesis, the objective of this study was to investigate the effect of tyrosol, a major compound in olive oil and white wine, on high fat diet-induced hepatic oxidative stress and the mechanisms involved. Mice (C57BL/6) were fed for 5 weeks with a control diet (10 % kcal fat), a high fat diet (60 % kcal fat, HFD) or a HFD supplemented with tyrosol. High fat diet feeding induced hepatic oxidative stress, as indicated by the significant increase in lipid peroxidation and NADPH oxidase activity. Tyrosol supplementation significantly increased hepatic CBS and CSE expression and H2S synthesis in high fat diet-fed mice. Such effects were associated with the attenuation of high fat diet-induced hepatic lipid peroxidation and the restoration of the redox equilibrium of the antioxidant glutathione. Tyrosol also inhibited palmitic acid-induced oxidative stress in hepatocytes (HepG2 cells). These results suggest that the antioxidant properties of tyrosol may be mediated through functional changes in CBS and CSE activity, which might contribute to the hepatoprotective effect of the Mediterranean diet.

EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells.

(-)-epigallocatechin-3-gallate (EGCG) is a well-known cancer chemopreventive agent. The potential mechanisms include regulation of multiple molecules. Carcinogenesis in lung cancer is related to the imbalance of tumor suppressor and oncogene. JWA is a structurally novel microtubule-binding protein and is a potential tumor suppressor. DNA topoisomerase IIα is a nuclear enzyme that governs DNA topology and is usually highly expressed in many types of cancer. It serves as a target of anticancer drugs. In the current study, the regulation of JWA and topoisomerase IIα by EGCG, and thereafter the mutual interaction between them was investigated. The results revealed that EGCG up-regulated JWA while decreased topoisomerase IIα expression in both human non-small cell lung cancer (NSCLC) cells and an NSCLC xenograft mice model. There was a negative correlation between JWA and topoisomerase IIα in NSCLC as well as in human NSCLC tissue specimens. Topoisomerase IIα overexpression reduced JWA at the translational level. Meanwhile, JWA-induced topoisomerase IIα degradation was regulated both in the transcriptional and post-translational level. Interestingly, JWA and topoisomerase IIα regulated each other in the cells arrested in G2/M. Furthermore, JWA and topoisomerase IIα synergistically affected NCI-H460 cells invasion. These results may serve a novel mechanism for cancer prevention.

Curcumin Reactivates Silenced Tumor Suppressor Gene RARß by Reducing DNA Methylation.

Reactivation of tumor suppressor genes by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Retinoic acid receptor ß (RARß), one member of the RAR receptor family, is considered as a tumor suppressor. Reduced expression of RARß has been reported in lung cancer and other solid tumors. DNA hypermethylation of the promoter region of RARß is a major mechanism for its silencing in tumors. Recently, curcumin has been considered as a potential DNA methyltransferase inhibitor. Herein, we demonstrated that curcumin significantly elevate RARß expression at the mRNA and protein levels in tested cancer cells. Additionally, curcumin decreased RARß promoter methylation in lung cancer A549 and H460 cells. Mechanistic study demonstrated that curcumin was able to downregulate the mRNA levels of DNMT3b. In a lung cancer xenograft node mice model, curcumin exhibited protective effect against weight loss because of tumor burden. Tumor growth was strongly repressed by curcumin treatment. As the results from in vitro, RARß mRNA were increased and DNMT3b mRNA were decreased by curcumin treatment compared with the mice in control group. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for lung cancer through reactivation of RARß.

EGCG Enhances Cisplatin Sensitivity by Regulating Expression of the Copper and Cisplatin Influx Transporter CTR1 in Ovary Cancer.

Cisplatin is one of the first-line platinum-based chemotherapeutic agents for treatment of many types of cancer, including ovary cancer. CTR1 (copper transporter 1), a transmembrane solute carrier transporter, has previously been shown to increase the cellular uptake and sensitivity of cisplatin. It is hypothesized that increased CTR1 expression would enhance the sensitivity of cancer cells to cisplatin (cDDP). The present study demonstrates for the first time that (-)-epigallocatechin-3-gallate (EGCG), a major polyphenol from green tea, can enhance CTR1 mRNA and protein expression in ovarian cancer cells and xenograft mice. EGCG inhibits the rapid degradation of CTR1 induced by cDDP. The combination of EGCG and cDDP increases the accumulation of cDDP and DNA-Pt adducts, and subsequently enhances the sensitivity of ovarian cancer SKOV3 and OVCAR3 cells to the chemotherapeutic agent. In the OVCAR3 ovarian cancer xenograft nude mice model, the combination of the lower concentration of cDDP and EGCG strongly repressed the tumor growth and exhibited protective effect on the nephrotoxicity induced by cisplatin. Overall, these findings uncover a novel chemotherapy mechanism of EGCG as an adjuvant for the treatment of ovarian cancer.

Mechanism of Dose-Dependent Regulation of UBE1L by Polyphenols in Human Bronchial Epithelial Cells.

Ubiquitin activating enzyme E1-like (UBE1L) is the activating enzyme for ISG15ylation (ISG15, interferon stimulated gene 15). UBE1L is thought to be a candidate tumor suppressor gene and has positive activity against stress responses such as viral infections. Both type I interferon and retinoic acid are known to induce UBE1L expression. However, the molecular mechanism of UBE1L regulation is unclear. Here, the effect of several chemopreventive polyphenols on UBE1L expression in human bronchial epithelial cells (Beas-2B) was investigated. Lower concentrations of curcumin, (-)-epigallocatechin-3-gallate (EGCG) and resveratrol upregulated UBE1L, while high concentrations of curcumin, EGCG and resveratrol downregulated UBE1L levels. Interestingly, curcumin, EGCG and resveratrol diminished intracellular reactive oxygen species (ROS) at lower concentrations but generated ROS at higher concentrations. The antioxidant N-acetylcysteine (NAC) increased UBE1L protein levels, while pro-oxidants such as hydrogen peroxide and tert-butyl hydroperoxide (tBHP) decreased UBE1L protein levels, indicating that the intracellular redox status is associated with UBE1L expression. Kinase inhibitors were used to examine the contribution of mitogen-activated protein kinase (MAPK) activity to the polyphenol-regulated UBE1L. Only the inhibition of c-Jun N-terminal kinase (JNK) significantly reduced UBE1L expression. Knockdown of nuclear factor erythroid-2 related factor-2 (Nrf2) caused a concomitant decrease in UBE1L protein levels. It is concluded from the above mentioned results that JNK/Nrf2 signal pathway is involved in the regulation of UBE1L via intracellular ROS status when cells came in contact with polyphenols.

Downregulation of cystathionine ß-synthase and cystathionine γ-lyase expression stimulates inflammation in kidney ischemia-reperfusion injury.

Inflammation plays a critical role in kidney ischemia-reperfusion injury but mechanisms of increased proinflammatory cytokine expression are not completely understood. Kidney has a high expression of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) that can synthesize hydrogen sulfide. CBE and CSE are also responsible for the synthesis of cysteine, an essential precursor for glutathione, an antioxidant. Reduced hydrogen sulfide and glutathione production is associated with multiple organ injury. Although pro- and anti-inflammatory effects of hydrogen sulfide have been reported, its role in ischemia-reperfusion-induced inflammation in the kidney has not been well addressed. The aim of this study was to investigate the effect of CBS and CSE-mediated hydrogen sulfide and glutathione production on kidney inflammatory response and the mechanism involved. The left kidney of Sprague-Dawley rat was subjected to 45-min ischemia followed by reperfusion for 24 h. Ischemia-reperfusion caused a significant decrease in CBS and CSE mRNA and protein levels with a concomitant reduction of glutathione and hydrogen sulfide production in the kidney while the expression of proinflammatory cytokine expression (MCP-1, IL-6) was elevated. Hypoxia-reoxygenation of proximal tubular cells led to a decrease in CBS and CSE expression and an increase in proinflammatory cytokine expression. Supplementation of glutathione or hydrogen sulfide donor (NaHS) effectively attenuated cytokine expression in tubular cells. These results suggested that ischemia-reperfusion impaired CBS and CSE-mediated glutathione and hydrogen sulfide production in the kidney, which augmented the expression of proinflammatory cytokines. Regulation of CBS and CSE expression may be therapeutically relevant in alleviating ischemia-reperfusion-induced inflammation and improving kidney function.

Curcumin and (-)-epigallocatechin-3-gallate attenuate acrylamide-induced proliferation in HepG2 cells.

Acrylamide, a proven rodent carcinogen, is present in carbohydrate-rich food heated at high temperatures. It can be metabolized into glycidamide mainly by cytochrome P450 2E1 (CYP2E1). The fact that acrylamide is a potential carcinogen to human-beings draws public attention recently. This study aimed to elucidate the effect of acrylamide at low doses on proliferation of HepG2 cells, and to test whether the two well-studied chemopreventive agents, curcumin and (-)-epigallocatechin-3-gallate (EGCG), would have antagonistic effects against acrylamide. The results showed that lower concentration of acrylamide (⩽100µM) significantly increased the proliferation of HepG2 cells, but not of the other cancer cells (MDA-231, HeLa, A549, and PC-3). Only in HepG2 cells, low concentration of acrylamide was able to induce CYP2E1 expression significantly. Knockdown of CYP2E1 restrained acrylamide to increase viability of HepG2 cells. In addition, acrylamide raised expression of epidermal growth factor receptor (EGFR), cyclin D1 and nuclear factor-κB (NF-κB), which contributed to cell proliferation. Both curcumin and EGCG effectively reduced acrylamide-induced proliferation, as well as protein expression of CYP2E1, EGFR, cyclin D1 and NF-κB. All these results suggest that low concentration of acrylamide may contribute to progression of hepatocellular carcinoma (HCC). Curcumin or EGCG could prevent acrylamide triggering this effect.

Folic acid supplementation attenuates high fat diet induced hepatic oxidative stress via regulation of NADPH oxidase.

Diets high in saturated fat and cholesterol facilitate weight gain, a predisposing factor that contributes to the onset of obesity and metabolic disorders. Hepatic oxidative stress is commonly reported in various animal models of obesity and has been associated with enhanced expression of NADPH oxidase. We have previously reported several antioxidant mechanisms through which folic acid confers protection during hyperhomocysteinemia-induced oxidative stress. The objective of the present study was to investigate whether folic acid supplementation ameliorates high-fat diet induced oxidative stress in the liver, and to identify the underlying mechanisms. Male C57BL/6J mice were fed a control diet, a high-fat diet, or a high-fat diet supplemented with folic acid for 12 weeks. A high-fat diet led to increased body mass, hepatic lipid peroxidation, and liver injury. There was a significant increase in hepatic NADPH oxidase activity, which was associated with enhanced expression of several NADPH-oxidase subunits. Folic acid supplementation had a protective effect against high-fat diet induced hepatic oxidative stress and liver injury. Further analysis revealed that the antioxidant effect of folic acid was attributed, in part, to transcriptional regulation of NADPH oxidase. These results suggested that folic acid supplementation may be hepatoprotective from liver injury associated with a high-fat diet.