Suppression Of Proliferation Of Laryngeal Squamous Cell Carcinoma By Stable Expression Of Anti-sense Perlecan CDNA

Part IExpression of Perlecan and basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor in Laryngeal Carcinoma Hep-2 Cells and Laryngeal Squamous Cell CarcinomaParagraph oneExpression of Perlecan and basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor in Laryngeal Carcinoma Hep-2 CellsAbstract Objective: To study the expression of perlecan and basic fibroblast growth factor (bFGF) and the four high affinity FGF receptors and vascular endothelial growth factor (VEGF) and both the Flt-1 and KDR high affinity VEGF receptors in human laryngeal carcinoma Hep-2 cells and its significance. Methods: In this study, we investigated the expression of perlecan and bFGF and the four high affinity FGF receptors and VEGF and both the Flt-1 and KDR high affinity VEGF receptors mRNA in human laryngeal carcinoma cells, Hep-2 cells, using techniques of semi-quantify RT-PCR, and investigated the expression of perlecan and bFGF and VEGF in the Hep-2 cells using techniques of immunohistochemistry. Results: It was showed that the expression level of perlecan and bFGF and VEGF mRNA was significantly increased in Hep-2 cells as compared with the normal cells, HaCaT cells (P < 0.01), and there was the high expression level of FGFR-1 and FGFR-2 and FGFR-4 and Flt-1 mRNA in the Hep-2 cells, but not FGFR-3 and KDR mRNA. Conclusion: These data raise the possibility that perlecan and bFGF and VEGF its receptors many play key roles in the growth, invasion and metastasis of human laryngeal carcinoma Hep-2 cells through either a paracrine or an autocrinemechanism.Paragraph twoExpression of Perlecan and basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor in Laryngeal Squamous Cell CarcinomaAbstract Objective: To study the expression of perlecan and basic fibroblast growth factor (bFGF) and the four high affinity FGF receptors and vascular endothelial growth factor (VEGF) and both the Flt-1 and KDR high affinity VEGF receptors in human laryngeal squamous cell carcinoma (LSCC) and its significance. Methods: In this study, we investigated the expression of perlecan and bFGF and the four high affinity FGF receptors and VEGF and both the Flt-1 and KDR high affinity VEGF receptors mRNA in LSCC, using techniques of semi-quantify RT-PCR, and investigated the expression of perlecan and bFGF and VEGF in the LSCC using techniques of immunohistochemistry. Results: It was showed that the expression level of perlecan and bFGF and VEGF mRNA was significantly increased in LSCC as compared with the normal human laryngeal mucous membrane (P < 0.01), and there was the high expression level of FGFR-1 and FGFR-2 and FGFR-4 and Flt-1 mRNA in the LSCC, but not FGFR-3 and KDR mRNA. The expression level of perlecan and bFGF and VEGF mRNA and protein were significantly increased in III and IV stage tissues of human LSCC as compared with the I and II stage tissues of LSCC (P < 0.01). Conclusion: These data raise the possibility that perlecan and bFGF and VEGF its receptors many play key roles in the growth, invasion and metastasis ofLSCC.Part IISuppression of Proliferation and Invasion of Hep-2 Cells by Stable Expression of Anti-Sense Perlecan cDNAAbstract Objective: To study the suppression of proliferation and invasion of laryngeal cancer Hep-2 cells by stable expression of anti-sense perlecan cDNA. Mehods: In this study, the plasmids of perlecan anti-sense cDNA (pAP) were transfected into laryngeal cancer Hep-2 cells by using cationic liposome (lipofectamine 2000). We investigated the expression of perlecan mRNA and protein in Hep-2 cells transfected with the pAP and with the vector alone, ph β Apr-neol using techniques of semi-quantify RT-PCR and western blot assay, and investigated the invasion of laryngeal cancer Hep-2 cells by stable expression of anti-sense perlecan cDNA using techniques of Transwell assay. The pAP and ph β Apr-neol transfected Hep-2 cells were grown in RPMI 1640 supplemented with low serum and basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in order to assay if perlecan affected proliferation in response to the growth factor using the metabolic MTT assay at 24 hour periods over 4 days. Results: It was showed that the expression of perlecan mRNA and protein were significantly reduced in pAP transfected Hep-2 cells as compared with the wild type cells and ph β Apr-neol transfected cells (P < 0.01). In the presence of 1 ng/ml of bFGF in low serum (0.1% FCS), or 20 ng/ml of VEGF in low serum (0.1% FCS), the pAP transfected cells showed a reduced proliferation rat (MTT assay) while the wild type cells and ph β Apr-neol transfected cells grew rapidly. The pAP transfected cells showed a significantly reduced invasion rat (Transwell assay) as compared with the wild type cells and ph β Apr-neol transfected cells (P < 0.01). Conclusion: The proliferation and invasion of Hep-2 cells could be inhibited significantly by perlecan anti-sense cDNA plasmids transfection. Part IIIGrowth inhibition effection of perlecan Anti-sense cDNA on human laryngeal carcinoma xnograft in nude miceAbstract Objective: To observe growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. Methods: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. Results: Volume of xnografts in the group transfected by the plasmids of perlecan anti-sense cDNA (pAP) were significant small as compared with other two groups made by the wild type cells and ph β Apr-neol cells (P < 0.01). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and ph β Apr-neol cells (P < 0.01). Conclusion: These data raise the possibility that perlecan anti-sense cDNA many play key roles in the growth of those xnografts in nude mice.