A Lipidomics Approach to Determine the Role of Lipids and Its Crosstalk with Autophagy in Lung Cancer Metastasis.

Non-small cell lung cancer (NSCLC) is among the most malignant tumors with high propensity for metastasis and is the leading cause of cancer-related death globally. Most patients present with regional and distant metastasis, associated with poor prognosis. Lipids may play an essential role in either activating or inhibiting detachment-induced apoptosis (anoikis), where the latter is a crucial mechanism to prevent metastasis, and it may have a cross-talk with autophagy. Autophagy has been shown to be induced in various human cancer metastasis, modulating tumor cell motility and invasion, cancer cell differentiation, resistance to anoikis, and epithelial to mesenchymal transition. Hence, it may play a crucial role in the transition of benign to malignant phenotypes, the core of metastasis initiation. Here, we provide a method we have established in our laboratory for detecting lipids in attached and detached non-small lung cancer cells and show how to analyze lipidomics data to find its correlation with autophagy-related pathways.

Valvular Prostaglandins Are Elevated in Severe Human Aortic Valve Stenosis.

BACKGROUND: Aortic valve stenosis (AVS) is the most common valvular disease in the developed world. AVS involves the progressive fibrocalcific remodeling of the aortic valve (AV), which impairs function and can ultimately lead to heart failure. Due to gaps in our understanding of the underlying mechanisms of AVS, there are no pharmacological treatments or dietary interventions known to slow AVS progression. Recent studies have begun to suggest oxylipins-a class of bioactive lipids-may be dysregulated in the valves of patients with AVS. METHODS: We utilized high-performance liquid chromatography-tandem mass spectrometry to conduct a targeted oxylipin analysis on human AV tissue and plasma from a cohort of 110 patients undergoing AV surgery. RESULTS: We identified 36 oxylipins in human AV tissue with all showing significant increase in patients with severe AVS. A multivariate model including patient characteristics and valvular oxylipins identified the arachidonic acid-COX (cyclooxygenase) pathway-derived prostanoids to be the most associated with AVS severity. Plasma oxylipin levels were measured in a subset of AV surgery patients and compared with a control group of healthy participants, showing distinct oxylipin profiles between control and disease. CONCLUSIONS: Our comprehensive analysis of oxylipins in the human AV identified the inflammatory and osteogenic regulating prostanoids to be positively correlated with AVS severity. This elucidation of prostanoid dysregulation warrants further research into COX inhibition to mitigate AVS.

Lipidomic Predictors of Coronary No-Reflow.

The 'no-reflow' phenomenon (NRP) after primary percutaneous coronary intervention (PCI) is a serious complication among acute ST-segment elevation myocardial infarction (STEMI) patients. Herein, a comprehensive lipidomics approach was used to quantify over 300 distinct molecular species in circulating plasma from 126 patients with STEMI before and after primary PCI. Our analysis showed that three lipid classes: phosphatidylcholine (PC), alkylphosphatidylcholine (PC(O)), and sphingomyelin (SM), were significantly elevated (p < 0.05) in no-reflow patients before primary PCI. The levels of individual fatty acids and total fatty acid levels were significantly lower (p < 0.05) in no-reflow subjects after PCI. The grouping of patients based on ECG ST-segment resolution (STR) also demonstrated the same trend, confirming the possible role of these differential lipids in the setting of no-reflow. Sphingomyelin species, SM 41:1 and SM 41:2, was invariably positively correlated with corrected TIMI frame count (CTFC) at pre-PCI and post-PCI. The plasma levels of SM 42:1 exhibited an inverse association (p < 0.05) consistently with tumor necrosis factor-alpha (TNF-α) at pre-PCI and post-PCI. In conclusion, we identified plasma lipid profiles that distinguish individuals at risk of no-reflow and provided novel insights into how dyslipidemia may contribute to NRP after primary PCI.

Impact of Reperfusion on Plasma Oxylipins in ST-Segment Elevation Myocardial Infarction.

ST-segment elevation myocardial infarction (STEMI) occurs as a result of acute occlusion of the coronary artery. Despite successful reperfusion using primary percutaneous coronary intervention (PPCI), a large percentage of myocardial cells die after reperfusion, which is recognized as ischemia/reperfusion injury (I/R). There are rapid changes in plasma lipidome during myocardial reperfusion injury. However, the impact of coronary artery reperfusion on plasma oxylipins is unknown. This study aimed to investigate alterations in the oxylipin profiles of STEMI patients during ischemia and at various reperfusion time points following PPCI. Blood samples were collected from patients presenting with STEMI prior to PPCI (Isch, n = 45) and subsequently 2 h following successful reperfusion by PPCI (R-2 h, n = 42), after 24 h (R-24 h, n = 44), after 48 h (R-48 h, n = 43), and then 30 days post PPCI (R-30 d, n = 29). As controls, blood samples were collected from age- and sex-matched patients with non-obstructive coronary artery disease after diagnostic coronary angiography. High-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) using deuterated standards was used to identify and quantify oxylipins. In patients presenting with STEMI prior to reperfusion (Isch group), the levels of docosahexaenoic acid (DHA)-derived oxylipins were significantly higher when compared with controls. Their levels were also significantly correlated with the peak levels of creatine kinase (CK) and troponin T(TnT) before reperfusion (CK: r = 0.33, p = 0.046, TnT: r = 0.50, p = 1.00 × 10-3). The total concentrations of oxylipins directly produced by 5-lipoxygenase (5-LOX) were also significantly elevated in the Isch group compared with controls. The ratio of epoxides (generated through epoxygenase) to diols (generated by soluble epoxide hydrolysis (sEH)) was significantly lower in the Isch group compared with the controls. Following reperfusion, there was an overall reduction in plasma oxylipins in STEMI patients starting at 24 h post PPCI until 30 days. Univariate receiver operating characteristic (ROC) curve analysis also showed that an elevated ratio of epoxides to diols during ischemia is a predictor of smaller infarct size in patients with STEMI. This study revealed a large alteration in plasma oxylipins in patients presenting with STEMI when compared with controls. Total oxylipin levels rapidly reduced post reperfusion with stable levels reached 24 h post reperfusion and maintained for up to 30 days post infarct. Given the shifts in plasma oxylipins following coronary artery reperfusion, further research is needed to delineate their clinical impact in STEMI patients.

Impact of myocardial reperfusion on human plasma lipidome.

The primary aim of the study is to investigate the temporal changes in plasma lipidome before and after reperfusion in patients with ST-segment elevation myocardial infarction (STEMI) and their association with myocardial injury. We found that 56% of the identified lipid species were significantly altered (corrected p< 0.05) in the first 24 h following reperfusion in patients with STEMI. Three lipid species, namely, acylcarnitine 18:2, TG 51:0, and LPC 17:1 were associated with a change in troponin concentration (delta troponin) and in-hospital cardiovascular events. Of these, acylcarnitine 18:2, and LPC 17:1 and their respective whole class levels, were significantly higher (p < 0.05) in the STEMI population than the age/sex-matched control subjects. Overall, our analyses showed a large shift in plasma lipidome in patients that undergo myocardial reperfusion. The differences found for acylcarnitines and LPC species and their association with both cardiac markers and cardiac outcomes need further validation.

Increase in Plasma Oxidized Phosphatidylcholines (OxPCs) in Patients Presenting With ST-Elevation Myocardial Infarction (STEMI).

Objective: ST-segment Elevation Myocardial Infarction (STEMI) occurs as a result of acute occlusion of the coronary artery. Despite successful reperfusion using percutaneous coronary intervention (PCI), a large percentage of myocardial cells die after reperfusion which is recognized as ischemia/reperfusion injury (I/R). Oxidized phosphatidylcholines (OxPCs) are a group of oxidized lipids generated through non-enzymatic oxidation and have pro-inflammatory properties. This study aimed to examine the roles of OxPCs in a clinical setting of myocardial I/R. Methods: Blood samples were collected from STEMI patients at presentation prior to primary PCI (PPCI) (Isch) and at 4 time-points post-PPCI, including 2 h (R-2 h), 24 h (R-24 h), 48 h (R-48 h), and 30 days (R-30 d) post-PPCI. As controls, blood samples were collected from patients with non-obstructive coronary artery disease after diagnostic coronary angiography. Aspiration thrombectomy was also performed in selected STEMI patients. High-performance lipid chromatography-electrospray mass spectrometry (LC-MS/MS) was used for OxPCs analysis. Results: Twenty-two distinct OxPC species were identified and quantified in plasma samples in patients presenting with STEMI. These compounds were categorized as fragmented and non-fragmented species. Total levels of OxPCs did not significantly differ between Isch and control groups. However, total levels of fragmented OxPCs increased significantly in the ischemic period compared with controls (Isch: 4.79 ± 0.94, Control: 1.69 ± 0.19 ng/µl of plasma, P < 0.05). Concentrations of non-fragmented OxPCs had significant reductions during ischemia compared to the control group (Isch: 4.84 ± 0.30, Control: 6.6 ± 0.51 ng/µl, P < 0.05). Levels of total OxPCs in patients with STEMI were not significantly different during reperfusion periods. However, fragmented OxPCs levels were elevated at 48 h post-reperfusion and decreased at 30 days following MI, when compared to R-2 h and R-24 h time points (Isch: 4.79 ± 0.94, R-2 h: 5.33 ± 1.17, R-24 h: 5.20 ± 1.1, R-48 h: 4.18 ± 1.07, R-30 d: 1.87 ± 0.31 ng/µl, P < 0.05). Plasma levels of two fragmented OxPCs, namely, POVPC and PONPC were significantly correlated with peak creatine kinase (CK) levels (P < 0.05). As with plasma levels, the dominant OxPC species in coronary aspirated thrombus were fragmented OxPCs, which constituted 77% of total OxPC concentrations. Conclusion: Biologically active fragmented OxPC were elevated in patients presenting with STEMI when compared to controls. PONPC concentrations were subsequently increased after PPCI resulting in reperfusion. Moreover, levels of POVPC and PONPC were also associated with peak CK levels. Since these molecules are potent stimulators for cardiomyocyte cell death, therapeutics attenuating their activities can result in a novel therapeutic pathway for myocardial salvage for patients undergoing reperfusion therapy.

Defining Acute Coronary Syndrome through Metabolomics.

As an emerging platform technology, metabolomics offers new insights into the pathomechanisms associated with complex disease conditions, including cardiovascular diseases. It also facilitates assessing the risk of developing the disease before its clinical manifestation. For this reason, metabolomics is of growing interest for understanding the pathogenesis of acute coronary syndromes (ACS), finding new biomarkers of ACS, and its associated risk management. Metabolomics-based studies in ACS have already demonstrated immense potential for biomarker discovery and mechanistic insights by identifying metabolomic signatures (e.g., branched-chain amino acids, acylcarnitines, lysophosphatidylcholines) associated with disease progression. Herein, we discuss the various metabolomics approaches and the challenges involved in metabolic profiling, focusing on ACS. Special attention has been paid to the clinical studies of metabolomics and lipidomics in ACS, with an emphasis on ischemia/reperfusion injury.

Proteomics Analysis Reveals Diverse Molecular Characteristics between Endocardial and Aortic-Valvular Endothelium.

The variations in the protein profile of aortic-valvular (AVE) and endocardial endothelial (EE) cells are currently unknown. The current study's objective is to identify differentially expressed proteins and associated pathways in both the endothelial cells. We used endothelial cells isolated from the porcine (Sus scrofa) aortic valve and endocardium for the profiling of proteins. Label-free proteomics was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our proteomics analysis revealed that 29 proteins were highly expressed, and 25 proteins were less expressed in the valve than the endocardial endothelium. The cell surface markers, such as CD63, ICAM1, PECAM1, PROCR, and TFRC, were highly expressed in EE. In contrast, CD44 was highly expressed in AVE. The pathway analysis showed that metabolic process-related proteins and extracellular matrix-related proteins were enriched in valves. Differential enrichment of signaling pathways was observed in the endocardium. The hemostasis function-related proteins were increased in both endothelial cells. The proteins and pathways enriched in aortic-valvular and endocardial endothelial cells revealed the distinct phenotype of these two closely related cells.

Oxolipidomics profile in major depressive disorder: Comparing remitters and non-remitters to repetitive transcranial magnetic stimulation treatment.

BACKGROUND: Repetitive Transcranial Magnetic Stimulation [rTMS] is increasingly being used to treat Major Depressive Disorder [MDD]. Given that not all patients respond to rTMS, it would be clinically useful to have reliable biomarkers that predict treatment response. Oxidized phosphatidylcholine [OxPC] and some oxylipins are important plasma biomarkers of oxidative stress and inflammation. Not only is depression associated with oxidative stress, but rTMS has been shown to have anti-oxidative effects. OBJECTIVES: To investigate whether plasma oxolipidomics profiles could predict treatment response in patients with treatment resistant MDD. METHODS: Fourty-eight patients undergoing rTMS treatment for MDD were recruited along with nine healthy control subjects. Plasma OxPCs and oxylipins were extracted and analyzed through high performance liquid chromatography coupled with mass spectrometry. Patients with a Hamilton Depression Rating Scale score [Ham-D] ≤7 post-treatment were defined as having entered remission. RESULTS: Fifty-seven OxPC and 32 oxylipin species were identified in our subjects. MDD patients who entered remission following rTMS had significantly higher pre-rTMS levels of total and fragmented OxPCs compared to non-remitters and controls [one-way ANOVA, p<0.05]. However, no significant changes in OxPC levels were found as a result of rTMS, regardless of treatment response [p>0.05]. No differences in plasma oxylipins were found between remitters and non-remitters at baseline. CONCLUSION: Certain categories of OxPCs may be useful predictive biomarkers for response to rTMS treatment in MDD. Given that elevated oxidized lipids may indicate higher levels of oxidative stress and inflammation in the brain, patients with this phenotype of depression may be more receptive to rTMS treatment.

IL-10 attenuates OxPCs-mediated lipid metabolic responses in ischemia reperfusion injury.

Oxidized phospholipids (OxPLs) promote inflammation as well as low density lipoprotein (LDL) uptake in a variety of physiological and pathological states. Given the anti-inflammatory role of the cytokine IL-10, we investigated its modulatory effect on the production of oxidized phosphatidylcholines (OxPCs) as well as lipid metabolic responses in global myocardial ischemia/reperfusion (I/R) injury. Increased OxPCs levels, by 1-Palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (POVPC), promoted oxidative stress (OS) and cell death. OxPCs-mediated-OS, resulted in oxidized low-density lipoprotein receptor 1 (LOX-1) activation and upregulated the expression of toll-like receptor 2 (TLR2). IL-10-induced increase in proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulated LOX-1 as well as TLR2 inflammatory responses. Under stress conditions, phosphorylation of sterol regulatory element binding protein 1c (SREBP 1c) was prevented by IL-10. The latter also prevented the generation of OxPCs and reduced their ratio (OxPCs/PCs) during injury. LOX-1 activation also promoted SREBP1c-mediated TGF-ßRII expression which was inhibited by IL-10. Both fragmented and non-fragmented OxPCs were elevated during I/R and this effect was attenuated by IL-10. The largest impact (two-threefold change at log2) was on PAzPC, (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine)-a fragmented OxPC. Thus it appears that among different OxPCs, IL-10 significantly reduces a single molecule (PAzPC)-mediated lipid metabolic responses in cardiomyocytes thereby mitigating inflammation and cell death.

Metabolomic Signature of Human Aortic Valve Stenosis.

This study outlines the first step toward creating the metabolite atlas of human calcified aortic valves by identifying the expression of metabolites and metabolic pathways involved at various stages of calcific aortic valve stenosis progression. Untargeted analysis identified 72 metabolites and lipids that were significantly altered (p < 0.01) across different stages of disease progression. Of these metabolites and lipids, the levels of lysophosphatidic acid were shown to correlate with faster hemodynamic progression and could select patients at risk for faster progression rate.

Metabolomic characterization of myocardial ischemia-reperfusion injury in ST-segment elevation myocardial infarction patients undergoing percutaneous coronary intervention.

AIM: The aim of the study was to discover the metabolomic changes in plasma that occur during human Ischemia-Reperfusion (I/R) injury and to evaluate the diagnostic utility of plasma metabolomic biomarkers for determination of myocardial injury. Deciphering the details of plasma metabolome in ST-segment elevation myocardial infarction (STEMI) patients before and after primary percutaneous coronary interventions (PPCI) would allow for better understanding of the mechanisms involved during acute myocardial ischemia and reperfusion in humans. We performed a detailed non-targeted metabolomic analysis of plasma from 27 STEMI patients who had undergone PPCI in the first 48 hrs employing a LC-MS approach. Plasma metabolome at ischemic condition was compared to multiple time points after PPCI which allowed us to focus on changes in the reperfusion phase. Classification of the differential metabolites based on chemical taxonomy identified a major role for lipids and lipid-derived molecules. Biochemical pathway analysis identified valine, leucine and isoleucine biosynthesis, vitamin B6 metabolism and glutathione metabolism as the most significant metabolic pathways representing early response to I/R injury. We also identified phenyl alanine, tyrosine, linoleic acid and glycerophospholipid metabolism as the most significant pathways representing late response to I/R injury. A panel of three metabolites pentadecanoic acid, linoleoyl carnitine and 1-linoleoylglycerophosphocholine was discovered to have diagnostic value in determining the extent of I/R injury based on cardiac biomarkers. Using a non-targeted LC-MS approach, we have successfully generated the most comprehensive data to date on significant changes in the plasma metabolome in STEMI patients who had undergone PPCI in the first 48 hrs showing that lipid metabolites represent the largest cohort of molecules undergoing significant change.

Oxylipin profile of human low-density lipoprotein is dependent on its extent of oxidation.

BACKGROUND AND AIMS: Atherosclerosis is usually the underlying cause of heart attacks, strokes and peripheral vascular diseases - collectively known as cardiovascular diseases. Oxidation of low density lipoprotein (LDL) and its lipid content has an important role in the formation of lipid-laden atherosclerotic plaques. Not much is known about the impact of oxidative stress on bioactive oxylipin molecules present in LDL. The aim of this study is to understand the changes in oxylipin molecules present in LDL characterized by varying degrees of LDL oxidation. METHODS: LDL was isolated from the pooled plasma of healthy normolipidemic volunteers and was subjected to in vitro copper-catalyzed oxidation for varying time intervals (0 h, 6 h, 12 h, 24 h and 30 h). At each time interval, oxylipins were isolated through solid phase extraction and quantified using a targeted LC/-MS/MS approach employing stable isotope dilution method. RESULTS: Our results demonstrate that different forms of oxidized LDL (OxLDL) are characterized by specific oxylipin distribution and concentration. Compared to non-oxidized LDL, there is a significant increase in oxylipin generation (p ≤ 0.05) in OxLDL subjected to 12 h and 24 h of oxidation. Though linoleate derived oxylipins are the most abundant in OxLDL extracts, the concentration of particular oxylipin species differed with different degrees of oxidation. Specifically, two pro-inflammatory linoleate-derived triols, namely 9,10,13-triHOME and 9,12,13-triHOME, exhibited a concentration increase of ~25 fold in 12h-OxLDL compared to non-oxidized LDL. Moreover, Partial least squares Discriminant Analysis (PLS-DA) identified 10 oxylipins, primarily prostaglandins, which could serve as additional biomarkers for oxidative stress or cardiovascular risk assessment. CONCLUSIONS: Our data suggests that oxidative stress induces profound changes in the oxylipin content of LDL and the pattern of change is based on the extent of oxidation.

Label-free quantitative proteomics analysis reveals distinct molecular characteristics in endocardial endothelium.

Endocardial endothelium, which lines the chambers of the heart, is distinct in its origin, structure, and function. Characterization studies using genomics and proteomics have reported molecular signatures supporting the structural and functional heterogeneity of various endothelial cells. However, though functionally very important, no studies at protein level have been conducted so far characterizing endocardial endothelium. In this study, we used endothelial cells from pig heart to investigate if endocardial endothelial cells are distinct at the proteome level. Using a high-throughput liquid chromatography-tandem mass spectrometry for proteome profiling and expression, we identified sets of proteins that belong to specific biological processes and metabolic pathways in endocardial endothelial cells supporting its specific structural and functional roles. The study also identified several transcription factors and cell surface markers, which may have roles in the specificity of endocardial endothelium. The detection of sets proteins preferentially expressed in endocardial endothelium offers new insights into its role in the regulation of cardiac function. Data are made available through ProteomeXchange with identifier PXD009194.

High throughput screening reveals no significant changes in protein synthesis, processing, and degradation machinery during passaging of mesenchymal stem cells 1.

Increasing reports of successful and safe application of bone marrow derived mesenchymal stem cells (BM-MSCs) for cell therapy are pouring in from numerous studies. However poor survival of transplanted cells in the recipient has impaired the benefits of BM-MSCs based therapies. Therefore cell product preparation procedures pertaining to MSC therapy need to be optimized to improve the survival of transplanted cells. One of the important ex vivo procedures in the preparation of cells for therapy is passaging of BM-MSCs to ensure a suitable number of cells for transplantation, which may affect the turnover of proteins involved in regulation of cell survival and (or) death pathways. In the current study, we investigated the effect of an increase in passage number of BM-MSCs in cell culture on the intracellular protein turnover (protein synthesis, processing, and degradation machinery). We performed proteomic analysis of BM-MSCs at different passages. There was no significant difference observed in the ribosomal, protein processing, and proteasomal pathways related proteins in BM-MSCs with an increase in passage number from P3 to P7. Therefore, expansion of MSCs in the cell culture in clinically relevant passages (Passage 3-7) does not affect the quality of MSCs in terms of intracellular protein synthesis and turnover.

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior.

BACKGROUND: Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3-7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation. METHODS: The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform. RESULTS: Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege. CONCLUSIONS: The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.

Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression.

BACKGROUND: Smurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. METHODS: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation. RESULTS: CNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines. CONCLUSIONS: Altogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.

VDAC1 and SERCA3 Mediate Progesterone-Triggered Ca2+ Signaling in Breast Cancer Cells.

Progesterone is a biphasic hormone whose confounding role in breast cancer cells involves an initial proliferative surge, followed by sustained growth arrest. Recently we reported that progesterone induces a time- and concentration-dependent release of reactive oxygen species and thus regulates the antiproliferative activity in the breast cancer cell line. Furthermore, the expression of p27, a crucial cell cycle control protein, was regulated by binding of progesterone on progesterone receptor B, thus leading to antiproliferative signaling via multiple signaling pathways including p53, PTEN, and antioxidant systems. Here, we performed an LC-MS/MS analysis of three different breast cancer cell lines. Bioinformatics data analysis and functional classification of proteins revealed a role of progesterone in calcium signaling in MCF-7 cells, and the major differentially expressed calcium regulators were S100A11, S100A10, calreticulin, VDAC1, SERCA3, and SERCA1. Later on we confirmed it by a cell-line-based system having a calcium cameleon sensor targeted at endoplasmic reticulum and found moderate calcium efflux from endoplasmic reticulum upon progesterone treatment. Real-time PCR, Western blot, and TMRM staining confirmed the role of calcium signaling regulators VDAC1 and SERCA3 in progesterone response. Taking together all of these results with our previous studies, we suggest that progesterone, by regulating important proteins involved in calcium signaling and transport, can modulate cell proliferation and cell death. Furthermore, our research may open new avenues for the hypothesis that surgery conducted during the luteal phase of the menstrual cycle might facilitate improved patient survival.

Nucleophosmin (NPM1)/B23 in the Proteome of Human Astrocytic Cells Restricts Chikungunya Virus Replication.

Chikungunya virus (CHIKV), a positive-stranded RNA virus, can cause neurological complications by infecting the major parenchymal cells of the brain such as neurons and astrocytes. A proteomic analysis of CHIKV-infected human astrocytic cell line U-87 MG revealed tight functional associations among the modulated proteins. The predominant cellular pathways involved were of transcription-translation machinery, cytoskeletol reorganization, apoptosis, ubiquitination, and metabolism. In the proteome, we could also identify a few proteins that are reported to be involved in host-virus interactions. One such protein, Nucleophosmin (NPM1)/B23, a nucleolar protein, showed enhanced cytoplasmic aggregation in CHIKV-infected cells. NPM1 aggregation was predominantly localized in areas wherein CHIKV antigen could be detected. Furthermore, we observed that inhibition of this aggregation using a specific NPM1 oligomerization inhibitor, NSC348884, caused a significant dose-dependent enhancement in virus replication. There was a marked increase in the amount of intracellular viral RNA, and ∼105-fold increase in progeny virions in infected cells. Our proteomic analysis provides a comprehensive spectrum of host proteins modulated in response to CHIKV infection in astrocytic cells. Our results also show that NPM1/B23, a multifunctional chaperone, plays a critical role in restricting CHIKV replication and is a possible target for antiviral strategies.

Bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine photodynamic therapy disrupts redox homeostasis and induce mitochondria-mediated apoptosis in human breast cancer cells.

Photodynamic therapy (PDT) is a clinically established and highly evolving treatment modality for cancer. PDT utilizes a light responsive drug called photosensitizer that selectively destroys tumor cells upon light irradiation. Squaraines are a class of dyes possessing all favorable characteristics of a photosensitizer and have been considered to be a potent candidate for next generation PDT. In this study we chose an iodo derivative of squaraine called diiodo-squaraine (bis(3, 5-diiodo-2,4,6-trihydroxyphenyl)squaraine) which has been reported for its tumor specificity but least studied for its cellular and molecular functions. Our studies revealed that the iodo derivative of squaraine possess maximum photodynamic activity in human breast cancer cells MDA- MB- 231 and had very little cytotoxicity in normal breast cells MCF-10A. We analyzed its pro and anti-apoptotic events initiated by oxidative stress exploring a proteomic approach and delineated other critical molecular pathways and key proteins involved in regulating the complex network of cellular response upon PDT. Our study showed that, diiodo- squaraines predominantly accumulate in mitochondria and induce mitochondria-mediated apoptosis. Our study also reveals the novel mechanistic role of diiodo-squaraines to induce oxidative stress there by activating both protective and death inducing pathways post PDT.