Small interfering RNAs targeting cyclin D1 and cyclin D2 enhance the cytotoxicity of chemotherapeutic agents in mantle cell lymphoma cell lines

Abstract

Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40-60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC(50) (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.

Figure 1. Cyclin D1 protein expression of Non-Hodgkin’s Lymphoma cell lines

Western blot analysis was performed on 8 cell lines derived from Mantle Cell Lymphoma (Jeko-1, Granta-519, JVM-2, Rec-1 and Z138), Burkitt’s Lymphoma (Raji, Daudi) and Diffuse Large B-cell Lymphoma (SUDHL-4, SUDHL-6).

Figure 2. Downregulation of Cyclin D1 in Jeko-1 (C,D) and Granta-519 (A,B) cells with four siRNAs (CCND1 224, CCND1 391, CCND1 778 and CCND1 trunc)

(A) Cyclin D1 mRNA levels of Granta-519 cells transfected with siRNAs targeting Cyclin D1. The mRNA levels were examined by real-time PCR and calculated relative to internal control RPLP0. (B) Cyclin D1 protein levels of Granta-519 cells transfected with four siRNAs. Protein levels were determined by Western blot analysis using α-tubulin as internal control. (C) Relative Cyclin D1 mRNA levels of Jeko-1 cells transfected with four different siRNAs. (D) Relative Cyclin D1 protein levels of Jeko-1 transfected cells.

Figure 3. Cyclin D2 upregulation after treatment with Cyclin D1 targeting siRNAs (CCND1 224, CCND1 391, CCND1 778, CCND1 trunc) in Granta-519 (A) and Jeko-1 (B)

Relative Cyclin D2 mRNA determined by real time PCR after treatment with 4 siRNAs targeting Cyclin D1. RPLP0 was employed as internal control.

Figure 4. Effect of siRNAs targeting CCND1 and D2 in combination (CCND1 778, CCND1 778+D2, CCND1 224+D2, siD1/D2) on Cyclin D1 and Cyclin D2 expression in Granta-519 cells

(A) Relative mRNA levels of Cyclin D1 and Cyclin D2 48h post transfection with CCND1 778 alone, CCND1 778 in combination with D2, CCND1 224 in combination with D2 siRNA and dual-targeting siD1/D2. RPLP0 was employed as internal control. (B) Cyclin D1 protein levels were detected with Western blotting 48h post transfection. Relative Cyclin D1 levels were calculated with α-tubulin as loading control.

Figure 5. Effect of siRNAs targeting CCND1 and D2 in combination (CCND1 778, CCND1 778+D2, CCND1 224+D2, siD1/D2) on Cyclin D1 and Cyclin D2 expression in Jeko-1 cells

(A) Relative mRNA levels of Cyclin D1 and Cyclin D2 48h post transfection with CCND1 778 alone, CCND1 778 in combination with D2, CCND1 224 in combination with D2 siRNA and dual-targeting siD1/D2. RPLP0 was employed as internal control. (B) Cyclin D1 protein levels were detected with Western blotting 48h post transfection. Relative Cyclin D1 levels were calculated with α-tubulin as loading control.