Dil Nyla, Banerjee Abhijit G
Departments of Medical Microbiology and Infectious Diseases, Winnipeg, MB, Canada.
Head Neck Oncol. 2012 Jun 8;4:11. doi: 10.1186/1758-3284-4-11.
Oral cancer accounts for roughly 3% of cancer cases in the world with about 350,000 newly reported cases annually and a 5-year survival rate of only 50%. Majority of oral cancers are squamous cell carcinomas that originate in the oral mucosal epithelial linings. We have previously shown that in human malignant squamous cells carcinoma (SCC-25) as well as in dysplastic oral keratinocytes (DOK), a small leucine-rich multifunctional proteoglycan decorin is aberrantly expressed and localized in the nucleus where it interacts with nuclear epidermal growth factor receptor (EGFR). Post-transcriptional silencing of nuclear decorin significantly reduced IL-8 and IL8-dependent migration and invasion in these dysplastic and malignant oral epithelia. The objective of this study was to further examine the effects of nuclear decorin silencing on angiogenesis and angiogenesis related mediators in this oral cancer progression cell line model.
We have used multiplex PCR, western blotting, and in vitro endothelial tube formation assay to study angiogenesis and related pathways in nuclear decorin silenced (stable knockdown) DOK and SCC-25 cells.
Nuclear decorin knockdown resulted in significant down regulation of IL-8 expression, however IL-10, and TGF-β expression was not affected in either DOK or SCC25 cells as measured by multiplex RT PCR. IL-8 receptor CXCR 1 and 2 expression was slightly lower in nuclear decorin silenced cells indicating a contributing mechanism in previously shown reduced IL-8 mediated migration and invasion phenotype in these cells. IL-8 is known to induce Matrix metalloproteinase 9 (MMP9) which not only plays a role in tumour migration and invasion but also induces angiogenic switch. We found MMP9 to be significantly reduced in nuclear decorin silenced dysplastic and malignant oral epithelia. Other potent angiogenic mediators, VEGF189 and ANG-1 were either significantly reduced or completely abrogated in these cells. Angiogenesis as measured by endothelial tube-like formations of HUVEC cells was reduced by almost 50 percent when HUVECs were incubated in the presence of conditioned medium form nuclear decorin silenced dysplastic and malignant cell lines as compared to respective controls.
Together these results indicate that aberrantly expressed nuclear localized decorin strongly influences angiogenic potential of dysplastic and malignant oral epithelial cells.
口腔癌约占全球癌症病例的3%，每年新报告病例约35万例，5年生存率仅为50%。大多数口腔癌是起源于口腔黏膜上皮的鳞状细胞癌。我们之前已经表明，在人类恶性鳞状细胞癌（SCC - 25）以及发育异常的口腔角质形成细胞（DOK）中，一种富含亮氨酸的小分子多功能蛋白聚糖核心蛋白聚糖异常表达并定位于细胞核，在细胞核中它与核表皮生长因子受体（EGFR）相互作用。核核心蛋白聚糖的转录后沉默显著降低了这些发育异常和恶性口腔上皮细胞中白细胞介素 - 8（IL - 8）以及IL - 8依赖性迁移和侵袭。本研究的目的是在这种口腔癌进展细胞系模型中进一步研究核核心蛋白聚糖沉默对血管生成及血管生成相关介质的影响。
我们使用多重PCR、蛋白质印迹法以及体外内皮管形成试验来研究核核心蛋白聚糖沉默（稳定敲低）的DOK和SCC - 25细胞中的血管生成及相关途径。
通过多重逆转录PCR检测，核核心蛋白聚糖敲低导致IL - 8表达显著下调，然而在DOK或SCC25细胞中，IL - 10和转化生长因子 - β（TGF - β）的表达均未受影响。在核核心蛋白聚糖沉默的细胞中，IL - 8受体CXCR 1和2表达略低，这表明了在之前所显示的这些细胞中IL - 8介导的迁移和侵袭表型降低的一种作用机制。已知IL - 8可诱导基质金属蛋白酶9（MMP9），其不仅在肿瘤迁移和侵袭中起作用，还可诱导血管生成开关。我们发现核核心蛋白聚糖沉默的发育异常和恶性口腔上皮细胞中MMP9显著降低。其他强效血管生成介质，血管内皮生长因子189（VEGF189）和血管生成素 - 1（ANG - 1）在这些细胞中要么显著降低要么完全缺失。与各自的对照相比，当人脐静脉内皮细胞（HUVEC）在核核心蛋白聚糖沉默的发育异常和恶性细胞系的条件培养基中孵育时，通过HUVEC细胞的内皮管样形成测量的血管生成减少了近50%。
这些结果共同表明，异常表达的核定位核心蛋白聚糖强烈影响发育异常和恶性口腔上皮细胞的血管生成潜能。
