Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

The anti-cancer activities of Vernonia amygdalina extract in human breast cancer cell lines are mediated through caspase-dependent and p53-independent pathways.

Breast cancer is currently the leading cause of cancer-related deaths among women globally. Notably, medicinal plant extracts may be a potential source for treatments of breast cancer. Vernonia amygdalina (VA) is a woody shrub reported to have not only diverse therapeutic effects but also anti-cancer properties. However, current research about the mechanisms of the anti-cancer potential of VA has been limited. This study aimed to investigate the mechanisms of action of VA that underlie its anti-cancer effects in human breast cancer cell lines (MCF-7 and MDA-MB-231 cells). Results from MTT assay revealed that VA inhibits the proliferation of MCF-7 and MDA-MB-231, in a time- and dose-dependent manner. The underlying mechanism of this growth inhibition involved the stimulation of cell-type specific G1/S phase cell cycle arrest in only MCF-7 cells, and not in MDA-MB-231 cells. While the growth arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of cyclin D1 and cyclin E, it was shown that VA causes cell cycle arrest through a p53-independent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Furthermore, this study revealed that VA induces apoptosis in the two cell lines, as indicated by the increase in Annexin V-positive cells and sub-G1 population, and that this VA-induced apoptosis occurred through both extrinsic and intrinsic apoptotic pathways. The apoptosis in MCF-7 cells was also likely to be caspase-dependent and not p53 transcriptional-dependent. Given that approximately 70% of diagnosed breast cancers express ER-α, a crucial finding was that VA inhibits the expression of ER-α and its downstream player, Akt, highlighting the potential clinical significance of VA. Moreover, VA exhibits synergism when combined with doxorubicin, suggesting that it can complement current chemotherapy. Overall, this study demonstrates the potential applications of VA as an anti-cancer drug for breast cancer treatment.

Anti-diabetic and anti-lipidemic effects of chlorogenic acid are mediated by ampk activation.

Chlorogenic acid (CGA) has been shown to stimulate glucose uptake in skeletal muscle through the activation of AMPK. However, its effect on other metabolic pathways and likewise its effects after long-term consumption have yet to be understood. We investigated the effects of CGA on glucose tolerance, insulin sensitivity, hepatic gluconeogenesis, lipid metabolism and skeletal muscle glucose uptake in Lepr(db/db) mice. Hepatoma HepG2 was used to investigate CGA's effect on hepatic glucose production and fatty acid synthesis. Subsequently, we attempted to evaluate whether these effects of CGA are associated with the activation of AMPK. In Lepr(db/db) mice, acute treatment with CGA lowered AUCglucose in an OGTT. Chronic administration of CGA inhibited hepatic G6Pase expression and activity, attenuated hepatic steatosis, improved lipid profiles and skeletal muscle glucose uptake, which in turn improved fasting glucose level, glucose tolerance, insulin sensitivity and dyslipidemia in Lepr(db/db) mice. CGA activated AMPK, leading to subsequent beneficial metabolic outcomes, such as suppression of hepatic glucose production and fatty acid synthesis. Inhibition and knockdown of AMPK abrogated these metabolic alterations. In conclusion, CGA improved glucose and lipid metabolism, via the activation of AMPK.

Hydrogen sulfide regulates Na+/H+ exchanger activity via stimulation of phosphoinositide 3-kinase/Akt and protein kinase G pathways.

Intracellular pH (pH(i)) is an important endogenous modulator of cardiac function. Inhibition of Na(+)/H(+) exchanger-1 (NHE-1) protects the heart by preventing Ca(2+) overload during ischemia/reperfusion. Hydrogen sulfide (H(2)S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H(2)S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 µM, produced sustained decreases in pH(i) in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl(4) prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester (KT5823) significantly attenuated NaHS-suppressed NHE-1 activity and/or NaHS-induced cardioprotection. Although KT5823 failed to affect NaHS-induced Akt phosphorylation, Akt inhibitor did attenuate NaHS-stimulated PKG activity. In conclusion, this work demonstrated for the first time that H(2)S produced cardioprotection via the suppression of NHE-1 activity involving a PI3K/Akt/PKG-dependent mechanism.

Polyphenols-rich Vernonia amygdalina shows anti-diabetic effects in streptozotocin-induced diabetic rats.

AIM OF THE STUDY: This study aims to investigate the hypoglycemic properties of Vernonia amygdalina Del. (VA) and its possible mechanisms of action in a single-dose STZ induced diabetic rat model. MATERIALS AND METHODS: A dose-response study was conducted to determine optimum dose for the hypoglycemic effect of VA in STZ-induced diabetic rats. The optimum dose (400 mg/kg) was used throughout the 28-day chronic study. Body weight, food and water intakes of the rats were monitored daily. Fasting blood serum, pancreas, liver and soleus muscle were collected for biochemical analyses. Chemical composition of VA was analysed using HPLC and LC-ESI-MS. RESULTS: The study reveals that ethanolic extract of VA contains high level of polyphenols mainly 1,5-dicaffeoyl-quinic acid, dicaffeoyl-quinic acid, chlorogenic acid and luteolin-7-O-glucoside. In an oral glucose tolerance test, 400 mg/kg VA exhibited a significant improvement in glucose tolerance of the STZ-induced diabetic rats. 28-day treatment with 400 mg/kg VA resulted in 32.1% decrease in fasting blood glucose compared to diabetic control. VA also caused significant decrease (18.2% and 41%) in triglyceride and total cholesterol level. Besides, VA showed protective effect over pancreatic ß-cells against STZ-induced damage, causing a slight increase in insulin level compared to diabetic control. VA administration also showed positive regulation of the antioxidant system, both enzymatic and non-enzymatic. Furthermore, VA was found to increase expression of GLUT 4 (24%) in rat skeletal muscle. Further tissue fractionation revealed that it can increase the GLUT 4 translocation (35.7%) to plasma membrane as well, suggesting that VA may stimulate skeletal muscle's glucose uptake. This observation is in line with the restoration in skeletal muscle glycogenesis of VA-treated group. However, no alteration was observed in GLUT 1 expression. In addition, VA also suppressed (40% inhibition) one of the key hepatic gluconeogenic enzymes, glucose-6-phosphatase (G6Pase). CONCLUSIONS: VA possesses antihyperglycemic effect, most probably through increasing GLUT 4 translocation and inhibiting hepatic G6Pase. The polyphenols in the extract may be the candidates that are responsible for the above-mentioned biological activities.

Leonurine protects middle cerebral artery occluded rats through antioxidant effect and regulation of mitochondrial function.

BACKGROUND AND PURPOSE: Oxidative stress is known to be involved in ischemic stroke. Intense interest is drawn to the therapeutic potential of Chinese herbs on ischemic stroke because many of them contain antioxidant properties. Leonurine, 1 of the active compounds from purified Herba Leonuri, was studied to evaluate its possible therapeutic effects on ischemic stroke. Method-Middle cerebral artery occlusion was selected as our model of study. The animals were pretreated with Leonurine orally for 7 days and the surgery was done. One day after surgery, 2,3,5-triphenyltetrazolium chloride staining and neurological deficit score were carried out to evaluate the functional outcome of animals, whereas levels of superoxide dismutase, glutathione peroxidase, and malondialdehyde were analyzed for oxidative stress analysis. For mitochondrial studies, 3 hours after surgery, mitochondria were isolated for analysis of reactive oxygen species production, adenosine triphosphate biosynthesis, oxygen consumption, and respiratory control ratio value. Result-In in vivo experiments, Leonurine pretreatment reduced infarct volume, improved neurological deficit in stroke groups, increased activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase, and decreased levels from the lipid peroxidation marker malondialdehyde. In terms of mitochondrial modulation, Leonurine inhibited mitochondrial reactive oxygen species production and adenosine triphosphate biosynthesis. Animal studies also demonstrated that the mitochondrial function and redox state were restored by Leonurine treatment. CONCLUSIONS: Leonurine has neuroprotective effects and carries a therapeutic potential of stroke prevention.

Scutellarin sensitizes drug-evoked colon cancer cell apoptosis through enhanced caspase-6 activation.

BACKGROUND: We have reported that resveratrol (RSV) and 5-fluorouracil (5-FU) evoked apoptosis through caspase-6 activation in wild-type (p53+/+) and knockout (p53(-/-)) HCT116 human colon cancer cells. In this study, we investigated the sensitization effects of scutellarin (SC), a compound isolated from the traditional Chinese herb Erigeron breviscapus, on RSV and 5-FU-evoked apoptosis of these cancer cells. MATERIALS AND METHODS: The drug-induced apoptosis was qualified by TUNEL staining under fluorescence microscopy, before being quantified by propiodium iodide staining through flow cytometric assay. RESULTS: SC (100 microM) sensitized RSV- (200 microM) and 5-FU (500 microM)-evoked apoptosis in p53+/+ but not p53(-/-) cells. RSV- and 5-FU-elicited caspase-6 activation was promoted by SC in a time-dependent manner. SC itself did not trigger apoptosis or caspase-6 activation at the concentration tested. CONCLUSION: SC is a novel sensitizing agent for both RSV- and 5-FU-evoked apoptosis, through the enhancement of caspase-6 activation in a p53-dependent manner.

Cerebral protection of purified Herba Leonuri extract on middle cerebral artery occluded rats.

AIM OF STUDY: Oxidative stress is involved in stroke. In particular, Chinese Herbal Medicine with antioxidant properties is believed to have potential therapeutic effect. In this study, neuroprotective effects of purified Herba Leonuri (pHL) were evaluated in Wistar rats undergone middle cerebral artery occlusion (MCAO). MATERIALS AND METHODS: The rats were treated with their respective treatments for 2 weeks prior to the MCAO, continually treated for another 7 days after MCAO. During the post-surgery treatment period, neurological deficit score was measured. At the end of treatment, animals were sacrificed and samples were collected for analysis of infarct volume, apoptosis and antioxidant analysis. RESULTS: Under the treatment of pHL, the infarct volume was reduced significantly from 20.75+/-0.03% to 15.19+/-0.02% (p<0.05). The neurological impairment was alleviated to 1.82 as compared to vehicle (2.43). Plasma antioxidant concentration was increased from 0.31+/-0.03 mM to 0.42+/-0.05 mM (p<0.05). DNA oxidative damage was reduced to 1.19+/-0.03 in stroke pHL treated group (p<0.05 as compared to vehicle group, 1.78+/-0.03). pHL could reduce the level of apoptosis and also the pro-apoptotic proteins, but increase the level of anti-apoptotic proteins. CONCLUSION: pHL is believed to have promising therapeutic effect for stroke treatment through antioxidant mechanisms.

Baicalin improves antioxidant status of streptozotocin-induced diabetic Wistar rats.

This study investigated the antioxidant and antidiabetic effects of baicalin, as well as its effects in combination with the antidiabetic drug metformin. Three groups of streptozotocin-induced diabetic rats were given the following treatments for 30 days: (1) 500 mg/kg metformin; (2) 120 mg/kg baicalin; (3) 500 mg/kg metformin + 120 mg/kg baicalin. In addition, vehicle-treated diabetic and nondiabetic controls were used in the experiment. The rats treated with baicalin and metformin + baicalin had significantly elevated (p < 0.05) hepatic activities of superoxide dismutase, catalase, and glutathione peroxidase compared with the vehicle- and metformin-treated groups. Plasma and hepatic lipid peroxide concentrations of the baicalin- and baicalin + metformin-treated groups were significantly reduced (p < 0.05). In addition, baicalin significantly reduced plasma and hepatic triglycerides and cholesterol levels. The study thus showed that baicalin mitigated oxidative stress as well as enhanced the antidiabetic effect of metformin by improving the antioxidant status.

Activation of macrophages by polysaccharide-protein complex from Lycium barbarum L.

Macrophages play crucial roles in innate immunity. This paper reports that a polysaccharide-protein complex isolated from Lycium barbarum (LBP) is able to activate macrophages. LBP was isolated from Lycium barbarum fruit and separated to five homogenous fractions, designated LBPF1, LBPF2, LBPF3, LBPF4 and LBPF5. It was found that LBP (50 mg/kg, i.p.) markedly upregulated the expressions of CD40, CD80, CD86 and MHC class II molecules on peritoneal macrophages. In vitro studies showed that LBP and LBPF1-5 activated transcription factors NF-kappaB and AP-1 by RAW264.7 macrophage cells, induced TNF-alpha, IL-1beta, IL-12p40 mRNA expression, and enhanced TNF-alpha production in a dose-dependent manner. Furthermore, LBP (50 mg/kg, i.p.) significantly enhanced macrophage endocytic and phagocytic capacities in vivo. These results indicate that LBP enhances innate immunity by activating macrophages. The mechanism may be through activation of transcription factors NF-kappaB and AP-1 to induce TNF-alpha production and upregulation of MHC class II costimulatory molecules.

The iron chelator desferrioxamine inhibits atherosclerotic lesion development and decreases lesion iron concentrations in the cholesterol-fed rabbit.

Several epidemiological studies have suggested that increased iron stores are associated with increased atherosclerotic events. In order to test the hypothesis that decreasing the vascular level of iron slows lesion growth, we examined the effects of the iron chelator Desferal (72 mg/kg/day, 5 days/week) on atherosclerosis and lesion iron content in cholesterol-fed New Zealand White rabbits. Rabbits were fed with a 1% w/w cholesterol diet for either 8 weeks (and for the last 5 weeks injected daily with Desferal) or 12 weeks (and for the last 9 weeks injected with Desferal). Controls were injected with saline. A significant reduction in average lesion area (p = 0.038) was observed in the 12-week treated animals compared with the 12-week controls. The average lesion iron level of the 12-week treated animals (58 ppm dry wt) was also significantly lower (p = 0.030) than in 12-week control animals (95 ppm dry wt), as measured using nuclear microscopy with the combination of scanning transmission ion microscopy, Rutherford back-scattering spectroscopy, and particle-induced X-ray emission. No reduction in lesion area or iron content was observed in the 8-week treated animals compared with controls, and no change in lesion zinc concentration was observed for either group. Our data strengthen the concept that iron contributes to the early stages of the development of atherosclerosis.

GST-pi expression correlates with oxidative stress and apoptosis in breast cancer.

Glutathione S-transferase (GST) is known to play a key role in the detoxification and reduction of reactive oxygen species (ROS). Thus, we assessed GST activity and GST-pi expression in relation to oxidative stress and apoptosis in breast cancer. Tumor tissues from 32 breast cancer patients were evaluated for GST activity and thiobarbituric acid reactive substances (TBARS) that are by-products of oxidative stress. Four-micron sections of formalin-fixed, paraffin embedded tumors were stained immunohistochemically with anti-GST-pi. Apoptotic cells were detected by in situ end labeling of DNA fragments using a commercial kit. TBARS levels were significantly higher in breast cancers of older patients. GST-pi expression was up-regulated in breast cancers that exhibited higher oxidative stress and associated with higher GST activity. Apoptosis in GST-pi negative tumors was not correlated with GST activity, but GST-pi positive tumors within the same range of oxidative stress showed a reduction in apoptosis as well as an increased GST activity. This correlation was absent in GST-pi positive tumors experiencing higher oxidative stress. GST-pi expression may influence the level of GST activity and delay apoptosis in breast cancer. However, GST-pi expression in tumors with higher levels of oxidative stress may not be sufficient to abrogate the deleterious effects of ROS.

Hypericin-mediated photodynamic therapy induces lipid peroxidation and necrosis in nasopharyngeal cancer.

Photoactivation of hypericin is known to generate singlet oxygen and superoxide anion radicals. Reactive oxygen species (ROS) produced by photodynamic therapy (PDT) has the capacity to induce oxidative damage and tumor destruction. We have previously shown that hypericin-PDT induces tumor shrinkage and regression in the human nasopharyngeal cancer (NPC)/HK1 murine tumor model. In this extended study, we show by electron microscopy that subcutaneously implanted HK1 NPC cells from Balb/c nude mice perished by cell necrosis with hypericin-PDT treatment. There was evidence of cytoplasmic swelling accompanied by loss of cell membrane integrity and autophagic vacuolization of cytoplasm but no nuclear changes. There was also no significant difference in the apoptotic index of control and PDT-treated tumors, when analyzed by in situ end labeling of DNA strand breakage to detect apoptosis. This further supports the observation that cell death in PDT-treated NPC/HK1 tumors was by necrosis. Lipid peroxidative stress analyzed by the malonaldehyde assay was significantly elevated in PDT-treated cells. However, PDT had no effect on the activity of superoxide dismutase, an intracellular antioxidant enzyme. The findings show that hypericin-PDT of nasopharyngeal tumors in vivo induces tumor necrosis with accompanying lipid peroxidation.

Glutathione S-transferase pi expression in nasopharyngeal cancer.

BACKGROUND: Glutathione S-transferase pi (GST-pi) is an enzyme that catalyzes the conjugation of electrophilic substrates and prevents oxidative damage. Although GST-pi expression has been analyzed in many cancers, the significance of GST-pi expression in nasopharyngeal cancer (NPC), a tumor with a high treatment failure rate, is still unclear. OBJECTIVE: To elucidate the significance of GST-pi expression in NPC. DESIGN: Evaluation of GST-pi expression in NPC tissue specimens and determination of its relationship with tissue iron (a pro-oxidant) and clinicopathological factors in NPC. MATERIALS AND METHODS: Immunohistochemical expression of GST-pi was carried out in 55 NPC and 4 normal nasopharyngeal tissue sections. Eleven nasopharyngeal biopsy specimens (4 normal and 7 NPC) were analyzed for tissue iron levels. The expression of GST-pi in NPC was correlated with corresponding tissue iron levels. The relationships between GST-pi expression with sex, race, tumor stage, cervical nodal status, and clinical staging were also analyzed. RESULTS: Glutathione S-transferase pi immunoreactivity was observed in all NPC sections, with the percentage of immunopositive cells ranging from 1.0% to 72.0%. Tissue iron levels were significantly higher in the NPC tissues compared with normal tissues (P =.001). A direct correlation was observed between GST-pi expression and total and nuclear iron levels in NPC (P =.01 and P =.047, respectively). A significant association was also observed between GST-pi expression and cervical nodal disease (P =.007). CONCLUSIONS: Nasopharyngeal tumor cells may respond to pro-oxidant conditions by modulating intracellular antioxidant defense. Glutathione S-transferase pi expression appears to be associated with lymphogenous metastasis in NPC.