Abstract
BackgroundInvolvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.Methodology/Principal FindingsMMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Conclusions/SignificanceDownregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.
Highlights
Integrins are a family of adhesion molecules involved in interactions between the cell and the surrounding extracellular matrix (ECM)
In the present study, we investigated the effect of downregulating matrix metalloproteinases (MMPs)-9, urokinase-type plasminogen activator receptor (uPAR) and ctahepsin B using both single as well as MMP-9/uPAR and MMP-9/cathepsin B bicistronic shRNA plasmid constructs on the expression of integrins and on the migrating and invasive potential of glioma xenograft cells
Bands related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at mRNA and protein levels associated with various treatments in 4910 cells indicate that similar amounts of mRNA/protein were loaded in all treatment groups
Summary
Integrins are a family of adhesion molecules involved in interactions between the cell and the surrounding extracellular matrix (ECM). Through interactions with the ECM, integrins control many cellular processes that occur in the progression of diseases such as cancer. Integrin signaling regulates diverse functions in tumor cells, including migration, invasion, proliferation and survival. The expression of particular integrins correlates with increased disease progression and decreased patient survival [8]. Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential
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