Glycosylation and Melanoma: Materials and Methods, RNA Extraction from Melanoma Cells (Essay Sample)

Melanoma
Name
Institution
Abstract
Melanoma is considered to be among the most aggressive tumors that affect humans; itemanates from the melanin-producing melanocytes. Since there is nocomplete operative therapy that can be administered at the high-stage melanoma, patients suffering from melanoma can only recover from the tumor when it is still at the early-stage through surgery where the thin layer of early-stage melanoma is removed. Hence, cancer biology forms one of the primary objectives of melanoma research that can assist in the development or enhancement of diagnostic techniques that quickly screen or detect early melanocytic lesions; in the process patients that are suffering from melanoma can have an opportunity survive or live longer. There is substantial evidence of aberrant glycosylation when melanoma advance to the higher stages leading to altered glycosylation of mucins, partial synthesis of glycans, and an increase of β1–6 GlcNAc branched N-glycans. N-glycosylation of proteins occurs in the Golgi apparatus, and endoplasmic reticulum; it is a complicated processthat involves the regulation of various enzymes. The GnT-V and glycosyltransferases GnT-III are considered to be part of the chief enzymes that are responsible for adding GlcNAc (N-acetylglucosamine) to N-glycan acceptors in a medial-Golgi compartment. During the alteration of malignant, there is an increase of β1, 6 branching of N-linked oligosaccharides produced due to the activityof GnT-V bondedon ahighly expressed MGAT-5 (GnT-V gene). The study aims at confirming the presence of MGAT-5a mRNA Sequence in MM253 antisense (AS) melanoma cells, which were transfected with human MGAT-5 cDNA, as well as to determine if the MM253 antisense cells are expressing an antisense sequence and the cells surface proteins. Two techniques were performed to investigate the expression of GnT-V, RNA analysis, such as RT-PCR, and protein analysis includes western blotting and flow cytometry.
Introduction
Melanoma
Melanoma is considered to be among the most aggressive tumours that affect humans; melanoma comes from melanocytes cells associated with the formation of melanin (Przybyło & Litynska, 2011).Since the majority of the cells are located in the skin, the majority of the melanoma cases usually emerge in the skin; that is, at least 95 percent of the tumors are found in the skin. However, in rare occasions, further more melanoma can begin to advance to other areas of the body; for instance, the membrane that covers the brain, in the eye, and spinal cord, lymph nodes and the digestive tract (Przybyło & Litynska, 2011).The Primary skin melanoma also called cutaneous melanoma can advance from the precursor melanocytic nevi; however, over 50 percent of cases are thought to increase de novo in the absence of prior pigments lesions (Przybyło & Litynska, 2011).
Melanoma is classified as one of the highly metastatic cancers with over 90 percent of the patients that exhibit the early primary tumour (stage 1) can survive for five years and drastically reduces to thirteen percent once the distant metastatic lesions appear at the fourth stage. The discrepancies in the rates of survival among the primary and the metastatic melanoma, possibly reveals several variations that have occurred in the physiology of cancer cell; hence, making the disease progress from one stage to the next. Melanoma can be shown to be an excellent prototype of epithelial neoplasm since its evolvement is noted by the five, renowned stages of histology (Przybyło & Litynska, 2011). In this respect, malignant melanoma is identified to be among the frequently studied tumours constituting of nearly five thousand free cell line strains that are availed for study.
Since the development of melanoma involves various stages, the presence of cells at different stages of the disease progression offers an opportunity to make multiple accounts on the molecular events that complement the gain of metastatic potential for the abnormal cells (Paweł Link). Hence, cancer biology forms one of the primary objectives of melanoma research that can assist in the development or enhancement of diagnostic techniques to quickly screen or detect early melanocytic lesions; in the process patients that are suffering from melanoma can have an opportunity survive or live longer (Przybyło & Litynska, 2011).
Glycosylation and Melanoma
One of the various molecular changes being linked to the neoplastic process and medicalfeatures for cutaneous melanoma is glycosylation; however, it is still undergoing an intensive investigation. Glycosylation is among the rich post-translational alterations of proteins; it is estimated that a half of all the proteins are glycosylated. Glycosylation is a process that is involved in various pathological and physiological events comprising of the protein stability, and folding, signal transduction, metastasis, migration, cell growth, tumor invasion, and differentiation (Przybyło & Litynska, 2011).
There is substantial evidence of aberrant glycosylation when melanoma advance to the higher stages leading to altered glycosylation of mucins, partial synthesis of glycans and an upsurge of β1–6 GlcNAc-branched N-glycans. Some of the well-categorized hallmarks of N- and O- glycosylation are linked with malignancy. For instance, the transformed glycosylation of mucins, the partial synthesis of glycans and therise of β1–6 GlcNAc- branched N-glycans that presents vastly branched structures such as tetra- and tri- antennary.
N-glycosylation of proteins occurs in the Golgi apparatus, and endoplasmic reticulum; it is a complicated process that involves the regulation of various enzymes, auxiliary proteins and sugar donor transporters (Przybyło & Litynska, 2011). The N-glycans of developed glycoproteins exhibit a diverse structure when undergoing different stages of biosynthesis; glycosyltransferases located in the Golgi apparatus catalyse the process of biosynthesis. The GlcNAc is added to N-glycan acceptors in the presence of key enzymes such as the glycosyltransferases GnT-V and GnT-III become part of the vital enzymes that ensures that GlcNAc is added to N-glycan acceptors in the Golgi compartment. Theβ1-4 connection between β-mannose residue and GlcNAc inside the core of oligosaccharide is formed in the presence of N-acetylglucosaminyltransferase III (GnT-III). The synthesis of β1-6 bonds amid the sugar residue of the donor and α1, 6-linked mannose of the core of N-glycan occurs in the presence of N-acetylglucosaminyltransferase V (GnT-V) as a catalyst (Monika).
The synthesis of β1, 6-branched complex-type N-glycans occurs because of the activity of GnT-V, that enhances GlcNAc being added to α6-D-mannoside located at the central structure, to develop tetra/tri- antennary oligosaccharides (Przybyło & Litynska, 2011). During the alteration of malignant, there is an increase of β1, 6-branching of N-linked oligosaccharides being produced due to the action of GnT-V bonded on the highly expressed MGAT-5 (GnT-V gene) that in response is controlled by the pathway signals found in the tumour cells. In the oncogenic alteration, the Ets family that is composed of the Ets-1 transcription factor that also controls various molecules related to the metastasis, and cell invasion and the Mgat5 expression.
The significance of...