Zinc Dependent Endopeptidase Character Analysis

N-cadherin is a protein encoded by the CDH2 gene. It interacts with the cellular cytoskeleton, and is often involved in cardiac muscle, as well as certain cancers. Being calcium dependent, it helps to maintain cellular structure and integrity. For example, it plays a role in trans-endothelial migration, which involves cell-cell adhesion [3]. The endothelial layer contains many different fibers, as well as pathways that allow attachment for the cadherin protein. Some cancer cells can eventually pass through the endothelium, causing the cancer to become malignant and spread. Cadherins in this case can be used to identify and track the spread of the cells, and further identify common routes of travel through the human vasculature.

Aim 2: To identify the proteins in the exosome of the metastatic cancer cells. The exosome or the secretory vesicle fraction will be concentrated from the extracellular media of metastatic, non-metastatic cancer cell and compare with the control cells. SILAC labelled proteomic analysis will be performed in the isolated exosomes to identify the proteins that are specific in metastatic cells.

Hyaluronan (HA) is an extracellular matrix component that is secreted by fibroblasts and also works in inhibiting cell division. In a research article by Dr. Tian et al., They propose a mechanism for the cancer resistance exhibited by naked mole rats. They observed that in naked mole rats, there are greater levels of high molecular weight HA excreted from their fibroblasts. This high molecular weight HA accumulates in the tissues of the naked mole rat because the enzyme that is responsible for degrading, hyaluronan synthase 2, has decreased activity. When experiments are performed to remove HA, by the two methods, knocking down HAS2 or causing the overexpression of the enzyme Hyal2 which is responsible for the degradation of HA, the cells of

In the present study the overall aim of this experimental series was to undertake the steps to successfully clone and express a specific fusion protein from the alga Chlamydomonas reinhardtii. The specific allocated RNA encoding gene that was extracted from Chlamydomonas reinhardtii named Ferrochelatase, will be expressed as a fusion protein into two individual strains of Escherichia coli. Once the gene Ferrochelatase was extracted from the Chlamydomonas reinhardtii mRNA using reverse transcriptase RT-PCR, this allowed the cDNA to become amplified. This cDNA was then cloned into a pET100/D-TOPO expression plasmid allowing the expression of Ferrochelatase to be expressed as a fusion protein in two strains of Escherichia coli named BL21 (DE3)

Zinc coordinates proteins with four different surroundings with different roles: catalytic, co-catalytic, structural and interface. In many proteins Zn2+ ions also serve to correct polypeptide folding. Furthermore, zinc is a cofactor in all six enzyme classes.

The mechanisms underlying melanoma invasion are presently poorly understood. The Hippo pathway (the Salvador-Warts-Hippo) is an evolutionarily conserved mechanism in charge of tissue and cell growth.42 Downstream in the Hippo pathway are YAP and TAZ effector proteins which are amplified in many cancers and promote epithelial-mesenchymal transition-a frequent hallmark of metastasis.43 In a model of skin reconstruct, YAP overexpression led to increased melanoma cell invasiveness, and YAP knockdown led to decreased metastasis potential.43 Thus, mutations in the Hippo pathway yield increased activation of YAP and TAZ, which promote metastasis regardless of BRAF mutation status in melanomas.42,44 Current research is examining verteprofin,44 a molecule that inhibits YAP function and whether or not it can successfully regulate Hippo effector functions and subsequently melanoma

Prostate cancer originates in the secretory epithelial compartment of the prostate. The major function of these cells is to secrete protein components of prostatic fluid such as MSMB. With progression to metastasis, cells proliferate faster and lose their differentiated secretory phenotype. Expression of the mRNA of tumor suppressors and many cytoskeletal proteins decline. On the other hand, expression of genes associated with cell cycle [cyclins and cyclin dependent kinases] and protection from apoptosis [BIRC5] increase. As tumors grow, they become hypoxic due to characteristics of the tumor specific blood supply.

Cancer, medically called ‘tumorigenesis’ (Thaker, Lutgendorf, & Sood, 2007, p.430) occurs when cells in the body orient themselves for malignant growth. Such cells show ‘self-sufficiency in growth signals’, are ‘insensitive to anti-growth signals’ and have ‘limitless replicative potential’ (Thaker, Lutgendorf, & Sood, 2007, p.430). Once a particular set of cells become malignant, the malignancy can spread to other set of cells in different organs due to ‘crosstalk’ between the affected cells and their surrounding ‘tissues’ and ‘micro-environments’(Thaker, Lutgendorf, & Sood, 2007, p.430).

The uncontrollable spread of cancer is the principal event which leads to the death in individuals with cancer and it is the greatest barrier of developing cures for cancer. Metastasis is the progressive spread of malignant cancer cells from the primary tumour to secondary organ in distant sites and this potential is dependent on the specific microenvironment which support them to complete each step of the metastatic process (Poste & Fidler 1980). To understand the molecular basis of metastasis, investigators have now separated the complex and highly selective metastasis process into series of steps to try and solve the problems cause by

Cancer is listed as the second most common cause of death in western countries; particularly, in adults. Though it has a long antiquity, its prevalence and incidence today is pervasive and the war on cancer has not been promising. Malignant neoplasia is characterized by uncontrolled growth and the ability to metastasize or spread from the original site. Cancer results from mutations that promote cell proliferation and inhibit cell adhesion (metastasis). According to the National Cancer Institute (2016), “Cancer can also spread regionally,

IGF-I functions have been shown to be modulated through the interaction of the IGF system and ECM proteins such as fibronectin (FN), vitronectin (VN), collagen (I-IV), laminins, and integrins 15,16. We previously demonstrated that IGF-I interacts with the ECM protein VN through insulin-like growth factor binding proteins (IGFBPs) forming IGF-I:IGFBP:VN trimeric complexes 17. These complexes were shown to induce enhanced cell adhesion, migration and protein synthesis in human keratinocytes 18 and furthermore survival, and anti-apoptosis in both normal and as well as breast cancer cells 19-21. VN has been shown to anchor growth factors in the ECM which in turn enhances cell adhesion and migration of tumour cells 22 and in melanoma, it stimulates cell migration and invasion 23,24 through integrin receptors such as αvβ3 which is over-expressed in metastatic stages 25.

Local invasion represents the first step of the metastatic cascade and requires profound changes in cell-cell adhesion, cell-ECM interactions, proteolysis of ECM components and acquisition of migratory properties. Cancer cells can deploy different types of cell migration, including collective migration and single cell migration50. Predominantly, most epithelial cancer cells use collective cell migration, however some cancer cells may co-opt the biological program known as the EMT to migrate as single cells. EMT is a critical process for multiple aspects of normal embryonic development51. Since its discovery, there has been the realization that there are many parallels in the mechanisms that govern EMT in embryonic development and in cancer

Metastasis is an extraordinarily complex process. A sequence of steps is executed by a cancer cell to successfully form distant colonies at a secondary site. Typically, cancer cells escape from the primary tumour, break through basement membrane and invade surrounding tissues, encounter the circulatory system and/or lymphatics and travel to distant sites. Finally, they are arrested in small capillaries while being able to grow and form new tumours.[1] Tumour cells acting through this multistep metastatic cascade need to acquire some communicational skills and for that reason they seem to have numerous interactions with the extracellular matrix (ECM).

The ability of cancerous cells to migrate from their primary site and invade a secondary site is a hallmark of malignant progression. The invasive potential of a tumor hinges on the rigidity of its epithelial microenvironment in vivo, or the stiffness of the matrix on which the cells are grown in vitro. The serine/threonine protein kinase Cα (PKCα) promotes cell movement and contributes to tumor invasion, partially due to its activity on two newly discovered substrates, CEP4 and α6-tubulin. We set out to characterize the role of these PKCα substrates in the invasive phenotype of an aggressive metastatic mammary carcinoma cell line LM2-4175. We hypothesized that phosphorylation-resistant mutants of CEP4 and α6-tubulin would decrease the invasive phenotype. To test this, we transiently transfected LM2-4175 cells with either wild type or phosphorylation-resistant CEP4 or α6-tubulin and studied their migratory efficiency in transwells fitted with matrices of differing stiffness. In parallel, cells were treated with a PKC activator. As predicted, migration was potentiated by stiffer matrices under all conditions, with the exception of cells transfected with phosphorylation resistant CEP4 and α6-tubulin where the invasion in the softer matrix was greater. Furthering our understanding of the individual contributions of substrates of PKCα holds clinical promise, as it will permit the development of novel targeted therapeutics to curb cell migration and metastasis.

During tumour progression, angiogenesis is constantly being induced leading to the formation of vasculature from otherwise quiescent endothelial cells (5,15,16). Tumour induced angiogenesis leads to the production of aberrant, distorted and leaky vasculature (16) which promote tumour growth by increasing the blood supply in targeted areas. A study observed that cancer cells expressing the oncoproteins Bcl-2 or Bcl-xl had a less proliferative and less angiogenic effect when compared to the parental cancer cells which lacked the apoptotic inhibiting proteins and where more proliferative (13), linking angiogenesis promotion with more proliferative cancers. There are a number of matrix/non-matrix derived inhibitors and promoters of angiogenesis which control the stimulation or inhibition of angiogenesis. It has been shown that overexpression of certain endogenous inhibitors suppresses angiogenesis and limiting tumour growth. In certain cases, not only is angiogenesis effected but carcinoma cell migration is decreased showing a direct anti-tumour effect (27).