Abstract
We identified nocodazole as a potent antimyeloma drug from a drug screening library provided by the Multiple Myeloma Research Foundation. Nocodazole is a benzimidazole that was originally categorized as a broad-spectrum anthelmintic drug with antineoplastic properties. We found that nocodazole inhibited growth and induced apoptosis of primary and multiresistant multiple myeloma cells cultured alone and in the presence of bone marrow stromal cells. Nocodazole caused cell-cycle prophase and prometaphase arrest accompanied by microtubular network disarray. Signaling studies indicated that increased expression of Bim protein and reduced X-linked inhibitor of apoptosis protein and Mcl-1L levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, triggered by the activation of c-jun-NH2 kinase (JNK) instead of p38 kinase or extracellular signal–regulated kinases. Treatment with JNK inhibitor decreased Bcl-2 phosphorylation and subsequently reduced nocodazole-induced cell death. Nocodazole combined with dexamethasone significantly inhibited myeloma tumor growth and prolonged survival in a human xenograft mouse model. Our studies show that nocodazole has potent antimyeloma activity and that targeting the microtubular network might be a promising new treatment approach for multiple myeloma. Mol Cancer Ther; 10(10); 1886–96. ©2011 AACR.
Introduction
Multiple myeloma is the second most prevalent hematologic malignancy and is uniformly fatal, very often as a result of development of drug resistance. To overcome the chemoresistance to current therapies and improve patient outcome, novel treatment agents are needed to target mechanisms whereby multiple myeloma cells grow and survive.
The coordinated processes of cell-cycle progression, cell growth, and apoptosis are dysfunctional in cancer (1, 2). During cell-cycle progression, microtubule assembly is a proven target for anticancer drug development because of its critical role for mitotic spindle formation and the separation of chromosomes during mitosis (3). It has been shown that the c-jun-NH2 kinase (JNK)/stress-activated protein kinase (SAPK) pathway is involved in cell-cycle regulation and that microtubule-interfering agents activate this pathway inducing G2–M arrest that results in apoptosis in a variety of human cancer cells (4).
The Bcl-2 family of proteins includes both pro- and antiapoptotic molecules and their ratio determines the fate of cells. Bcl-2 protein is regulated at transcriptional and posttranslational levels including phosphorylation within the flexible loop regulatory domain. These modifications induce conformational changes in the Bcl-2 protein and regulate its active forms in response to cell death signaling (5, 6). It has been further found that Bcl-2 is phosphorylated/inactivated by JNK/SAPK (7), suggesting that Bcl-2 protein is a target of microtubule-damaging agents, resulting in G2–M cell-cycle block (8, 9).
Benzimidazoles, including albendazole, fenbendazole, mebendazole, and nocodazole, have been used as anthelmintics and fungicides on the basis of their antimicrotubule activity (10) and have been reported to elicit promising antitumor effect (11–13). Although nocodazole has been recently categorized as an antineoplastic agent, the antimyeloma effects and its underlying mechanism of action have not been examined yet. By using multiplex cytokine array on a chemical library containing 1,000 compounds provided by the Multiple Myeloma Research Foundation, we identified nocodazole as a potent antimyeloma agent and showed that benzimidazoles, and especially nocodazole, significantly reduce the secretion of cytokines essential for multiple myeloma survival (14). In the present study, we show that benzimidazoles induce cell death in multiple myeloma cell lines and in primary CD138+ myeloma cells and overcome drug resistance. The induction of apoptosis by nocodazole was associated with G2–M phase of cell-cycle arrest and was induced by JNK-mediated Bcl-2 phosphorylation in both primary and multiple myeloma cell lines. Subsequent induction of apoptosis by nocodazole could be abrogated by a specific JNK inhibitor. Nocodazole, alone or combined with dexamethasone, effectively suppressed myeloma tumor growth in a human multiple myeloma xenograft mouse model, suggesting that nocodazole is a potent and promising new antimyeloma agent.
Materials and Methods
Reagents and cell culture
U0126 and PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], SB203580 (p38-MAPK inhibitor), and SP600125 (JNK inhibitor) were purchased from Calbiochem. Fetal calf serum (FCS) and other cell culture reagents were purchased from Sigma-Aldrich. Albendazole, fenbendazole, mebendazole, and nocodazole (Fig. 1B) were prepared in dimethyl sulfoxide as stock solutions, stored at −20°C, and subsequently diluted in RPMI-1640 medium before use. All drugs were provided by Prestwick Chemicals and obtained within a grant awarded by the Multiple Myeloma Research Foundation.
Effects of benzimidazoles on multiple myeloma cell growth and viability. A, U266 multiple myeloma cells were treated with concentrations of benzimidazole for 48 hours. H929 multiple myeloma cells were treated with the indicated concentrations of benzimidazoles for 48 hours. DNA synthesis was measured by 3H-thymidine incorporation. A, bottom, RPMI-8226 multiple myeloma cells were treated with benzimidazoles for 16 hours: albendazole, fenbendazole, mebendazole, and nocodazole. Percentages of the apoptotic cell death with typical apoptotic nuclear morphology were determined by staining the cells with Hoechst 33258 fluorochrome. B, chemical structure of nocodazole. Primary CD138+ and CD138− bone marrow mononuclear cells were treated with indicated concentrations of nocodazole for 48 and 72 hours. Cell viability was determined using trypan blue staining. C, bone marrow stromal cells were grown in 96-well plates in Dulbecco's Modified Eagle's Medium containing 10% FCS. H929 multiple myeloma cells were added to bone marrow stromal cells (BMSC) and cultured for another 48 hours in the presence or absence of the indicated concentrations of nocodazole. DNA synthesis was measured by 3H-thymidine incorporation. D, RPMI-8226 cell line (RPMI-8226/S) and its chemoresistant sublines (RPMI-8226/Dox40, RPMI-8226/MR20, or RPMI-8226/LR5) and MM.1S and its dexamethasone-resistant subline (MM.1R) were treated with indicated concentrations of nocodazole for 48 hours. Cell proliferation was measured by 3H-thymidine incorporation. All experiments were conducted in triplicates. Results are shown as mean ± SD. *, P <.05.
Effects of benzimidazoles on multiple myeloma cell growth and viability. A, U266 multiple myeloma cells were treated with concentrations of benzimidazole for 48 hours. H929 multiple myeloma cells were treated with the indicated concentrations of benzimidazoles for 48 hours. DNA synthesis was measured by 3H-thymidine incorporation. A, bottom, RPMI-8226 multiple myeloma cells were treated with benzimidazoles for 16 hours: albendazole, fenbendazole, mebendazole, and nocodazole. Percentages of the apoptotic cell death with typical apoptotic nuclear morphology were determined by staining the cells with Hoechst 33258 fluorochrome. B, chemical structure of nocodazole. Primary CD138+ and CD138− bone marrow mononuclear cells were treated with indicated concentrations of nocodazole for 48 and 72 hours. Cell viability was determined using trypan blue staining. C, bone marrow stromal cells were grown in 96-well plates in Dulbecco's Modified Eagle's Medium containing 10% FCS. H929 multiple myeloma cells were added to bone marrow stromal cells (BMSC) and cultured for another 48 hours in the presence or absence of the indicated concentrations of nocodazole. DNA synthesis was measured by 3H-thymidine incorporation. D, RPMI-8226 cell line (RPMI-8226/S) and its chemoresistant sublines (RPMI-8226/Dox40, RPMI-8226/MR20, or RPMI-8226/LR5) and MM.1S and its dexamethasone-resistant subline (MM.1R) were treated with indicated concentrations of nocodazole for 48 hours. Cell proliferation was measured by 3H-thymidine incorporation. All experiments were conducted in triplicates. Results are shown as mean ± SD. *, P <.05.
The following primary antibodies were purchased from Cell Signaling Technology: phospho-c-Jun NH2-terminal kinase, phospho-p38 and phospho-p44/42 MAPKs, phospho-Bcl-2, Bax, Bim, X-linked inhibitor of apoptosis (XIAP), survivin, and Bid. Mcl-1L and death receptor 4 (DR4) antibodies were purchased from Santa Cruz Biotechnology, and actin and β-tubulin conjugated with Alexa Fluor 555 antibodies were purchased from Sigma-Aldrich.
Multiple myeloma cell lines H929 and U266 were purchased from American Type Culture Collection in May 2010 and August 2009, respectively. Dr. William Dalton (H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL) kindly provided the human multiple myeloma cell line RPMI-8226/S and its sublines RPMI-8226/Dox40 (resistant to doxorubicin), RPMI-8226/MR20 (resistant to mitoxantrone), and RPMI-8226/LR5 (resistant to melphalan) in August 2009. Dexamethasone-sensitive and -resistant cell lines MM.1S and MM.1R were provided by Dr. Klaus Podar (Dana Farber Cancer Institute, Harvard Medical School, Boston, MA) in August 2009. These cell lines were tested for their drug resistance, but no cell line authentication was done by the authors. Multiple myeloma cell lines were cultured in RPMI-1640 medium with 10% FCS, 2 mmol/L glutamine, and 100 U/mL penicillin–streptomycin (Sigma-Aldrich) at 37°C with 5% CO2. The chemoresistant multiple myeloma cell lines were cultured in the presence of doxorubicin, mitoxantrone, melphalan, or dexamethasone, and resistance was confirmed by cell proliferation assays (Supplementary Fig. S1).
Bone marrow mononuclear cells were obtained from untreated multiple myeloma patients and were isolated by separation on Ficoll-Hypaque gradients as described previously (15). CD138+ bone marrow cells from multiple myeloma patients were purified by CD138 (syndecan-1) microbeads using a magnetic cell sorting system (Miltenyi Biotec) as described previously (16) and used as primary multiple myeloma cells. Unlabeled cells were used as CD138− cells. All studies were approved by the Institutional Review Board of the University of Pittsburgh, and all subjects signed approved consent forms.
Cell proliferation assay (3H-thymidine incorporation)
U266, H929, RPMI-8226 (3 × 104 cells per well), MM.1S cell line (6 × 104 cells per well), and their resistant sublines were cultured in 96-well culture plates (Costar) in RPMI-1640 medium containing 10% FCS with or without drugs for 48 hours at 37°C with 5% CO2. For bone marrow stromal cell coculture experiments, human primary bone marrow stromal cells (3 × 103 cells per well) were cultured for 24 hours in Dulbecco's Modified Eagle's Medium containing 10% FCS in 96-well plates. Then, multiple myeloma cells (3 × 104 cells per well) were seeded on bone marrow stromal cells and cultured for 48 hours in the presence or absence of nocodazole at different concentrations. Cells were pulsed with 1 μCi/well 3H-thymidine during the last 8 to 10 hours of culture, harvested onto glass fiber filter mats (Wallac) with an automatic cell harvester and counted using a Wallac TriLux Beta plate scintillation counter.
Cell-cycle analyses
After incubation with nocodazole (100 nmol/L for 0–24 hours or 0–160 nmol/L for 16 hours), human multiple myeloma cells were harvested and washed with cold PBS, fixed in 70% ethanol at −20°C, treated with DNase-free RNase A (Sigma-Aldrich), and stained with 50 μg/mL propidium iodide (Sigma) at 37°C for 30 minutes. Analyses were conducted on a BD FACSCalibur flow cytometer and analyzed using ModFit LT 2.0 and Cell Quest software (BD Biosciences).
Assessment of apoptotic cell death and cell viability
Apoptosis was assessed morphologically by evaluation of nuclear condensation and fragmentation using Hoechst 33258 staining as described previously (17). Treated multiple myeloma cells were incubated with 2.5 μg/mL Hoechst 33258 (Molecular Probes, Life Technologies Corp.) for 20 minutes followed by examination under a fluorescence microscope (Olympus CKX41). Cells (∼200 per condition) were randomly selected and assessed. Hoechst 33258–positive cells with apoptotic bodies or condensed and fragmented nuclei were considered and counted as apoptotic cells.
Viability of cells was determined by trypan blue staining, which distinguishes the membrane-defective dead cells from the viable cells.
Immunofluorescence staining and electron microscopy
Multiple myeloma cells (H929 or RPMI-8226) were fixed in PBS containing 3% formaldehyde for 15 minutes and washed in PBS. Cytospin slides were prepared using Cytospin (Thermo Shandon Inc.) and permeabilized in 0.3% Triton X-100/PBS (30 minutes at 4°C). After blocking with 2.5% bovine serum albumin in PBS, the samples were incubated with Alexa Fluor 555–conjugated antibody against β-tubulin overnight at 4°C. Counterstaining with Hoechst 33258 for nuclear location and integrity was conducted before sealing the slides with Fluoro-Gel (Electron Microscopy Sciences). Slides were examined under a filter-combined fluorescence microscope (Olympus CKX41) equipped with a ×20/0.40 numeric aperture objective lens (Olympus America, Inc.).
For electron microscopy, multiple myeloma cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L PBS (pH = 7.4) followed by 1% OsO4. After dehydration, sections were stained with uranyl acetate and lead citrate and examined under a JEM 1011 (JEOL) electron microscope. To examine and quantify cellular morphologic changes, digital phase-contrast images were recorded.
SDS-PAGE and Western blotting
Western blotting was conducted as previously described (18). Briefly, cells were harvested, lysed with radioimmunoprecipitation assay buffer (Pierce) containing phosphatase and protease inhibitors (Halt Protease Inhibitor Cocktail Kit; Pierce). Lysates were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membrane. Following probing with specific primary antibodies plus horseradish peroxidase–conjugated secondary antibody, the protein bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Human xenograft mouse model
Beige/nude/X-linked immunodeficient mice were purchased from Charles River Laboratories at 6 to 8 weeks of age with a weight between 20 and 25 grams. For human tumor xenograft studies, 1 × 107 H929 myeloma cells in 50 μL RPMI-1640 together with an equal volume of Matrigel basement membrane matrix (BD Biosciences) were injected subcutaneously as described previously (19). When tumors were palpable, mice were assigned randomly to treatment groups receiving 5 or 20 mg/kg nocodazole intraperitoneally 3 times weekly on Monday, Wednesday, and Friday. Combination studies with dexamethasone were carried out by treating mice with 12 mg/kg nocodazole (n = 5) intraperitoneally on Monday, Wednesday, and Friday; 2 mg/kg dexamethasone (n = 6) subcutaneously on Monday and Friday; or their combination (n = 5). The control group (n = 6) received the same volume of vehicle alone with the same treatment frequency. Mice were weighed twice weekly and observed daily for diarrhea or any changes in behavior and condition. Tumor sizes were measured whenever the drug was given using a caliper and calculated by the formula: 0.5 × width2 × length, representing the 3-dimensional volume of an ellipse. Animals from both cohorts were euthanized when their tumors reached 2 cm in one diameter and/or were ulcerated (endpoint). Survival was evaluated from the first day of treatment until animals were euthanized. This was defined as endpoint of the study.
All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.
Statistical analyses
All quantitative data are presented as mean ± SD of at least triplicates. Statistical differences were determined by Student's t test. Kaplan–Meier survival analysis and log-rank test were conducted to estimate the survival rates and survival differences. Results were considered significantly different if P < 0.05.
Results
Nocodazole inhibits myeloma cell growth and overcomes chemoresistance
We tested the direct effects of benzimidazoles on human multiple myeloma cell proliferation and cell death induction. Albendazole, fenbendazole, and nocodazole significantly inhibited multiple myeloma cell proliferation with an IC50 value of 25 to 94 nmol/L for U266 multiple myeloma cells and an IC50 value of 63 to 380 nmol/L for H929 multiple myeloma cells. Furthermore, benzimidazoles induced nuclear fragmentation, a typical sign of cell apoptosis, with an IC50 value of 30 to 250 nmol/L in RPMI-8226 myeloma cells (Fig. 1A). On the basis of our finding that nocodazole exhibited the strongest antimyeloma effect, we focused our further studies on nocodazole (Fig. 1B, left). Following 48- and 72-hour exposure to nocodazole, the viability of primary CD138+ multiple myeloma cells decreased in a dose-dependent manner as monitored by trypan blue staining (IC50 = 125–185 nmol/L). The viability of non–multiple myeloma (CD138−) cells obtained from the same patients was affected only at much higher concentration (IC50 ≥ 500 nmol/L), indicating the relative selectivity of nocodazole for malignant plasma cells (Fig. 1B, right). In addition, the strong inhibition of proliferation of multiple myeloma cells treated with nocodazole was not reversed if multiple myeloma cells were cocultured with bone marrow stromal cells (Fig. 1C).
Because drug resistance is a prevalent problem in multiple myeloma treatment (20), we determined whether nocodazole overcomes drug resistance in multiple myeloma. First, we confirmed the chemoresistant phenotypes of RPMI-8226 sublines (8226/Dox40, 8226/MR20, or 8226/LR5) by culturing the RPMI-8226 cells with doxorubicin, mitoxantrone, or melphalan and analyzing cell proliferation (Supplementary Fig. S1). The drug-resistant sublines exhibited similar responses to nocodazole treatment (IC50 = 25–65 nmol/L) as their parental cell lines. Similar results were observed with dexamethasone-sensitive and -resistant cell lines MM.1S and MM.1R (Fig. 1D), suggesting that nocodazole overcomes chemoresistance.
Nocodazole-induced prometaphase arrest is associated with disruption of microtubular network array in multiple myeloma cells
Treatment of H929 multiple myeloma cells with nocodazole resulted in G2–M phase cell-cycle arrest. After treatment with nocodazole for 7 and 16 hours, the percentage of G2 population increased to 50% and 35%, respectively, in comparison with control (21%). Prolonged treatment was associated with increased apoptosis of multiple myeloma cells, as evidenced by the increased population of pro-G0 phase cells as well as the appearance of polyploid cells (Fig. 2A, top). In accordance with the time-dependent induction of G2 arrest, we also observed a dose-dependent G2 arrest with an increase of G2 population to 40% at 80 nmol/L and 56% at 160 nmol/L in comparison with control (19%; Fig. 2A, bottom).
Nocodazole induces prometaphase arrest associated with disruption of the microtubular network. A, H929 multiple myeloma cells were treated with nocodazole as indicated. Cell cycle was analyzed by propidium iodide staining and flow cytometry, measuring the cellular DNA content. Results are representative of 2 independent experiments. B, H929 multiple myeloma cells were treated with nocodazole (100 nmol/L) for 0 to 16 hours and immunofluorescence staining against tubulin was conducted. Nuclear location and integrity were detected by counterstaining with Hoechst 33258. Disarray of microtubular network is shown by white arrows and prometaphase nuclei are shown by yellow arrows. C, H929 multiple myeloma cells treated with nocodazole (100 nmol/L) were subjected to electron microscopy study. Prometaphase nuclei morphology and apoptotic bodies are detected after 16 and 24 hours of nocodazole treatment.
Nocodazole induces prometaphase arrest associated with disruption of the microtubular network. A, H929 multiple myeloma cells were treated with nocodazole as indicated. Cell cycle was analyzed by propidium iodide staining and flow cytometry, measuring the cellular DNA content. Results are representative of 2 independent experiments. B, H929 multiple myeloma cells were treated with nocodazole (100 nmol/L) for 0 to 16 hours and immunofluorescence staining against tubulin was conducted. Nuclear location and integrity were detected by counterstaining with Hoechst 33258. Disarray of microtubular network is shown by white arrows and prometaphase nuclei are shown by yellow arrows. C, H929 multiple myeloma cells treated with nocodazole (100 nmol/L) were subjected to electron microscopy study. Prometaphase nuclei morphology and apoptotic bodies are detected after 16 and 24 hours of nocodazole treatment.
To identify mitotic phase arrest in nocodazole-treated multiple myeloma cells, we examined microtubule dynamic change and chromosome status as well as nuclear membrane integrity. Immunofluorescence staining of the vehicle-treated cells for tubulin showed the regular structure of microtubules with cells in anaphase/telophase. However, nocodazole-treated multiple myeloma cells displayed a disordered microtubule structure. Spindle formation was disrupted and cells exhibited an irregular staining pattern for tubulin along with some small punctate areas (Fig. 2B). The mitosis-like chromosomes with disordered orientation were condensed and scattered inside the cells. The peak of destruction of the microtubular network was observed after 16 hours of nocodazole treatment (Fig. 2B and Supplementary Fig. S2A). Electron microscopy studies indicated that the nuclear membrane had collapsed and disappeared (Fig. 2C), which is a typical phenomenon of prophase and prometaphase, suggesting that the cells arrested in prophase/prometaphase.
We found that nocodazole also caused significant (P < 0.05) morphologic elongation of multiple myeloma cells (Fig. 2B and C and Supplementary Fig. S2A–C). Most of the elongated cells only had one irregular nucleus, consisting of clusters of condensed chromosomes, suggesting that the cells failed to progress into mitosis. Importantly, immunofluorescence staining for tubulin showed that the disordered structure of microtubules was only observed in the elongated cells (Fig. 2B and Supplementary Fig. S2A). These data suggest that nocodazole induces damage of the microtubular network and disrupts the interaction of microtubules and kinetochore, resulting in the prevention of the formation of the mitotic spindle and the arrest in mitosis prophase/prometaphase.
JNK-mediated Bcl-2 phosphorylation contributes to nocodazole-induced cell death
To study the molecular mechanism underlying nocodazole-induced myeloma cell death, we focused on the apoptosis-regulatory protein Bcl-2. Bcl-2 has been suggested to regulate microtubule integrity (21) and is rapidly phosphorylated and inactivated in response to exposure of cells to microtubular disrupting agents. Bcl-2 phosphorylation was strongly induced in a dose- and time-dependent manner at both Ser70 and Thr56 sites whereas total Bcl-2 protein was unaffected by nocodazole treatment in myeloma cells (Fig. 3A–C). Importantly, upregulation of Bcl-2 phosphorylation and proapoptotic BH3-only protein Bim isoforms (BimEL, BimL, and BimS) was also observed when primary CD138+ myeloma cells were treated with nocodazole (Fig. 3B and C, left). On the contrary, nocodazole exposure had no significant effect on the Bcl-2 phosphorylation and Bim isoforms in nonmyeloma CD138− cells (Fig. 3B and C, right). Nocodazole decreased the protein levels of antiapoptotic XIAP and Mcl-1L but had no effect on proapoptotic molecules such as Bid and Bax as well as death receptor 4 and the apoptosis inhibitor, survivin (Fig. 3A and D).
Nocodazole induces phosphorylation of Bcl accompanied by a decreased expression of antiapoptotic proteins such as Mcl-1L and XIAP. RPMI-8226 (A and D) and CD138+ primary multiple myeloma cells and CD138− cells (B and C) were treated with vehicle control or nocodazole at indicated concentrations for indicated times. Whole-cell lysates were subjected to Western blot assay and detected with indicated antibodies. β-Actin served as loading control.
Nocodazole induces phosphorylation of Bcl accompanied by a decreased expression of antiapoptotic proteins such as Mcl-1L and XIAP. RPMI-8226 (A and D) and CD138+ primary multiple myeloma cells and CD138− cells (B and C) were treated with vehicle control or nocodazole at indicated concentrations for indicated times. Whole-cell lysates were subjected to Western blot assay and detected with indicated antibodies. β-Actin served as loading control.
Several protein kinases have been identified to be critical for Bcl-2 phosphorylation (5). We found that nocodazole phosphorylates JNK and p38-MAPK and decreases the phosphorylation of the extracellular signal–regulated kinases (ERK) in a dose- and time-dependent manner (Fig. 4A). JNKs, in contrast to p38-MAPK or ERKs, contribute primarily to the induction of Bcl-2 phosphorylation. To confirm whether only JNK induces phosphorylation of Bcl-2 in nocodazole-treated multiple myeloma cells, we treated RPMI-8226 multiple myeloma cells with nocodazole in the presence of specific MAPK inhibitors. Inhibition of MEK-ERK (UO126 and PD98059) and p38-MAPK (SB202190) pathways had no effect on the phosphorylation of Bcl-2 protein. Only the JNK inhibitor (SP600125) was able to abrogate Bcl-2 phosphorylation at both Ser70 and Thr56 sites (Fig. 4B). In accordance with this is the finding that only JNK inhibitor SP600125 prevented nocodazole-induced cell nuclear fragmentation. Compared with vehicle control, nocodazole-induced cell death was significantly decreased by 50% and 64% when JNK pathway was blocked by SP600125 at 10 μmol/L and 20 μmol/L, respectively. Other MAPK inhibitors failed to rescue multiple myeloma cells from nocodazole-induced cell death (Fig. 4C).
Nocodazole induces multiple myeloma cell apoptosis via JNK-induced Bcl-2 phosphorylation. A, RPMI-8226 multiple myeloma cells were treated with nocodazole at indicated concentrations for indicated times. Whole-cell lysates were subjected to Western blot assay and analyzed with indicated antibodies. B, RPMI-8226 multiple myeloma cells were incubated with MAPK (U0126, 25 μmol/L; PD98059, 25 μmol/L; SB203580, 10 μmol/L); and JNK inhibitor (SP600125, 20 μmol/L) for 30 minutes and with nocodazole for another 16 hours. Cell lysates were subjected to Western blot assay with indicated antibodies. C, RPMI-8226 multiple myeloma cells were treated with nocodazole 100 nmol/L in combination with different phosphorylation inhibitors as indicated. Apoptotic cell death characterized by typical apoptotic nuclear morphology was determined by Hoechst 33258 fluorochrome staining. *, P <.02; and **, P <. 01. NC, negative control.
Nocodazole induces multiple myeloma cell apoptosis via JNK-induced Bcl-2 phosphorylation. A, RPMI-8226 multiple myeloma cells were treated with nocodazole at indicated concentrations for indicated times. Whole-cell lysates were subjected to Western blot assay and analyzed with indicated antibodies. B, RPMI-8226 multiple myeloma cells were incubated with MAPK (U0126, 25 μmol/L; PD98059, 25 μmol/L; SB203580, 10 μmol/L); and JNK inhibitor (SP600125, 20 μmol/L) for 30 minutes and with nocodazole for another 16 hours. Cell lysates were subjected to Western blot assay with indicated antibodies. C, RPMI-8226 multiple myeloma cells were treated with nocodazole 100 nmol/L in combination with different phosphorylation inhibitors as indicated. Apoptotic cell death characterized by typical apoptotic nuclear morphology was determined by Hoechst 33258 fluorochrome staining. *, P <.02; and **, P <. 01. NC, negative control.
A high amount of phosphorylated Bcl-2 protein was detected specifically in G2–M phase within 16 hours of nocodazole exposure (Figs. 2A and 3A). These results in combination with the known role of Bcl-2 phosphorylation in all cell cycles indicate that nocodazole-induced Bcl-2 phosphorylation is associated with accumulation and arrest of cells in G2–M phase that can be abrogated by JNK inhibitor treatment.
Nocodazole alone and in combination with dexamethasone inhibits multiple myeloma tumor growth in vitro and in vivo
To further investigate the effects of nocodazole, we tested nocodazole in combination with other compounds. As shown in Fig. 5A, combination of nocodazole with lenalidomide, dexamethasone, or a novel histone deacetylase inhibitor inhibitor KD5170 (17) resulted in a significant (P < 0.05) inhibition of proliferation compared with either drug alone. Analysis of nuclear fragmentation as a marker for cell death indicated that dexamethasone (20 nmol/L) alone induced less than 20% cell death. Low concentrations (15 and 30 nmol/L) of nocodazole alone induced 10% and 16% nuclear fragmentation. However, combination of 20 nmol/L dexamethasone with 15 or 30 nmol/L nocodazole significantly increased the percentage of apoptotic cells to 67% and 92%, respectively (P < 0.01; Fig. 5B).
Nocodazole in combination with other agents exhibits significantly stronger antimyeloma effects. A, H929 multiple myeloma cells were treated for 48 hours with lenalidomide, dexamethasone, histone deacetylase inhibitor (HDACi) KD5170 alone or in combination with nocodazole. DNA synthesis was measured by 3H-thymidine incorporation. B, H929 multiple myeloma cells were treated with nocodazole alone or in combination with dexamethasone for 24 hours at the indicated concentrations. Cell death was determined using Hoechst 33258 fluorochrome staining. Results are shown as mean ± SD (n = 3). *, P < 0.05 compared with the respective drug alone use.
Nocodazole in combination with other agents exhibits significantly stronger antimyeloma effects. A, H929 multiple myeloma cells were treated for 48 hours with lenalidomide, dexamethasone, histone deacetylase inhibitor (HDACi) KD5170 alone or in combination with nocodazole. DNA synthesis was measured by 3H-thymidine incorporation. B, H929 multiple myeloma cells were treated with nocodazole alone or in combination with dexamethasone for 24 hours at the indicated concentrations. Cell death was determined using Hoechst 33258 fluorochrome staining. Results are shown as mean ± SD (n = 3). *, P < 0.05 compared with the respective drug alone use.
To assess the antimyeloma effect of nocodazole in vivo, we used the human xenograft mouse model using human multiple myeloma cells (H929). H929 multiple myeloma cells grow vigorously and behave as an aggressive tumor in vivo. To titrate a treatment dosage, we used high (20 mg/kg) and low (5 mg/kg) concentrations of nocodazole without dexamethasone. Nocodazole (20 mg/kg) injected intraperitoneally 3 times weekly showed significant inhibition of tumor growth as early as the tenth day of treatment (P < 0.02) compared with that of the control group (Supplementary Fig. S3). At 20 mg/kg nocodazole, no severe side effects, such as weight loss or diarrhea, were observed but the mice showed mild dry skin with scales. At 5 mg/kg, mice showed no physical changes. For further in vivo testing, an intermediate dose of 12 mg/kg nocodazole was chosen. Because our in vitro data showed that combination of nocodazole and dexamethasone significantly enhances apoptosis of multiple myeloma cells (Fig. 5B), combination of nocodazole and dexamethasone was tested in the abovementioned mouse model. Mice were treated with an intermediate dose of nocodazole (12 mg/kg intraperitoneally 3 times weekly), 2 mg/kg dexamethasone (subcutaneously twice weekly), their combination, or vehicle only. Either drug alone had the tendency to inhibit tumor growth. Only in the combination group, a significant reduction of tumor volume was observed as early as day 8 that persisted for the entire experiment (Fig. 6A). Kaplan–Meier survival analysis indicated that the combination treatment significantly (P < 0.01) prolonged survival time in comparison with vehicle treatment and either drug alone. In the vehicle control group, 100% of the animals had to be sacrificed (endpoint) by day 15 whereas in the combination group, only 20% of mice had reached the endpoint (P < 0.01; Fig. 6B). No adverse toxicity was observed throughout the treatment period for all the treatment groups as monitored by body weight, behavior, and activity of mice.
Nocodazole significantly inhibits tumor growth and prolongs survival in the human xenograft mouse model. H929 multiple myeloma cells mixed with Matrigel were injected subcutaneously into the right flank of beige/nude/X-linked immunodeficient mice. A, once tumors became palpable, mice were treated (intraperitoneally) with vehicle solution, dexamethasone alone (Dex, 2 mg/kg), nocodazole (Noco, 12 mg/kg) alone, or the combination of dexamethasone and nocodazole (n = 5 each group). Results are shown as mean ± SD of tumor volume. *, P < 0.02; **, P < 0.01 compared with vehicle-treated mice. B, survival rate was evaluated using Kaplan–Meier method and log-rank analysis. Combination group (Noco + Dex) showed significantly increased survival (P < 0.01) compared with control.
Nocodazole significantly inhibits tumor growth and prolongs survival in the human xenograft mouse model. H929 multiple myeloma cells mixed with Matrigel were injected subcutaneously into the right flank of beige/nude/X-linked immunodeficient mice. A, once tumors became palpable, mice were treated (intraperitoneally) with vehicle solution, dexamethasone alone (Dex, 2 mg/kg), nocodazole (Noco, 12 mg/kg) alone, or the combination of dexamethasone and nocodazole (n = 5 each group). Results are shown as mean ± SD of tumor volume. *, P < 0.02; **, P < 0.01 compared with vehicle-treated mice. B, survival rate was evaluated using Kaplan–Meier method and log-rank analysis. Combination group (Noco + Dex) showed significantly increased survival (P < 0.01) compared with control.
Discussion
Despite the development of novel therapeutic approaches and combination therapies, multiple myeloma remains an incurable malignancy with a median survival of 3 to 5 years (22). Multiple myeloma is characterized by aberrant proliferation of terminally differentiated plasma cells and the inability to undergo apoptosis. Some benzimidazole compounds were originally approved by Food and Drug Administration as broad-spectrum anthelmintic drugs with known toxicity profiles. Recently Spagnuolo and colleagues described the antimyeloma effect of flubendazole, that also is a benzimidazole (13). Monaghan and colleagues reported that tubulin polymerization inhibitor CYT997 synergizes with bortezomib to produce potent antimyeloma activity (23). Although nocodazole has been categorized as a benzimidazole with antineoplastic activity, its effects on multiple myeloma cells are unknown. In this study, we focused on the therapeutic potential of nocodazole against multiple myeloma in vitro and in vivo. The concentrations we used were in the range of therapeutic achievable levels (24, 25). A study by Woodtli and colleagues revealed that plasma levels of 0.30 μmol/L mebendazole are the optimal therapeutic levels for treatment of inoperable echinococcosis (26). In our studies, the similar concentration of 0.25 μmol/L mebendazole induced apoptosis shown by nuclear fragmentation in 65% of the multiple myeloma cells (Fig. 1B). Recently, we have shown that nocodazole inhibits the production of cytokines essential for multiple myeloma growth and survival (14). Here, we show that nocodazole also inhibits growth and survival of primary multiple myeloma cells and of multiple myeloma cell lines alone and in coculture with bone marrow stromal cells by inducing cell-cycle arrest and subsequent apoptosis. Inhibition of cell proliferation by nocodazole occurred independent from MDR of the multiple myeloma cell lines, suggesting that nocodazole overcomes cell adhesion–mediated resistance and MDR to conventional therapies. In addition, its relative selective cytotoxicity to multiple myeloma cell lines and primary myeloma cells, but not to nonmalignant bone marrow mononuclear cells, suggests a favorable therapeutic index. Our in vitro data on combination studies show that nocodazole with compounds, such as dexamethasone, significantly increases the antimyeloma effect (Fig. 5B), but further studies are needed to explore additive or even synergistic effect. In vivo nocodazole combined with dexamethasone significantly inhibited tumor growth and prolonged survival in a human xenograft myeloma mouse model. Furthermore, our data show that in cycling cells, nocodazole affects the microtubule assembly and causes mitotic arrest, which in turn leads to apoptosis. The higher selectivity of nocodazole toward tumor cells might result from targeting cycling cells compared with the non- or slow-cycling cells. At prometaphase, the phase of mitosis following prophase and preceding metaphase, the nuclear envelope fragments and disappears. The role of prometaphase is completed when all of the microtubules have attached to their kinetochores, at which point metaphase begins (27). In nocodazole-treated multiple myeloma cells, the microtubule fails to attach to the kinetochore and activate the spindle assembly checkpoint. This causes a predominant arrest in prometaphase and cells die after prolonged arrest in mitosis, suggesting that mitotic cell death occurs in nocodazole-treated myeloma cells (28–30). Based on the fact that cells treated with nocodazole arrest with G2 or M phase DNA content, nocodazole is frequently used to synchronize the cell division cycle (24, 31–33), and our findings of myeloma cell death after prolonged arrest of cells in mitotic prometaphase is not unexpected (34).
Further analysis of prodeath BH3-only molecules showed that Bim isoform proteins were increased in both myeloma cell lines and primary CD138+ cells. Bim is usually sequestered in the cytosol by binding with the dynein light chain-1 and is released from the microtubules in response to apoptotic stimuli via microtubular damage (35). This is in accordance with our data indicating that nocodazole-induced microtubular network damage leads to the release of Bim and apoptosis induction. The functional significance of XIAP and Mcl-1L downregulation by nocodazole indicates a link between cell-cycle arrest and cell apoptosis (36, 37). Furthermore, downregulating XIAP has been shown to promote persistent JNK1 activation (38), which may induce Bcl-2 phosphorylation. Our data show that nocodazole-induced death of myeloma cells is associated with JNK activation and Bcl-2 phosphorylation. This is in accordance with data showing that activation of JNK by drug treatment induces Bcl-2 phosphorylation on specific residues including Ser70, which leads to Bcl-2 inactivation and apoptosis (6, 9, 21). Furthermore, it was also suggested that Bcl-2 phosphorylation might represent a preapoptotic phase after microtubule damage and subsequently dephosphorylation initiates apoptosis (39). We found that phosphorylation of Bcl-2 at Ser70, and to a less degree at Thr56, was markedly induced after treatment with nocodazole. Specific inhibition of JNK1/2 prevented nocodazole-induced Bcl-2 phosphorylation (Fig. 4B). In accordance with this, nocodazole-induced myeloma cell apoptosis was also reduced by concomitant treatment with JNK1/2 inhibitor (Fig. 4C). The connection of JNK activation and Bcl-2 phosphorylation was further shown by comparing the overlap between kinetics and dose dependence of these kinase-driven events (Figs. 3 and 4A). Furthermore, it has been shown that JNK-induced Bcl-2 phosphorylation diminished the binding activity of Bcl-2 to both multidomain and BH3-only proapoptotic family members (40, 41), thereby facilitating the proapoptotic activity of these members. Therefore, our results are consistent with findings from other investigators showing that the JNK pathway is responsible for phosphorylation of Bcl-2 in the same loop region in response to microtubule-damaging agents (42, 43).
In summary, our studies show that nocodazole targets the multiple myeloma cell and its microenvironment (14). Nocodazole mediates its antimyeloma activity through sequential microtubular network damage and cell-cycle arrest. JNK-mediated Bcl-2 phosphorylation results in multiple myeloma cell apoptosis. Nocodazole overcomes drug resistance, decreases tumor growth, and extends survival in vivo in human xenograft mice model. The known toxicity profile and selective activity against myeloma cells provide the rationale for considering nocodazole as future treatment for multiple myeloma.
Disclosure of Potential Conflicts of Interest
There are no conflicts of interest to disclose.
Authors' Contributions
R. Feng designed and carried out experiments, analyzed data, and wrote the manuscript; S. Li and C. Lu carried out experiments; C. Andreas conducted bone marrow collection; D.B. Soltz assisted in electron microscopy studies; M.Y. Mapara analyzed data and reviewed the manuscript; and S. Lentzsch designed the experiments, analyzed data, and wrote the manuscript.
Acknowledgments
We would like to thank Dr. Jie Han and E. Michael Meyer for assistance with flow cytometric studies. The authors thank Dr. William Dalton (H. Lee Moffitt Cancer Center & Research Institute, University of South Florida) for providing the human multiple myeloma cell line RPMI-8226/S and its sublines RPMI-8226/Dox40, RPMI-8226/MR20, and RPMI-8226/LR5. Dexamethasone-sensitive and -resistant cell lines MM.1S and MM.1R were obtained from Dr. Klaus Podar (Dana Farber Cancer Institute, Harvard Medical School). We thank Ms. Rita Bhutta for excellent preparation of the manuscript.
Grant Support
The current study was supported in part by research funding from Multiple Myeloma Research Foundation to S. Lentzsch.
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