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To investigate the anti-tumor effect of licochalcone A (LCA) on proliferation and migration in cholangiocarcinoma (CCA) cells and to elucidate their underlying mechanisms.Human CCA cells, KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452 were used to study effect of LCA on proliferation and migration by a cytotoxicity assay, wound healing assay. Reactive oxygen species levels were evaluated using DHE-fluorescent probes. Proteins associated with cancer survival and progression were analyzed by immune blotting assay.LCA suppressed proliferation and induced cell death in CCA cells including KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452. The CCAs cells were suppressed in association with LCA-induced accumulation of intracellular reactive oxygen species (ROS). Increased formation of ROS was causally related with suppression of Nrf2 and its down-stream antioxidant and cytoprotective enzymes. These effects may lead to the expression of Bax and release of cytochrome c and ensuring cell death. Interestingly, LCA could also inhibit cell migration and cell cycle arrest at low concentrations. These effects were associated with down-regulation of NF-kB, STAT3 and their down-stream proteins, cyclin D1, VEGF, and ICAM-1.These results suggest that LCA has potential therapeutic activity in suppression of CCA cells.

Two new compounds, a rearranged limonoid (harriforanoid) and a benzopyran (harriforanone) were isolated from the leaves and stems of Harrisonia perforata (Blanco) Merr., together with three known limonoids and fourteen known chromones. The structures of the compounds were elucidated using spectroscopic analysis. The experimental and calculated ECD spectra were used to characterize the absolute configuration of harriforanone. The cytotoxicity of each compound was assessed towards cholangiocarcinoma (KKU-100) and human liver cancer (HepG2) cell lines. However, none of these compounds exhibited any activity (IC50 > 50 μM).

Purpose: To explore the effect of alendronate on cell death and migration of cholangiocarcinoma (CCA). Methods: Migration and cell death of CCA cells were determined using sulforhodamine B (SRB), colony formation, wound healing, and gelatin zymography assays. The mechanism of action of alendronate was studied with reverse-transcriptase polymerase reaction (RT-PCR) for gene expression and by Western blotting analysis for protein expression. Results: Alendronate stimulated KKU-100 cell death in dose- and time-dependent manner, with low IC50 value, and significantly inhbited colony formation at doses of 5 - 100 µM. Moreover, alendronate at doses of 250 - 1000 µM significantly stimulated CCA apoptosis via reactive oxygen species (ROS) generation, and enhanced caspase 3 activity at a dose of 1000 µM. Moreover, at a dose of 250 µM, it significantly inhibited cell growth through induction of caspase 3 and p53, and reduction of protein expression levels of NF-ĸB. Furthemore, alendronate altered mevalonate (MVA) pathway via downregulation of Rac1 protein expression. In contrast, it significantly inhibited CCA cell migration, and reduced MMP 2 and MMP 9 levels at doses of 25 - 100 µM. Conclusion: Alendronate may be useful as a novel drug for prevention and chemotherapy of CCA.

Objective Cholangiocarcinoma (CCA) is a noxious malignancy of epithelium of the bile duct with a low response rate to chemotherapy. The epidermal growth factor receptor (EGFR) signaling pathway is implicated in the development of cancerous cells, especially CCA. In this study, we report detailed biological profiling of 13f identified from our earlier hit expansion studies. The aim of this work was to expand our understanding of 13f via more detailed investigations of its mechanism of action against KKU-100, KKU-452 and KKU-M156 CCA cells, as well as in comparison to the EGFR inhibitor Gefitinib and non-specific chemotherapeutic agents such as Cisplatin. Methods Inhibiting EGFR-Kinase, cytotoxicity, clonogenic assay, wound healing and apoptosis were performed. Levels of total expression of EGFR and EGFR phosphorylation proteins were detected. Results 13f was confirmed as an inhibitor of EGFR with an IC50 value against the tyrosine kinase of EGFR of 22 nM and IC50 values for 48 h incubation period were 1.3 ± 1.9, 1.5 ± 0.4 and 1.7 ± 1.1 µM of KKU-100, KKU-452 and KKU-M156, respectively through dose- and time-dependent induction of early apoptosis of CCA cells. The compound also suppressed the clonogenic ability of KKU-100 and KKU-M156 cells stronger than Gefitinib, while potently inhibiting EGF-stimulated CCA cell migratory activity in KKU-452 cells. It was observed that under normal conditions EGFR was activated in CCA cells. EGF-stimulated basal expression of EGFR in KKU-452 cells was suppressed following 13f treatment, which was significantly greater than that of the marketed EGFR inhibitor Gefitinib. Conclusion In summary, our study showed that 13f has potent anti-cancer activities including antiproliferation, clonogenic ability and migration through the modulation of EGFR signaling pathway in CCA for the first time. The compound represents an interesting starting point as a potential chemotherapeutic agent in ongoing efforts to improve response rate in CCA patients. .

Objective: To evaluate the potential therapeutic effect of Sang-Yod rice bran hydrolysates (SRH) and in combination with lisinopril against hypertension, endothelial dysfunction, vascular remodeling, and oxidative stress in rats with nitric oxide deficiency-induced hypertension. Methods: Hypertension was induced in male Sprague-Dawley rats by administration of a nitric oxide synthase inhibitor, Nω- nitro-L-arginine methyl ester (L-NAME) in drinking water for 6 weeks. Hypertensive rats were administered daily with SRH (500 mg/kg/day), lisinopril (1 mg/kg/day), or the combination of SRH and lisinopril by gastric lavage for the last 3 weeks of L-NAME treatment. Hemodynamic status, vascular reactivity to vasoactive agents, and vascular remodeling were assessed. Blood and aortic tissues were collected for measurements of oxidative stress markers, plasma angiotensin-converting enzyme (ACE) activity, plasma angiotensin II, and protein expression. Results: L-NAME induced remarkable hypertension and severe oxidative stress, and altered contents of smooth muscle cells, elastin, and collagen of the aortic wall. SRH or lisinopril alone reduced blood pressure, restored endothelial function, decreased plasma ACEs and angiotensin II levels, alleviated oxidant markers and glutathione redox status, and restored the vascular structure. The effects were associated with increased expression of endothelial nitric oxide synthase and decreased expression of gp91phox and AT1R expression. The combination of SRH and lisinopril was more effective than monotherapy. Conclusions: SRH alone or in combination with lisinopril exert an antihypertensive effect and improve endothelial function and vascular remodeling through reducing oxidative stress and suppressing elevated renin-angiotensin system.

Objective Cholangiocarcinoma (CCA) is a malignant tumor with aggressive metastatic property resulted from dysregulation of metastasis-regulated signaling pathways. The aim of this study was to investigate the effect of cucurbitacin B on metastatic behavior of CCA cells through modulation of focal adhesion kinase (FAK) protein. Methods KKU-452 cells were treated with a specific FAK inhibitor, FAK inhibitor-14, or cucurbitacin B at various concentrations for 24 h. Cell viability was assessed by sulforhodamine B assay. The migratory and invasive abilities of the cells were investigated using wound healing and transwell invasion assays, respectively. The fibronectin-coated plate was used for adhesion assay. The effects of the test compounds on FAK activation and the expression of metastasis-associated proteins were determined by Western blot analysis. The amount of MMP-9 was evaluated using a commercial ELISA Kit. Results FAK inhibitor-14 and cucurbitacin B at concentrations which minimally affected KKU-452 cell viability could suppress FAK activation, evidently by decreased level of phospho-FAK protein after exposure to the compound. At these conditions, cucurbitacin B suppressed metastatic behavior including migration, invasion and adhesion abilities of CCA cells similar to FAK inhibitor-14. Further molecular studies demonstrated that FAK inhibitor-14 and cucurbitacin B downregulated the expression of metastasis-associated proteins including MMP-9, ICAM-1 and VEGF. Consequently, exposure to cucurbitacin B inhibited the production of MMP-9 enzyme in CCA cells similar to FAK inhibitor-14 treatment. Conclusion FAK participated in regulation of metastatic behavior of KKU-452 CCA cells. Cucurbitacin B suppressed FAK activation in the cells which was associated with inhibition of metastasis essential steps and their related metastatic proteins. The compound may be developed as a novel therapeutic agent for CCA metastasis therapy. .

PURPOSE Cholangiocarcinoma (CCA) remains a disease with poor prognosis and limited therapeutic options. Identification of driver genetic alterations may lead to the discovery of more effective targeted therapies. CCAs harboring FGFR2 fusions have recently demonstrated promising responses to FGFR inhibitors, highlighting their potential relevance as predictive biomarkers. CCA incidence is high in the northeast of Thailand and its neighboring countries because of chronic infection with the liver fluke Opisthorchis viverrini (Ov). However, there are currently no available data on the prevalence of FGFR alterations in fluke-associated CCA in endemic countries. MATERIALS AND METHODS In this study, we performed anchored multiplex polymerase chain reaction target enrichment RNA sequencing of FGFR1-3, validated by fluorescence in situ hybridization and Sanger sequencing, in 121 Ov-associated and 95 non–Ov-associated CCA tumors. RESULTS Compared with non–fluke-associated CCA (11/95; 11.6%), FGFR2 fusions were significantly less common in fluke-associated CCA (1/121; 0.8%; P = .0006). All FGFR fusions were detected exclusively in intrahepatic CCAs and were mutually exclusive with KRAS/ERBB2/BRAF/FGFR mutations, pointing to their potential roles as oncogenic drivers. CONCLUSION FGFR2 fusions are rare in fluke-associated CCA, underscoring how distinct etiologies may affect molecular landscapes in tumors and highlighting the need to discover other actionable genomic alterations in endemic fluke-associated CCA.

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.

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism. Methods: Rice bran hydrolysates were prepared from pigmented rice (Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells (PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells (HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin (PHA)- stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of mTOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

Objectives Strategies that induce apoptosis of malignant cells are recognized as effective cancer treatments. This study evaluated the apoptosis-inducing ability of momordin Ic against cholangiocarcinoma (CCA) cells and the respective underlying mechanisms. Methods Quantification of apoptotic cells was performed using Annexin V/7-AAD double dye staining followed by flow cytometry. The effect of momordin Ic on the expression of epidermal growth factor receptor (EGFR) and its downstream signalling molecules was determined via Western blot analysis. The RT2 Profiler PCR Array was used to determine the expression of cell death-associated genes. Expression levels of apoptosis-related proteins were examined using an apoptosis antibody array. Key findings Momordin Ic potently limited the ability of CCA cells to thrive by promoting apoptotic cell death. This apoptosis-inducing activity was accompanied with suppression of expression of EGFR, p-EGFR, c-Myc and other downstream EGFR signalling-related molecules. Additional molecular analyses demonstrated that momordin Ic modified the expression profile of cell death-associated genes in CCA cells. Moreover, significant upregulation of apoptosis-activating proteins and downregulation of apoptosis-inhibiting protein were also observed after exposure to momordin Ic. Conclusions These results suggest that momordin Ic has a potential therapeutic opportunity for CCA treatment by acting as an EGFR suppressant.

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Pamidronate has been hypothesized to effectively inhibit cancer cell growth and metastasis in bone tissue. Furthermore, pamidronate (Pami) exerts various direct effects against several cancer cell types, including growth and migration. The present study aimed to determine the underlying mechanism of Pami's effect on the proliferation and migration of cholangiocarcinoma (CCA) cells. KKU-100 cells were used to determine the effects of Pami on cell death and migration. The following were assessed: Sulforhodamine B, colony formation, apoptosis via flow cytometry, reactive oxygen species (ROS) production and caspase-3 activity. In addition, the effects of the test compound on the mevalonate (MVA) signaling pathway were determined via western blotting and reverse transcription-quantitative PCR. Cell migration was observed via wound healing, Matrigel and gelatin zymography. The results indicated that Pami induced CCA cell death and inhibited colony formation in a dose-dependent manner, with IC50 values of 444.67±44.05 µM at 24 h and 147.33±17.01 µM at 48 h. Furthermore, Pami treatment suppressed colony formation at a lower concentration than growth inhibition with IC50 values of 5.36±0.31 µM. The mechanism of growth inhibition was determined to potentially be associated with increased ROS generation and stimulated caspase-3 enzyme activity, leading to the induction of apoptosis. Furthermore, Pami treatment interfered with the MVA signaling pathway by reducing Rac1 protein levels and modulating the gene and protein expression of RhoA. Furthermore, Pami suppressed CCA cell migration by decreasing matrix metalloproteinase (MMP)2 and MMP9 levels. Additionally, Pami treatment activated CCA cell death and inhibited CCA migration at low concentrations. Pami significantly decreased the protein expression levels of Rac1 in the MVA signaling pathway and may therefore be beneficial for developing a novel chemotherapeutic method for CCA.

Four new dimeric styrenes, 1-4, were isolated from an EtOAc crude extract of the seeds of Atalantia monophylla. The biosynthetic pathway of 1 is proposed to involve a [2 + 2] cycloaddition, while 2-4 may be generated via a polar mechanism with a carbocation as the key intermediate. The structures of 1-4 were defined from spectroscopic analysis; experimental and calculated ECD spectra were used to characterize their absolute configurations. When tested against two different cancer cell lines, 1-4 were not determined to be cytotoxic (IC50 > 10 μM).

Chemotherapy is a palliative treatment for unresectable patients with cholangiocarcinoma (CCA). However, drug resistance is a major cause of the failure of this treatment. Derrischalcone (DC), a novel chalcone isolated from Derris indica fruit, has been shown pharmacologically active; though, the effect of DC on CCA is unknown. The present study investigated the cytotoxic, antiproliferative, anti-migration, and anti-invasion effects and underlying mechanisms of DC on CCA KKU-M156 and KKU-100 cells. Cytotoxicity and apoptosis were evaluated by acridine orange and ethidium bromide fluorescent staining. Reactive oxygen species (ROS) was measured by dihydroethidium assay. Cell proliferation and reproductive cell death were assessed by sulforhodamine B staining and colony-forming assay. Migration and invasion were determined by wound healing and transwell chamber assays. Protein expressions associated with cell death, proliferation, migration, and invasion were analyzed by western immunoblotting. We found that DC induced cytotoxicity and apoptosis in association with ROS formation and oxidative stress. Treatment with N-acetylcysteine suppressed ROS formation and attenuated DC-induced cytotoxic and apoptotic effects. DC increased the expression of p53, p21, Bax, and cytochrome c proteins in association with cell death. DC-induced antiproliferation, colony formation, anti-migration, and anti-invasion were associated with the suppression of Akt/mTOR/cyclin D1 and FAK signaling pathways. These findings suggest that the multi-targeting strategies with DC may be a novel treatment for cancer therapy.

Lead (Pb) and cadmium (Cd) are environmental pollutants and nonessential elements in the body. Both metals induce the development of hypertension which is associated with oxidative stress. Curcumin (CUR) is a polyphenolic compound with strong antioxidant activity. The present study evaluated the effect of CUR on oxidative stress, alteration of vascular responsiveness and hypertension induced by exposure to either Pb, Cd or the combination of Pb and Cd. Male Sprague-Dawley rats were exposed to low level of lead acetate (100 mg/L) and/or cadmium chloride (10 mg/L) in the drinking water for 16 weeks. The control animals received deionized water as drinking water. CUR (100 mg/kg) or propylene glycol as vehicle was intragastrically administered once daily for the last 4 weeks. Exposure to Pb, Cd or the combination induced increases in blood pressure and peripheral vascular resistance, and decreased the blood pressure response to intravenous infusion to acetylcholine. Supplementation with CUR significantly reduced blood pressure, alleviated oxidative stress, and increased plasma nitrate/nitrite and glutathione in the blood. The effects of CUR were associated with the improvement of vascular responsiveness, upregulation of the endothelial nitric oxide synthase and downregulation of the NADPH oxidase expression. Furthermore, CUR reduced the metal levels in blood, aorta, liver and kidney. Altogether, exposure to the combination of Pb and Cd aggravated hypertension and oxidative stress, and CUR effectively ameliorated these adverse events in metal exposed animals. Data indicate that CUR may be useful as a dietary supplement for protection against the noxious effects of the heavy metals.

To investigate the oncogenic role of FGFR2 in carcinogenesis in cholangiocarcinoma (CCA) cells. In addition, the feasibility of using FGFR inhibitors in combination with standard chemotherapy was also explored for the chemosensitizing effect in CCA cells.Five CCA cell lines were used to screen FGFR2 expression by Western immunoblotting. Two CCA cell lines, KKU-100 and KKU-213A, were knocked down of the FGFR2 gene using siRNA. Cell viability was assessed by the MTS cell proliferation assay. Reproductive cell death was assessed by clonogenic assay. The effects on cell migration and invasion were analyzed by the Transwell chamber method. Cell cycle analysis was performed by flow cytometry. Cell angiogenesis was assessed by HUVEC tube formation and human angiogenesis antibody array analysis. Proteins associated with proliferative and metastatic properties were evaluated by Western blotting.Knockdown of FGFR2 suppressed cell growth and colony formation in CCA cells in association with G2/M cell cycle arrest and downregulation of STAT3, cyclin A and cyclin B1. Silencing FGFR2 enhanced the suppressive effect of gemcitabine (Gem) on cell migration and invasion. The combination of infigratinib, an FGFR inhibitor, and Gem, interrupted cell growth, migration, and invasion via downregulation of FGFR/AKT/mTOR pathways and the EMT-associated proteins vimentin and slug. Moreover, the combination also suppressed tube formation together with decreased expression of the proangiogenic factor VEGF.Inhibition of FGFRs by infigratinib enhanced the antitumor effect of Gem in CCA cells through downregulation of the FGFR/AKT/mTOR, FGFR/STAT3 and EMT signaling pathways.

Inadequate responses to traditional chemotherapeutic agents in cholangiocarcinoma (CCA) emphasize a requirement for new effective compounds for the treatment of this malignancy. This study aimed to investigate the antiproliferative property of cucurbitacin B on KKU-100 CCA cells. The determination of underlying molecular mechanisms was also carried out. The results revealed that cucurbitacin B suppressed growth and replicative ability to form colonies of CCA cells, suggesting the antiproliferative effect of this compound against the cells. Flow cytometry analysis demonstrated that the interfering effect of cucurbitacin B on the CCA cell cycle at the G2/M phase was accountable for its antiproliferation property. Accompanied with cell cycle disruption, cucurbitacin B altered the expression of proteins involved in the G2/M phase transition including downregulation of cyclin A, cyclin D1, and cdc25A, and upregulation of p21. Additional molecular studies demonstrated that cucurbitacin B suppressed the activation of focal adhesion kinase (FAK) which consequently resulted in inhibition of its kinase-dependent and kinase-independent downstream targets contributing to the regulation of cell proliferation including PI3K/PDK1/AKT and p53 proteins. In this study, the transient knockdown of FAK using siRNA was employed to ascertain the role of FAK in CCA cell proliferation. Finally, the effect of cucurbitacin B on upstream receptor tyrosine kinases regulating FAK activation was elucidated. The results showed that the inhibitory effect of cucurbitacin B on FAK activation in CCA cells is mediated via interference of EGFR and HER2 expression. Collectively, cucurbitacin B might be a promising drug for CCA treatment by targeting FAK protein.

Rice bran hydrolysates contain highly nutritional proteins and beneficial phytochemicals. Sang-Yod rice bran hydrolysates (SRH) extracted from red pigmented rice is a rich source of nutrients and phenolic compounds. The present study evaluated the antihypertensive effect of SRH and its safety in Sprague-Dawley rats. Hypertension was induced in male rats by administration of L-NAME (50 mg/kg/day) in drinking water for three weeks, and the antihypertensive effect of SRH was evaluated. Treatment of SRH (250 or 500 mg/kg) significantly reduced arterial blood pressure and improved hemodynamic parameters. The antihypertensive effect was associated with decreased oxidative stress, suppressed p47phox NADPH oxidase expression, increased nitric oxide bioavailability and decreased angiotensin II level and ACE activity. The SRH was shown to be safe after feeding male and female rats with a rodent diet containing 1.5% SRH for 90 days. Overall, these findings suggest that SRH is safe and may help to prevent hypertension.

Twelve lignan derivatives were synthesized from deoxypodophyllotoxin isolated from Hernandia nymphaeifolia. Cytotoxicity evaluation against cholangiocarcinoma, KKU-100, and HepG2 cell lines showed that compounds 3, 9, 10, and 13 exhibited stronger cytotoxicity than the starting material, 1, with IC50 ranging from 0.42 to 2.01 μM. Compound 10 displayed interesting activity by showing IC50 values of 0.75 and 0.46 μM against KKU-100 and HepG2 cell lines, respectively. From these observation, 10 seems to be useful as a lead compound for the development of anticancer agents.

Aims Transcription factor Nrf2, which regulates the expression of cytoprotective and antioxidant enzymes, contributes to proliferation and resistance to chemotherapy in cancer. The inhibition of Nrf2 can sensitize cholangiocarcinoma (CCA) cells to the cytotoxicity of several chemotherapeutic agents. In this study, we investigated the mechanism of this chemosensitizing effect. Main methods KKU-100 cells were used in the study. Nrf2 expression was knocked down by siRNA and expression was validated by reverse transcription and polymerase chain reaction. Cytotoxicity was assessed by sulforhodamine B method. Intracellular reactive oxygen species (ROS) was examined by fluorescent dye, dichlorofluorescin diacetate method and mitochondrial transmembrane potential was assessed by JC1 dye assay. Key findings Cytotoxicity of cisplatin (Cis) in KKU-100 cells was enhanced by knockdown of Nrf2 expression. The enhanced cytotoxic effect was abolished by treatment with N-acetylcysteine, TEMPOL and MnTBAP. Cells with Nrf2 knockdown or Cis treatment increased production of ROS, and ROS was markedly enhanced by a combination of Nrf2 knockdown and Cis. The increased ROS formation was associated with a decrease in mitochondrial transmembrane potential (Δψm), where this decrease was prevented by antioxidant compounds. The loss of Δψm and cell death were prevented by cyclosporine, an inhibitor of mitochondrial permeability transition pore (MPTP). Luteolin inhibited Nrf2 and markedly enhanced cytotoxicity in combination with Cis. Significance Inhibition of Nrf2 is a feasible strategy in enhancing antitumor activity of chemotherapeutic agents and improving efficacy of chemotherapy in CCA.

Carthamus tinctorius L. (CT) is widely used in Asian countries as a beverage and in folk medicine. The effects of CT extract on hemodynamics, vascular remodeling, the renin-angiotensin system (RAS) and oxidative stress in the two-kidney, one clip (2K-1C) hypertensive rat model were investigated. Renovascular hypertension was induced in male Sprague-Dawley rats and were treated with CT extract (500mg/kg/day) or captopril (5mg/kg/day) or vehicle for four weeks. CT extract or captopril reduced blood pressure, hindlimb vascular resistance, and increased hindlimb blood flow in 2K-1C hypertensive rats (p

Hesperidin, a flavonoid derived from citrus fruits, possesses several beneficial effects including anti-oxidation and anti-inflammation. The aim of this study was to investigate the effects of hesperidin on the renin-angiotensin system (RAS) cascade that mediated oxidative stress and sympathoexcitation in two-kidney, one-clipped (2K-1C) hypertensive rats. 2K-1C hypertension was induced in male Sprague-Dawley rats. Hypertensive rats were treated with hesperidin at 20[Formula: see text]mg/kg or 40[Formula: see text]mg/kg or losartan at 10[Formula: see text]mg/kg beginning at three weeks after surgery and then continued for four weeks ([Formula: see text]/group). Hesperidin reduced blood pressure in a dose-dependent manner in hypertensive rats compared to untreated rats ([Formula: see text]). Increased plasma angiotensin converting enzyme (ACE) activity and angiotensin II levels, as well as, upregulated AT1 receptor protein expression in aortic tissues were attenuated in hypertensive rats treated with hesperidin. Hesperidin suppressed oxidative stress markers and NADPH oxidase over-expression, and restored plasma nitric oxide metabolites in 2K-1C rats. This was associated with improvement of the vascular response to acetylcholine in isolated mesenteric vascular beds and aortic rings from 2K-1C rats treated with hesperidin ([Formula: see text]). Enhancement of nerve-mediated vasoconstriction related to high plasma noradrenaline in the 2K-1C group was alleviated by hesperidin treatment ([Formula: see text]). Furthermore, losartan exhibited antihypertensive effects by suppressing the RAS cascade and oxidative stress and improved vascular dysfunction observed in 2K-1C rats ([Formula: see text]). Based on these results, it can be presumed that hesperidin is an antihypertensive agent. Its antihypertensive action might be associated with reducing RAS cascade-induced NOX2 over-expression and sympathoexcitation in 2K-1C hypertensive rats.

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 AT1R protein expression, and suppressed AT2R 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 gp91phox 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-AT1R-NADPH oxidase-NF-κB pathway.

Nω-Nitro-l-arginine methyl ester (l-NAME)-induced hypertension and cardiovascular remodeling are associated with oxidative stress and inflammation. Garcinia mangostana Linn., has been reported to have antioxidant and anti-inflammatory properties. This study investigated whether G. mangostana pericarp extract (GME) could prevent l-NAME-induced hemodynamic alterations, cardiovascular remodeling, oxidative stress and inflammation in rats. Male Sprague-Dawley rats were given 40mg/kg/day of l-NAME in drinking water to induce hypertension, and were simultaneously treated with GME at a dose of 200mg/kg/day. Rats that received l-NAME for five weeks had high blood pressure, left ventricular hypertrophy and thickening of aortic wall. Vascular superoxide production, plasma malondialdehyde (MDA), and plasma tumor necrosis factor alpha (TNF-α) were significantly increased in l-NAME-hypertensive rats (p

Excessive iron can generate reactive oxygen species (ROS), leading to oxidative stress that is closely associated with cardiovascular dysfunction. Iron overload was induced in male ICR mice by injection of iron sucrose (10mg/kg/day) for eight weeks. Iron overload was evidenced by increased serum iron indices. The mice developed increased blood pressure, impaired vascular function and blunted response of the autonomic nervous system. These effects were accompanied by increased malondialdehyde levels in various tissues, increased nitric oxide metabolites in plasma and urine, and decreased blood glutathione. Tetrahydrocurcumin (THU, 50mg/kg/day), deferiprone (or L1, 50mg/kg/day) or both was orally administered throughout the period of iron sucrose injection. The treatments significantly alleviated the deleterious cardiovascular effects of iron overload, and were associated with modulation of nitric oxide levels. An imbalance between endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) expression in response to iron overload was normalized by THU, L1 or the combination treatment. Moreover, the treatment decreased the upregulated expression levels of gp91phox, p47phox and HO-1. The combination of THU and L1 exerted a greater effect than THU or L1 monotherapy. These results suggest beneficial effects of THU and L1 on iron-induced oxidative stress, hypertension, and vascular dysfunction.

Four new dimeric styrenes

Oxidative stress caused as a result of iron overload is implicated in clinical manifestation of beta-thalassemia/haemoglobin E (β-Thal/HbE). In this study, we investigated the cellular adaptation against oxidative stress in β-Thal/HbE patients. Twenty-four paediatric β-Thal/HbE patients and 22 healthy controls were recruited in the study. Blood samples from patients exhibited iron overload, elevation of lipid peroxidation, and marked diminution in the reduced glutathione (GSH) level. However, expression of glutamate-cysteine ligase catalytic (GCLC) subunit, a key enzyme in GSH biosynthesis, was up-regulated when compared with that in controls. GCLC protein levels were correlated with serum iron. There was an enhanced binding activity of the oligonucleotide probe for Nrf2-driven antioxidant response element (ARE) to nuclear protein from blood mononuclear cells of thalassemia subjects. In conclusion, β-Thal/HbE patients exhibit elevated plasma levels of GCLC expression and Nrf2-ARE binding activity, which may account for their adaptive survival response to oxidative stress.

Aim of Study: Cholangiocarcinoma (CCA) is an aggressive cancer with considerable metastatic potential. Various cytokines secreted by tumor cells or cells in the tumor environment can promote the metastasis of CCA. The aim of the present study was to investigate the effect of myricetin on the inhibition of cytokine-induced migration and invasion and the associated cellular mechanisms in human CCA cells. Materials and Methods: CCA KKU-100 cells were treated with a pro-inflammatory cytokine mixture consisting of interleukin-6, interferon-γ, and tumor necrosis factor-α. The migratory and invasive ability of KKU-100 cells were determined using a wound-healing assay and transwell invasion assay. The effect of myricetin on cytokine-induced STAT3 activation in CCA cells was determined using Western blot analysis. The real-time polymerase chain reaction was performed to determine messenger RNA expression. Results: Myricetin significantly inhibited cytokine-induced migration and invasion of KKU-100 cells. Detailed molecular analyses revealed that myricetin suppressed the activation of the STAT3 pathway, evidently by a decrease of the active phospho-STAT3 protein expression after myricetin treatment. The cytokine-mediated upregulation of metastasis- and inflammatory-associated genes, which are downstream genes of STAT3 including the intercellular adhesion molecule-1, matrix metalloproteinase-9, inducible nitric oxide synthase, and cyclo-oxygenase 2 (COX-2), were also significantly abolished by myricetin treatment. Moreover, the anti-migratory and anti-invasive activities of a widely prescribed COX inhibitor, indomethacin, were also revealed. Conclusion: This finding reveals the anti-metastatic effect of myricetin against CCA cells which is mediated partly through suppression of the STAT3 pathway. This compound could be potentially useful as a therapeutic agent against CCA.

Objective Endothelial dysfunction associated with reduction in nitric oxide (NO) bioavailability plays an important role in development of hypertension. Consumption of a diet rich in antioxidants appears to lower the risk for hypertension. Virgin rice bran oil (VRBO) possesses antioxidant, anti-inflammatory, and hypocholesterolemic activities. However, to our knowledge, the antihypertensive effect of VRBO has not been investigated. The aim of this study was to examine the antihypertensive effect of VRBO in Nω-nitro- l -arginine methyl ester (L-NAME)-induced hypertensive rats and its underlying mechanisms. Methods Hypertension was induced in rats by administration of L-NAME, after which VRBO, lisinopril (Lis), or VRBO + Lis was administered. Studies were then conducted on the hemodynamics of vascular responses to vasoactive substances, plasma angiotensin-converting enzyme (ACE), plasma nitrate/nitrite, oxidative stress, and inflammatory markers. Results L-NAME administration induced hemodynamic changes including elevation of blood pressure, increased peripheral vascular resistance, and endothelial dysfunction. Reduction in plasma nitrate/nitrite, overproduction of vascular superoxide, and increases in plasma ACE, malondialdehyde, protein carbonyl, and plasma tumor necrosis factor-α were observed in L-NAME hypertensive rats. The changes were associated with a marked decrease in endothelial NO synthase expression, increased expression of gp91phoxand vascular cell adhesion molecule-1, and activation of nuclear factor-κB in aortic tissues. Administration of either VRBO or Lis significantly mitigated all of these deleterious effects. The combination of VRBO and Lis was more effective than either treatment alone. Conclusions The antihypertensive effect of VRBO may be mediated by restoration of hemodynamics, increased NO bioavailability, and alleviation of oxidative stress and inflammation. VRBO has an additive effect on antihypertensive medication.

Altered expression of a cytosolic flavoenzyme NAD(P)H:quinone oxidoreductase-1 (NQO1) has been seen in many human tumors. Its remarkable overexpression in cholangiocarcinoma (CCA; an aggressive malignancy of the biliary duct system) was associated with poor prognosis and short survival of the patients. Inhibition of NQO1 has been proposed as a potential strategy to improve the efficacy of anticancer drugs in various cancers including CCA. This study investigated novel NQO1 inhibitors and verified the mechanisms of their enzyme inhibition. Among the different chemical classes of natural NQO1 inhibitors are coumarins, flavonoids, and triterpenoids. Coumarins are a group of particularly potent NQO1 inhibitors. The mechanisms and kinetics of enzyme inhibition of coumarin, aesculetin, umbelliferone, and scopoletin using the cell lysates as a source of NQO1 enzyme best fit with an uncompetitive inhibition model. Among the NOQ1 inhibitors tested in KKU-100 CCA cells, scopoletin and umbelliferone had the strongest inhibitory effect on this enzyme, while aesculetin and coumarin barely affected intracellular NQO1. All coumarins were further tested for cytotoxicity and anti-migration activity. At modest cytotoxic doses, scopoletin and umbelliferone greatly inhibited the migration of KKU-100 cells, whereas coumarin and aesculetin barely reduced cell migration. The anti-migration effect of scopoletin was associated with decreased ratio of matrix metalloproteinase 9/tissue inhibitors of metalloproteinases 1 ( MMP9/ TIMP1) mRNA. These findings suggest that natural compounds with potent inhibitory effect on intracellular NQO1 have useful anti-migration effects on CCA cells. In order to prove that the potent NQO1 inhibitor, scopoletin, is clinically useful in the enhancement of CCA treatment, additional in vivo studies to elucidate the mechanism of these effects are needed.