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. 2006 May 15;107(10):4053-62.
doi: 10.1182/blood-2005-08-3434. Epub 2006 Jan 17.

Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells

Teru Hideshima  1 , Laurence Catley, Hiroshi Yasui, Kenji Ishitsuka, Noopur Raje, Constantine Mitsiades, Klaus Podar, Nikhil C Munshi, Dharminder Chauhan, Paul G Richardson, Kenneth C Anderson
Affiliations

Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells

Teru Hideshima et al. Blood. .

Abstract

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agents which _targets cell membranes and inhibits Akt activation. Here we show that baseline phosphorylation of Akt in multiple myeloma (MM) cells is completely inhibited by perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate] in a time- and dose-dependent fashion, without inhibiting phosphoinositide-dependent protein kinase 1 phosphorylation. Perifosine induces significant cytotoxicity in both MM cell lines and patient MM cells resistant to conventional therapeutic agents. Perifosine does not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous interleukin-6 (IL-6) nor insulinlike growth factor 1 (IGF-1) overcomes Perifosine-induced cytotoxicity. Importantly, Perifosine induces apoptosis even of MM cells adherent to bone marrow stromal cells. Perifosine triggers c-Jun N-terminal kinase (JNK) activation, followed by caspase-8/9 and poly (ADP)-ribose polymerase cleavage. Inhibition of JNK abrogates perifosine-induced cytotoxicity, suggesting that JNK plays an essential role in perifosine-induced apoptosis. Interestingly, phosphorylation of extracellular signal-related kinase (ERK) is increased by perifosine; conversely, MEK inhibitor synergistically enhances Perifosine-induced cytotoxicity in MM cells. Furthermore, perifosine augments dexamethasone, doxorubicin, melphalan, and bortezomib-induced MM cell cytotoxicity. Finally, perifosine demonstrates significant antitumor activity in a human plasmacytoma mouse model, associated with down-regulation of Akt phosphorylation in tumor cells. Taken together, our data provide the rationale for clinical trials of perifosine to improve patient outcome in MM.

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Figures

Figure 1.
Figure 1.
Perifosine inhibits Akt phosphorylation and induces cytotoxicity in MM cells. (A) Baseline phosphorylation of Akt, STAT3, and ERK in MM cell lines and tumor cells from MM patients assessed by Western blotting. Lane 1 indicates MM.1S; 2, MM.1R; 3, U266; 4, INA-6; 5, RPMI8226; 6, LR5; 7, Dox40; 8, OPM1; 9, OPM2; and 10 to 12, patient tumor cells. (B) MM.1S cells were cultured with perifosine (10 μM) for the indicated periods. Whole-cell lysates were subjected to Western blotting using anti–p-PDK1, anti–p-Akt, anti-Akt, anti–p-FKHRL1, anti–p-GSK3α/β, anti–p-MEK, anti–p-ERK, anti-ERK2, and α-tubulin Abs. (C) MM.1S cells were cultured with perifosine (1-10 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-ERK, and anti-ERK2 Abs. (D) MM.1S cells were cultured with perifosine (5 μM) for 6 hours prior to stimulation with IL-6 (20 ng/mL). Whole-cell lysates were immunoprecipitated with anti-Akt Ab. The immunoprecipitates were washed and subjected to in vitro kinase assay according to manufacturer's protocol. (E) MM.1S (▪), MM.1R (□), U266 (▴), INA-6 (▵), RPMI8226 (•), LR5 (○), Dox40 (♦), OPM1 (⋄), and OPM2 (*) cells were cultured with perifosine (1.5-100 μM) for 24 hours (i) and 48 hours (ii). (F) MM.1S cells stably transfected with Myr-Akt (▪) and control construct (•) were cultured with perifosine (0.6-10 μM) for 24 hours. Whole-cell lysates from control and Myr-Akt–transfected cells were subjected to Western blotting using anti–p-Akt and Akt Abs. (G-H) Freshly isolated tumor cells from 4 patients with MM (G) and PBMCs from 3 healthy donors (H) were cultured with increasing doses of perifosine for 48 hours. Cytotoxicity was assessed by MTT assay (E-H); data represent mean (± SD) of quadruplicate cultures.
Figure 2.
Figure 2.
Perifosine induces JNK/caspase-dependent MM cell apoptosis. (A) MM.1S and RPMI8226 cells were cultured with perifosine (5 and 10 μM) for 24 hours. Cells were then subjected to cell-cycle profiling by PI staining and flow cytometry. Percentage indicates sub-G1 phase cells. (B) MM.1S cells were cultured with perifosine (5 and 10 μM) for 8 hours. (C) MM.1S cells were also cultured with perifosine (10 μM) for 6 and 12 hours. Cells were then lysed and subjected to Western blotting using caspase-8, caspase-9, and PARP Abs. (C) MM.1S cells were cultured for 24 hours with perifosine (5 and 7.5 μM) in the presence of control media (□), and with 25 μM of Z-IETD-FMK (formula image), Z-LEHD-FMK (formula image), or Z-VAD-FMK (▪). (D) MM.1S cells were cultured with perifosine (10 μM) for the indicated periods. Cells were then lysed and subjected to Western blotting using anti–p-p38 MAPK, anti-p38 MAPK, anti–p-JNK, anti-JNK, and α-tubulin Abs. (E) MM.1S cells were cultured with perifosine (5 μM and 10 μM) for 8 hours, in the presence or absence of SP600125 (10 μM). Cells were then lysed and subjected to Western blotting using anti–p-JNK, anti-JNK, and caspase-8 Abs. (F) MM.1S cells were cultured for 24 hours with control media (□), or with 2.5 μM (formula image), 5 μM (formula image), and 7.5 μM(▪) perifosine, in the presence or absence of SP600126 (10 μM). (G) MM.1S cells were transiently transfected with control (•) or JNK2 siRNA expression plasmid (▪). Whole-cell lysates were subjected to Western blotting using anti-JNK2 and α-tubulin Abs. (H) MM.1S cells were cultured for 24 hours with control media (□) or perifosine (5 and 7.5 μM) in the presence or absence of control media (□), 200 nM (formula image), or 400 nM (▪) SCIO469. Cytotoxicity was assessed by MTT assay; data represent mean (± SD) of quadruplicate cultures.
Figure 3.
Figure 3.
Neither growth factors nor adherence to BMSCs protect against perifosine-induced MM cell cytotoxicity. (A) MM.1S cells were cultured for 48h with control media (white bars); with 50 nM (light blue bars), 100 nM (medium blue bars), and 200 nM (dark blue bars) Dex; or with 2.5 μM (light orange bars), 5 μM (medium orange bars), and 10 μM (dark orange bars) perifosine, in the presence or absence of IL-6 (10 ng/mL). Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (B) MM.1S cells were cultured with control media or perifosine (5 μM) for 6 hours. Cells were then stimulated with IL-6 (10 ng/mL) or IGF-1 (25 ng/mL) for 10 minutes. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-MEK, anti–p-ERK, anti-ERK1/2, and p-STAT3 Abs. (C) MM.1S cells were cultured with control media (white bars); and with 1.25 μM (light orange bars), 2.5 μM (medium orange bars), and 5 μM (dark orange bars) perifosine for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed by [3H]-thymidine uptake; data represent means (± SD) of quadruplicate cultures. (D) MM.1S cells were cultured with control media or perifosine (5 μM) for 6 hours in the presence or absence of BMSCs. MM.1S cells were harvested, lysed, and subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-ERK, anti-ERK1/2, and p-STAT3 Abs.
Figure 4.
Figure 4.
Inhibition of ERK signaling augments Perifosine-induced cytotoxicity. (A) MM.1S and MM.1R cells were cultured with perifosine (5 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-MEK, anti–p-ERK, and anti-ERK1/2 Abs. (B-C) MM.1S (B) and MM.1R (C) cells were cultured for 14 hours with control media and with 2.5 μM, 5 μM, and 7.5 μM perifosine in the absence (□) or presence of 5 μM (light blue bars) or 10 μM (dark blue bars) MEK1/2 inhibitor U0126. Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (D) MM.1S cells were cultured for 8 hours with control media, perifosine (5 μM), U0126 (5 μM), or perifosine (5 μM) plus U0126 (5 μM). Cells were then lysed and subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-ERK, anti-ERK1/2, anti–p-JNK, caspase-8, and PARP Abs. (E) Freshly isolated tumor cells from MM patients (n = 2) were cultured for 24 hours with control media (□) and with 5 μM (formula image) or 10 μM(▪) perifosine in the presence or absence of U0126 (5 μM). Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (F) MM.1S cells were cultured for 48 hours with control media (□) and with 1.25 μM (light orange bars), 2.5 μM (medium orange bars), or 5 μM (dark orange bars) perifosine with or without U0126 (2.5 μM) and in the presence or absence of BMSCs. Cell proliferation was assessed by [3H]-thymidine uptake; data represent means (± SD) of quadruplicate cultures.
Figure 5.
Figure 5.
Perifosine enhances cytotoxicity of conventional (Dex, Mel, Dox) agents. MM.1S cells were cultured for 12 hours with control medium (□) and with 2.5 μm(▪) or 5 μm (▪) perifosine in the presence or absence of (A) Dex, 50 nm and 100 nm; (B) Mel, 2-5 μm and 5 μm; and (C) Dox, 50 nm and 100 nm. Cytotoxicity was assessed by MTT assay; data represent the mean plus or minus SD of quadruplicate cultures.
Figure 6.
Figure 6.
Perifosine enhances cytotoxicity of novel (bortezomib) agents. (A) MM.1S cells were cultured for 24 hours with control media (□) and with 2.5 μM (formula image) or 5 μM (▪) perifosine in the presence or absence of bortezomib (5 nM and 7.5 nM). Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (B) MM.1S cells were cultured with bortezomib (10 nM) for 4 hours and 8 hours. Whole-cell lysates were subjected to Western blotting with p-Akt and Akt Abs. (C) MM.1S cells were cultured with bortezomib (10 nM) for 8 hours. Whole-cell lysates were immunoprecipitated with anti-Akt Ab. The immunoprecipitates were washed and subjected to in vitro kinase assay, according to manufacturer's protocol. The reaction mixtures were immunoblotted with anti–p-GSK3α/β. (D) MM.1S cells were cultured with bortezomib (5, 10, and 20 nM) in the presence (5 μM) or absence of perifosine. Whole-cell lysates were subjected to Western blotting with p-Akt, Akt, p-ERK, and ERK2 Abs. (E) MM.1S cells were cultured for 8 hours with control media, perifosine (5 μM), bortezomib (10 nM), or perifosine (5 μM) plus Bortezomib (10 nM). Cells were then lysed and subjected to Western blotting using anti–p-JNK1/2, caspase-8, and PARP Abs.
Figure 7.
Figure 7.
Perifosine inhibits human MM cell growth in vivo. (A-B) Immunodeficient BNX mice were inoculated subcutaneously in the flank with 3 × 107 MM.1S cells in 100 μL RPMI 1640 medium and 100 μL Matrigel. Oral perifosine was administered daily (red line; 36 mg/kg, n = 7) and weekly (green line; 250 mg/kg, n = 7) starting after the development of measurable tumor. Perifosine significantly inhibited MM tumor growth (A) (P = .04) and increased survival (B) (P = .001) compared with the control group treated with vehicle (blue line; normal saline, n = 6) only. Error bars in panel A represent ± 1 SD of 7 mice in the treated groups and 6 mice in the control group. (C) Significant growth inhibition of subcutaneous tumor was observed after 4 weekly treatments with perifosine (250 mg/kg). (D) Tumor tissues from mice treated with control vehicle or with weekly perifosine were harvested; whole-tissue lysates were subjected to Western blotting using anti–p-Akt and Akt Abs.
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