Abstract
BackgroundOral 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.MethodsWe 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.ResultsNuclear 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.ConclusionsTogether these results indicate that aberrantly expressed nuclear localized decorin strongly influences angiogenic potential of dysplastic and malignant oral epithelial cells.
Highlights
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%
Effect of nuclear decorin silencing on cytokines/Chemokines expression in dysplastic and malignant oral epithelia To evaluate the effect of aberrantly expressed nuclear decorin on the expression level of TGF-β, IL-10, and IL-8 simultaneously, dysplastic oral keratinocytes (DOK) and SCC-25 wild type (WT), decorin silenced and control cells were allowed to grow in culture for 24 h, RNA was extracted and reverse transcribed to cDNA and subjected to multiplex PCR
Nuclear decorin silencing down regulates IL-8 receptors in DOK and SCC-25 cells We determined the expression of IL-8 receptor (CXCR1&2) along with other chemokines stromal cell-derived factor (SDF-1) and their receptors (CXCR4) in a multiplex PCR system
Summary
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%. 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). Angiogenesis is a progressive physiological process during which specific mechanisms are induced to overcome the natural angiostatic state (in most adult tissues) of the vasculature leading to the angiogenic switch [5,6] Some proteases such as matrix metalloproteinase 9 (MMP-9) are known to induce this switch [7]. Augmented endogenous VEGF expression has been shown in various cancers and has been associated with poor prognosis and metastasis in oral squamous cell carcinoma [12] Another important mediator of angiogenesis is angiopoietin-1 (ANG-1). During the process of angiogenesis VEGF and angiopoietins function in concert, with VEGF acting early during vessel formation, and ANG-1 acting later during vessel remodelling, maturation and stabilization [13]
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