| Objective:Cytohesins are guanine nucleotide exchange factors (GEFs) for ADP ribosylation factors (ARFs) that belong to the family of small Ras-like GTPases. It is report that Cytohesins as cytoplasmic ErbB receptor activators in lung cancer. Here, we report the effection and mechanism of Cytohesins in colorectal cancer cells proliferation.Methods:We detected the four homologous members (cyhesin-1, Cytohesin-2/ARNO, Cytohesin-3/Grp-1, Cytohesin-4) of Cytohesins in colorectal cancer cell lines HT29, SW620, SW480and HCT116by RT-PCR and Immunofluorescence. After finding ARNO is major member of Cytohesins in colorectal cancer cell lines, we detected ARNO, pEGFR and pIGF-IR in colorectal cancer tissues compared normal tissue by immunohistory. We investigated the impact of Cytohesins in EGFR and IGF-IR pathway by making use of SecinH3and ARNO-siRNA. In this assay we detected EGFR pathway including:ARNO, p-EGFR, pIRS1, pShc and pERK1/2, and also detected ARNO, pIGF-IR, pIRS and pAKT for IGF-IR pathway. In a cellular context, we block the Cytohesins in HT29, HCT116cell lines. The proliferate activity and migration of colorectal cancer cell line (HT29) blocked by SecinH3or transfected with ARNO-siRNA were detected by MTT and Transwell assay. The cell cycles of HT29were detected by Flow Cytometry. The epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor (IGF-IR) associated signal pathways were detected by Western blotting. Tumors were generated by s. c. injections of HT29cells into nu/nu athymic male mice. After tumor establishment, mice were treated by daily i.p. SecinH3. During the treatment, we measured the diameter of tumor, and then immunohistochemical strained tumors for Ki-67to detect the proliferation inhibition.Result:All Cytohesins of four members were detected in colorectal cancer lines by RT-PCR and Immunofluorescence in those tumor cells, and Cytohesin-2/ARNO was detected the highest expressed one. In immunohistory assay, ARNO, pEGFR and pIGF-IR in colorectal cancer tissues expressed higher than normal tissues. When we blocked Cytohesins by SecinH3or ARNO-siRNA, we found the downstreams of EGFR and IGF-IR signaling reduced, including:ARNO, p-EGFR, pIRS1, pShc and pERK1/2in EGFR pathway and ARNO, pIGF-IR, pIRS and pAKT in IGF-IR pathway. The proliferate activity of HT29cells blocked by SecinH3and transfected with ARNO-siRNA were significantly lower than that of cells taken by DMSO or transfected with negative siRNA at72h and48h. SecinH3and ARNO-siRNA could stop HT29in G1date. The HT29tumor xenografts were generated in nude mice, we inject SecinH3daily. We found it was obviously inhibited after nine days of secinH3injection. Finally we fund the proliferation reduced in cancer cell of mice tumor by detection of Ki67.Conclusion:Cytohesins higher expressed in colorectal cancer than normal colorectal tissues, play an important role in colorectal cancer proliferation, migration and growth. The mechanism of Cytohesins in colorectal cancer cells is relative with activation of IGF-IR and EGFR signaling. Cytohesins might serve as a new molecular target therapy for the treatment of colorectal cancer. |
