Limits...
A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells.

Dil N, Banerjee AG - Head Neck Oncol (2011)

Bottom Line: This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively.Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines.Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Oral Biology, University of Manitoba, Health Sciences Center, Winnipeg, Canada. dil@cc.umanitoba.ca

ABSTRACT

Background: Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression.

Materials and methods: We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines.

Results: More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia.

Conclusions: Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.

Show MeSH

Related in: MedlinePlus

Decorin silencing does not affect DOK or SCC-25 cell growth/proliferation. WT, control and decorin silenced DOK and SCC-25 cells were cultured for 24 h. During the last hour of culture, 20 μl of CellTiter 96® Aqueous One Solution Reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) was added to the media (100 μl per well), and color changes were recorded by absorbance at 490 nm. Data are presented as mean ± SE of three replicates of one representative experiment of three.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198745&req=5

Figure 2: Decorin silencing does not affect DOK or SCC-25 cell growth/proliferation. WT, control and decorin silenced DOK and SCC-25 cells were cultured for 24 h. During the last hour of culture, 20 μl of CellTiter 96® Aqueous One Solution Reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) was added to the media (100 μl per well), and color changes were recorded by absorbance at 490 nm. Data are presented as mean ± SE of three replicates of one representative experiment of three.

Mentions: To evaluate the effect of aberrantly expressed nuclear decorin on the cellular proliferation rates of dysplastic and malignant oral epithelial cells, DOK and SCC-25 WT cells, DCN-shRNA transfectants and ctrl-shRNA transfectants were allowed to grow in culture for 24, 48 and 72 h and proliferation was assessed by MTS assay. Compared with WT or control-shRNA cells, decorin silenced DOK and SCC-25 cells did not show any change in cell proliferation rates at 24 hrs (Figure 2). Similar results were obtained at 48 and 72 h time points (data not shown). This might be due to sequestration of decorin in the nucleus and consequent inability to interact with membrane receptors to which extracellular decorin is known to bind to effect tumor growth in other cancers.


A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells.

Dil N, Banerjee AG - Head Neck Oncol (2011)

Decorin silencing does not affect DOK or SCC-25 cell growth/proliferation. WT, control and decorin silenced DOK and SCC-25 cells were cultured for 24 h. During the last hour of culture, 20 μl of CellTiter 96® Aqueous One Solution Reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) was added to the media (100 μl per well), and color changes were recorded by absorbance at 490 nm. Data are presented as mean ± SE of three replicates of one representative experiment of three.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198745&req=5

Figure 2: Decorin silencing does not affect DOK or SCC-25 cell growth/proliferation. WT, control and decorin silenced DOK and SCC-25 cells were cultured for 24 h. During the last hour of culture, 20 μl of CellTiter 96® Aqueous One Solution Reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) was added to the media (100 μl per well), and color changes were recorded by absorbance at 490 nm. Data are presented as mean ± SE of three replicates of one representative experiment of three.
Mentions: To evaluate the effect of aberrantly expressed nuclear decorin on the cellular proliferation rates of dysplastic and malignant oral epithelial cells, DOK and SCC-25 WT cells, DCN-shRNA transfectants and ctrl-shRNA transfectants were allowed to grow in culture for 24, 48 and 72 h and proliferation was assessed by MTS assay. Compared with WT or control-shRNA cells, decorin silenced DOK and SCC-25 cells did not show any change in cell proliferation rates at 24 hrs (Figure 2). Similar results were obtained at 48 and 72 h time points (data not shown). This might be due to sequestration of decorin in the nucleus and consequent inability to interact with membrane receptors to which extracellular decorin is known to bind to effect tumor growth in other cancers.

Bottom Line: This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively.Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines.Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Oral Biology, University of Manitoba, Health Sciences Center, Winnipeg, Canada. dil@cc.umanitoba.ca

ABSTRACT

Background: Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression.

Materials and methods: We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines.

Results: More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia.

Conclusions: Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.

Show MeSH
Related in: MedlinePlus