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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 Matrigelâ„¢ 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.

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Related in: MedlinePlus

Nuclear Decorin associates with EGFR. Nuclear lysates were extracted from DOK and SCC-25 cells and were immunoprecipitated (IP) with decorin antibody. Immunocomplexes were subjected to SDS-PAGE and were analysed by immunoblotting (IB) with anti-decorin and anti-EGFR antibodies.
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Figure 6: Nuclear Decorin associates with EGFR. Nuclear lysates were extracted from DOK and SCC-25 cells and were immunoprecipitated (IP) with decorin antibody. Immunocomplexes were subjected to SDS-PAGE and were analysed by immunoblotting (IB) with anti-decorin and anti-EGFR antibodies.

Mentions: We have previously shown that decorin is aberrantly expressed and localized to the nucleus in DOK and SCC-25. However, decorin is not known to carry nuclear localization signal. In an effort to identify possible binding partners of decorin in the nucleus of these dysplastic and malignant oral epithelial cells, we explored weather nuclear decorin physically associates with nuclear EGFR. Nuclear lysates were obtained from DOK and SCC-25 cells. Interestingly, immunoprecipitation with decorin antibody co-precipitated EGFR in both dysplastic (DOK) and malignant (SCC-25) oral epithelial cells (Figure 6). However, similar immunoprecpitation with decorin antibody did not co-precipitate either biglycan or LRRFIP2, the other two suspect NLS carrying proteins (data not shown). Our results suggest that nuclear localized decorin interacts with nuclear localized EGFR in these dysplastic and malignant oral epithelial cells. Intriguingly, EGFR (trans) activation has been shown to mediate IL-8 production in epithelial cells [28,29].


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)

Nuclear Decorin associates with EGFR. Nuclear lysates were extracted from DOK and SCC-25 cells and were immunoprecipitated (IP) with decorin antibody. Immunocomplexes were subjected to SDS-PAGE and were analysed by immunoblotting (IB) with anti-decorin and anti-EGFR antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Nuclear Decorin associates with EGFR. Nuclear lysates were extracted from DOK and SCC-25 cells and were immunoprecipitated (IP) with decorin antibody. Immunocomplexes were subjected to SDS-PAGE and were analysed by immunoblotting (IB) with anti-decorin and anti-EGFR antibodies.
Mentions: We have previously shown that decorin is aberrantly expressed and localized to the nucleus in DOK and SCC-25. However, decorin is not known to carry nuclear localization signal. In an effort to identify possible binding partners of decorin in the nucleus of these dysplastic and malignant oral epithelial cells, we explored weather nuclear decorin physically associates with nuclear EGFR. Nuclear lysates were obtained from DOK and SCC-25 cells. Interestingly, immunoprecipitation with decorin antibody co-precipitated EGFR in both dysplastic (DOK) and malignant (SCC-25) oral epithelial cells (Figure 6). However, similar immunoprecpitation with decorin antibody did not co-precipitate either biglycan or LRRFIP2, the other two suspect NLS carrying proteins (data not shown). Our results suggest that nuclear localized decorin interacts with nuclear localized EGFR in these dysplastic and malignant oral epithelial cells. Intriguingly, EGFR (trans) activation has been shown to mediate IL-8 production in epithelial cells [28,29].

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 Matrigelâ„¢ 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