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Phenformin (Phen), a potent activator of AMPK, is effective against some resistant cancers. This study evaluated the inhibition of proliferation, migration, invasion, and angiogenesis by Phen in aggressive cancer cells and investigated the underlying mechanism of the inhibition. Cholangiocarcinoma (CCA) KKU-156 and KKU-452 cells were used in this study. The results showed that Phen suppressed cell proliferation and induced apoptosis in both cells. Phen suppressed migration and invasion of cancer cells in wound healing and transwell chamber assays, respectively. The effects were associated with depletions of glutathione (GSH) and decreased glutathione redox ratio which represents cellular redox state. The redox stress was linked with the loss of mitochondrial transmembrane potential, as evaluated by JC-1 assay. The effect of Phen on angiogenesis was performed using HUVEC cultured cells. Phen alone did not affect tube formation of HUVEC cells. However, conditioned media from CCA cell cultures treated with Phen suppressed the tube-like structure formation. The antitumor effect of Phen was associated with AMPK activation and suppression of mTOR phosphorylation, HIF-1A, and VEGF protein expression. In conclusion, Phen inhibits cell proliferation, migration, invasion, and angiogenesis probably through AMPK-mTOR and HIF-1A-VEGF pathways. Phen may be repurposed as chemoprevention of cancer.

Oxidative stress plays a critical role in the pathophysiology of hypertension (HT) and the progression of atherosclerotic coronary artery disease (CAD). Genetic variations in superoxide dismutase (SOD), glutathione peroxidase 3 (GPX3), paraoxonase 1 (PON1) and glutathione S-transferase theta 1 (GSTT1) may modulate their gene functions, affecting protein functions. These changes could have an impact on the pathogenesis of HT and progression of CAD. The present study investigated the associations of individual and combined antioxidant-related gene polymorphisms with the incidence of HT and severity of CAD. Two study populations were enrolled. The HT-associated study comprised 735 control and 735 hypertensive subjects (mean age 59.3 ± 9.0 years), matched for age and sex. The CAD study, hospital-based subjects (mean age 62.1 ± 9.5 years), included 279 CAD patients and 165 non-CAD subjects. Gene polymorphisms were identified in genomic DNA using polymerase chain reaction (PCR)-based technique. Genetic variations were assessed for their associations with HT and severity of CAD. Antioxidant gene variants, SOD3 rs2536512-GG, GPX3 rs3828599-GG, PON1 rs705379-TT, and GSTT1 -/- and +/- , were independently associated with the incidence of HT. A combination of four HT-associated genotypes, as a genetic risk score (GRS), revealed an association of GRS 5 and GRS ≥ 6 with increased susceptibility to HT and CAD, and further with multivessel coronary atherosclerosis (multivessel CAD) compared with GRS 0-2 [respective ORs(95% CI) for GRS ≥ 6 = 2.37 (1.46-3.85), 3.26 (1.29-8.25), and 4.36 (1.36-14.0)]. Combined polymorphisms in these four antioxidant-related genes were associated with the incidences of HT and CAD, and with the severity of coronary atherosclerosis.

Phenethyl isothiocyanate (PEITC) is a potential cancer prevention agent that is found in cruciferous vegetables. Previous studies have shown that the effect of PEITC-induced cell death declines rapidly after administration. The metabolic fate of PEITC is modulated by glutathione S-transferases (GST). In this study, we investigated whether GST activity modulates PEITC-induced cytotoxicity on cholangiocarcinoma (CCA) cells. The sensitivity of KKU-M214 and KKU-100 cells to PEITC was associated with GST activity. Two GST inhibitors, ethacrynic acid (EA) and cibacron blue, potentiated the cytotoxic effect of PEITC in CCA cells. PEITC-induced glutathione (GSH) depletion and redox stress, whereas EA itself or in combination with PEITC did not alter GSH redox status. The enhanced cytotoxic effect of EA may be due to inhibition of GST activity. This idea was validated by using siRNA directed against GSTP1 mRNA in KKU-M214 cells, and GSTP1 and GSTT1 mRNA in KKU-100 cells. These GST isoforms were abundantly expressed in the cell lines. Knockdown of GSTs in CCA cell lines potentiated the cytotoxic effect of PEITC. The present study shows that the antitumor effect of PEITC was potentiated by the suppression of GST activity. The inhibition of GST could be a crucial strategy to potentiate chemotherapeutic effect of PEITC on CCA.

Asiatic acid, a triterpenoid compound derived from Centella asiatica, has been demonstrated to have antioxidant and anti-inflammatory effects. The present study evaluated the effects of asiatic acid on hemodynamic alterations, renin-angiotensin system (RAS), oxidative stress, and inflammation in 2K-1C hypertensive rats. Renovascular hypertension was induced in male Sprague-Dawley rats and treated with vehicle, asiatic acid (30 mg/kg/day), or captopril (5 mg/kg/day) for 4 weeks. We observed that 2K-1C hypertensive rats exhibited hemodynamic alterations such as high blood pressure, heart rate, hindlimb vascular resistance, and low hindlimb blood flow. Signs of RAS activation, such as increased plasma angiotensin II and serum angiotensin-converting enzyme activity, enhanced AT 1 R protein expression, and suppressed AT 2 R expression was observed in 2K-1C hypertensive rats. Overproduction of vascular superoxide, high levels of plasma MDA, low levels of plasma nitric oxide metabolites (NOx), and upregulation of gp91 phox protein expression were observed in hypertensive rats. Furthermore, inflammation was observed in hypertensive rats, as evidenced by increased plasma TNF-α, NF-κB, and phospho-NF-κB protein expression. Asiatic acid or captopril alleviated hemodynamic alterations, RAS activation, oxidative stress, and inflammation in 2K-1C hypertensive rats. These findings indicate that asiatic acid is an antihypertensive agent that ameliorates hemodynamic alterations in 2K-1C hypertensive rats. This effect may involve one or both of the following mechanisms: the direct effect of asiatic acid on RAS activation, oxidative stress and inflammation, and/or asiatic acid acting as an ACE inhibitor agent to inhibit the Ang II-AT 1 R-NADPH oxidase-NF-κB pathway.

Phenethyl isothiocyanate (PEITC) is a natural isothiocyanate with anticancer activity against many drug-resistant cancer cells. A body of evidence suggests that PEITC enhances oxidative stress leading to cancer cell death. Cholangiocarcinoma (CCA) is an aggressive bile duct cancer with resistance to chemotherapeutic drugs. PEITC rapidly kills KKU-100 CCA cells with concurrent induction of cellular glutathione depletion, superoxide formation, and loss of mitochondrial transmembrane potential. The loss was associated with increased Bax and decreased Bcl-xl proteins followed by the release of cytochrome c and the activation of caspase-9 and -3. Although TEMPOL could prevent superoxide formation, it did not prevent the disruption of glutathione (GSH) redox, mitochondrial dysfunction, and cell death. On the other hand, N-acetylcysteine could prevent the events and cell death. It was concluded that disruption of GSH redox but not superoxide formation may be an initial step leading to mitochondrial injury. PEITC could be a promising chemopreventive agent for CCA.

Patients with thalassemia major are susceptible to cardiovascular complications by mechanisms not fully understood. Although overt cardiovascular complications usually occur after puberty, their underlying pathogenesis may begin much earlier. This study investigated whether there were early changes in vascular endothelial function and arterial stiffness in young patients with beta-thalassemia and hemoglobin E, and whether these changes were associated with oxidative stress and expression of antioxidant genes. The study recruited 30 pediatric patients and 30 age-matched control subjects. Compared with the control subjects, the patients had increased levels of oxidant biomarkers including malondialdehyde, protein carbonyl, and non-transferrin-bound iron and a decreased glutathione redox ratio. There were clear signs of vascular endothelial dysfunction and increased arterial stiffness, as shown by marked suppression of forearm blood flow after reactive hyperemia and increased pulse-wave velocity in the trunk and legs. The changes in FBF were associated with oxidative stress markers and free iron. An adaptive antioxidant gene response was activated in blood mononuclear cells, as shown by upregulation of GCLC and Bach-1 mRNA but downregulation of heme oxygenase-1 and thioredoxin mRNA. The results highlight the vascular changes seen even in young patients during treatment. These changes were associated with oxidative stress and suggest an adaptive response that serves to protect host cells from further oxidative damage.

Inhibition of nitric oxide synthesis with N ( ω )-nitro-L-arginine methyl ester (L-NAME) induces marked hypertension and oxidative stress. Curcumin (CUR) has been shown strong antioxidant property. Tetrahydrocurcumin (THU), a major metabolite of CUR, possesses several pharmacological effects similar to CUR; however, it is less studied than CUR. We investigated whether CUR and THU could prevent vascular dysfunction and inhibit development of hypertension in L-NAME-treated rats. Male Sprague-Dawley rats were administered with L-NAME (50 mg/kg/day) in drinking water for 3 weeks. CUR or THU (50 and 100 mg/kg/day) was fed to animals simultaneously with L-NAME. L-NAME administration induced increased arterial blood pressure and elevated peripheral vascular resistance accompanied with impaired vascular responses to angiotensin II and acetylcholine. CUR and THU significantly suppressed the blood pressure elevation, decreased vascular resistance, and restored vascular responsiveness. The improvement of vascular dysfunction was associated with reinstating the marked suppression of eNOS protein expression in the aortic tissue and plasma nitrate/nitrite. Moreover, CUR and THU reduced vascular superoxide production, decreased oxidative stress, and increased the previously depressed blood glutathione (GSH) and the redox ratios of GSH in L-NAME hypertensive rats. The antihypertensive and some antioxidant effects of THU are apparently more potent than those of CUR. This study suggests that CUR and THU prevented the development of vascular dysfunction induced by L-NAME and that the effects are associated with alleviation of oxidative stress.

Thalassemia major is characterized by anemia, iron overload, and oxidant damage to major organs, especially the cardiovascular system. Oxidative stress is ultimately involved in endothelial dysfunction, a condition which is evident in adults suffering from various cardiovascular diseases including thalassemia. We investigated endothelial function in pediatric patients with hemoglobin E-beta thalassemia (HbE-beta thalassemia), who have been exposed to excessive iron and oxidative stress for much shorter period than adults with thalassemia. We recruited 22 blood transfusion-dependent HbE-beta thalassemia patients aged 11.8 +/- 2.9 years and 20 healthy controls aged 12.1 +/- 1.7 years. Oxidant status was determined, and endothelial function was assessed by a forearm blood flow technique. Oxidative stress was increased in the thalassemic patients, as blood glutathione (GSH) and ratios of reduced GSH to GSH disulfide were markedly reduced, and superoxide anion released from blood cells was highly elevated. Oxidative stress response, assessed by gamma-glutamylcysteine ligase activity, was increased approximately twofold in thalassemia patients. Basal forearm blood flow was significantly increased in patients compared with controls (7.3 +/- 1.8 vs 6.0 +/- 1.8 ml/100 ml tissue/min, respectively), whereas forearm vasodilatory response to reactive hyperemia was depressed by 50% in patients compared with controls. Endothelial function is impaired in young thalassemia patients, and impaired endothelial function is associated with oxidant stress.