Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response.

PURPOSE: Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. EXPERIMENTAL DESIGN: In this case control study, small EVs are isolated from serum of 58 subjects (all male, 33 (15-70) in years) including drug naïve active tuberculosis (ATB: n = 22), non-tuberculosis (NTB: n = 18), and healthy subjects (n = 18). Serum small EVs proteome analysis is carried out using isobaric tag for relative and absolute quantification (iTRAQ) experiments and an independent sample (n = 36) is used for validation. RESULTS: A set of 132 and 68 proteins are identified in iTRAQ-I (ATB/Healthy) and iTRAQ-II (ATB/NTB) experiments, respectively. Four proteins (KYAT3, SERPINA1, HP, and APOC3) show deregulation (log2 -fold change > ±0.48, p < 0.05) in ATB with respect to healthy controls and Western blot data corroborated mass spectrometry findings. CONCLUSIONS AND CLINICAL RELEVANCE: These important proteins, involved in neutrophil degranulation, plasma heme scavenging, kynurenine, and lipid metabolism, show deregulation in ATB patients. Identification of such a protein panel in circulating small EVs besides providing novel insights into their role in tuberculosis may prove to be useful targets to develop host-directed therapeutic intervention.

The HBx gene of hepatitis B virus can influence hepatic microenvironment via exosomes by transferring its mRNA and protein.

The cellular secretory vesicles known as 'exosomes' have emerged as key player in intercellular transport and communication between different eukaryotic in order to maintain body homeostasis. Many pathogenic viruses utilize exosome pathway to efficiently transfer bioactive components from infected cells to naïve cells. Here, we show that HBx can tweak the exosome biogenesis machinery both by enhancing neutral sphingomyelinase2 activity as well as by interacting with exosomal biomarkers such as neutral sphingomyelinase2, CD9 and CD81. The nano particle tracking analysis revealed enhanced secretion of exosomes by the HBx-expressing cells while confocal studies confirmed the co-localization of HBx with CD9 and CD63. Importantly, we observed the encapsulation of HBx mRNA and protein in these exosomes besides some other qualitative changes. The exosomal cargo secreted by HBx-expressing cells had a profound effect on the recipient hepatic cells including creation of a milieu conducive for cellular-transformation. Thus, the present study unfolds a novel role of HBx in intercellular communication by facilitating horizontal transfer of viral gene products and other host factors via exosomes in order to support viral spread and pathogenesis.

The traditional Tibetan medicine Yukyung Karne exhibits a potent anti-metastatic activity by inhibiting the epithelial to mesenchymal transition and cell migration.

BACKGROUND: In Traditional Tibetan medicine, Yukyung Karne has been used for the treatment of ovarian cancer. Though Yukyung Karne has been reported to be clinically effective, the molecular mechanism of its anti-metstatic action remains elusive. METHODS: The cytotoxic property of Yukyung Karne was evaluated by crystal violet staining while its ability to induce ceramide production was analyzed by sphingomyelinase assay. The anti-metastatic property was investigated using adhesion, invasion, migration and colony formation assays. The effect of Yukyung Karne on the expression of extracellular matrix components, and epithelial and mesenchymal markers were evaluated by confocal microscopy and western blotting. RESULTS: Yukyung Karne exhibited a strong anti-metastatic property by significantly reducing the invasion, migration and colony formation ability of ovarian cancer cells. Besides it inhibited the levels of biomarkers involved in epithelial to mesenchymal transition such as down-regulation of vimentin and N-cadherin and up-regulation of epithelial E-cadherin. Yukyung Karne also induced the neutral sphingomyelinase II (nSMNaseII) enzyme activity that is known to hydrolyze sphingomyelins into pro-apoptotic intracellular molecule ceramide. CONCLUSIONS: The study provides some compelling evidences supporting the anti-metastatic potential of Yukyung Karne which strongly suggests its possible usage as a promising alternative medicine. Thus, Yukyung Karne may be used as an anticancer and anti-metastatic agent along with other conventional anticancer therapeutics to increase their efficacy.

Artesunate Ameliorates Functional Limitations in Freund's Complete Adjuvant-Induced Monoarthritis in Rat by Maintaining Oxidative Homeostasis and Inhibiting COX-2 Expression.

Drugs exhibiting anti-inflammatory and analgesic properties have been clinically used in the management of pain and impairment of joint functions in arthritis. In view of available studies on the beneficial effect of artesunate in various inflammatory conditions, the present study was carried out to evaluate its efficacy in ameliorating functional limitations of arthritis and to understand the underlying mechanisms. The study was carried out in rat model of Freund's complete adjuvant-induced monoarthritis where artesunate was found to produce a dose-dependent reduction in joint inflammation, improvement in functional parameters like stair climbing ability, motility, and suppression of mechanical allodynia at the doses of 50 and 150 mg/kg. Our study shows that protection afforded by artesunate was brought about by decreasing the levels of nitric oxide, influx of neutrophils, maintenance of oxidative homeostasis, inhibition of COX-2 expression, and apoptosis. Further, histological analysis of the arthritic joints also substantiated the anti-inflammatory property of artesunate. Thus, our study shows that artesunate has a potential for use in the treatment of arthritis.

Molecular insights into the anti-cancer properties of traditional Tibetan medicine Yukyung Karne.

BACKGROUND: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment of ovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the present study, the anticancer property of YK was investigated in cell culture. METHODS: The growth inhibitory property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while VEGF levels in culture supernatant were measured by a colorimetric method. RESULTS: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which is a well-known anti-cancer therapeutic drug. CONCLUSIONS: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justify its usage in primary as well as adjunct therapy for ovarian cancer.

Pro-apoptotic and anticancer properties of Thapring - A Tibetan herbal formulation.

AIM OF THE STUDY: To evaluate the pro-apoptotic and anti-tumorigenic properties of Thapring - a Traditional Tibetan Medicine - in hepatoma cells and in a transgenic mouse model of hepatocellular carcinoma. MATERIAL AND METHODS: The pro-apoptotic action and growth inhibition property of Thapring were assessed in Huh7, HepG2 and A549 cell lines using flow cytometry and MTT assay, respectively. Confocal microscopy for colocalization of cytochrome c and mitochondria was done using dsRed mitotracker in Huh7 cells. The activation of p38 MAP kinase and p53 pathway was evaluated by Western blotting. Serological studies for liver function, vascular endothelial growth factor and superoxide dismutase were assessed in the serum of X15-myc transgenic mice. Immuno-histochemical studies for Bcl2 and p21(Waf1) expression were also carried out in the liver section of the above mice. RESULTS: Treatment with Thapring inhibited proliferation and accumulation of hepatoma cells in G1 phase. There was increased cytochrome c release from mitochondria and decreased Bcl2 levels - the key markers of apoptotic cell death. Besides activation of p38 MAP kinase and increased p53 expression were also observed. Oral administration of Thapring in transgenic mice lowered serum VEGF levels and conferred hepatoprotection as evident from normal serum ALT levels. Further, immunohistochemical analysis of the liver samples revealed reduced expression of anti-apoptotic protein Bcl2 and over-expression of cell cycle regulator p21(Waf1). CONCLUSIONS: The ability of Thapring to impose growth arrest and trigger pro-apoptotic death in cell culture as well as ameliorative effects in vivo provides scientific basis for its usefulness as traditional medicine and its clinical application in adjunct/combination therapy along with other known anticancer drugs.

Chemopreventive and anti-cancer properties of the aqueous extract of flowers of Butea monosperma.

ETHNOPHARMACOLOGICAL RELEVANCE: Butea monosperma (Lam.) (Fabaceae) popularly known as 'flame of the forest' has been widely used in the traditional Indian medical system of 'Ayurveda' for the treatment of a variety of ailments including liver disorders. AIM OF THE STUDY: To evaluate the antioxidative, anti-inflammatory, hepatoprotective and anti-cancer activities of the aqueous extract of Butea monosperma flowers. MATERIALS AND METHODS: Dried flowers of Butea monosperma were extracted with water. The extract was tested for its anti-proliferative, pro-apoptotic and anti-carcinogenic effects in hepatoma cell lines. The chemopreventive and anti-angiogenic effects of the extract were evaluated by its daily oral administration in a HBV-related X15-myc mouse model of hepatocellular carcinoma (HCC). RESULTS: Treatment with the aqueous extract inhibited cell proliferation and accumulation of cells in G1 phase. This was accompanied by a marked reduction in the levels of activated Erk1/2 and SAPK/JNK and induction of apoptotic cell death. Oral administration of the extract in transgenic mice conferred hepatoprotection as is evident from normal serum ALT levels and improved liver histopathology and lowered serum VEGF level. CONCLUSIONS: The ability of aqueous extract of Butea monosperma flowers to impose growth arrest and trigger pro-apoptotic death in cell culture strongly correlated with its strong chemopreventive effect in vivo when given orally.

Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma.

AIM: To evaluate the anticancer property of the dried latex (DL) of Calotropis procera, a tropical medicinal plant, in the X15-myc transgenic mouse model of hepatocellular carcinoma and to elucidate its mechanism of action in cell culture. METHODS: The young transgenic mice were orally fed with the aqueous suspension of DL (400 mg/kg for 5 d/wk) for 15 wk and their liver was examined for histopathological changes at 20 wk. Serum levels of vascular endothelial growth factor (VEGF) were also measured in these animals. To characterize the active fraction, DL was extracted with petroleum ether followed by methanol. The methanolic extract was sub-fractionated on a silica gel G column using a combination of non-polar and polar solvents and eleven fractions were obtained. Each fraction was analysed for cytotoxic effect on hepatoma (Huh7) and non-hepatoma (COS-1) cell lines and non-transformed hepatocytes (AML12) using tetrazolium (MTT) assay. Finally, the mechanism of cell death was investigated by measuring the levels of Bcl2, caspase 3 and DNA fragmentation. RESULTS: DL treatment of mice showed a complete protection against hepatocarcinogenesis. No adverse effect was observed in these animals. The serum VEGF level was significantly lowered in the treated mice as compared to control animals. Cell culture studies revealed that the methanolic extract of DL as well as its fraction 8 induced extensive cell death in both Huh-7 and COS-1 cells while AML12 cells were spared. This was accompanied by extensive fragmentation of DNA in Huh-7 and COS-1 cells. No change in the levels of canonical markers of apoptosis such as Bcl2 and caspase 3 was observed. CONCLUSION: DL of C. procera has the potential for anti-cancer therapy due to its differentiable targets and non-interference with regular pathway of apoptosis.