Abstract
Multiple myeloma is the second most common hematological malignancy and remains incurable. Glucocorticoids (GC) are among the most commonly used therapy for myeloma by inducing growth arrest and apoptosis in myeloma cells. While GC treatments are usually effective at first, all patients will eventually develop resistance. Understanding the mechanism of GC resistance is an important step towards developing new therapy to overcome this significant clinical problem. In order to study the mechanism underlying the GC resistance phenotype in myeloma, we compared the gene expression profiles of the clinically relevant myeloma clones derived from the same patient (MM.1) which are either sensitive or resistant to GC-induced apoptosis and growth inhibition. By cDNA microarray analysis, we identified NALP7 and NALP11 among the few genes that showed most significant differential levels of expression between the sensitive and resistant myeloma lines. Both genes belong to the NALP (NATCH-, LRR- and pyrin domain-containing proteins) family of proteins which have been implicated in the innate immune response. However, their function and regulation in myeloma has never been explored. Using a two-step quantitative real-time RT-PCR analysis, we have demonstrated that the NALP7 expression was significantly reduced whereas the NALP11 expression was markedly increased in GC-resistant MM.1 cells compared to the GC-sensitive MM.1 cells. We then investigated the biological significance of NALP gene differential expression in myeloma GC-resistance. To test whether suppressing NALP7 expression would lead to GC-resistance, we transfected the sensitive MM.1 cells with NALP-7 siRNA which effectively knocked down NALP7 mRNA level by over 80%. The knock-down of NALP-7, however, did not appear to affect the GC sensitivity of the transfected myeloma cells as dexamethasone (dex) treatment effectively caused growth arrest and apoptosis in transfected cells as in the control cells. We have also tested whether suppressing NALP11 expression would overcome the resistance to GC treatment by transfecting the GC-resistant MM.1 cells with NALP11 siRNA. While NALP11 expression was effectively knocked down to less than 20%, the transfected cells remain resistant to GC treatment as in the control cells, suggesting that NALP11 expression is not required to maintain the GC-resistant phenotype. We further investigated the upstream signaling cascades influencing NALP7 and NALP11 expression. Interestingly, dex treatment suppressed the expression of both NALP7 and NALP11 genes in the GC-sensitive MM.1 cells in a time- and dose-dependent manner. Co-treatment with glucocorticoid receptor (GR) antagonist RU486 blocked the down-regulation of NALP7 and NALP11 by dex, suggesting that a functional GR is required to mediate this action. In GC-resistant MM.1 cells which have much reduced levels of wild type GR, NALP7 and NALP11 mRNA levels were not affected by dex treatment, suggesting further that the suppression of NALP7 by GC requires sufficient levels of GR. Our studies demonstrated that the differential expression of two NALP family genes, NALP7 and NALP11, correlates with GC sensitivity in myeloma, and that both genes are regulated by glucocorticoid signaling. The biological significance of their differential regulation in myeloma remains to be determined.
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