Abstract
Abstract 4589
Objective:
BAG3, a member of BAG family, is shown to sustain cell survival and underlies resistance to chemotherapy in human cancer cells, through down-modulation of apoptosis. It can also enhance cell adhesion and migration to promote tumoral invasion. However, the role of BAG3 in human chronic lymphatic leukemia (CLL) remains unclear. Our study aims to discover the roles of BAG3 in the apoptosis and migration of primary CLL cells.
Method:
In our study, Peripheral blood mononuclear cells were freshly isolated from 18 previously untreated patients. The primary CLL cells were cultured for 6 hours, following, three siRNA (siRNA-1, siRNA-2 and siRNA-3) targeting with different regions of BAG3 mRNA and one non-targeting siRNA were transfected into CLL cells by using Lipofectamine 2000. 6 samples were collected for checking the transfection efficiency by real-time PCR and western blot after 24 or 48 hours. The other 6 samples were exposured with or without fludarabine 12 hours later after tansfection; with another 24 or 48 hours, both cells were harvested, stained with annexin V/PI,and then apoptosis were analyzed using a FACSCalibur flow cytometer.on the other hand, transwell assay were performed in another 6 samples 12 hours after tansfection. to analysis cell migration, the cells were collected and counted 24 hours later.
Result:
By using the siRNA technique, we successfully down-regulated the expression of BAG3 by 3 different siRNAs. BAG3 mRNA was decreased □‘6 folds (p < 0.05). In order to analyze the effect of BAG3 on CLL cell apoptosis, we used flow cytometry to measure cell apoptosis. Here, the apoptosis rates of CLL cells were increased □‘2 folds after 24 hours transfection by siRNA-2 and siRNA-3 (p < 0.05), and □‘2.5 folds after 48 hours transfection by BAG3 siRNA-1, siRNA-2 and siRNA-3 (p < 0.05). These indicate that BAG3 makes an anti-apoptotic, and knocking-down of it increases the cell apoptosis. To assess the role of BAG3 in the migration of CLL, we used transwell assay to measure cell migration. We found that the migration of CLL cells was decreased 28% by BAG3 siRNA-1 treatment (p < 0.05), 47% by siRNA-2 (p < 0.01) and 50% by siRNA-3 (p < 0.01), respectively. These data showed that BAG3 enhances the CLL migration, and down-regulation of it can inhibit cell migration. Furthermore, fludarabine and BAG3 siRNA were co-treated with CLL cells, to check whether knocking-down of BAG3 can reverse the CLL cells’ resistance against fludarabine. However, the apoptosis rates are not different between co-treated cells and fludarabine-treated cells, which indicated knocking-down of BAG3 may not reverse the fludarabine resistance, though it can promote cell apoptosis and inhibit cell migration.
Conclusion: Based on these observations, we conclude that BAG3 may be a potential therapeutic target of human CLL, and inhibition of BAG3 expression can induce cell apoptosis and inhibit cell migration.
Disclosures:
No relevant conflicts of interest to declare.
TWO DIFFERENT COMPLEMENT RECEPTORS ON HUMAN LYMPHOCYTES