Role Of ADP-ribosylation Factor 1 (ARF1) In Cell Adhesion Of Colorectal Cell Lines In Vitro

BACKGROUND & OBJECTIVE: Tumor cells' ability to adhere to other tumor cells, host cells or EMC (extracellular matrix), influences its invasiveness and metastasis. Adhesion of the tumor cells plays a dual role in invasiveness: on one hand, the tumor cells have to get rid of local adhesion, in this way, adhesion can inhibit invasiveness. On the other hand, the cells have to draw assistance from the adhesion for moving, because they gain pull strength from a series of the adhesion and losing adhesion. If the cell adhesion is too firmly, the cells cannot separate themselves from the primary focus and move. So the procedure of metastasis and invasiveness is a procedure of adhesion and losing adhesion. There are two kinds of cell adhesion: ?cell-cell adhesion; ?cell-matrix adhesion. Former was divided into two categories according to the cell type taking part in adhesion. One is homotypic adhesion, which is among the same kind of cells; the other is heterotypic adhesion, which is among over two kinds of cells. The weaker the tumor cells' homotypic adhesion is, and the stronger their cell-matrix adhesion is, then the stronger their ability of metastasis is. To discover the regularity of the colorectal cancer cells adhesion, and further to disclose the molecular biological mechanism of their invasiveness and metastasis, three colorectal cancer cell lines--LoVo, SW480, LST-R1(laterally spreading tumor-rectum 1), were selected to study. They were derived from different pathological tissue resource: LoVo cells were established from a fragment of a metastatic tumor nodule in the left supraclavicular region; SW480 cells were established from a primary adenocarcinoma of the colon, LST-R1 cells were established by our institute, from a rectal LST (laterally spreading tumor), which still grows horizontally along with the surface of mucosa instead of invading submucosa vertically, when its diameter is over 10mm.MATERIAL & METHODS: We screened the differentially expressed genes among the rectal LST cell strain and the other two colorectal cell lines, LoVo and SW480 by cDNA microarray. Then, we selected ARF1 (ADP-ribosylation factor 1) that is overexpressed in LST from the microarray result and confirmed its overexpression through RT-PCR (Reverse transcription polymerase chain reaction) and Western blotting at mRNA and protein expression levels. Immunocytochemistry (ICH) was use to detect the distribution of target protein in the cells. At last, the protein expression of ARF1 was changed by RNAi (RNA interference) and transfecting the target gene into the cells. Then, we observed the effect of ARF1 on the cell adhesion of LST-R1.RESULTS: Fifty-eight genes were found up-regulated and thirty-nine genes down-regulated in human expression profile cDNA microarray in the CLST cells compared to LoVo and SW480 cells. As far as degree of difference is concerned, difference between LoVo and LST-R1 is more significant than that between SW480 and LST-R1 since the former multiple is bigger than the latter, and the scatter graph of couple sample can tell the difference between them directly. As far as the differential genes classification is concerned, quantity of four kinds of differential genes—cell signal transduction, metabolism, development, and differentiation, is much more than others. Next, we chose ARFl to study. Although differential degree of it among three cell line is medium, it has three kinds of function including signal transduction, development, and differentiation, which belong to the major classification of differential genes. Furthermore, its function is closed to cell adhesion. ARFl that is overexpressed in the LST-R1 at the mRNA level by RT-PCR displayed higher protein level in LoVo but not in SW480 cells by Western blotting. ICH showed that ARFl exists in cytoplasm of the three colorectal cancer cell lines.The ARFl RNAi expression vector was constructed successfully in this study. After transfecting the vector and siRNA (small interference RNA) into LST-R1 respectively, Western blotting confirms that protein expression of ARFl was interfered successfully. Furthermore, we established the ARFl's expression vector, pEGFP-cl/ARFl, which coexpresses enhanced green fluorescent protein. Therefore, distribution of target protein in cells can be observed after transfecting it into the cells, which coincide with that of ICH. That is ARFl localized in cytoplasm, especially more around nuclear. Although ARFl RNAi expression vector or ARFl siRNA was transfected into LST-R1, its homotypic adhesion was weakened, whereas cell-matrix adhesion was enhanced. However, after transfecting pEGFP-cl/ARFl into it, its both homotypic adhesion and cell-matrix adhesion were enhanced.CONCLUSIONS: LST-R1 has specific gene expression profile compared to LoVo and SW480 cells. There are ninety-nine genes was differentially expressed in LST-R1 versus them through cDNA microarray analyses. And fifty-eight genes were found up-regulated and thirty-nine genes down-regulated in LST-R1 cells compared to LoVo and SW480 cells.ARFl is overexpressed in LST-R1 and mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation. Focal adhesions are the most important bridge linking cells with EMC, and stress fiber is closed to mobility. Therefore, ARFl influences mobility and adhesion of cells indirectly. In this study, the result of RNAi indicates that: Low expression of ARFl makes homotypic adhesion weaken, and cell-matrix adhesion enhanced. However, increase of ARFl expression makes both kinds of adhesion enhanced. Change of cell-matrix adhesion seems contradictory to the result of RNAi. Perhaps, ARFl overexpressed too high and for too long time. Then, there might be an adaptable mechanism that is unknown so far, occurred in the cells.The characteristic, that LST grows horizontally along with the surface of mucosa, maybe for itsstrong homotypic adhesion and weak cell-matrix adhesion. Then, LST will not invade submucosa early. Therefore, the fact that ARF1 was up-regulated in LST-R1 cells, leads to LST's homotypic adhesion strong and cell-matrix adhesion weak and change its direction of mobility. Then, LST is only able to grow horizontally along with the surface of mucosa early.