SiRNAs Against Cyclin D1/D2 Improves Cytotoxicity of Chemotherapy In Mantle Cell Lymphoma

Abstract

AbstractAbstract 1816 Introduction:Mantle cell lymphoma is an aggressive B cell neoplasm with a median survival of 3 years. Cyclin D1 overexpression is the genetic hallmark of MCL and regulates cell cycle progression. However, the significance of cyclin D1 in the pathogenesis and treatment of MCL still remains to be defined. The aim of this study is to determine whether down regulation of cyclin D1 with siRNA will lead to enhanced therapeutic effect of chemotherapy in MCL. We used siRNA technology in three well characterized MCL cell lines, and tested traditional chemotherapy agents (doxorubicin and etoposide) as a model system. Material and Methods:We designed three different siRNA targeting cyclin D1 (si-224, 391, 778), one siRNA against cyclin D2 (si-D2), and a dual targeting siRNA against both cyclin D1 and D2 (si-D1/D2). The siRNAs used were 27 mer asymetric duplexes with a 2nd 3′ overhang. Granta-519 cells were transfected by lipofection (Lipofectamin RNAimax, Invitrogen), Z-138 and Jeko-1 cells were transfected with electroporation (BioRad). Western Blot analysis and real time PCR were performed to examine the down regulatory efficiency of the siRNAs on cyclin D1 mRNA and protein. Chemotherapeutics doxorubicin and etoposide were tested for enhancement of cytotoxicity by siRNA. The effect on cell viability of cyclin D1 reduction in combination with chemotherapeutics was analyzed by MTS assay. Results:We achieved cyclin D1 mRNA and protein down regulation in all 3 MCL cell lines, although the efficiency of knockdown varied among the siRNAs and the cell lines of interests. (Table 1) Si-224 has the best activity in Granta-519 while si-778 has the best activity in Jeko-1.Table 1Granta-519 mRNAGranta-519 proteinJeko-1 mRNAJeko-1 proteinZ-138 mRNAZ-138 proteinsi-224−40%−40%−65%−40%−45%−20%si-391−35%−0%−70%−25%−25%−0%si-778−40%−25%−80%−70%−55%−25%We determined the cytotoxic effect of chemotherapy alone as well as in combination with siRNAs by MTS assays. The combination of chemotherapeutic with our siRNAs decreased the IC50 of both doxorubicine and etoposide. In Granta 519, si-224 decreased the IC 50 of doxorubicin by 32% and etoposide by 28%. In Jeko-1, si-778 decreased the IC 50 of doxorubicin by 49% but no effect on etoposide was seen. The magnitude of cyclin D1 down regulation seems to correlates with the percentages changes in IC 50.Klier et al previously reported that knockdown of cyclin D1 leads to an upregulation of cyclin D2 in MCL. Hence we mixed si-224 as well as si-778 targeting cyclin D1 with a si-D2 against cyclin D2 in combination with doxorubicine and etoposide in Granta-519. We also designed a dual-targeting siRNA against CCND1 and CCND2 (si-D1/D2). Targeting both cyclin D1 and D2 decreased the IC 50 of doxorubicin further than targeting cyclin D1 alone. Si224/D2 decreased the IC 50 of doxorubicin by 57% (si-224 alone 32%) and etoposide by 39% (si-224 alone 28%), and si778/D2 decreased the IC 50 of doxorubicine by 58% (si-778 alone 49%). The dual-targeting siRNA showed a decrease in IC 50 of doxorubicin by 45% and etoposide by 48%. Conclusions:Down regulation of cyclin D1 in MCL with siRNA improves the IC 50 of chemotherapeutic agents. Dual inhibition of both cyclin D1 and D2 further enhances the cytotoxic effect of doxorubicine and etoposide. Besides being a cell cycle regulator, cyclin D1 also seems to regulate chemosensitivity in MCL. Footnotes:This work was supported by grants from the Tower Cancer Research Foundation and Tim Nesvig Lymphoma Research Fund and Fellowship, Think Cure, Keck-foundation, SPORE. Disclosures:No relevant conflicts of interest to declare.