Smiti V. Gupta

Inhibition Of Cell Growth And Induction Of Apoptosis In Non-Small Cell Lung Cancer Cells By Delta-Tocotrienol Is Associated With Notch-1 Down-Regulation

Lung cancer is the leading cause of death among all cancers. Non‐small cell lung cancer accounts for 80% of lung cancer with a 5‐year survival rate of 16%. Notch pathway, especially Notch‐1 is up‐regulated in a subgroup of non‐small cell lung cancer patients. Since Notch‐1 signaling plays an important role in cell proliferation, differentiation, and apoptosis, down‐regulation of Notch‐1 may exert anti‐tumor effects. The objective of this study was to investigate whether delta‐tocotrienol, a naturally occurring isoform of Vitamin E, inhibits non‐small cell lung cancer cell growth via Notch signaling. Treatment with delta‐tocotrienol resulted in a dose and time dependent inhibition of cell growth, cell migration, tumor cell invasiveness, and induction of apoptosis. Real‐time RT‐PCR and western blot analysis showed that antitumor activity by delta‐tocotrienol was associated with a decrease in Notch‐1, Hes‐1, Survivin, MMP‐9, VEGF, and Bcl‐XL expression. In addition, there was a decrease in NF‐κB‐DNA binding activity. These results suggest that down‐regulation of Notch‐1, via inhibition of NF‐κB signaling pathways by delta‐tocotrienol, could provide a potential novel approach for prevention of tumor progression in non‐small cell lung cancer. J. Cell. Biochem. 112: 2773–2783, 2011.

Delta-tocotrienol suppresses Notch-1 pathway by upregulating miR-34a in nonsmall cell lung cancer cells.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating various cellular functions by transcriptional silencing. miRNAs can function as either oncogenes or tumor suppressors (oncomirs), depending on cancer types. In our study, using miRNA microarray, we observed that downregulation of the Notch‐1 pathway, by delta‐tocotrienol, correlated with upregulation of miR‐34a, in nonsmall cell lung cancer cells (NSCLC). Moreover, re‐expression of miR‐34a by transfection in NSCLC cells resulted in inhibition of cell growth and invasiveness, induction of apoptosis and enhanced p53 activity. Furthermore, cellular mechanism studies revealed that induction of miR‐34a decreased the expression of Notch‐1 and its downstream targets including Hes‐1, Cyclin D1, Survivin and Bcl‐2. Our findings suggest that delta‐tocotrienol is a nontoxic activator of mir‐34a which can inhibit NSCLC cell proliferation, induce apoptosis and inhibit invasion, and thus offering a potential starting point for the design of novel anticancer agents.

Garcinol Inhibits Cell Proliferation and Promotes Apoptosis in Pancreatic Adenocarcinoma Cells

Garcinol, or polyisoprenylated benzophenone, isolated from the rind of fruiting bodies of Garcinia indica, has been used in traditional medicine for its potential antiinflammatory and antioxidant properties. The objective of this study was to investigate the effect of garcinol on pancreatic cancer (PaCa) cell viability and proliferation. For this, 2 human PaCa cell lines, BxPC-3 and Panc-1, with wild and mutant k-ras, respectively, were treated with garcinol (0–40 μM). Garcinol significantly (P < 0.05) inhibited cell growth (trypan blue exclusion) by induction of apoptosis in a dose- and time-dependent manner. Flow cytometric analysis revealed G0–G1 phase cell cycle arrest in both cell lines. The molecular mechanism of garcinols action on PaCa cells was investigated by targeting signaling moieties involved in apoptosis (X-IAP, cIAP, caspase-3, 9, and PARP cleavage), transcription factor NF-κB, believed to contribute toward a chemoresistance phenotype in pancreatic tumors, and molecules associated with neovascularization and metastasis (MMP-9, VEGF, IL-8, and PGE2). Garcinol significantly (P < 0.05) augmented antiproliferative, proapoptotic, antimetastatic, and antiangiogenic effects in both PaCa cell types relative to untreated cells. These effects were more pronounced in Panc-1. This is the first report on the therapeutically relevant effect of garcinol in PaCa. Further studies are warranted, based on our findings.

Synergistic effect of garcinol and curcumin on antiproliferative and apoptotic activity in pancreatic cancer cells.

Pancreatic cancer (PaCa) is a major health concern due to its aggressiveness and early metastasis. Current treatments for PaCa are limited by development of resistance against therapy. As an alternative strategy, we assessed the combinatorial effect of dietary compounds, garcinol and curcumin, on human PaCa cells (BxPC-3 and Panc-1). A significant (P < 0.05) dose-dependent reduction in cell viability and increase in apoptosis were observed in both cell lines as compared to untreated controls. A combination index (CI) value < 1, for a two-way comparison of curcumin and garcinol, suggests synergism. The potency (Dm) of the combination of garcinol and curcumin was 2 to 10 fold that of the individual agents. This indicates that curcumin and garcinol in combination exhibit a high level of synergism, with enhanced bioactivity, thereby reducing the required effective dose required for each individually. This combinatorial strategy may hold promise in future development of therapies against PaCa.

Potential Role of Garcinol as an Anticancer Agent

Garcinol, a polyisoprenylated benzophenone, is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Although the fruit has been consumed traditionally over centuries, its biological activities, specifically its anticancer potential is a result of recent scientific investigations. The anticarcinogenic properties of garcinol appear to be moderated via its antioxidative, anti-inflammatory, antiangiogenic, and proapoptotic activities. In addition, garcinol displays effective epigenetic influence by inhibiting histone acetyltransferases (HAT 300) and by possible posttranscriptional modulation by mi RNA profiles involved in carcinogenesis. In vitro as well as some in vivo studies have shown the potential of this compound against several cancers types including breast, colon, pancreatic, and leukemia. Although this is a promising molecule in terms of its anticancer properties, investigations in relevant animal models, and subsequent human trials are warranted in order to fully appreciate and confirm its chemopreventative and/or therapeutic potential.

Antibacterial and cytotoxic activity of isoprenylated coumarin mammea A/AA isolated from Mammea africana

ETHNOPHARMACOLOGICAL RELEVANCE The stem bark of Mammea africana is widely distributed in tropical Africa and commonly used in traditional medicine. This study aims to identify the active compound in Mammea africana and to evaluate its antimicrobial and antiproliferative activity. MATERIALS AND METHODS Methanol extract from the bark of the Mammea africana was separated by liquid-liquid extraction, followed by open column chromatography. A principal antimicrobial compound was purified by high performance liquid chromatography (HPLC) and its structure was elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The antibacterial activity of the purified compound was determined using the broth microdilution method against 7 common pathogenic bacteria. The compound was also evaluated for cytotoxicity by cell proliferation assay (MTS) using the mouse embryonic fibroblast cell line NIH 3T3 and the non-small cell lung cancer cell line A549. RESULTS The purified active compound was determined to be mammea A/AA and was found to be highly active against Campylobacter jejuni (MIC=0.5 μg/ml), Streptococcus pneumoniae (MIC=0.25 μg/ml), and Clostridium difficile (MIC=0.25 μg/ml). The compound exhibited significant antiproliferative activities against both NIH 3T3 and A549 cell lines. CONCLUSION Mammea A/AA isolated from Mammea africana exerts specific inhibitory activity against Campylobacter jejuni, Streptococcus pneumoniae, and Campylobacter difficile. Mammea A/AA was also found to exhibit significant cytotoxicity against both cancer and normal cell lines.

Quantitative Chemiluminescent Immunoassay for NF‐κB–DNA Binding Activity

Abstract Nuclear factor‐κB (NF‐κB) is a ubiquitous redox‐sensitive transcription factor involved in the pro‐inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF‐κB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimers disease. The conventional method for detection of NF‐κB ‐DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time‐consuming and non‐quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF‐κB DNA‐binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF‐κB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3–5 h post sample preparation, (2) Greater sensitivity: 10 pg NF‐κB/well, (3) Quantitative: linear range: 10–1000 pg NF‐κB; r2 = 0.999 (4) High throughput adaptability: 96‐well plate format can analyze up to 40 samples in duplicate, (5) Safety: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF‐κB which is translocated into the nucleus and total (bound + unbound) NF‐κB present in the cytosol/cell.

Differences in metabolomic profiles of male db/db and s/s, leptin receptor mutant mice

Leptin, a protein hormone secreted by adipose tissue, plays an important role in regulating energy metabolism and the immune response. Despite similar extremes of adiposity, mutant mouse models, db/db, carrying spontaneous deletion of the active form of the leptin receptor (LEPR-B) intracellular signaling domain, and the s/s, carrying a specific point mutation leading to a dysfunctional LEPR-B-STAT3 signaling pathway, have been shown to have robust differences in glucose homeostasis. This suggests specific effects of leptin, mediated by non-STAT3 LEPR-B pathways. Differences in the LEPR-B signaling pathways in these two LEPR-B mutant mice models are expected to lead to differences in metabolism. In the current study, the hypothesized differences in metabolism were investigated using the metabolomics approach. Proton nuclear magnetic resonance spectroscopy ((1)HNMR) was conducted on 24 h urine samples in deuterium oxide using a 500 MHz instrument at 25°C. Principle Component Analysis showed clear separation of urine NMR spectra between the groups (P < 0.05). The CHENOMX metabolite database was used to identify several metabolites that differed between the two mouse models. Significant differences (P < 0.05) in metabolites associated with the glycine, serine, and homocysteine metabolism were observed. The results demonstrate that the metabolomic profile of db/db and s/s mice are fundamentally different and provide insight into the unique metabolic effects of leptin exerted through non-STAT3 LEPR-B pathways.

Effects of exchanging 4%en between dietary stearic and palmitic acid on hamster plasma lipoprotein metabolism

This study was designed to determine whether the exchange of specific fatty acids (palmitic (16 : 0) for stearic (18 : 0)), would exert differential effects on plasma and lipoprotein lipids, when diets contained ~30%en from fat with adequate levels of linoleic acid (18 : 2). Thirty-two male Golden Syrian hamsters were fed isocaloric purified diets with comparable amounts of 18 : 2 (~10.5%en). The 18 : 0-rich diet (50% cocoa butter, 41% safflower oil, 9% sunflower oil) provided 4.8%en 16 : 0 and 5.3%en from 18 : 0, while the 16 : 0-rich diet (59% palm oil, 36% safflower oil, 5% olive oil) provided 8.7%en from 16 : 0 and 1.2%en from 18 : 0, resulting in a 16 : 0/18 : 0 exchange of ~4%en. Both diets contained negligible amounts of lauric and myristic acid (<0.2%en), ~9.5%en from oleic acid and 77 mg cholesterol/1000 kcal. Animals were fed their respective diets for 4 weeks at which point various lipid and lipoprotein parameters were meassured. There were no significant difference between dietary groups for any of the measured parameters, which included body weights, food consumption, plasma lipids, lipoprotein lipid and apoprotein concentrations, as well as lipoprotein compositions. Additionally, estimated diameters of various lipoprotein particles were not affected by the fatty acid exchanges employed. Thus these data suggest that when total fat is restricted to 30%en and 18 : 2 levels are ~10%en, a 4%en exchange between 16 : 0 and 18 : 0 (representing intakes of ~9 g/d/2000 kcal diet) produces comparable plasma lipids.This study was designed to determine whether the exchange of specific fatty acids (palmitic (16:0) for stearic (18:0)), would exert differential effects on plasma and lipoprotein lipids, when diets contained approximately 30%en from fat with adequate levels of linoleic acid (18:2). Thirty-two male Golden Syrian hamsters were fed isocaloric purified diets with comparable amounts of 18:2 (approximately 10.5%en). The 18:0-rich diet (50% cocoa butter, 41% safflower oil, 9% sunflower oil) provided 4.8%en 16:0 and 5.3%en from 18:0, while the 16:0-rich diet (59% palm oil, 36% safflower oil, 5% olive oil) provided 8.7%en from 16:0 and 1.2%en from 18:0, resulting in a 16:0/18:0 exchange of approximately 4%en. Both diets contained negligible amounts of lauric and myristic acid (< 0.2%en), approximately 9.5%en from oleic acid and 77 mg cholesterol/1000 kcal. Animals were fed their respective diets for 4 weeks at which point various lipid and lipoprotein parameters were measured. There were no significant difference between dietary groups for any of the measured parameters, which included body weights, food consumption, plasma lipids, lipoprotein lipid and apoprotein concentrations, as well as lipoprotein compositions. Additionally, estimated diameters of various lipoprotein particles were not affected by the fatty acid exchanges employed. Thus these data suggest that when total fat is restricted to 30%en and 18:2 levels are approximately 10%en, a 4%en exchange between 16:0 and 18:0 (representing intakes of approximately 9 g/d/2000 kcal diet) produces comparable plasma lipids.

Metabolomics connects aberrant bioenergetic, transmethylation, and gut microbiota in sarcoidosis

AbstractSarcoidosis is a systemic granulomatous disease of unknown etiology. Granulomatous inflammation in sarcoidosis may affect multiple organs, including the lungs, skin, CNS, and the eyes, leading to severe morbidity and mortality. The underlying mechanisms for sustained inflammation in sarcoidosis are unknown. We hypothesized that metabolic changes play a critical role in perpetuation of inflammation in sarcoidosis. 1H nuclear magnetic resonance (NMR)-based untargeted metabolomic analysis was used to identify circulating molecules in serum to discriminate sarcoidosis patients from healthy controls. Principal component analyses (PCA) were performed to identify different metabolic markers and explore the changes of associated biochemical pathways. Using Chenomx 7.6 NMR Suite software, we identified and quantified metabolites responsible for such separation in the PCA models. Quantitative analysis showed that the levels of metabolites, such as 3-hydroxybutyrate, acetoacetate, carnitine, cystine, homocysteine, pyruvate, and trimethylamine N-oxide were significantly increased in sarcoidosis patients. Interestingly, succinate, a major intermediate metabolite involved in the tricyclic acid cycle was significantly decreased in sarcoidosis patients. Application of integrative pathway analyses identified deregulation of butanoate, ketone bodies, citric cycle metabolisms, and transmethylation. This may be used for development of new drugs or nutritional modification.