Simultaneous downregulation of uPAR and MMP-9 induces apoptosis via upregulation of MAP4K and JUN and retards nuclear localization of CD44ICD and NF   κ   B in human glioma xenograft cells Free

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Glioblastoma multiforme is a highly malignant, primary central nervous system neoplasm that is extremely refractory to therapy. Glioblastoma are resistant to treatment due to the tendency of the tumor cells to invade normal brain tissue. We have previously demonstrated that the simultaneous downregulation of uPAR and MMP-9 resulted in the regression of pre-established intracranial tumors in nude mice. In this study, we have attempted to further evaluate the molecular mechanisms involved in glioma tumor regression via the simultaneous downregulation of uPAR and MMP-9. The results from the RT-PCR real time arrays show that the simultaneous downregulation of uPAR and MMP-9 caused a 23-fold upregulation of MAP4K, a 5-fold increase in JUN, and a 5-fold decrease in CDKN1A, which preceded caspase 9-mediated apoptosis. To determine the involvement of CD44 in uPAR and MMP-9 downregulated cells, CD44 levels were measured in cytoplasmic and nuclear extracts; these analyses indicated that the levels of CD44ICD in the nucleus were reduced when compared to controls. CD44 has been shown to be involved in multiple functions and is also suspected to have transcriptional activity. From our Western blots of nuclear and extracellular fractions of CD44 under various treatment conditions using antibody for total CD44, we were able to show localization of CD44ICD in the nucleus. We were also able to detect CD44 in the conditioned media indicating its cleavage by various proteases such as MT1-MMP and MMP-9. Total CD44 levels decreased in uPAR and MMP-9 downregulated cells, and the CD44ICD domain in the nucleus was almost undetectable. We also observed that caspase 9 activation was initiated and accompanied the dephosphorylation of ERK1/2, and nuclear and cytoplasmic levels of NFκB p65 and 50 were decreased. Mobility shift assay with oligos specific for NFκB further confirmed NFκB downregulation. The real time RT-PCR array results showed that MEK levels did not change and the levels of MAP3K2 decreased, whereas the levels of MAP4K1 levels increased, thereby indicating the activation of the JUN pathway. Previous studies have speculated that MAP4K1 may play a role in response to environmental stress. It is also speculated that the decrease in the kinase activity of these molecules may be related to cell surface components associated with the target molecules uPAR and MMP-9. Taken together, it is evident that glioma cells, which overexpress uPAR and MMP-9, have the potential to undergo apoptosis upon the downregulation of uPAR and MMP-9. Hence, the simultaneous targeting of uPAR and MMP-9 holds promise for glioma therapy.