Girija Ramaswamy

SILAC-based quantitative proteomic analysis of gastric cancer secretome

Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood‐based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer.

Serum carotene, vitamin A, and vitamin C levels in breast cancer and cancer of the uterine cervix

Levels of carotene, vitamin A, and vitamin C measured in the serum of patients with cancer of the breast and uterine cervix were compared with levels in healthy controls and patients with benign diseases of the breast and cervix. Serum ascorbate levels were significantly lower in patients with benign diseases of the breast and cervix than in controls. In cancer patients, there was a significant trend of lower serum vitamin levels with increasing stage of the disease.

Serum levels of bone alkaline phosphatase in breast and prostate cancers with bone metastasis

BALP activity in the sera of metastatic patients of breast and prostate malignancy has increased significantly. Our studies with patients in India conform the earlier reports that BALP may have a useful complementary role in the early diagnosis of bone metastases.

Calcium calmodulin dependent kinase kinase 2 - a novel therapeutic target for gastric adenocarcinoma

Gastric cancer is one of the most common gastrointestinal malignancies and is associated with poor prognosis. Exploring alterations in the proteomic landscape of gastric cancer is likely to provide potential biomarkers for early detection and molecules for targeted therapeutic intervention. Using iTRAQ-based quantitative proteomic analysis, we identified 22 proteins that were overexpressed and 17 proteins that were downregulated in gastric tumor tissues as compared to the adjacent normal tissue. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was found to be 7-fold overexpressed in gastric tumor tissues. Immunohistochemical labeling of tumor tissue microarrays for validation of CAMKK2 overexpression revealed that it was indeed overexpressed in 94% (92 of 98) of gastric cancer cases. Silencing of CAMKK2 using siRNA significantly reduced cell proliferation, colony formation and invasion of gastric cancer cells. Our results demonstrate that CAMKK2 signals in gastric cancer through AMPK activation and suggest that CAMKK2 could be a novel therapeutic target in gastric cancer.

Identification of differentially expressed serum proteins in gastric adenocarcinoma.

UNLABELLED Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu-Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. BIOLOGICAL SIGNIFICANCE Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well classified patient samples would prove useful in identifying novel blood based biomarkers for gastric cancers. This article is part of a Special Issue entitled: Proteomics in India.

CORRELATION STUDY OF CARCINO EMBRYONIC ANTIGEN & CANCER ANTIGEN 15.3 IN PRETREATED FEMALE BREAST CANCER PATIENTS

Carcino Embryonic Antigen (CEA) and Cancer Antigen 15.3 (CA15.3) are the most common tumor markers in breast cancer patients. Measurement of circulating tumor markers is a non-invasive quantitative method. Serum levels of CEA and CA 15.3 were studied in female breast cancer patients prior to treatment. To evaluate the utility of these markers, 207 Breast carcinoma patients belonging to all the stages were considered. Healthy age matched 75 female individuals formed the control group. The serum levels of CEA and CA 15.3 were analyzed by Enzyme Linked Immunosorbent Assay (ELISA). Results were taken and compared with stages, tumor size, node and grade. The serum CA 15.3 levels were significant in all the study parameters whereas serum CEA levels showed no significant changes with any of the parameters. Measurement of serum CA 15.3 levels showed significant correlation (24.8%) with advanced stages and larger tumor sizes, whereas serum CEA levels did not show any significant correlation in breast cancer patients prior to treatment.

A network map of the gastrin signaling pathway

Gastrin is the primary hormone that induces gastric acid secretion (Edkins 1906). In humans, the gene encoding for gastrin is located on chromosome 17q21 (Lund et al. 1986). This hormone is produced by the G-cells of the antrum of stomach as preprogastrin, which comprises of 101 amino acids and is cleaved between Ala-21 and Ser-22 to yield progastrin (Reeve et al. 1984). Progastrin is then sequentially cleaved by prohormone convertase and carboxypeptidase E to yield glycine-extended gastrins- a 35-amino acid gastrin-34-Gly (G34-Gly) or an 18-amino acid gastrin-17-Gly (G17-Gly) in endocrine cells (Varro et al. 1995; Lacourse et al. 1997). G34-Gly and G17-Gly are amidated at their carboxyl terminal groups by the enzyme peptidyl alpha-amidating mono-oxygenase to form amidated gastrin. Gastrin interacts with the membrane-bound G-protein coupled cholecystokinin receptor group consisting of Cholecystokinin A receptor (CCKAR) and Cholecystokinin B receptor (CCKBR). CCKAR possesses high affinity for cholecystokinin and has a negligible affinity for gastrin (Ferrand and Wang 2006; Dufresne et al. 2006). However, CCKBR has a high affinity for gastrin and their carboxyl amidated analogues. CCKBR is expressed in the brain, smooth muscle cells, and parietal cells (Kopin et al. 1992; Berna et al. 2007). In addition to this, gastrin has also been reported to interact with annexin 2, and thereby exerts proliferation effects in gastrointestinal cancers (Singh et al. 2007; Singh 2007; Sarkar et al. 2012). The release of gastrin is induced by gastrin-releasing peptide, a neurotransmitter which acts on its basolateral receptor in the G-cells. Binding of gastrin to CCKBR present in parietal and enterochromaffin-like (ECL) cells (Schmitz et al. 2001; Kulaksiz et al. 2000)induces gastric acid secretion by parietal cells as well as histamine release by ECL cells (Dockray et al. 2005). The released histamine reaches parietal cells by paracrine diffusion where it binds H2 receptors and induces gastric acid secretion. The secretion of gastric acid inhibits the release of gastrin hormone rendering a negative feedback control, further preventing excess acid secretion. In addition, low pH value in the stomach inhibits gastrin release through stimulating somatostatin secretion by antral D-cells (Bloom et al. 1974). Gastrin is a well-known growth factor for the gastrointestinal tract. Gastrin stimulates proliferation of gastric mucosal cells (Hansen et al. 1976), maturation of parietal cells and enterochromaffin-like cells (Jain and Samuelson 2006), and promotes islet differentiation in the pancreas (Wang et al. 1993). It has also been shown to stimulate proliferation of gastric (Ishizuka et al. 1992) and colon cancer cells (Watson et al. 1989). Gastrin modulates invasion (Wroblewski et al. 2002), apoptosis (Przemeck et al. 2008; Todisco et al. 2001) and migration (Noble et al. 2003) in epithelial cells. Both glycine-extended gastrin and amidated gastrin have been reported to induce angiogenesis (Clarke et al. 2006; Lefranc et al. 2004). Moreover, the hypergastrinemic state has been associated with diverse physiological disorders in humans and other mammals. These include atrophic gastritis (Lehy et al. 2000), pernicious anemia (Orlando et al. 2007), excess acid secretion leading to duodenal ulcer disease in Helicobacter pylori infection (Berna et al. 2006; Jensen 2002; Scarpignato et al. 1996) and gastrinoma (Kloppel and Anlauf 2007). Recently, a number of research groups have explored the feasibility of using gastrin to treat various diseases. Gastrin has been used in combination therapy with epidermal growth factor to increase beta-cell mass which reversed hyperglycemia in diabetic mice (Suarez-Pinzon et al. 2005). Gastrin-stimulated beta cell neogenesis when used in combination with glucagon-like peptide 1 in human pancreatic duct cell transplanted in immunodeficient diabetic mice (Suarez-Pinzon et al. 2008). Gastrins and their receptors have been suggested as potential targets to treat gastrointestinal and pancreatic cancers (Rengifo-Cam and Singh 2004). Gastrin has also been shown to have therapeutic promise as it inhibits the growth of cholangiocarcinoma cells by promoting apoptosis (Kanno et al. 2001). On account of the functional significance of different natural forms of gastrin, we have assembled signaling pathway reactions induced by them upon binding to gastrin receptor(s) in different human cell types. These manually curated signaling events are made available through NetPath (http://www.netpath.org/) (Kandasamy et al. 2010) in different formats for analysis by the scientific community.

Evaluation of immunohistochemistry and enzyme linked immunosorbent assay for HER-2/neu expression in breast carcinoma

HER-2 is overexpressed in approximately 20–30% of invasive Breast Cancer. ECD of the HER-2 protein is frequently cleaved and released into the circulation, where it can be detected by ELISA in up to 45% of patients with metastatic breast cancer. The objective of our study was to compare the current methods for the detection of HER-2 protein. Tissue HER-2 levels were studied in 100 breast cancer patients by IHC and compared with serum HER-2 levels by ELISA. IHC frequency was 29%. Serum HER-2 ECD was positive in 42% of patients. A statistically significant correlation was observed. HER-2 detected by IHC correlates significantly with serum HER-2 levels detected by ELISA. Thus, ELISA is a reliable and economical tool to assess the HER-2 status in tumors, when breast tissue sample is not available.

EFFECT OF VITAMIN B 12 AND FOLATE ON HOMOCYSTEINE LEVELS IN COLORECTAL CANCER

Folate and cobalamin (Vitamin B12) are two essential micronutrients involved in one-carbon metabolism, which affects heart disease, neural tube defects and cancer. Methylenetetrahydrofolate reductase, the key enzyme involved in one carbon metabolism produces methyl tetrahydrofolate from methylene tetrahydrofolate, which in turn donates methyl group to homocysteine to generate methionine. There exist two common low function polymorphic variants of the methylenetetrahydrofolate reductase gene involving nucleotides 677 C→T and 1298 A→C, which are associated with hyperhomocysteinemia. These polymorphisms are also linked with increased risk for certain cancers such as breast cancer and at the same time providing a protective effect on colorectal cancer. In this case control study, we have evaluated levels of folic acid, vitamin B12 and homocysteine in patients with colorectal cancer. Folate and homocysteine levels did not differ significantly between the two groups; however an increasing trend was noticed with increase in homocysteine levels. Vitamin B12 levels were increased in cases compared to control group.

Association between plasma homocysteine and riboflavin status in Acute Lymphoblastic Leukemia in children

Remethylation of homocysteine to methionine is dependent on an adequate supply of one or more of the B vitamins like folate, vitamin B12 and vitamin B6. Plasma total homocysteine (tHcy) is also influenced by genetic factors such as polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. MTHFR is a flavo enzyme and a key player in folate metabolism and changes in its activity could modify the susceptibility to Acute Lymphoblastic Leukemia (ALL). In this case — control study we have examined the effect of riboflavin status as measured by erythrocyte glutathione reductase activation coefficient (EGRAC) on homocysteine levels along with vitamin B12 and folate in pediatric ALL. Folate and B12 levels were significantly lower among cases as compared to controls while EGRAC and tHcy did not differ significantly among the groups. The multivariate regression analysis revealed that in the ALL group EGRAC significantly influences tHcy levels suggesting that riboflavin availability may be a predictor of tHcy levels in patients with ALL. This finding may have implications for tHcy lowering therapy.