Samar Masoumi-Moghaddam

The developing story of Sprouty and cancer

Sprouty proteins are evolutionarily conserved modulators of MAPK/ERK pathway. Through interacting with an increasing number of effectors, mediators, and regulators with ultimate influence on multiple targets within or beyond ERK, Sprouty orchestrates a complex, multilayered regulatory system and mediates a crosstalk among different signaling pathways for a coordinated cellular response. As such, Sprouty has been implicated in various developmental and physiological processes. Evidence shows that ERK is aberrantly activated in malignant conditions. Accordingly, Sprouty deregulation has been reported in different cancer types and shown to impact cancer development, progression, and metastasis. In this article, we have tried to provide an overview of the current knowledge about the Sprouty physiology and its regulatory functions in health, as well as an updated review of the Sprouty status in cancer. Putative implications of Sprouty in cancer biology, their clinical relevance, and their proposed applications are also revisited. As a developing story, however, role of Sprouty in cancer remains to be further elucidated.

Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy

BackgroundBromelain and N-acetylcysteine are two natural, sulfhydryl-containing compounds with good safety profiles which have been investigated for their benefits and application in health and disease for more than fifty years. As such, the potential values of these agents in cancer therapy have been variably reported in the literature. In the present study, the efficacy of bromelain and N-acetylcysteine in single agent and combination treatment of human gastrointestinal carcinoma cells was evaluated in vitro and the underlying mechanisms of effect were explored.MethodsThe growth-inhibitory effects of bromelain and N-acetylcysteine, on their own and in combination, on a panel of human gastrointestinal carcinoma cell lines, including MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T, were assessed by sulforhodamine B assay. Moreover, the influence of the treatment on the expression of a range of proteins involved in the regulation of cell cycle and survival was investigated by Western blot. The presence of apoptosis was also examined by TUNEL assay.ResultsBromelain and N-acetylcysteine significantly inhibited cell proliferation, more potently in combination therapy. Drug-drug interaction in combination therapy was found to be predominantly synergistic or additive. Mechanistically, apoptotic bodies were detected in treated cells by TUNEL assay. Furthermore, Western blot analysis revealed diminution of cyclins A, B and D, the emergence of immunoreactive subunits of caspase-3, caspase-7, caspase-8 and cleaved PARP, withering or cleavage of procaspase-9, overexpression of cytochrome c, reduced expression of anti-apoptotic Bcl-2 and pro-survival phospho-Akt, the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1. These results were more prominent in combination therapy.ConclusionWe report for the first time to our knowledge the growth-inhibitory and cytotoxic effects of bromelain and N-acetylcysteine, in particular in combination, on a panel of gastrointestinal cancer cell lines with different phenotypes and characteristics. These effects apparently resulted from cell cycle arrest, apoptosis and autophagy. Towards the development of novel strategies for the enhancement of microscopic cytoreduction, our results lay the basis for further evaluation of this formulation in locoregional approaches to peritoneal surface malignancies and carcinomatosis.

Depletion of mucin in mucin-producing human gastrointestinal carcinoma: Results from in vitro and in vivo studies with bromelain and N-acetylcysteine

Aberrant expression of membrane-associated and secreted mucins, as evident in epithelial tumors, is known to facilitate tumor growth, progression and metastasis, and to provide protection against adverse growth conditions, chemotherapy and immune surveillance. Emerging evidence provides support for the oncogenic role of MUC1 in gastrointestinal carcinomas and relates its expression to an invasive phenotype. Similarly, mucinous differentiation of gastrointestinal tumors, in particular increased or de novo expression of MUC2 and/or MUC5AC, is widely believed to imply an adverse clinicopathological feature. Through formation of viscous gels, too, MUC2 and MUC5AC significantly contribute to the biology and pathogenesis of mucin-secreting gastrointestinal tumors. Here, we investigated the mucin-depleting effects of bromelain (BR) and N-acetylcysteine (NAC), in nine different regimens as single or combination therapy, in in vitro (MKN45, KATOIII and LS174T cell lines) and in vivo (female nude mice bearing intraperitoneal MKN45 and LS174T) settings. The inhibitory effects of the treatment on cancer cell growth and proliferation were also evaluated in vivo. Our results suggest that a combination of BR and NAC with dual effects on growth and mucin products of mucin-expressing tumor cells is a promising candidate towards the development of novel approaches to gastrointestinal malignancies with the involvement of mucin pathology. This capability supports the use of this combination formulation in locoregional approaches for reducing the adverse effects of the aberrantly secreted gel-forming mucins, as in pseudomyxoma peritonei and similar pathologies with ectopic production of mucin.

Vascular endothelial growth factor expression correlates with serum CA125 and represents a useful tool in prediction of refractoriness to platinum-based chemotherapy and ascites formation in epithelial ovarian cancer

There is an increasing need for the identification of novel biological markers and potential therapeutic targets in epithelial ovarian cancer (EOC). Given the critical role of growth factors in the biology of EOC, we aimed in the present study to evaluate the intratumoral expressions of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and their clinical relevance in a cohort of 100 patients with EOC. All patients received platinum-based chemotherapy after surgery. A comparative immunohistochemical study of normal ovarian and EOC tissues showed that both growth factors were expressed at higher levels in tumor samples. In our statistical analysis, while no association existed between the FGF expression status and the clinicopathological characteristics of patients, intratumoral VEGF was identified as a potential biomarker for the prediction of ascites formation. In addition, the expression status of VEGF appeared to independently predict overall survival and response to chemotherapy. Furthermore, a direct association was demonstrated between the pre-treatment VEGF expression and serum CA125 after three cycles of chemotherapy. In sum, we report for the first time to our knowledge the correlation between intratumoral VEGF and serum CA125 in EOC. Our data also shows the prognostic value of VEGF expression in EOC. These results suggest the potential value of intratumoral VEGF in patient stratification. Dual inhibition of VEGF and CA125 might bring about a better outcome for patients with EOC.

Sprouty 2 protein, but not Sprouty 4, is an independent prognostic biomarker for human epithelial ovarian cancer.

Sprouty proteins are evolutionary‐conserved modulators of receptor tyrosine kinase signaling, deregulation of which has been implicated in the pathophysiology of cancer. In the present study, the expression status of Spry2 and Spry4 proteins and its clinical relevance in human epithelial ovarian cancer (EOC) were investigated retrospectively. We examined the immunohistochemical expression of Spry2 and Spry4 in matched tumor and normal tissue samples from 99 patients. The expression of ERK, p‐ERK, Ki67, fibroblast growth factor‐2, vascular endothelial growth factor and interleukin‐6 and their correlation with Sprouty homologs were also evaluated. Moreover, the correlation between Spry2 and Spry4 and the clinicopathological characteristics were analyzed along with their predictive value for overall survival (OS) and disease‐free survival (DFS). Our data indicated significant downregulation of Spry2 and Spry4 in tumor tissues (p < 0.0001). A significant inverse correlation was evident between Spry2 and p‐ERK/ERK (p = 0.048), Ki67 (p = 0.011), disease stage (p = 0.013), tumor grade (p = 0.003), recurrence (p < 0.001) and post‐treatment ascites (p = 0.001), individually. It was found that Spry2 low‐expressing patients had significantly poorer OS (p = 0.002) and DFS (p = 0.004) than those with high expression of Spry2. Multivariate analysis showed that high Spry2 (p = 0.018), low stage (p = 0.049) and no residual tumor (p =0.006) were independent prognostic factors for a better OS. With regard to DFS, high Spry2 (p = 0.044) and low stage (p = 0.046) remained as independent predictors. In conclusion, we report for the first time significant downregulation of Spry2 and Spry4 proteins in human EOC. Spry2 expression was revealed to significantly impact tumor behavior with predictive value as an independent prognostic factor for survival and recurrence.

Intratumoral interleukin-6 predicts ascites formation in patients with epithelial ovarian cancer: A potential tool for close monitoring

BackgroundThe implication of IL-6 in the pathogenesis of epithelial ovarian cancer (EOC) is well documented. Accordingly, the clinicopathological significance of this cytokine in patients’ ascites fluid or serum has largely been investigated. Since the main source of IL-6 secreted into the biological fluids is the tumor tissue, this study was designed to investigate the status and possible clinical relevance of the IL-6 expression in an array of EOC tissue specimens.MethodsTissue samples obtained from ninety-eight consecutive patients with EOC were studied using immunohistochemistry. Clinicopathological characteristics and treatment related factors were collected from patient files. The relationship between the expression of the protein of interest and the study endpoints of disease-free survival (DFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. For evaluating the predictive value of IL-6, logistic regression and cox proportional hazards models were employed.ResultsAn upregulation of IL-6 expression was observed in EOC tissues as compared with the normal samples (p < 0.0001). As regards the clinical relevance, IL-6 failed to predict OS, DFS and response to the platinum-based chemotherapy in EOC patients. In multivariate analysis, however, IL-6 was identified as an independent predictive factor for the development of post-treatment ascites (p:0.033).ConclusionsHaving the capability to predict the ascites formation, IL-6 might serve as a biomarker and a useful tool in EOC for monitoring purposes. IL-6 targeting for the prevention of the ascites development is a potential avenue for further investigation.

The expression of the Sprouty 1 protein inversely correlates with growth, proliferation, migration and invasion of ovarian cancer cells

BackgroundOur recent study on a panel of human ovarian cancer cells revealed that SKOV-3 cells barely express the Sprouty isoform 1 (Spry1) while 1A9 cells maintain it at a level similar to normal ovarian cells. Here we investigated the functional outcomes of induced alterations in the expression of Spry1 in the two cell lines in vitro.MethodsUsing the Spry1 specific plasmid and siRNA, the expression of Spry1 was induced and conversely silenced in SKOV-3 and 1A9 cells, respectively. The functional outcome was investigated by means of proliferation, MTT, scratch-wound, migration and invasion assays and selection of the stable clones. Mechanism of the effect was explored by Western blot.ResultsIn the Spry1-transfected SKOV-3 cells, a significant reduction in growth and proliferation was evident. Stable clones of the Spry1-transfected SKOV-3 were almost undetectable after day 14. The number of migrated and invaded cells and the percentage of the scratch closure were significantly lower in the Spry1-transfected group. Spry1 silencing in 1A9 cells, on the other hand, led to a significant increase in cell growth and proliferation. The number of migrated and invaded cells and the percentage of the scratch closure significantly increased in Spry1-silenced 1A9 group. Mechanistically, overexpression of Bax, activation of caspases 3, 7, 8 and 9, cleavage of PARP and attenuation of Bcl-2 and Bcl-xl were observed along with reduced activation of Erk and Akt and increased amount and activity of PTEN in the Spry1-transfected SKOV-3 cells.ConclusionsHere, we report the inverse correlation between the expression of Spry1 and growth, proliferation, invasion and migration of ovarian cancer cells.

Is mucin a determinant of peritoneal dissemination of gastrointestinal cancer? Analysis of mucin depletion in two preclinical models

BackgroundMucinous gastrointestinal cancers may indicate a higher propensity for widespread peritoneal seeding than their non-mucinous counterparts. We hypothesized that mucin content of gastrointestinal cancer cells and tumors is an indicator of cell viability and a determinant of the peritoneal tumor burden and tested our hypothesis in relevant experimental models.MethodsMKN45 and LS174T models of human gastrointestinal cancer were treated with known mucin-depleting agents in vitro and in vivo, their mucin production was evaluated with Western blot immunohistochemistry, PAS staining and ELISA, and its correlation with cell viability and peritoneal tumor burden was analyzed.ResultsA relationship was found between the viability of cancer cells and their mucin levels in vitro. In agreement, when treated animal models were categorized into low- and high-burden groups (based on the weight and number of the peritoneal nodules), tumoral mucin levels were found to be significantly higher in the latter group.ConclusionsTumoral mucin is apparently among the factors that dictate the pattern and extent of the peritoneal spread of gastrointestinal cancer, where it allows for enhanced dissemination and redistribution. If further tested and validated, our hypothesis could lay the basis for the development of novel mucin-targeted strategies.

Mucins and Tumor Biology

Mucins are a diverse family of high molecular weight, heavily glycosylated proteins that are expressed both physiologically by normal epithelial cells and aberrantly by tumor cells. Under physiological conditions, mucins are differentially expressed by specialized epithelial cells of mucosal surfaces throughout the body in a relatively organ- and cell type-specific manner. Membrane-associated mucins communicate information about extracellular conditions, mediate intracellular signal transduction, and contribute to morphological and behavioral characteristics of the epithelial cells. Secreted mucins provide a physical barrier for epithelial cells lining the gastrointestinal and respiratory tracts and form the ductal surfaces of organs such as liver, breast, pancreas, and kidney. In the gastrointestinal tract, secreted mucins not only protect and lubricate the lining of the alimentary canal for enhanced digestive functionality but also contribute to the specialized tasks of these organs. In the stomach, the mucous layer forms a protective layer over the surface epithelium and acts as a selective diffusion barrier for HCl. The intestinal mucin participates in the front line of the enteric host defense generated by the alliance of the epithelial cells, immune cells, and resident microbiota. On the other hand, mucins have long been implicated in tumor biology and the pathogenesis of cancer, particularly adenocarcinomas. At the simplest level, tumor cells use mucins in much the same way as normal epithelia to control their local microenvironment and to protect themselves from adverse growth conditions. The mucus layer covering tumor cells is believed to serve as an impenetrable physicochemical barrier that helps them evade immune and inflammatory responses. Moreover, aberrant composition and structure of tumor-produced mucins enhance their growth and survival in otherwise inhospitable conditions, and confer on tumor cells potential ligands for interaction with other receptors at the cell surface, contributing to their survival during invasion and metastasis. Through ligand–receptor interactions and morphogenetic signal transduction, mucins are believed to regulate differentiation, proliferation, and adhesion of malignant cells. In addition, mucins contribute to the invasive and metastatic properties of adenocarcinomas by simultaneously configuring the adhesive and anti-adhesive properties of tumor cell surface. In addition, many lines of evidence support the involvement of both membrane-associated and secreted mucins in diverse biological mechanisms underlying resistance to chemotherapy, including their implications in the formation of a physical barrier, resistance to apoptosis, drug metabolism, cell stemness, and epithelial–mesenchymal transition (EMT). Furthermore, mucins make specific contributions to the biology and clinical features of some peritoneal malignancies, including PC from mucinous gastrointestinal adenocarcinomas and PMP. In this chapter, classification of this protein family, and molecular and biological features of different family members and their role in health (Corfield, Biochimica et Biophysica Acta 1850: 236–252, 2015) and cancer (Hollingsworth and Swanson, Nature Reviews Cancer 4: 45–60, 2004) are reviewed.

Peritoneal Dissemination of Gastrointestinal Tumors

The term peritoneal surface malignancy (PSM) applies to a wide range of epithelial or, less frequently, mesenchymal neoplasms that originate from the primitive structure of the peritoneum (primary PSM), or spread over the peritoneum as metastases from tumors of intra-abdominal, retroperitoneal, or extra-abdominal organs (secondary PSM). Primary PSMs, including peritoneal mesothelioma and primary peritoneal carcinoma, are rare. Peritoneal dissemination of colorectal, gastric, and ovarian cancers, known as peritoneal carcinomatosis (PC), represents the most frequent types of secondary PSM (Treatment of peritoneal surface malignancies, Milan, 2015). As the focus of this monograph, three secondary PSM entities, including colorectal and gastric PCs and the pseudomyxoma peritonei (PMP) syndrome, are revisited in this chapter. Hematogenous and lymphatic metastases of gastrointestinal cancers follow a well-defined pattern of distribution, with the regional lymph nodes and the liver being the initial sites for cancer spread. Peritoneal dissemination is a third mechanism that can result from serosal invasion or visceral perforation by tumor, or may develop as an iatrogenic complication following diagnostic or therapeutic interventions. Distinctions in gene expression profiles and the involvement of certain molecular pathways are believed to determine the metastatic site of gastrointestinal cancer (peritoneum versus liver) (Varghese et al., Annals of Surgical Oncology 14: 3460–3471, 2007). The past assumption that peritoneal dissemination is a random process has been challenged by meticulous observations that have identified different patterns and mechanisms governing the dissemination of tumor cells. In this regard, anatomic site of the primary tumor, histologic type of tumor, changes in intra-abdominal pressure, gravity, peritoneal surface motion (peristalsis), peritoneal fluid resorption, viscosity and volume of fluid within the abdomen, peritoneal adhesions, and fibrin entrapment are among factors that affect the pattern of spread (Carmignani et al., Cancer and Metastasis Reviews 22: 465–472, 2003). Also classified as a secondary PSM with an appendiceal primary origin, PMP represents the prototype for the peritoneal neoplasms associated with mucin secretion and accumulation. PSM is usually associated with poor prognosis regardless of the origin of the primary tumor. By virtue of the recent advances, however, the treatment paradigm has shifted from palliative to curative (Sugarbaker, Langenbeck’s Archives of Surgery 384: 576–587, 1999a; Esquivel, Surgical Oncology Clinics of North America 21: xv–xviii, 2012; Mohamed et al., Current Oncology 18: e84–e96, 2011). The classification of PSM is outlined in Table 1.1.