Syzygium cumini ameliorates high fat diet induced glucose intolerance, insulin resistance, weight gain, hepatic injury and nephrotoxicity through modulation of PTP1B and PPARγ signaling.

Metabolic disorders are majorly associated with insulin resistance and an impaired glucose tolerance. Since, many of the currently available drugs exhibit adverse effects and are resistant to therapies, natural products are a promising alternate in the alleviation of complex metabolic disorders. In the current study, Syzygium cumini methanolic extract (SCE) was investigated for its anti-diabetic and anti-adipogenic potential using C57BL/6 mice fed on high fat diet (HFD). The HFD fed obese mice were treated with 200 mg/kg SCE and compared with positive controls Metformin, Pioglitazone and Sodium Orthovanadate. The biometabolites in SCE were characterized using Fourier transform infrared and gas chromatography and mass spectroscopy. A reduction in blood glucose levels with improved insulin sensitivity and glucose tolerance was observed in SCE-treated HFD obese mice. Histopathological and biochemical investigations showed a reduction in hepatic injury and nephrotoxicity in SCE-administered HFD mice. Results showed inhibition of PTP1B and an upregulation of IRS1 and PKB-mediated signaling in skeletal muscle. A significant decrease in lipid markers such as TC, TG, LDL-c and VLDL-c levels were observed with increased HDL-c in SCE-treated HFD mice. A significant decrease in weight and adiposity was observed in SCE-administered HFD mice in comparison to controls. This decrease could be due to the partial agonism of PPARγ and an increased expression of adiponectin, an insulin sensitizer. Hence, the dual-modulatory effect of SCE, partly due to the presence of 26% Pyrogallol, could be useful in the management of diabetes and its associated maladies.

Hyperhomocysteinemia-related serum metabolome alterations not normalized by short-term folic acid treatment.

INTRODUCTION: Hyperhomocysteinemia (HHCys) is an independent risk factor for various diseases such as cardiovascular diseases, Alzheimer's, and cancers. Folate deficiency is one of the significant reasons for HHCys. However, it is not known whether folate deficiency with HHCys is associated with any serum metabolites. OBJECTIVES: Our objective was to identify the metabolic alterations in people having folate deficiency with HHCys and check whether a short-term folic acid therapy could reverse those metabolic changes. METHODS: The study enrolled 34 participants aged between 18 and 40 years having folate deficiency (< 4.6 ng/mL) with HHCys (> 15 µmol/L) and 21 normal healthy individuals. A short-term intervention of oral folic acid (5 mg/day) was done in the HHCys group for 30 days. Untargeted metabolomics analysis of serum was performed in all study subjects before and after the folic acid treatment. Different univariate methods and the multivariable-adjusted linear regression models were employed to determine an association between homocysteine level and metabolite profile. RESULTS: Metabolomics analysis data showed that many metabolites involved in the biochemical pathways of lipid metabolisms such as polyunsaturated fatty acids, glycerolipids, and phospholipids were downregulated in the HHCys group. Short-term oral folic acid therapy significantly reduced their serum homocysteine level. However, the metabolic pathway alterations observed in folate-deficient HHCys-condition were unaltered even after the folic acid treatment. CONCLUSIONS: Our study revealed that people who have a folic acid deficiency with HHCys have an altered metabolite profile related to lipid metabolism, which cannot be reversed by short-term folic acid therapy.

Role of STAT3 Phosphorylation in Ethanol-Mediated Proliferation of Breast Cancer Cells.

PURPOSE: In this study, we investigated the molecular mechanism involved in ethanol (EtOH)-mediated proliferation of breast cancer cells. METHODS: EtOH concentration was optimized by studying its effect on cell proliferation in MCF-7 and MDA MB-231 cells. We used flow cytometry and immunoblot analysis to evaluate the increased proliferation caused by the optimized concentrations of EtOH. The mechanism of EtOH-mediated proliferation was determined using reactive oxygen species (ROS) release assay, reverse transcription polymerase chain reaction, and immunoblot studies. Gene silencing followed by quantitative real-time polymerase chain reaction studies and inhibitor studies indicated the involvement of signal transducer and activator of transcription 3 (STAT3) in EtOH-mediated breast cancer proliferation. RESULTS: Exposure to EtOH caused an increase in cell proliferation and an accumulation of cells in S-phase in MCF-7 (347 µM EtOH) and MDA MB-231 (173 µM EtOH) cells. Additionally, increased release of ROS and the expression of pro-inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, confirmed that the proliferation was induced by the ROS-linked inflammatory response in breast cancer. The proinflammatory response was followed by phosphorylation of STAT3. The importance of STAT3 activation in EtOH-mediated proliferation was confirmed through the silencing of STAT3, followed by an investigation on the expression of cyclins and matrix metalloproteinases. Finally, studies using specific inhibitors indicated that the EtOH-mediated effect on STAT3 activation could be regulated by phosphoinositide-3-kinase and Janus kinase 2. CONCLUSION: The study demonstrates the involvement of STAT3 signaling in EtOH-mediated breast cancer proliferation.

Monitoring of changes in lipid profiles during PLK1 knockdown in cancer cells using DESI MS.

The importance of the polo-like kinase 1 (PLK1) gene is increasing substantially both as a biomarker and as a target for highly specific cancer therapy. This is due to its involvement in multiple points of cell progression and carcinogenesis. PLK1 inhibitors' efficacy in treating human cancers has been limited due to the lack of a specific targeting strategy. Here, we describe a method of targeted downregulation of PLK1 in cancer cells and the concomitant rapid detection of surface lipidomic perturbations using desorption electrospray ionization mass spectrometry (DESI MS). The efficient delivery of siRNA targeting PLK1 gene selectively to the cancer cells is achieved by targeting overexpressed cell surface epithelial cell adhesion molecule (EpCAM) by the EpDT3 aptamer. The chimeric aptamer (EpDT3-siPLK1) showed the knockdown of PLK1 gene expression and PLK1 protein levels by quantitative PCR and western blotting, respectively. The abundant surface lipids, phosphatidylcholines (PCs), such as PC(32:1) (m/z 754.6), PC(34:1) (m/z 782.6), and PC(36:2) (m/z 808.6), were highly expressed in MCF-7 and WERI-RB1 cancer cells compared to normal MIO-M1 cells and they were observed using DESI MS. These overexpressed cell surface lipids in the cancer cells were downregulated upon the treatment of EpDT3-siPLK1 chimera indicating a novel role of PLK1 to regulate surface lipid expression in addition to the efficient selective cancer targeting ability. Our results indicate that DESI MS has a potential ability to rapidly monitor aptamer-mediated cancer therapy and accelerate the drug discovery process. Graphical abstract Binding of aptamer chimera to the cells and changes in lipid profile.

DEPTOR promotes survival of cervical squamous cell carcinoma cells and its silencing induces apoptosis through downregulating PI3K/AKT and by up-regulating p38 MAP kinase.

DEPTOR is an endogenous inhibitor of mTOR complexes, de-regulated in cancers. The present study reveals a vital role for DEPTOR in survival of cervical squamous cell carcinoma (SCC). DEPTOR was found to be overexpressed in both cervical SCC cells and tissues and it's silencing in cervical SCC cells induced apoptosis, mainly by up-regulation of p38 MAPK and by inhibiting PI3K/AKT pathway via a feed-back inhibition from mTORC1-S6K. DEPTOR silencing resulted in reduced expression of the nitric oxide synthases iNOS and eNOS, as well as increased activation of ERK1/2 and p38 MAP kinases. Activation of AKT signaling by overexpression of constitutively active-AKT (CA-AKT) failed to overcome the apoptosis caused by DEPTOR silencing. Similarly pharmacological inhibition of ERK also failed to control apoptosis. However pharmacological inhibition of p38 MAPK rescued the cells from apoptosis, indicating the major role of p38 MAPK in cell death induced by DEPTOR silencing. DEPTOR was also found to regulate ERK1/2 in an AKT dependent manner. DEPTOR knockdown induced cell death in SiHa cells overexpressing the anti-apoptotic Bcl-2 and Bcl-xL, indicating strong survival role of DEPTOR in these cells. DEPTOR overexpression activated PI3K/AKT by relieving the negative feed-back inhibition from mTORC1-S6K. DEPTOR regulation was also observed to be independent of HPV E6/E7 oncoproteins, but it might be a molecular co-factor contributing to cervical carcinogenesis. In summary, DEPTOR is found to promote survival of cervical SCC cells and its reduction induced apoptosis via differential effects on PI3K/AKT and p38 MAPK and can be a potential target in cervical SCC.

Role of STAT3 Phosphorylation in Ethanol-Mediated Proliferation of Breast Cancer Cells / 한국유방암학회지

PURPOSE: In this study, we investigated the molecular mechanism involved in ethanol (EtOH)-mediated proliferation of breast cancer cells. METHODS: EtOH concentration was optimized by studying its effect on cell proliferation in MCF-7 and MDA MB-231 cells. We used flow cytometry and immunoblot analysis to evaluate the increased proliferation caused by the optimized concentrations of EtOH. The mechanism of EtOH-mediated proliferation was determined using reactive oxygen species (ROS) release assay, reverse transcription polymerase chain reaction, and immunoblot studies. Gene silencing followed by quantitative real-time polymerase chain reaction studies and inhibitor studies indicated the involvement of signal transducer and activator of transcription 3 (STAT3) in EtOH-mediated breast cancer proliferation. RESULTS: Exposure to EtOH caused an increase in cell proliferation and an accumulation of cells in S-phase in MCF-7 (347 µM EtOH) and MDA MB-231 (173 µM EtOH) cells. Additionally, increased release of ROS and the expression of pro-inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, confirmed that the proliferation was induced by the ROS-linked inflammatory response in breast cancer. The proinflammatory response was followed by phosphorylation of STAT3. The importance of STAT3 activation in EtOH-mediated proliferation was confirmed through the silencing of STAT3, followed by an investigation on the expression of cyclins and matrix metalloproteinases. Finally, studies using specific inhibitors indicated that the EtOH-mediated effect on STAT3 activation could be regulated by phosphoinositide-3-kinase and Janus kinase 2. CONCLUSION: The study demonstrates the involvement of STAT3 signaling in EtOH-mediated breast cancer proliferation.