Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells

Abstract

Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G(2)/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients.

Figure 1.

Synergism between R115777 and paclitaxel or docetaxel to inhibit MM cell proliferation. RPMI8226/S cells were treated with drugs for 72 hours before MTT assays. Results represent means and standard deviations of 4 independent experiments. (A,C) R115777 enhanced the cytotoxic effects of paclitaxel or docetaxel. (B,D) Isobologram based on data from panels A and C, showing synergism between R115777 and paclitaxel or docetaxel.

Figure 2.

R115777 synergizes with paclitaxel to induce apoptosis. RPMI8226/S cells were treated with paclitaxel (1 nM), or R115777 (50 nM), or both for 36 hours before TUNEL assays. (A) Representative pictures of TUNEL slides of cells with different drug treatments are shown. (B) Means and standard deviations of 3 independent experiments.

Figure 3.

R115777 synergizes with paclitaxel to induce cytochrome c (cyt C) and caspase-3. (A) R115777 (100 nM) synergized with paclitaxel (2 nM) in caspase-3 activation. RPMI8226/S cells were treated with drugs for 48 hours before caspase-3 and caspase-8 activities were assayed. Results represent means and standard deviation of 3 independent experiments. (B) R115777 (100 nM) synergized with paclitaxel (2 nM) in causing cytochrome c release. RPMI8226/S cells were treated with drugs for 72 hours before harvesting for Western blot analysis. (C) Effect of treatment of paclitaxel and R115777 on apoptotic and prosurvival Bcl2 family members. RPMI8226/S cells were treated with drugs at indicated concentrations for 72 hours before harvesting for Western blot analysis.

Figure 4.

R115777 synergizes with paclitaxel to cause mitotic arrest. RPMI8226/S cells were treated with drugs for 48 hours before being stained with PI and analyzed by flow cytometry. Representative cell-cycle profiles of cells with different drug treatments are shown.

Figure 5.

Synergism between R115777 and paclitaxel or docetaxel in R115777-resistant MM cells. RPMI8226/R5 cells were treated with drugs for 72 hours before MTT assays. Results represent means and standard deviations of 4 independent experiments. (A,C) R115777 enhanced the cytotoxic effects of paclitaxel or docetaxel. (B,D) Isobologram based on data from panels A and C, showing synergism between R115777 and paclitaxel or docetaxel.

Figure 6.

R115777 enhances paclitaxel antitumor activity in the SCID-hu bone mouse model of MM growth. SCID mice were implanted with human fetal bones injected with RPMI8226/S cells. After the tumors formed, the mice were treated with paclitaxel or R115777 or both for 2 weeks. The implanted bones were then analyzed by histology staining. Representative pictures from each group are shown.

Figure 7.

Combination of paclitaxel or docetaxel with R115777 is beneficial in the induction of apoptosis in MM patient bone marrow MNCs. MNCs were isolated from bone marrow aspirates and incubated with paclitaxel (P), docetaxel (D), and R115777 (R) at different concentrations (μM) for 48 to 72 hours. Cells were then stained with CD138-PE, annexin V-APC, and 7-AAD, and subjected to flow cytometric analysis as described in “Materials and methods.” Apoptosis in CD138⁺ and CD38^- cells was then analyzed. (A) Dot graphs showing annexin-V staining profiles of cells from patient no. 5 treated with different drugs at different concentrations. CD138⁺ cells were gated for analysis. (B) Summary of annexing staining results of MNCs from 6 patients after treatment with different drugs at different concentrations. Net increases in apoptotic cells after subtracting the apoptotic cells in the untreated group from CD138⁺() and CD138^- (□) cells are shown.