Proteomic patterns of cultured breast cancer cells and epithelial mammary cells.

Breast cancer is one of the leading causes of death from cancer among women in western countries. The different types of breast cancer are grouped into invasive and noninvasive forms. Among the invasive types, ductal infiltrating carcinoma (DIC) is the most common and aggressive form. Using an in vitro model consisting of a DIC-derived cell line (8701-BC) and a nontumoral mammary epithelial cell line (HB2), we used the proteomics approach to search for homology and differences in protein expression patterns between tumoral and nontumoral phenotypes. Within an analysis window comprising 1,750 discernible spots we have currently catalogued 140 protein spots of potential interest. Fifty-eight of them were identified by gel matching with reference maps, immunodetection, or N-terminal microsequencing and classified into four functional groups. Twelve proteins were found differentially expressed in two cell lines: four were uniquely present in the neoplastic cell proteome and eight in epithelial cells. In addition, 53 proteins displayed different relative expression levels between the two cell lines, that is, 44 were more elevated in cancer cells and 9 in HB2 cells. Among proteins with greater relative abundance in cancer cells we identified glycolytic enzymes (or their isoforms), which may indicate that the known metabolic dysregulation in cancer can reflect oncogenic-related defects of glycolytic gene expression

Proteome analysis of breast cancer cells (8701-BC) cultured from primary ductal infiltrating carcinoma: relation to correspondent breast tissues

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Type V collagen induces apoptosis of 8701-BC breast cancer cells and enhances m-calpain expression

We previously reported that ductal infiltrating carcinomas (DIC) of the human breast display profound modifications of the stromal architecture, associated with anomalous collagen composition. The major alterations observed in the interstitial collagen were an abnormal ratio between type I and type III collagens, the appearence of an onco-foetal form of collagen (OF/LB) and a relative increase of type V collagen content. Biological assays performed by culturing a DIC-derived cell line (8701-BC) onto type V collagen substrate demonstrated that the latter was able to restrain cell growth and to inhibit cell motility and invasion "in vitro", differently from what found with other collagen species tested. To search for molecular mechanisms underlying the observed inhibitory effect of collagen type V on breast cancer cells. As a reference model, we used culture substrates prepared with type IV collagen, which represents the physiological substrate for cells of epithelial origin. Apoptosis was studied by both fluorescence microscopical analyses of cell viability and DNA fragmentation assays in preparations of 8701-BC cells grown onto either type V and type IV collagen. Differences in gene expression following cell adhesion onto the two substrates were analyzed by a "differential display" PCR (DD-PCR) technique and Western blot. In this paper we demonstrate that the inhibitory effect exerted by type V collagen is consistently associated with the switching-on of a death program by a significant proportion of the cell culture, concomitant with the formation of cohesive cell islands displaying a progressive decrease of cell spreading. DD-PCR and Western blot assays demonstrated a consistent association of type V collagen-promoted apoptosis with the up-regulation of the large subunit of m-calpain (L-mC) at both mRNA and protein level. Cell exposure to calpain inibitor I decreased the amount of DNA fragmentation by 30%. The present data substantiate our previous postulates that in cases of breast DIC the zonal increase of type V collagen contribute to the assembly of a "non-permissive" micro-environment for tumor cells, antagonist to other local permissive substrates. It is therefore conceivable that the spatio-temporal derangement of stromal components may actively modulate neoplastic cell behavior and metastatic propensity, thus contributing to the selection and development of more or less malignant tumor phenotypes

A contribution to breast cancer cell proteomics: detection of new sequences

Ductal infiltrating carcinoma (DIC) of the breast is the most common and potentially aggressive form of cancer. Knowledge of proteomic profiles, attained both in vivo and in vitro, is fundamental to acquire as much information as possible on the proteins expressed in these pathologic conditions. We used the breast cancer cell line 8701-BC, established from a primary DIC, with the aim of contributing to the databases on mammary cancer cells, which in turn will be very useful for the identification of differentially expressed proteins in normal and neoplastic cells. Within an analysis window comprising about 1750 discernible spots, we have at present catalogued 84 protein spots. The proteins for which an identity was assigned were identified essentially using gel comparison, N-terminal (Nt) microseqencing and immune detection. Among the protein spots Nt-microsequenced, sixteen corresponded to known proteins, four resulted as modified, relative to matching sequences deposited on databases, and seven were unknown. These modified or novel sequences are thus of potential interest to the knowledge of breast cancer proteomics and its applications

Decorin transfection induces proteomic and phenotypic modulation in breast cancer cells 8701-BC

Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decori

Caveolin-1, breast cancer and ionizing radiation

Breast cancer (BC) recovery has increased in recent years thanks to efforts of Omics-based research in this field. However, despite the important results obtained, BC remains a complex multifactorial pathology that is difficult to treat appropriately. Caveolin-1 (CAV1), the basic constituent protein of specialized plasma membrane invaginations called caveolae, is emerging as a potential therapeutic biomarker in BC. This factor may modulate BC response to chemotherapy and radiation therapy. In addition, recent reports describe the key role of CAV1 during cell response to oxidative stress. The aim of the present review was to describe the biological roles of CAV1 in BC considering its contrasting dual functions as an oncogene and as a tumor suppressor. In addition, we report on how CAV1 may contribute to tumor cell response to ionizing radiation treatment. Finally, new roles of CAV1 in BC both on epithelium and stroma may be useful as prognostic indicators for patient treatment and help clinicians in the selection of the best personalized therapy

Integrated multi-omics investigations of metalloproteinases in colon cancer: Focus on MMP2 and MMP9

Colorectal cancer (CRC) develops by genetic and epigenetic alterations. However, the molecular mechanisms underlying metastatic dissemination remain unclear and could benefit from multi-omics investigations of specific protein families. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in ECM remodeling and the processing of bioactive molecules. Increased MMP expression promotes the hallmarks of tumor progression, including angiogenesis, invasion, and metastasis, and is correlated with a shortened survival. Nevertheless, the collective role and the possible coordination of MMP members in CRC are poorly investigated. Here, we performed a multi-omics analysis of MMP expression in CRC using data mining and experimental investigations. Several databases were used to deeply mine different expressions between tumor and normal tissues, the genetic and epigenetic alterations, the prognostic value as well as the interrelationships with tumor immune-infiltrating cells (TIICs). A special focus was placed on to MMP2 and MMP9: their expression was correlated with immune markers and the interaction network of co-expressed genes disclosed their implication in epithelial to mesenchymal transition (EMT) and immune response. Finally, the activity levels of MMP2 and MMP9 in a cohort of colon cancer samples, including tissues and the corresponding sera, was also investigated by zymography. Our findings suggested that MMPs could have a high potency, as they are targeted in colon cancer, and might serve as novel biomarkers, especially for their involvement in the immune response. However, further studies are needed to explore the detailed biological functions and molecular mechanisms of MMPs in CRC, also in consideration of their expression and different regulation in several tissues

Proteomic Profiling of Colon Cancer Tissues: Discovery of New Candidate Biomarkers

Colon cancer is an aggressive tumor form with a poor prognosis. This study reports a comparative proteomic analysis performed by using two-dimensional differential in-gel electrophoresis (2D-DIGE) between 26 pooled colon cancer surgical tissues and adjacent non-tumoral tissues, to identify potential target proteins correlated with carcinogenesis. The DAVID functional classification tool revealed that most of the differentially regulated proteins, acting both intracellularly and extracellularly, concur across multiple cancer steps. The identified protein classes include proteins involved in cell proliferation, apoptosis, metabolic pathways, oxidative stress, cell motility, Ras signal transduction, and cytoskeleton. Interestingly, networks and pathways analysis showed that the identified proteins could be biologically inter-connected to the tumor-host microenvironment, including innate immune response, platelet and neutrophil degranulation, and hemostasis. Finally, transgelin (TAGL), here identified for the first time with four different protein species, collectively down-regulated in colon cancer tissues, emerged as a top-ranked biomarker for colorectal cancer (CRC). In conclusion, our findings revealed a different proteomic profiling in colon cancer tissues characterized by the deregulation of specific pathways involved in hallmarks of cancer. All of these proteins may represent promising novel colon cancer biomarkers and potential therapeutic targets, if validated in larger cohorts of patients

Retrospective Proteomic Screening of 100 Breast Cancer Tissues

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The specimens were selected from patients with invasive ductal carcinoma of the breast, the most frequent and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers potentially useful for clinical applications. The proteomic screening; by 2D-IPG and mass spectrometry; allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients; and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is reasonably supported by concurrent events: the inhibition of apoptosis and stimulation of cellular proliferation, and the increased expression of glycolytic enzymes with multiple functions. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. We suggest that this approach on large-scale 2D-IPG proteomics of breast cancer is currently a valid tool that offers the opportunity to evaluate on the same assay the presence and recurrence of individual proteins, their isoforms and short forms, to be proposed as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast

Anticancer activity of biogenerated silver nanoparticles: An integrated proteomic investigation

Silver nanoparticles (AgNPs), embedded into a specific polysaccharide (EPS), were biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs-EPSaer) and anaerobic conditions (AgNPs-EPSanaer). Both AgNPs-EPS matrices were tested by MTT assay for cytotoxic activity against human breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell lines, revealing AgNPs-EPSaeras the most active, in terms of IC50, with a more pronounced efficacy against breast cancer cell lines. Therefore, colony forming capability, morphological changes, generation of reactive oxygen species (ROS), induction of apoptosis and autophagy, inhibition of migratory and invasive capabilities and proteomic changes were investigated using SKBR3 breast cancer cells with the aim to elucidate AgNPs-EPSaermode of action. In particular, AgNPs-EPSaerinduced a significant decrease of cell motility and MMP-2 and MMP-9 activity and a significant increase of ROS generation, which, in turn, supported cell death mainly through autophagy and in a minor extend through apoptosis. Consistently, TEM micrographs and the determination of total silver in subcellular fractions indicated that the Ag+ accumulated preferentially in mitochondria and in smaller concentrations in nucleus, where interact with DNA. Interestingly, these evidences were confirmed by a differential proteomic analysis that highlighted important pathways involved in AgNPs-EPSaertoxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment triggering cell death trough apoptosis and/or autophagy activation