S. Odorico1, R. Jaskula-Sztul1,2, A. Harrison1, S. Clarkson1, S. Golden1, H. Jin1,2, H. Chen1,2 1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Alabama,Surgery,Birmingham, Alabama, USA
Introduction: Carcinoids are neuroendocrine tumors that often metastasize and other than surgery, no curative options are available. We have shown that Thailandepsin-A (TDP-A), a novel histone deacetylase inhibitor, can induce apoptosis in carcinoid tumor cells in vitro and reduce tumor size in vivo. However, treatment with TDP-A alone achieved only cytostatic effect. The suppression of anti-apoptotic proteins with concomitant TDP-A treatment could induce cytotoxic effect of both drugs. Therefore, our goal was to investigate a potential synergistic effect on apoptosis in carcinoid cells in vitro with dual treatment of TDP-A and siRNA silencing of three different anti-apoptotic markers, cyclin D1, MCL1 and BCL2.
Method: siRNA of three anti-apoptotic oncogenes, cyclin D1, MCL1 and BCL2, were transfected into gastrointestinal carcinoid cells (BON) using Lipofectamine. Treatment with TDP-A 7 nM (previously tested IC50 value of the drug) occurred 4 hours following siRNA transfection. Cells were incubated for 48 hours before harvesting. qRT-PCR analysis was completed to assess the efficiency of siRNA silencing and flow cytometry was analyzed to determine the apoptotic induction.
Results: In flow cytometric analysis, we assessed pre-apoptotic and apoptotic percentages of total cells after 48 hours of treatment with either siRNA alone, TDP-A alone or siRNA and TDP-A. Figure 1A represents a summary of pre-apoptotic and apoptotic percentages of total cells observed in all samples. We observed a synergistic induction of apoptosis when MCL1 siRNA and TDP-A were applied concurrently. Figure 1B depicts flow cytometric images visualizing the synergistic induction of apoptosis. For qRT-PCR, we observed reduction in relative gene expression in all target genes following transfection of siRNA as compared to scrambled siRNA transfection.
Conclusion: While BCL2 and cyclin D1 siRNA did indeed knock down the expression of their target genes, no synergistic induction of apoptosis occurred with the treatment of both the siRNA and TDP-A. However, combined MCL1 siRNA and TDP-A treatment resulted in a synergistic induction of apoptosis in BON carcinoid cells. These results suggest selective gene silencing of MCL1 enhanced TDP-A induced apoptosis in carcinoid cells in vitro, and warrants further investigation into the relationship between MCL1 knockdown and concomitant treatment of TDP-A.
