Ergosterol and its metabolites as agonists of Liver X receptor and their anticancer potential in colorectal cancer.

Aberrant cholesterol homeostasis is a well-recognized hallmark of cancer and is implicated in metastasis as well as chemotherapeutic resistance, the two major causes of cancer associated mortality. Liver X receptors (LXRs) are the key transcription factors that induce cholesterol efflux via enhancing the expression of ABCA1 and ABCG1. Therefore, a comprehensive analysis of several novel sterols namely ergosta-7,22,24(28)-trien-3ß-ol (Erg1), ergosta-5,22,25-trien-3-ol (Erg2), ergosta-5,7,22,24(28)-tetraen-3ß-ol (Erg3), and ergosta-7,22-dien-3ß-ol (Erg4) as LXR agonists has been performed. Molecular docking studies have shown that these sterols possess higher binding affinities for LXRs as compared to the reference ligands (GW3965 and TO901317) and also formed critical activating interactions. Molecular dynamic (MD) simulations further confirmed that docking complexes made of these sterols possess significant stability. To assess the extent of LXR activation, ABCA1 promoter was cloned into luciferase reporter plasmid and transfected into HCT116 cells. It was observed that treatment with Erg, Erg2 and Erg4 led to a significant LXR activation with an EC50 of 5.64 µM, 4.83 and 3.03 µM respectively. Furthermore, a significant increase in mRNA expression of NR1H2 and LXR target genes i.e. ABCA1, ABCG1 and ApoE was observed upon Erg treatment. Flow cytometric analysis have revealed a significant increase in the accumulation of ABCA1 upon Erg treatment. Cytotoxicity studies conducted on colorectal cancer cell and normal epithelial cell line showed that these sterols are selectively toxic towards cancer cells. Taken together, our findings suggests that ergosterol activates LXRs, have significant anticancer activity and could be a likely candidate to manage aberrant cholesterol homeostasis.

Dietary n-3 and n-6 polyunsaturated fatty acids differentially modulate the adiponectin and leptinmediated major signaling pathways in visceral and subcutaneous white adipose tissue in high fat diet induced obesity in Wistar rats.

Obesity is a chronic metabolic disease that involves excessive accumulation of fat in white adipose tissue (WAT). Apart from storing excess fats, WAT also serves as an important endocrine organ secreting adipocytokines such as adiponectin and leptin. Adiponectin and leptin bind to their transmembrane receptors adiponectin receptor 1 (AdipoR1)/adiponectin receptor 2 (AdipoR2) and Ob-R, respectively, and mediate their effect on metabolism by regulating multiple downstream targets. Dietary fat is considered the main culprit behind obesity development. Numerous preclinical studies have highlighted role of essential polyunsaturated fatty acids (PUFAs), particularly n-3 PUFAs, in prevention of obesity. Despite emerging data, there still is no clear understanding of the mechanism of action of n-3 PUFAs and n-6 PUFAs on adipose tissue function in two functionally and anatomically different depots of WAT: visceral and subcutaneous. We designed this study using a high fat diet (HFD) fed rodent model of obesity to test our hypothesis that n-3 and n-6 PUFAs possibly differentially modulate adipokine secretion and downstream metabolic pathways such as peroxisome proliferator-activated receptor-γ (PPAR-γ), protein kinase B (AKT)-forkhead box O1 (FOXO1), and Janus kinase-signal transducer and activator of transcription in obesity. The results of the current study showed that n-3 PUFAs upregulate the expression of AdipoR1/R2 and ameliorate the effects of HFD by modulating adipogenesis via PPAR-γ and by improving glucose tolerance and lipid metabolism via AKT-FOXO1 axis in fish oil fed rats. However, n-6 PUFAs did not show any remarkable change compared with HFD fed animals. Our study highlights that n-3 PUFAs modulate expression of various targets in adiponectin and leptin signaling cascade, bringing about an overall reduction in obesity and improvement in adipose tissue function in HFD induced obesity.

Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

Despite numerous reports on the altered sphingolipids metabolism in human cancers, their clinical significance in breast cancer remains obscure. Previously, we identified the high levels of sphingolipids, ceramide phosphates and sphingosine phosphates, and the genes involved in their synthesis, CERK and SPHK1, in breast cancer patients. The present study aimed to determine the correlations of CERK and SPHK1 with clinical outcomes as well as metastasis and drug resistance markers. Both local and TCGA cohorts were analysed. High-confidence regulatory interaction network was constructed to find association of target genes with metastasis and drug resistance. Furthermore, correlations of CERK and SPHK1 with selected metastasis and drug resistance markers were validated in both cohorts. Overexpression of CERK and SPHK1 was associated with nodal metastasis, late tumor stage and high proliferation potency. In addition, increased CERK expression was also indicative of poor patient survival. Computational network analysis revealed the association of CERK and SPHK1 with known metastasis markers MMP-2 and MMP-9 and drug resistance markers ABCC1 and ABCG2. Correlation analysis confirmed the associations of target genes with these markers in both local as well as TCGA cohort. The above findings suggest clinical utility of CERK and SPHK1 as potential biomarkers in breast cancer patients and thus could provide novel leads in the development of therapeutics.

Targeting fuel pocket of cancer cell metabolism: A focus on glutaminolysis.

Advances in cell metabolism over the past few decades have demonstrated glutamine as an essential nutrient for cancer cell survival and proliferation. Glutamine offers a remarkable capacity to fuel diverse metabolic pathways in cancer cells including the Krebs cycle, maintenance of redox homeostasis, and synthesis of cellular building blocks such as nucleic acids, fatty acids, glutathione, and other amino acids. The increase in glutaminolysis has further been linked to the accumulation of oncometabolites such as 2HG (2-Hydroxyglutarate), succinate, fumarate, etc., thereby contributing to tumorigenesis via regulating epigenetic modification of imprinted genes. Therefore, therapeutic targeting of glutaminolysis in cancer cells is worth exploring for possible treatment strategies for cancer management. In this review, we have discussed the detailed mechanism of glutamine uptake, transport, and its instrumental role in rewiring the metabolic adaptation of cancer cells in the tumor microenvironment under nutrient deprivation and hypoxia. Furthermore, we have attempted to provide an updated therapeutic intervention of glutamine metabolism as a treatment strategy for cancer management.

Elucidation of underlying molecular mechanism of 5-Fluorouracil chemoresistance and its restoration using fish oil in experimental colon carcinoma.

Latest strategies for cancer treatment primarily focus on the use of chemosensitizers to enhance therapeutic outcome. N-3 PUFAs have emerged as the strongest candidate for the prevention of colorectal cancer (CRC). Our previous studies have demonstrated that fish oil (FO) rich in n-3 PUFAs not only increased therapeutic potential of 5-Fluorouracil(5-FU) in colon cancer but also ameliorated its toxicity. Henceforth, the present study is designed to elucidate mechanistic insights of FO as a chemosensitizer to circumvent drug resistance in experimental colon carcinoma. The colon cancer was induced by 1,2-dimethylhydrazine(DMH)/dextran sulfate sodium(DSS) in male Balb/c mice and these animals were treated with 5-FU(12.5 mg/kg b.w.), FO(0.2 ml), or 5-FU + FO(12.5 mg/kg b.w + 0.2 ml) orally for 14 days. The molecular mechanism of overcoming 5-FU resistance using FO in colon cancer was delineated by estimating expression of cancer stem cell markers using flowcytometric method and drug transporters by immunohistochemistry and immunoblotting. Additionally, distribution profile of 5-FU and its cytotoxic metabolite, 5-FdUMP at target(colon), and non-target sites (serum, kidney, liver, spleen) was assessed using high-performance liquid chromatography(HPLC) method. The observations revealed that expression of CSCs markers was remarkably reduced after using fish oil along with 5-FU in carcinogen-treated animals. Interestingly, the use of FO alongwith 5-FU also significantly declined the expression of drug transporters (ABCB1,ABCC5) and consequently resulted in an increased cellular uptake of 5-FU and its metabolite, 5-FdUMP at target site (colon). It could be possibly associated with change in permeability of cell membrane owing to the alteration in membrane fluidity. The present study revealed the mechanistic insights of FO as a MDR revertant which successfully restored 5-FU-mediated chemoresistance in experimental colon carcinoma.

Expression of miR-18a-5p, miR-144-3p, and miR-663b in colorectal cancer and their association with cholesterol homeostasis.

OBJECTIVE: Though cholesterol accumulation is an established hallmark of a tumor cell, the relationship between the two is still not clear. Previously, we identified 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), Sterol Regulatory Element BindingTranscription Factor 2 (SREBF2), Nuclear Receptor Subfamily 1 Group H Member 3 (NR1H3), and Nuclear Receptor Subfamily 1 Group H Member 2 (NR1H2) as the key cholesterol homeostasis genes involved in colorectal cancer (CRC). In the present study, we aimed to identify microRNAs regulating these key genes in CRC. METHODS: miR-18a-5p, miR-144-3p, and miR-663b were selected as the miRNAs targeting NR1H2, HMGCR, and SREBF2, respectively, based on the bioinformatic prediction tools and literature review. Their expression was evaluated in the local and The Cancer Genome Atlas (TCGA) cohorts. Receiver Operating Characteristic Curves and Kaplan Meier analysis were performed to elucidate their diagnostic and prognostic potential. Pearson or Spearman's correlations were used to evaluate the relationship between miRNAs and their target genes. Protein-protein interaction networks and Gene Ontology analyses were performed to investigate the potential molecular mechanism of these miRNAs. RESULTS: Deregulated expression of miR-18a-5p, miR-144-3p, and miR-663b was associated with various clinicopathological features. miR-18a-5p exhibited an inverse correlation with NR1H2. miR-18a-5p and miR-144-3p also had a significant direct correlation with miR-33a-5p, an important modulator of cholesterol homeostasis. These miRNAs also exhibited high centrality in the mirna-protein interaction network. miR-144-3p and miR-663b exhibited the potential to be used as diagnostic biomarkers. CONCLUSIONS: miR-18a-5p and miR-144-3p exhibited the potential to modulate cholesterol homeostasis in CRC. miR-663b is an interesting candidate in CRC pathophysiology.

Piperlongumine targets NF-κB and its downstream signaling pathways to suppress tumor growth and metastatic potential in experimental colon cancer.

NF-κB is the principle transcription factor and plays the central role in orchestrating chronic inflammation by regulating levels of cytokines, chemokines and growth factors. Piperlongumine (PL), a major alkaloid in the fruit of Piper longum Linn. has gained worldwide attention for its anticancer properties, however, its mechanism of action in the chemoprevention of colon cancer has not been investigated yet. Therefore, the present study was designed to elucidate the underlying molecular mechanism of PL in preventing DMH/DSS induced experimental colon cancer in mice. In the current study well established DMH/DSS induced experimental colon cancer mouse model was used to demonstrate the chemopreventive potential of PL. The expression of NF-κB and its downstream target proteins was evaluated mainly through western blotting. In addition, CAM assay, immunohistochemical staining and gelatin zymography was used to show anti-angiogenic and anti-invasive potential of PL. Additionally, important tumor biomarkers such as TSA, LASA, LDH and IL-6 levels were also estimated. The results of current study showed that PL was capable to inhibit NF-κB activation as well as its nuclear translocation. PL administration to DMH/DSS treated mice also inhibited the NF-κB downstream signaling cascades such as including COX-2 pathway, JAK/STAT pathway, ß-catenin, Notch signaling pathway, angiogenesis and epithelial to mesenchymal transition pathway. The findings of the present study have claimed PL as promising chemopreventive agent for colon cancer with pleiotropic action. The current study emphasizes that regular consumption of PL can be an effective approach in the prevention of colon cancer in humans.

Fish oil and corn oil induced differential effect on beiging of visceral and subcutaneous white adipose tissue in high-fat-diet-induced obesity.

Obesity is characterised by excessive accumulation of fat in white adipose tissue (WAT) which is compartmentalised into two anatomically and functionally diverse depots - visceral and subcutaneous. Advice to substitute essential polyunsaturated fatty acids (PUFAs) for saturated fatty acids is a cornerstone of various obesity management strategies. Despite an array of reports on the role of essential PUFAs on obesity, there still exists a lacuna on their mode of action in distinct depots i.e. visceral (VWAT) and subcutaneous (SWAT). The present study aimed to evaluate the effect of fish oil and corn oil on VWAT and SWAT in high-fat-diet-induced rodent model of obesity. Fish oil (FO) supplementation positively ameliorated the effects of HFD by regulating the anthropometrical and serum lipid parameters. FO led to an overall reduction in fat mass in both depots while specifically inducing beiging of adipocytes in SWAT as indicated by increased UCP1 and PGC1α. We also observed an upregulation of AMPKα and ACC1/2 phosphorylation on FO supplementation in SWAT suggesting a role of AMPK-PGC1α-UCP1 axis in beiging of adipose tissue. On the other hand, corn oil supplementation did not show any improvements in adipose tissue metabolism in both the depots of adipose tissue. The results were analysed using one-way ANOVA followed by Tukey's test in Graphpad Prism 5.0. Combined together our results suggest that n-3 PUFAs exert their anti-obesity effect by regulating adipokine secretion and inducing beiging of SWAT, hence increasing energy expenditure via thermogenic upregulation.

Electrochemical biosensor for miRNA-21 based on gold-platinum bimetallic nanoparticles coated 3-aminopropyltriethoxy silane.

We report an electrochemical biosensor based on gold platinum bimetallic nanoparticles (AuPtBNPs)/3-aminopropyltriethoxy silane (APTS) nanocomposite coated fluorine-doped tin oxide (FTO) as a biosensing platform for hybridization-based detection of miRNA-21. Field Emission-Scanning Electron Microscopy (FE-SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and electrochemical measurements were carried out to ensure the successful construction of the biosensor. The amount of cDNA immobilized on electrode surface and hybridization time required for the miRNA-21 sensing were optimized. The biosensing platform showed detection limit of 0.63 fM with wide linear range i.e. 1 fM-100 nM for miRNA-21 detection. The biosensing strategy demonstrates a good recovery yield from 90.18% to 94.6% in serum samples. It offers good selectivity for its complementary miRNA compared to the non-complementary miRNAs. Other analytical features of the biosensor such as stability, reusability and reproducibility were also tested, providing appropriate results.

LC-HRMS based approach to identify novel sphingolipid biomarkers in breast cancer patients.

Perturbations in lipid metabolic pathways to meet the bioenergetic and biosynthetic requirements is a principal characteristic of cancer cells. Sphingolipids (SPLs) are the largest class of bioactive lipids associated to various aspects of tumorigenesis and have been extensively studied in cancer cell lines and experimental models. The clinical relevance of SPLs in human malignancies however is still poorly understood and needs further investigation. In the present study, we adopted a UHPLC-High resolution (orbitrap) Mass spectrometry (HRMS) approach to identify various sphingolipid species in breast cancer patients. A total of 49 SPLs falling into 6 subcategories have been identified. Further, integrating the multivariate analysis with metabolomics enabled us to identify an elevation in the levels of ceramide phosphates and sphingosine phosphates in tumor tissues as compared to adjacent normal tissues. The expression of genes involved in the synthesis of reported metabolites was also determined in local as well as TCGA cohort. A significant upregulation in the expression of CERK and SPHK1 was observed in tumor tissues in local and TCGA cohort. Sphingomyelin levels were found to be high in adjacent normal tissues. Consistent with the above findings, expression of SGMS1 in tumor tissues was downregulated in TCGA cohort only. Clinical correlations of the selected metabolites and their performance as biomarkers was also evaluated. Significant ROC and positive correlation with Ki67 index highlight the diagnostic potential and clinical relevance of ceramide phosphates in breast cancer.

An electrochemical aptasensor for analysis of MUC1 using gold platinum bimetallic nanoparticles deposited carboxylated graphene oxide.

A simple electrochemical strategy has been designed for the analysis of MUC1 using electrodeposited gold platinum bimetallic nanoparticles (Au-PtBNPs) on the surface of carboxylated graphene oxide (CGO)/FTO electrode as a signal amplification platform. The carboxylic groups of CGO were activated with EDS-NHS linker and subsequently immobilized with streptavidin for further deposition of biotin labelled aptamer. All the modification steps were characterized by FE-SEM, EDS mapping, FT-IR, contact angle measurements and electrochemical methods. After incubating with target protein MUC1, the aptaelectrode produced some concentration dependent responses which were measured electrochemically by DPV assay. The prepared aptasensor exhibits wide linear range from 1 fM-100 nM with detection limit of 0.79 fM under optimal experimental conditions. The performance of this aptaelectrode was also evaluated showing good selectivity, storage stability (15 days), reproducibility and reusability (up to 3 times). Furthermore, the applicability of the aptasensor for spiked serum samples showed recovery range from 92% to 97%.

Clinical relevance of cholesterol homeostasis genes in colorectal cancer.

Accumulation of cholesterol is a well-known feature in cancer. Preclinical studies suggest the contribution of various cholesterol regulators in CRC. However, their clinical relevance remains poorly understood. The aim of the present study is to evaluate the expression of these modulators in CRC and elucidate their diagnostic and prognostic value. mRNA levels of HMGCR, SREBF2, NR1H3 and NR1H2 were downregulated in tumors in local and TCGA cohort. The expression of LDLR, ABCA1 and SCARB1 was not consistent in the two cohorts. Western Blot analysis showed the increased levels of LDLR and reduced levels of LXR in early stage patients. Tumoral SREBP2 levels were enhanced in early stage whereas decreased in late stage. The individual expression of HMGCR, SREBF2, NR1H3 and NR1H2 did not have the potential to be used as independent prognostic marker, however, the combined expression of these genes associated with poor clinical outcome independent of lymph node metastasis, distant metastasis and advanced stage. This work sheds light on deregulation of cholesterol uptake and efflux pathways and provides novel leads in the development of biomarkers and therapeutic regimens that can detect and target CRC at initial stages.

Role of cholesterol homeostasis and its efflux pathways in cancer progression.

Tumor cells show high avidity for cholesterol in order to support their inherent nature to divide and proliferate. This results in the rewiring of cholesterol homeostatic pathways by influencing not only de novo synthesis but also uptake or efflux pathways of cholesterol. Recent findings have pointed towards the importance of cholesterol efflux in tumor pathogenesis. Cholesterol efflux is the first and foremost step in reverse cholesterol transport and any perturbation in this pathway may lead to the accumulation of intracellular cholesterol, thereby altering the cellular equilibrium. This review addresses the different mechanisms of cholesterol efflux from the cell and highlights their role and regulation in context to tumor development. There are four different routes by which cholesterol can be effluxed from the cell namely, 1) passive diffusion of cholesterol to mature HDL particles, 2) SR-B1 mediated facilitated diffusion, 3) Active efflux to apo A1 via ABCA1 and 4) ABCG1 mediated efflux to mature HDL. These molecular players facilitating cholesterol efflux are engaged in a complex interplay with different signaling pathways. Thus, an understanding of the efflux pathways, their regulation and cross-talk with signaling molecules may provide novel prognostic markers and therapeutic targets to combat the onset of carcinogenesis.

A voltammetric hybridization assay for microRNA-21 using carboxylated graphene oxide decorated with gold-platinum bimetallic nanoparticles.

An electrochemical hybridization assay is described for the determination of microRNA-21. Fluorine tin oxide (FTO) sheets were coated with carboxylated graphene oxide followed by deposition of gold-platinum bimetallic nanoparticles by using chronoamperometry at a potential of -0.2 V for 350 s. The capture probe was immobilized on the surface of the modified FTO sheets by biotin-avidin interaction. On exposure to microRNA-21, hybridization occurs, and that can be detected at a relatively low working potential of 0.25 V by using ferri/ferro-cyanide as an electrochemical probe. The various modifications of the FTO sheets were characterized by means of FE-SEM, FT-IR, contact angle studies and electrochemical techniques. The effects of pH value, EDC-NHS activation time, concentration of capture probe and incubation time were optimized. The sensor has a wide linear response that extends from 1 fM to 1 µM of microRNA-21, and the detection limit is 1 fM. The sensor is stable for about 15 days (by retaining 90% of its initial activity) and can be reused for about 3 times (85% of initial activity) after regeneration with 50 mM NaOH solution. The sensor was applied to the analysis of spiked human serum and gave recoveries between 90 and 111%. Graphical abstract Carboxylated graphene oxide (CGO) coated on a fluorine tin oxide (FTO) electrode was decorated with Au-Pt bimetallic nanoparticles (Au-PtBNPs). The Au-PtBNPs/CGO/FTO electrode surface was used for immobilizing streptavidin and biotinylated capture probe which can electrochemically detect microRNA-21 based on its sequence complementarity.

Piperlongumine, a piper alkaloid targets Ras/PI3K/Akt/mTOR signaling axis to inhibit tumor cell growth and proliferation in DMH/DSS induced experimental colon cancer.

Colorectal cancer (CRC) is the most common carcinoma of the digestive tract. The slow growing nature of CRC offers a great opportunity for prevention strategies. The concept of chemoprevention of colorectal cancer using plant derived natural products is gaining substantial attention because it is an inherently safe and cost-effective alternative to conventional cancer therapies. Piperlongumine (PL), a natural alkaloid present in Piper longum Linn has been reported to exhibit notable anticancer effects in various in vitro studies. Nonetheless, the chemopreventive potential of PL has not been studied in experimentally induced colon cancer yet. Ras/PI3K/Akt/mTOR signaling axis plays a central role in promoting tumor cell growth, proliferation and survival by inhibiting apoptosis. In the present study, we demonstrated, for the first time, the chemopreventive effects of PL in DMH + DSS induced colon carcinogenesis animal model. We showed that PL displayed potent antineoplastic activity against colon cancer cell growth by targeting Ras proteins and PI3K/Akt signaling cascade. PL mediated inhibition of tumor cell growth was associated with inhibition of Ras protein levels and its preferred companion protein PI3K levels that led to suppressed activity of Akt/NF-κB, c-Myc and cyclin D1. It was also found that PL arrested the cell cycle progression at G2/M phase and induced mitochondrial apoptotic pathway by downregulating Bcl-2 levels. Furthermore, the results of liver and kidney toxicity suggested that PL exhibit no toxicity in animals. Our results suggest that PL may be an effective chemopreventive agent for colon cancer.

Differential ratios of fish/corn oil ameliorated the colon carcinoma in rat by altering intestinal intraepithelial CD8+ T lymphocytes, dendritic cells population and modulating the intracellular cytokines.

Intraepithelial lymphocytes (IELs) impart a crucial role in maintaining intestinal homeostasis, yet their role in colon cancer pathogenesis remains unknown. Here, we posited that the modulation of intestinal immune response via dietary interventions might be an implacable strategy in restraining colon carcinoma. In the above context, we studied the effect of differential ratios of fish oil (FO) and corn oil (CO) on the gut immune response in experimentally induced colon cancer. Male Wistar rats were divided into six groups: Group I obtained purified diet while Groups II and III were fed on the diet supplemented with differential ratios of FO and CO i.e. 1:1 and 2.5:1, respectively. The groups were further subdivided into control and carcinogenic group, treated with ethylenediaminetetraacetic acid (EDTA) or N,N'-dimethylhydrazine dihydrochloride (DMH), respectively. Initiation phase comprised the animals sacrificed 48 h after the last injection whereas, the post -initiation phase was constituted by animals sacrificed 12 weeks after the treatment regimen. CD8+ T cells, CD8/αß TCR cells, dendritic cells increased significantly on treatment with DMH as compared to control. However, on treatment with differential ratios of FO and CO these cells decreased significantly. The intracellular cytokine i.e. interferon gamma (IFN-γ) and cytotoxic granules component i.e Perforin and Granzyme decreased significantly in the initiation phase but in the post-initiation phase IFN-γ and Perforin increased considerably on carcinogen treatment as compared to the control group. On treatment with FO and CO in the initiation phase the IFN-γ, Perforin and Granzyme expression increased significantly. However, in the post-initiation phase treatment with differential ratios of FO and CO led to a significant decrease in the IFN-γ, Perforin and increase in Granzyme was observed in these groups. Altogether, FO supplementation appeared to activate the immune response that may further attenuate the process of carcinogenesis, in a dose and time-dependent manner.

Apoptosis mediated chemosensitization of tumor cells to 5-fluorouracil on supplementation of fish oil in experimental colon carcinoma.

5-Fluorouracil has been considered as a cornerstone therapy for colorectal cancer; however, it suffers from low therapeutic response rate and severe side effects. Therefore, there is an urgent need to increase the clinical efficacy of 5-fluorouracil. Recently, fish oil rich in n-3 polyunsaturated fatty acids has been reported to chemosensitize tumor cells to anti-cancer drugs. This study is designed to understand the underlying mechanisms of synergistic effect of fish oil and 5-fluorouracil by evaluation of tumor cell-associated markers such as apoptosis and DNA damage. The colon cancer was developed by administration of N,N-dimethylhydrazine dihydrochloride and dextran sulfate sodium salt. Further these animals were treated with 5-fluorouracil, fish oil, or a combination of both. In carcinogen-treated animals, a decrease in DNA damage and apoptotic index was observed. There was also a decrease in the expression of Fas, FasL, caspase 8, and Bax, and an increase in Bcl-2. In contrast, administration of 5-fluorouracil and fish oil as an adjuvant increased both DNA damage and apoptotic index by activation of both extrinsic and intrinsic apoptotic pathways as compared to the other groups. The increased pro-apoptotic effect by synergism of 5-fluorouracil and fish oil may be attributed to the incorporation of n-3 polyunsaturated fatty acids in membrane, which alters membrane fluidity in cancer cells. In conclusion, this study highlights that the induction of apoptotic pathway by fish oil may increase the susceptibility of tumors to chemotherapeutic regimens.

Identification of differentially expressed proteins in retinoblastoma tumors using mass spectrometry-based comparative proteomic approach.

In India, retinoblastoma is among the top five childhood cancers. Children mostly present with extraocular extension and high risk features that results in unsatisfactory treatment and low survival rate. In addition, lack of potential therapeutic and prognostic targets is another challenge in the management of retinoblastoma. We studied comparative proteome of retinoblastoma patients (HPV positive and negative (n=4 each) and controls (n=4), in order to identify potential retinoblastoma-specific protein targets. 2D-DIGE coupled MALDI-TOF/TOF mass spectrometry identified 39 unique proteins. Highly deregulated proteins were GFAP,RBP3,APOA1,CRYAA,CRABP1,SAG and TF. Gene ontology (Panther 7.0) revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). 8 proteins were significantly upregulated in HPV positive vis-a-vis HPV negative cases. Patient group exhibited 12 upregulated and 18 downregulated proteins compared to controls. Pathway and network analysis (IPA software) revealed CTNNB1 as most significantly regulated signalling pathway in HPV positive than HPV negative retinoblastoma. The trends in transcriptional change of 9 genes were consistent with those at proteomic level. The Western blot analysis confirmed the expression pattern of RBP3,GFAP and CRABP1. We suggest GFAP,RBP3,CRABP1,CRYAAA,APOA1 and SAG as prospective targets that could further be explored as potential candidates in therapy and may further assist in studying the disease mechanism. SIGNIFICANCE: In this study we evaluated tumor tissue specimens from retinoblastoma patients and identified 39 differentially regulated proteins compared to healthy retina. From these, we propose RBP3, CRABP1, GFAP, CRYAA, APOA1 and SAG as promising proteomic signatures that could further be explored as efficient prognostic and therapeutic targets in retinoblastoma. The present study is not only a contribution to the ongoing endeavour for the discovery of proteomic signatures in retinoblastoma, but, may also act as a starting point for future studies aimed at uncovering novel targets for further therapeutic interventions and improving patient outcomes.

Transglutaminase-2: evolution from pedestrian protein to a promising therapeutic target.

The ability of cancer cells to metastasize represents the most devastating feature of cancer. Currently, there are no specific biomarkers or therapeutic targets that can be used to predict the risk or to treat metastatic cancer. Many recent reports have demonstrated elevated expression of transglutaminase 2 (TG2) in multiple drug-resistant and metastatic cancer cells. TG2 is a multifunctional protein mostly known for catalyzing Ca2+-dependent -acyl transferase reaction to form protein crosslinks. Besides this transamidase activity, many Ca2+-independent and non-enzymatic activities of TG2 have been identified. Both, the enzymatic and non-enzymatic activities of TG2 have been implicated in diverse pathophysiological processes such as wound healing, cell growth, cell survival, extracellular matrix modification, apoptosis, and autophagy. Tumors have been frequently referred to as 'wounds that never heal'. Based on the observation that TG2 plays an important role in wound healing and inflammation is known to facilitate cancer growth and progression, we discuss the evidence that TG2 can reprogram inflammatory signaling networks that play fundamental roles in cancer progression. TG2-regulated signaling bestows on cancer cells the ability to proliferate, to resist cell death, to invade, to reprogram glucose metabolism and to metastasize, the attributes that are considered important hallmarks of cancer. Therefore, inhibiting TG2 may offer a novel therapeutic approach for managing and treatment of metastatic cancer. Strategies to inhibit TG2-regulated pathways will also be discussed.

Proteomic analysis of differentially expressed proteins in vitreous humor of patients with retinoblastoma using iTRAQ-coupled ESI-MS/MS approach.

There is close proximity of vitreous humor with the tumor bulk in eyes with retinoblastoma. This renders vitreous humor a promising source to evaluate disease-specific protein targets in retinoblastoma. We studied the differential proteome of vitreous fluid in retinoblastoma tumors (n = 4) as compared to controls (n = 4). The vitreous humor was depleted off the high abundant fraction using MARS-6 affinity column. Subsequently, the tryptic peptides were derivatised with iTRAQ labels. The labelled peptides were pooled and subjected to fractionation using bRPLC. This was followed by protein identification and quantification using electrospray ionisation mass spectrometry (ESI-MS/MS) approach. The identified proteins were subjected to bioinformatics analysis utilizing PANTHER 7.0 and IPA software. Four hundred and thirty-one non-redundant (362 upregulated and 69 downregulated) proteins (≥2 unique peptides, ± 1.5 folds, p < 0.05) were identified. The majority of the proteins were cytoplasmic (40 %), majorly involved in catalytic (32.7 %) and binding activities (26.3 %). Highly deregulated proteins included MMP2, TNC, CD44, SUZ12 and CRABP1. The protein expression of GFAP, CRABP1, MMP2 and TNC was validated by western blotting. Pathway and network analyses revealed p38MAPK and Akt signalling to be the most significantly regulated pathways in retinoblastoma. This is the first report of differential vitreous proteome of retinoblastoma and highlights novel protein targets, such as MMP2, TNC and CRABP1. Further investigations into unravelling the biological role of the proteins and their prospects of being utilised as potential candidates in therapeutics are warranted.