Ocimum gratissimum Aqueous Extract Induces Apoptotic Signalling in Lung Adenocarcinoma Cell A549

Ocimum gratissimum (OG) is widely used as a traditional herb for its antibacterial activity in Taiwan. Recently, antitumor effect of OG on breast cancer cell is also reported; however, the effects of OG on human pulmonary adenocarcinoma cell A549 remain unclear. Therefore, we aimed to investigate whether aqueous OG extract (OGE) affects viability of A549 cells and the signals induced by OGE in A549 cells. Cell viability assays revealed that OGE significantly and dose-dependently decreased the viability of A549 cell but not that of BEAS-2B cell. Morphological examination and DAPI staining indicated that OGE induced cell shrinkage and DNA condensation for A549 cells. Further investigation showed that OGE enhanced activation of caspase-3, caspase-9 and caspase-8 and increased protein level of Apaf-1 and Bak, but diminished the level of Bcl-2. Additionally, OGE inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) yet enhanced the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38). In conclusion, our findings indicate that OGE suppressed the cell viability of A549 cells, which may result from the activation of apoptotic signaling and the inhibition of anti-apoptotic signaling, suggesting that OGE might be beneficial to lung carcinoma treatment

Ocimum gratissimum Aqueous Extract Protects H9c2 Myocardiac Cells from H2O2-Induced Cell Apoptosis through Akt Signalling

Increased cell death of cardiomyocyte by oxidative stress is known to cause dysfunction of the heart. O. gratissimum is one of the more well-known medicinal plants among the Ocimum species and widely used in treatment of inflammatory diseases. In this study, we hypothesized that aqueous extract of O. gratissimum leaf (OGE) may protect myocardiac cell H9c2 from oxidative injury by hydrogen peroxide (H2O2). Our results revealed that OGE pretreatment dose-dependently protects H9c2 cells from cell death when exposed to H2O2. Additionally, DNA condensation induced by H2O2 was also reduced by OGE pretreatment, suggesting that Ocimum gratissimum extract may attenuate H2O2-induced chromosome damage. Further investigation showed that OGE pretreatment inhibited H2O2-induced activation of caspase-3 and caspase-9, as well as H2O2-induced upregulation of proapoptotic Apaf-1 and the release of cytosolic cytochrome c, but has little effect on the activation of caspase-8. Additionally, OGE pretreatment significantly upregulated Bcl-2 expression and Akt phosphorylation, and slightly affected the phosphorylation of mitogen-activated protein kinases including p38 MAPK and JNK. Taken together, our findings revealed that Ocimum gratissimum extract effectively inhibited the mitochondrial pathway and upregulated Bcl-2 expression, which may be important in protecting H9c2 cells from H2O2-induced cell death

Melatonin acts synergistically with pazopanib against renal cell carcinoma cells through p38 mitogen-activated protein kinase-mediated mitochondrial and autophagic apoptosis

Background Mounting evidence indicates that melatonin has possible activity against different tumors. Pazopanib is an anticancer drug used to treat renal cell carcinoma (RCC). This study tested the anticancer activity of melatonin combined with pazopanib on RCC cells and explored the underlying mechanistic pathways of its action. Methods The 786-O and A-498 human RCC cell lines were used as cell models. Cell viability and tumorigenesis were detected with the MTT and colony formation assays, respectively. Apoptosis and autophagy were assessed using TUNEL, annexin V/propidium iodide, and acridine orange staining with flow cytometry. The expression of cellular signaling proteins was investigated with western blotting. The in vivo growth of tumors derived from RCC cells was evaluated using a xenograft mouse model. Results Together, melatonin and pazopanib reduced cell viability and colony formation and promoted the apoptosis of RCC cells. Furthermore, the combination of melatonin and pazopanib triggered more mitochondrial, caspase-mediated, and LC3-II-mediated autophagic apoptosis than melatonin or pazopanib alone. The combination also induced higher activation of the p38 mitogen-activated protein kinase (p38MAPK) in the promotion of autophagy and apoptosis by RCC cells than melatonin or pazopanib alone. Finally, tumor xenograft experiments confirmed that melatonin and pazopanib cooperatively inhibited RCC growth in vivo and predicted a possible interaction between melatonin/pazopanib and LC3-II. Conclusion The combination of melatonin and pazopanib inhibits the growth of RCC cells by inducing p38MAPK-mediated mitochondrial and autophagic apoptosis. Therefore, melatonin might be a potential adjuvant that could act synergistically with pazopanib for RCC treatment

Identification and Immunologic Characterization of an Allergen, Alliin Lyase, from Garlic (Allium Sativum)

Background: Garlic (Allium sativum) is one of the most common relishes used in cooking worldwide. Very few garlic allergens have been reported, and garlic allergy has been rarely studied. Objective: The aim of the study was to identify allergenic proteins in garlic and to investigate their importance in allergies to other Allium species (leek, shallot, and onion). Methods: A crude extract of garlic proteins was separated by SDS- PAGE and 2-dimensional electrophoresis; immunoblotting was then performed with the use of individual and pooled sera from patients with garlic allergy, and the major IgE-binding proteins were analyzed by amino acid sequencing and mass spectrometry. The putative allergens were further purified by chromatography; the antigenicity, allergenicity, and IgE- binding cross- reactivity of the purified protein were then studied by immunoblotting, periodate oxidation, skin tests, and IgE- binding inhibition assays. Results: A major allergen, alliin lyase, was identified by mass spectrometry and Edman sequencing and purified to homogeneity through the use of a simple 2-step chromatographic method. Skin tests showed that the purified protein elicited IgE-mediated hypersensitive responses in patients with garlic allergy. Periodate oxidation showed that carbohydrate groups were involved in the antigenicity, allergenicity, and cross-reactivity. Garlic alliin lyase showed strong cross-reactivity with alliin lyases from other Allium species, namely leek, shallot, and onion. Conclusions: Alliin lyase was found to be a major garlic allergen in a garlic-allergic group of patients in Taiwan. The wide distribution of alliin lyase in Allium suggests it may be a new cross-reactive allergen

Perilla frutescens leaf extract inhibits mite major allergen Der p 2-induced gene expression of pro-allergic and pro-inflammatory cytokines in human bronchial epithelial cell BEAS-2B.

Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens

Aqueous Mulberry Leaf Extract Ameliorates Alcoholic Liver Injury Associating with Upregulation of Ethanol Metabolism and Suppression of Hepatic Lipogenesis

Excessive alcohol intake is a major cause of chronic liver damage and is highly associated with the development of a spectrum of hepatic disorders, including steatohepatitis, liver cirrhosis, and liver cancer. Thus, we aimed to explore the hepatoprotective effects of an aqueous mulberry leaf extract (AME) on alcoholic fatty liver disorder (AFLD) by using a mouse model fed with excessive ethanol. Compared with the normal diet, the ethanol diet significantly increased the body weight of the mice, while the AME supplement reduced the weight gain caused by the ethanol diet. The ethanol diet also attenuated the activity of alcohol dehydrogenase and antioxidant enzymes but increased lipid peroxidation in the liver, which were reversed by AME supplementation. Additionally, AME supplementation diminished the ethanol diet-induced hepatic leukocyte infiltration and expressions of IL-6 and TNFα. Moreover, AME supplementation also reduced the ethanol-diet-induced lipid accumulation and expression of 1-acylglycerol-3-phosphate acyltransferase, acetyl-CoA carboxylase, low-density lipoprotein receptor, and sterol regulatory element-binding protein-1/2 in the liver. Collectively, AME supplementation improved liver lipid accumulation and proinflammatory response in mice induced by the ethanol diet, which was associated with the upregulation of ethanol-metabolizing enzymes and the downregulation of lipogenesis components

PFE elevated cytosolic IκBα and diminished nuclear NF-κB in DP2-stimulated BEAS-2B cells.

<p>After 16-starvation, cells were pretreated with 5, 15, 30 and 50 µg/mL PFE for 1 h, and then stimulated with 20 µg/mL DP2 for 30 min. The treated cells were lysed for (A) determination of cytosolic IκBα, or (B) distribution of cytosolic and nuclear NF-κB by immunoblot using specific antibodies and chemiluminescence development. Quantitative data was performed by densitometric analysis and obtained from three independent experiments. Level of GAPDH and histone H1 was used as cytosolic and nuclear control, respectively. #, <i>p</i><0.05 as compared to GST alone; *, <i>P</i><0.05 as compared to DP2 alone.</p