Farnesyltransferase inhibitor R115777 inhibits cell growth and induces apoptosis in mantle cell lymphoma

Abstract

Introduction: The cytotoxic activity of the farnesyltranseferase inhibitor R115777 was evaluated in cell lines representative of mantle cell lymphoma (MCL).

Methods: Cell growth, proliferation, and apoptosis were analyzed in four human MCL cell lines (Granta, NCEB, REC, and UPN1) in presence of R115777, alone or in combination with vincristin, doxorubicin, bortezomib, cisplatin and cytarabine. Inhibition of farnesylation was determined by the appearance of prelamin A. The antitumor activity of R115777, administered p.o. at 100, 250 and 500 mg/kg, was determined in vivo in nude mice xenografted with UPN1 cells.

Results: R115777 inhibited the growth of MCL cell lines in vitro with inhibitory concentrations ranging between 2 and 15 nM. A fifty percent decrease of cell viability was observed at concentrations comprised between 0.08 and 17 microM. Apoptosis, evaluated by annexin V and activated caspase 3 staining, was induced in all cell lines, in 40 to 71% of the cells depending on the cell lines. In addition, R115777 significantly increased the cytotoxic effect of vincristine, doxorubicin, bortezomib, cisplatin and cytarabine (p=0.001, p=0.016, p=0.006, p=0.014 and p=0.007 respectively). Exposure of MCL cell lines to R115777 during 72 hours resulted in inhibition of protein farnesylation. R115777 administered p.o. twice daily for 8 consecutive days to mice bearing established s.c. UPN1 xenograft displayed cytostatic activity at the 500 mg/kg dosage.

Conclusion: We have demonstrated that inhibition of farnesyltransferase by R115777 was associated with growth inhibition and apoptosis of MCL cell lines in vitro and tumor xenograft stability in vivo.

Figure 1. Levels of both FNTA and FNTB mRNAs in MCL

Using quantitative real-time PCR, the relative amounts of FNTA and FNTB mRNAs were analysed in 39 MCL samples and MCL cell lines compared to that observed in normal B-cells. FNTA primers were 5′-CTT CCC TTT GCC TGT GTT GTA-3′ and 5′-GTA GCA GCA GCA CCC AAG GA-3′. The FNTA probe was 5′-AAG TGC ATC ACA CAG GTA TTG CTT TTT AAC AAG AAC-3′. FNTB primers were 5′-TGG ATG TGA GAA GCG CAT ACT G-3′ and 5′-TCA AAG AGG TCT GGA GTG ATG ATG-3′. The FNTB probe was 5′-TGC CTC CGT AGC CTC GCT GAC C-3′.

Figure 2. Effects of R115777 on lamin A farnesylation

Increases of unfarnesylated precursor of lamin A in MCL cells treated with R115777 at cytostatic concentrations (R) compared with cells treated with DMSO (C). “U” and “P”, unprocessed and processed proteins, respectively.

Figure 3. Caspase-3 activation and annexin V staining in MCL cell lines treated with R115777

Activation of caspase-3 was assessed by indirect immunofluorescence. A/percentage of cells stained with caspase-3 activated antibody in presence of DMSO (vehicle) or R115777 at cytotoxic concentrations. B/Percentage of cells stained with annexin V in presence of DMSO (vehicle) or R115777 in the 5 MCL cell lines analyzed.

Figure 4. Combination of vincristine, doxorubicin, bortezomib, cisplatine and cytarabine with R115777

Histograms represent the concentration of each drug needed to reduce NCEB cell viability by 50% when vincristine (VCR), doxorubicin (doxo), bortezomib, cisplatin (CDDP) and cytarabine (AraC) were used alone or in combination with R115777 (FTi).

Figure 5. Tumor growth curves of UPN1 human MCL xenografts

Mice were treated with either H₂O (NT), or with varying doses of R115777 administrated twice daily at 100, 250 or 500mg/kg for 8 consecutive days.