Introduction

   Although the modern medical technology has been developed remarkably, the survival rate of squamous cell carcinoma (SCC) has not been improved that much. SCC is a highly invasive cancer, and has high risk of regional and distant metastasis. Tissue microenvironments are thought to play an important role in the characteristics of SCC, highly invasive and highly metastatic.¹⁾ Basement membrane zone (BMZ) is associated with secretion, assembly and remodeling of extracellular matrix proteins into an organized structure.²⁾ BMZ has functions as follows; first, a scaffold on which epithelial cells and tissues can differentiate, divide, migrate, and develop, second, a scaffold on which epithelial cells can invade and metastasize, third, whether cells will proliferate, growth arrest, or undergo programmed cell death, forth, whether cells will migrate or remain stationary.³⁾ BMZ proteins produced by carcinoma, are required for tumor growth and cellular or tissue invasion. Intramolecular BMZ associations are crucial in supporting carcinoma development. BMZs have traditionally been viewed as protective structures to defend against cancer spread.⁴⁾ Recent study, however, have shown that the roles of the BMZ and extracellular matrix in cancer invasion and tumor development are more complex. Tumor-associated enzymes of the BMZ, prerequisite to invasion and growth, have significant effect on modifying tumor-derived BMZ.⁵⁾

Structure and Function

   Laminins are large extracelluar glycoproteins, and important component of all BMZs. Laminins are Heterotrimers containing α, β, γ chains. Five α chains, three β chains and three γ chains have been identified.⁶⁾ Laminins typically form a cross-shaped structure, when viewed by rotary shadowing electron microscopy (Fig. 1).⁷⁾ Laminin 332, is a member of laminin family. Laminin 332 was formerly termed laminin 5, kainin, nicelin, ladsin and epiligrin.⁵⁾ Laminin 332 is one of 16 different laminin isoforms. Laminin 332 is comprised of α3, β3 and γ2 chains. The main function of Laminin 332 in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external disruptive forces.⁸⁾ 

Role and Expression in Tumors

   Laminin 332 performs primarily an adhesive function in mature normal tissues, but seems to have a more complex role in cell migration and tumor invasion.⁵⁾ Laminin 332 is known to stimulate the migration of various cells including carcinoma cells.^9,10,11) Laminin 332 seems to be associated with squamous cell carcinoma, prostate cancers, melanoma, colon cancer, breast cancer, pancrease cancer and lung cancer.^{5,12,13,14,15,16,17,18,19,20)} Laminin 332 expression is noted to accumulate at the interface of the tumor with the surrounding stroma or in the cytoplasm of cancer cells (Fig. 2). Besides a few exceptions, basal cell carcinoma, advanced breast cancer and prostate cancer, tumors derived from tissues that normally express laminin 332 show high levels of laminin 332 expression, whereas tumors derived from tissues that do not normally express laminin 332 do not express laminin 332.⁵⁾ Laminins act as major BMZ ligands for epithelial and carcinoma cells, supporting spreading, attachment, migration and invasion.²⁾ One of the unique functions of laminin 332 in epidermal cells is its ability to interact with two major epithelial integrin receptors, α3β1 and α6β4. The bindings of these two separate structures promote two different pathways (Table 1).²¹⁾ Laminin 332 expression has been shown to correlate well with tumor invasiveness and poor patient's prognosis.^{22,23,24,25,26)} The correlation seems to be due to two roles of Laminin 332; first, Laminin 332 is known to stimulate the migration of various cells including carcinoma cells, second, Laminin 332 modulates extracellular proteolytic processing. The interaction of laminin 332 with focal adhesions (α3β1 integrin) and SACs (α6β4 integrin) is known to be involved in the stimulating the various cells. The γ2 chain in domain III near the junction with domain IV, and the α3 chain in the G3 domain near the junction with G4 are shown to be involved extracellular proteolytic activity.^5,10,27) A specific antibody against Laminin 332 such as G45 antibody treatment was reported to induce SCC tumor apoptosis, decreased SCC tumor proliferation, and markedly impaired human SCC tumorigenesis in vivo without affecting normal tissue adhesion.²⁸⁾ 

Purification Process

   Conditioned medium is harvested from 184A1 cell line culture. A reactive blue column is prepared with 20mL reactive blue from Sigma-Aldrich^®. Collected conditioned medium is mixed with proteinase inhibitor cocktail from Sigma-Aldrich^®. Conditioned medium is filtered to remove cell debris and precipitate. Filtered conditioned medium is gone through a reactive blue column (Fig. 3). After the conditioned medium has gone through the reactive blue column, the medium is divided into gradient fractions with a Buffer Gradient Apparatus. Optical density of each fraction is measured to verify the even gradient. Each fraction is incubated overnight with MCF7 cells for motility test. MCF cells with various fractions are stained with crystal violet, and high motility fractions are determined (Fig. 4). The high motility groups are centrifuged for concentrating protein with Centriplus YM-30, Millipore^® tubes. Concentrated protein is gone through a size exclusion column, Sephacryl S-400 HR, Sigma-Aldrich^®. The column is connected to fraction collector. Pro-tein bands are identified at about 105, 135, and 165 kD, three strands of Laminin 332 with a gel running. This identified sample is gone through a gelatin column, Sepharose, Sigma-Aldrich^® to remove other proteins such as fibronectin.

Conclusion

   Laminin 332 performs primarily an adhesive function in mature normal tissues, but seems to have a more complex role in cell migration and tumor invasion. Laminin 332 seems to act as a potent driving factor to promote invasion and tumorigenesis. As the laminin-associated interactions take place extracellularly, they may be particularly amenable to antibody intervention as a mode of cancer therapy. Inhibitory therapy for specific site of the Laminin 332 is thought to be a promising method for anticancer therapy of SCC.

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