Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hideshima, T.
Right arrow Articles by Anderson, K. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hideshima, T.
Right arrow Articles by Anderson, K. C.
Clinical Cancer Research Vol. 12, 5887-5894, October 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

MLN120B, a Novel I{kappa}B Kinase ß Inhibitor, Blocks Multiple Myeloma Cell Growth In vitro and In vivo

Teru Hideshima1, Paola Neri1, Pierfranchesco Tassone1, Hiroshi Yasui1, Kenji Ishitsuka1, Noopur Raje1, Dharminder Chauhan1, Klaus Podar1, Constantine Mitsiades1, Lenny Dang2, Nikhil Munshi1, Paul Richardson1, David Schenkein2 and Kenneth C. Anderson1

Authors' Affiliations: 1 Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts and 2 Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Requests for reprints: Kenneth C. Anderson, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: 617-632-2144; Fax: 617-632-2140; E-mail: kenneth_anderson{at}dfci.harvard.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The purpose of this study is to delineate the biological significance of I{kappa}B kinase (IKK) ß inhibition in multiple myeloma cells in the context of bone marrow stromal cells (BMSC) using a novel IKKß inhibitor MLN120B.

Experimental Design: Growth-inhibitory effect of MLN120B in multiple myeloma cells in the presence of cytokines [interleukin-6 (IL-6) and insulin-like growth factor-I (IGF-1)], conventional agents (dexamethasone, melphalan, and doxorubicin), or BMSC was assessed in vitro. In vivo anti-multiple myeloma activity of MLN120B was evaluated in severe combined immunodeficient (SCID)–hu model.

Results: MLN120B inhibits both baseline and tumor necrosis factor-{alpha}–induced nuclear factor-{kappa}B activation, associated with down-regulation of I{kappa}B{alpha} and p65 nuclear factor-{kappa}B phosphorylation. MLN120B triggers 25% to 90% growth inhibition in a dose-dependent fashion in multiple myeloma cell lines and significantly augments tumor necrosis factor-{alpha}–induced cytotoxicity in MM.1S cells. MLN120B augments growth inhibition triggered by doxorubicin and melphalan in both RPMI 8226 and IL-6-dependent INA6 cell lines. Neither IL-6 nor IGF-1 overcomes the growth-inhibitory effect of MLN120B. MLN120B inhibits constitutive IL-6 secretion by BMSCs by 70% to 80% without affecting viability. Importantly, MLN120B almost completely blocks stimulation of MM.1S, U266, and INA6 cell growth, as well as IL-6 secretion from BMSCs, induced by multiple myeloma cell adherence to BMSCs. MLN120B overcomes the protective effect of BMSCs against conventional (dexamethasone) therapy.

Conclusions: Our data show that the novel IKKß inhibitor MLN120B induces growth inhibition of multiple myeloma cells in SCID-hu mouse model. These studies provide the framework for clinical evaluation of MLN120B, alone and in combined therapies, trials of these novel agents to improve patient outcome in multiple myeloma.

Multiple myeloma remains incurable due to both intrinsic and acquired drug resistance (1–3). Importantly, the bone marrow microenvironment induces growth, survival, and drug resistance in multiple myeloma cells via at least two different mechanisms: adhesion of multiple myeloma cells to fibronectin confers cell adhesion–mediated drug resistance (4, 5) and cytokines [i.e., interleukin-6 (IL-6), insulin-like growth factor-I (IGF-I), and vascular endothelial growth factor] in the bone marrow milieu induce mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase /Akt, and/or Janus kinase 2/signal transducers and activators of transcription 3 signaling cascades. Akt signaling has been shown to mediate multiple myeloma cell resistance to conventional therapeutics (6–10).

Nuclear factor-{kappa}B (NF-{kappa}B) is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 subunits; it is constitutively present in the cytosol and inactivated by its association with I{kappa}B family inhibitors (11). I{kappa}B{alpha} therefore has a crucial role in regulating NF-{kappa}B activation. For example, various growth-promoting and/or anti-apoptosis-promoting cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha} refs. 12–14) and IGF-1 (8), trigger I{kappa}B protein phosphorylation in its NH2 terminus by the multisubunit I{kappa}B kinase (IKK) complex followed by NF-{kappa}B activation in multiple myeloma cells. Once phosphorylated, I{kappa}B is targeted for ubiquitination and degradation by the 26S proteasome, allowing translocation of NF-{kappa}B into the nucleus where it binds to specific DNA sequences in the promoters of target genes, thereby stimulating transcription.

Although the precise role of NF-{kappa}B activation in pathogenesis of multiple myeloma has not been fully characterized, we have shown previously that multiple myeloma cell adhesion to bone marrow stromal cells (BMSC) induces NF-{kappa}B-dependent up-regulation of transcription of IL-6 (14, 15). In addition, TNF-{alpha} secreted by multiple myeloma cells activates NF-{kappa}B in BMSCs, thereby directly up-regulating IL-6 transcription and secretion in BMSCs, and also activates NF-{kappa}B in multiple myeloma cells, thereby up-regulating intracellular adhesion molecule-1 (CD54) and vascular cell adhesion molecule-1 (CD106) expression on both multiple myeloma cells and BMSCs, thereby increasing multiple myeloma cell to BMSC binding (12). Because IL-6 is a major growth and survival factor in multiple myeloma cells (16, 17) and adherence of multiple myeloma cells to fibronectin confers resistance to drug-induced apoptosis, specific blockade of NF-{kappa}B signaling represents a novel therapeutic strategy in multiple myeloma.

We and others have shown already that a variety of novel agents with both preclinical and early clinical anti-multiple myeloma activity, including proteasome inhibitor bortezomib (PS-341; ref. 13), thalidomide and immunomodulatory derivatives (18), histone deacetylase inhibitors (19), transforming growth factor-ß inhibitor (20), lysophosphatidic acid acyltransferase-ß inhibitor (21), and 1'-acetoxychavicol acetate (22) inhibit both NF-{kappa}B activation and multiple myeloma cell growth. Importantly, we have also shown that IKKß inhibitor PS-1145 blocks multiple myeloma cell growth in the context of BMSCs, associated with down-regulation of IL-6 secretion from BMSCs (14).

MLN120B is a novel specific and stable small-molecule inhibitor of IKKß (23). In this study, we show that MLN120B, a more selective and stable novel IKKß inhibitor, blocks TNF-{alpha}-induced NF-{kappa}B activation in multiple myeloma cells through inhibition of I{kappa}B{alpha} phosphorylation and degradation of I{kappa}B{alpha}. Importantly, MLN120B inhibits both IL-6 secretion from BMSCs triggered by multiple myeloma cell adhesion and proliferation of multiple myeloma cells adherent to BMSCs. Most importantly, MLN120B inhibits multiple myeloma cell growth in a clinically relevant severe combined immunodeficient (SCID)–hu mouse model. These studies therefore confirm a central role for IKKß in regulating growth and survival of multiple myeloma cells in the bone marrow milieu and further suggest the potential utility of novel therapeutics targeting IKKß or NF-{kappa}B in multiple myeloma.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Multiple myeloma–derived cell lines. Dexamethasone-sensitive (MM.1S) and dexamethasone-resistant (MM.1R) human multiple myeloma cell lines were provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). RPMI 8226 and U266 human multiple myeloma cells were obtained from American Type Culture Collection (Rockville, Maryland). IL-6-dependent INA6 cell line was kindly provided by Dr. Renate Burger (University of Kiel, Kiel, Germany). Melphalan-resistant RPMI-LR5 and doxorubicin-resistant RPMI-Dox40 cell lines were provided by Dr. William Dalton (H. Lee Moffitt Cancer Center, Tampa, FL). All multiple myeloma cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 2 µmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (Life Technologies, Grand Island, NY).

BMSC cultures. Bone marrow specimens were obtained from patients with multiple myeloma. Mononuclear cells separated by Ficoll-Hypaque density sedimentation were used to establish long-term BMSC cultures as described previously (14). When an adherent cell monolayer had developed, cells were harvested in Hank's buffered saline solution containing 0.25% trypsin and 0.02% EDTA, washed, and collected by centrifugation.

Reagents. An IKKß inhibitor MLN120B (C19H15ClN4O2, MW = 367; Millennium Pharmaceuticals, Cambridge, MA) was dissolved in DMSO and stored at –20°C; it was diluted in culture medium immediately before use; MLN120B control medium contained <0.1% DMSO. For the animal studies, MLN120B was dissolved in vehicle (0.5% methylcellulose) at a final concentration of 15 mg/mL. TNF-{alpha}, IL-6, and IGF-I were purchased from R&D Systems (Minneapolis, MN). Dexamethasone, melphalan, and doxorubicin were purchased from Sigma Chemical.

DNA synthesis. Multiple myeloma cells (3 x 104 per well) were incubated in 96-well culture plates (Costar, Cambridge, MA) in the presence of medium, MLN120B, and/or other agents for 72 hours at 37°C. DNA synthesis was measured by [3H]thymidine ([3H]TdR; Perkin-Elmer, Boston, MA) uptake. Cells were pulsed with [3H]TdR (0.5 µCi/well) during the last 8 hours of 48-hour cultures. All experiments were done thrice in quadruplicate.

Cytotoxic assay. The inhibitory effect of MLN120B on multiple myeloma cell growth was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye absorbance as described previously (13). All experiments were done thrice in quadruplicate. Absorbance was measured at 570/630 nm using a spectrophotometer (Molecular Devices Corp., Sunnyvale CA).

Immunoblotting. Multiple myeloma cells were cultured with MLN120B, harvested, washed, and lysed using lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% NP40, 5 mmol/L EDTA, 5 mmol/L NaF, 2 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, 5 µg/mL aprotinin]. Whole-cell lysates were subjected to Western blotting using phosphorylated I{kappa}B{alpha}, I{kappa}B{alpha}, phosphorylated p65 NF-{kappa}B, and p65 NF-{kappa}B antibodies (Cell Signaling, Beverly, MA).

Electrophoretic mobility shift analysis. Electrophoretic mobility shift analyses (EMSA) were carried out as in our previous studies (12, 14). Briefly, multiple myeloma cells were preincubated with MLN120B (1.25-20 µmol/L for 60 minutes) before stimulation with TNF-{alpha} (5 ng/mL) for 20 minutes. Cells were then pelleted, resuspended in 400 µL hypotonic lysis buffer [20 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1 mmol/L EDTA, 0.2% Triton X-100, 1 mmol/L Na3VO4, 5 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, 5 µg/mL aprotinin], and kept on ice for 20 minutes. After centrifugation at 4°C, the nuclear pellet was extracted with 100 µL hypertonic lysis buffer [20 mmol/L HEPES (pH 7.9), 400 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L Na3VO4, 5 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, 5 µg/mL aprotinin] on ice for 20 minutes. After centrifugation at 4°C, the supernatant was collected as nuclear extract. Double-stranded NF-{kappa}B consensus oligonucleotide probe (5'-GGGGACTTTCCC-3'; Santa Cruz Biotechnology, Santa Cruz, CA) was end labeled with [{gamma}-32P]ATP (50 µCi at 222 TBq/mmol/L; Perkin-Elmer). Binding reactions containing 1 ng oligonucleotide and 5 µg nuclear protein were conducted at room temperature for 20 minutes in a total volume of 10 µL binding buffer [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 4% glycerol (v/v), and 0.5 µg poly(deoxyinosinic-deoxycytidylic acid) (Pharmacia, Peapack, NJ)]. The samples were loaded onto a 4% polyacrylamide gel, transferred to Whatman paper (Whatman International, Maidstone, United Kingdom), and visualized by autoradiography.

Effect of MLN120B on paracrine multiple myeloma cell growth in the bone marrow. To evaluate growth stimulation and signaling in multiple myeloma cells adherent to BMSCs, 3 x 104 MM.1S cells were cultured in BMSC-coated 96-well plates for 48 hours in the presence or absence of MLN120B. DNA synthesis was measured as described above. The DuoSet ELISA (R&D Systems) was used to measure IL-6 in supernatants of 48-hour cultures of BMSCs, with or without MM.1S cells, in the presence or absence of MLN120B.

SCID-hu mouse model. Human fetal long bone grafts were implanted into SCID mice (SCID-hu mice) as described previously (24, 25). Approximately 4 weeks following bone implantation, 2.5 x 106 INA6 multiple myeloma cells in 50 µL PBS was injected directly into human bone within SCID-hu hosts. Soluble human IL-6 receptor (shuIL-6R) released from INA6 cells was assessed in mouse sera by ELISA (R&D Systems) as in our prior studies (26). Mice were treated orally with vehicle alone or MLN120B 50 mg/kg (twice daily) for 3 weeks after detection of measurable shuIL-6R in mouse sera.

Statistical analysis. Statistical significance of differences observed in drug-treated versus control cultures was determined using the Wilcoxon signed-ranks test. The minimal level of significance was P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
MLN120B inhibits I{kappa}B{alpha} phosphorylation and NF-{kappa}B activation in multiple myeloma cells. We first examined baseline NF-{kappa}B activation in multiple myeloma cell lines by EMSA. Although there is variation, constitutive NF-{kappa}B activation was observed in all multiple myeloma cell lines (Fig. 1A ). Because IKKß is believed to be upstream kinase, which phosphorylates I{kappa}B{alpha}, we next examined whether MLN120B could sufficiently block phosphorylation of I{kappa}B{alpha}. MLB120B inhibits baseline phosphorylating of I{kappa}B{alpha} in RPMI 8226 and INA6 cells; however, no significant inhibition was observed in MM.1S cells (Fig. 1B). Consistent with inhibitory effect on I{kappa}B{alpha} phosphorylation, NF-{kappa}B inhibition was triggered by MLN120B in RPMI 8226 and INA6 cells in a dose-dependent fashion, whereas no inhibition was recognized in MM.1S cells (Fig. 1C).


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1. MLN120B inhibits I{kappa}B{alpha} phosphorylation and NF-{kappa}B activation in multiple myeloma cells. A, to assess baseline NF-{kappa}B activity, nuclear extracts from MM.1S, MM.1R, RPMI 8226, RPMI-LR5, RPMI-Dox40, U266, and INA6 cells were subjected to EMSA. B, MM.1S, RPMI 8226, and INA6 cells were cultured with MLN120B (5 and 10 µmol/L for 90 minutes), and whole-cell lysates were subjected to Western blot for detection of phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}) and I{kappa}B{alpha}. C, MM.1S, RPMI 8226, and INA6 cells were cultured with MLN120B (5 and 20 µmol/L for 90 minutes), and nuclear extracts were subjected to EMSA. D, MM.1S cells were pretreated with MLN120B (1.25-20 µmol/L for 90 minutes) and then stimulated by TNF-{alpha} [TNF-{alpha} (+); 5 ng/mL for 20 minutes]. The cells were lysed, electrophoresed, and then immunoblotted with anti-phosphorylated I{kappa}B{alpha} and anti-I{kappa}B{alpha} antibodies. E, MM.1S cells were pretreated with MLN120B (5 and 10 µmol/L for 90 minutes) and then stimulated with TNF-{alpha} (5 ng/mL for 20 minutes); nuclear extracts were subjected to EMSA.

 
Because NF-{kappa}B activation requires phosphorylation, ubiquitination, and degradation of I{kappa}B{alpha}, we next examined whether MLN120B could inhibit phosphorylation and degradation of I{kappa}B{alpha} using Western blotting. MM.1S cells pretreated with either DMSO control medium or MLB120B (1.25-20 µmol/L for 90 minutes) were stimulated with TNF-{alpha} (5 ng/mL for 20 minutes). TNF-{alpha}-induced phosphorylation and degradation of I{kappa}B{alpha} were completely abrogated by MLN120B in a dose-dependent fashion. Phosphorylation of p65 NF-{kappa}B induced by TNF-{alpha} was also blocked by MLN120B (Fig. 1D). We next examined whether MLN120B also could block TNF-{alpha}-induced NF-{kappa}B activation in MM.1S cells. As seen in Fig. 1E, MLN120B completely abrogated TNF-{alpha}-induced NF-{kappa}B activation in a dose-dependent fashion in MM.1S cells. No blockade of phosphorylation of extracellular signal-regulated kinase, signal transducers and activators of transcription 3, Akt, p38 mitogen-activated protein kinase, or c-Jun NH2-terminal kinase by MLN120B was observed (data not shown). These results indicate that MLN120B potently blocks phosphorylation and degradation of I{kappa}B{alpha}, thereby blocking NF-{kappa}B activation in multiple myeloma cells.

MLN120B inhibits proliferation of multiple myeloma cell lines. To study the growth-inhibitory effect of MLN120B on multiple myeloma cells, we next did both MTT assay and [3H]TdR uptake in MM.1S, MM.1R, U266, RPMI 8226, RPMI-LR5, RPMI-Dox40, and INA6 cell lines cultured for 72 hours in the presence of MLN120B (2.5-40 µmol/L). Five percent to fifty percent and 18% to 70% inhibition in proliferation was observed at doses >20 µmol/L MLN120B, assessed by MTT assay (Fig. 2A ) and [3H]thymidine uptake (Fig. 2B), respectively. We further examined cell cycle profile by propidium iodide staining using flow cytometry; no accumulation of sub-G0-G1 phase cells was recognized (data not shown), suggesting that MLN120B does not trigger apoptosis.


Figure 2
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. MLN120B inhibits proliferation of multiple myeloma cell lines. MM.1S, MM.1R, RPMI 8226, RPMI-LR5, RPMI-Dox40, U266, and INA6 cells were cultured for 72 hours in the presence of MLN120B (2.5-40 µmol/L). DNA synthesis and cell proliferation were assessed by MTT assay (A) and [3H]thymidine uptake (B), respectively. C, U266 cells were cultured with DMSO control medium or MLN120B (10 µmol/L) for 48 hours in the presence or absence of IL-6 or IGF-I (5 and 10 ng/mL). D, MM.1S cells were culture with DMSO control medium or MLN120B (10 µmol/L) for 24 hours in the presence or absence of 0.25 ng/ml (gray columns) and 0.5 ng/ml (black columns) TNF-{alpha}. Cell growth was assessed by MTT assay. Points, mean of quadruplicate cultures; bars, SD.

 
Because we and others have shown that IL-6 (7, 16, 17, 27, 28) and IGF-I (29–31) are major growth and antiapoptotic factors in multiple myeloma, we next examined whether MLN120B could overcome the growth-stimulatory effects of these cytokines. U266 cells were cultured with 10 µmol/L MLN120B in the presence or absence of IL-6 or IGF-I (5 and 10 ng/mL) for 48 hours. Importantly, MLN120B almost completely abrogated the growth-stimulatory effect of these cytokines (Fig. 2C). TNF-{alpha} is known to stimulate both NF-{kappa}B and cell death signaling cascades. We therefore hypothesized that MLN120B could induce cytotoxicity triggered by TNF-{alpha}, by blocking NF-{kappa}B activity in M cells. When MM.1S cells were cultured with TNF-{alpha} (0.25 and 0.5 ng/mL) in the presence of MLN120B (10 µmol/L) for 48 hours, MLN120B significantly augmented TNF-{alpha}-induced cytotoxicity (Fig. 2D). Similar results were observed in INA6 cells (data not shown). These results show that MLN120B triggers growth inhibition, which is not overcome by growth and antiapoptotic factors (IL-6 or IGF-I).

MLN120B enhances cytotoxicity of conventional (doxorubicin, melphalan, and dexamethasone) agents. We next examine whether MLN120B could enhance cytotoxicity of conventional therapies. RPMI 8226 (Fig. 3A ) and INA6 (Fig. 3B) cells were cultured with doxorubicin (125 and 250 nmol/L), melphalan (5 and 10 µmol/L), or dexamethasone (125 and 250 nmol/L) for 48 hours in the presence (10 µmol/L) or absence of MLN120B. MTT assay at 48 hours revealed that doxorubicin, melphalan, and dexamethasone alone each significantly inhibited RPMI 8226 and INA6 cell growth in a dose-dependent fashion; moreover, MLN120B additively enhanced their growth-inhibitory effects. Importantly, no additive or synergistic effect of MLN120B with these agents was observed in MM.1S cells. This result was consistent with that MLN120B does not inhibit baseline NF-{kappa}B activity in this cell line. Freshly isolated tumor cells from bone marrow aspirates of multiple myeloma patients were also cultured with doxorubicin (250 nmol/L) or dexamethasone (500 nmol/L) in the presence or absence of MLN120B (5-20 µmol/L) for 48 hours. As is true in multiple myeloma cell lines, MLN120B augmented cytotoxicity of doxorubicin and dexamethasone in a dose-dependent fashion in primary tumor cells (Fig. 3C). These results indicate that inhibition of IKK activity enhances conventional agent-triggered cytotoxicity in both multiple myeloma cell lines and primary tumor cells.


Figure 3
View larger version (25K):
[in this window]
[in a new window]
  		Fig. 3. MLN120B enhances cytotoxicity of conventional [dexamethasone (Dex), melphalan (Mel), doxorubicin (Dox)] agents. A, RPMI 8226 and (B) INA6 cells were cultured for 48 hours with control medium (white columns) or 10 µmol/L (black columns) MLN120B in the presence or absence of doxorubicin (125 and 250 nmol/L), melphalan (5 and 10 µmol/L), or dexamethasone (125 and 250 nmol/L). C, freshly isolated tumor cells from multiple myeloma patients (n = 2) were cultured for 48 hours with control medium (white columns) and with 2.5 (light gray columns), 5 (dark gray columns), or 10 (black columns) µmol/L MLN120B in the presence or absence of doxorubicin (250 nmol/L) or dexamethasone (500 nmol/L). Cytotoxicity was assessed by MTT assay; Columns, mean of quadruplicate cultures; bars, SD.

 
MLN120B inhibits paracrine multiple myeloma cell growth associated with down-regulation of IL-6 in the bone marrow microenvironment. We have shown that the bone marrow microenvironment confers growth and drug resistance in multiple myeloma cells (9, 14). We therefore next studied the effect of MLN120B on paracrine multiple myeloma cell growth in the bone marrow milieu. We first examined the direct inhibitory effect of MLN120B on baseline IL-6 secretion from patient BMSCs (n = 3) using ELISA as in our previous studies (14). MLN120B significantly (64-71%; P < 0.01) down-regulated IL-6 secretion from BMSCs in a dose-dependent fashion (Fig. 4A ). Furthermore, MLN120B also significantly (P < 0.01) inhibited TNF-{alpha}-induced up-regulation of IL-6 secretion from BMSCs (Fig. 4B). Because we have shown that IKK inhibitor PS-1145 inhibits multiple myeloma cell growth in the presence of BMSCs associated with down-regulation of IL-6 (14), we similarly examined growth-inhibitory effect of MLN120B on multiple myeloma cells in the presence of BMSCs. Multiple myeloma cell lines (MM.1S and INA6) were cultured with MLN120B (2.5-10 µmol/L) for 48 hours in the presence or absence of BMSCs. Adhesion of MM.1S (Fig. 4C) and INA6 (Fig. 4D) cells enhanced (1.7- to 2-fold; P < 0.01) IL-6 secretion from BMSCs; however, MLN120B (10 µmol/L) significantly (P < 0.01) inhibited this effect. Importantly, proliferation of MM.1S (3-fold; Fig. 4E) and INA6 (3.8-fold; Fig. 4F) triggered by adherence to BMSCs was significantly (P < 0.01) inhibited by MLN120B in a dose-dependent fashion. Because binding of multiple myeloma cells to BMSCs triggers increased secretion of IL-6 in culture supernatants (13, 14), these results are consistent with the observed inability of exogenous IL-6 to overcome the growth-inhibitory effects of MLN120B (Fig. 2).


Figure 4
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4. MLN120B inhibits paracrine multiple myeloma cell growth associated with down-regulation of IL-6 in the bone marrow microenvironment. A, BMSC from three multiple myeloma patients ({diamondsuit}, {blacksquare}, and bullet) were cultured for 48 hours in the presence (5-20 µmol/L) or absence of MLN120B. B, BMSCs were cultured with DMSO control (white columns) or 10 µmol/L (black columns) MLN120B for 24 hours in the presence (0.25 and 0.5 ng/mL) or absence of TNF-{alpha}. MM.1S (C) and INA6 (D) cells were cultured for 24 hours with DMSO control (white columns) or 10 µmol/L (black columns) MLN120B in the presence or absence of BMSCs. IL-6 in culture supernatant was measured by ELISA. MM.1S (E) and INA6 (F) cells were cultured for 48 hours with DMSO control medium (white columns) and with 2.5 (light gray columns), 5 (dark gray columns), or 10 µmol/L (black columns) MLN120B in the presence or absence of BMSCs. DNA synthesis and cell proliferation were assessed by [3H]thymidine uptake.

 
MLN120B abrogates the protective effect of BMSCs against dexamethasone-induced apoptosis. To define the functional sequelae of MLN120B-related NF-{kappa}B blockade in MM.1S cells, we examined its inhibitory effect on growth of multiple myeloma cells, in the presence of dexamethasone as well as with or without BMSCs. Dexamethasone significantly inhibited MM.1S cell growth in a dose-dependent fashion, and BMSCs completely abrogated dexamethasone-induced cytotoxicity. Importantly, MLN120B triggers dose-dependent growth inhibition even in the presence of BMSCs (Fig. 5 ). These data suggest that MLN120B retains their antitumor activity against multiple myeloma cells in the bone marrow milieu.


Figure 5
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5. MLN120B inhibits paracrine multiple myeloma cell growth. MM.1S cells were cultured for 24 hours in BMSC-coated or noncoated plates in control medium (white columns), as well as with 10 µmol/L MLN120B, in the presence of DMSO control medium (white columns) and 125 (gray columns) or 500 nmol/L (black columns) dexamethasone. DNA synthesis was assessed by [3H]thymidine uptake. Columns, mean of quadruplicate cultures; bars, SD.

 
MLN120B inhibits human multiple myeloma cell growth in vivo. Having shown the signaling mechanisms mediating the anti-multiple myeloma effects of MLN120B in vitro, we next determined whether MLN120B mediates anti-human multiple myeloma cell activity in vivo using a novel SCID-hu model, in which multiple myeloma cells grow in vivo in the context of the human bone marrow microenvironment (26, 32). Eight SCID mice were implanted with human fetal bone chips (SCID-hu), into which human IL-6-dependent INA6 cells were directly injected. These mice were treated orally with either MLN120B (50 mg/kg) or vehicle control twice daily for 3 weeks. As seen in Fig. 6 , treatment with MLN120B induced a reduction of shuIL-6R, marker of tumor growth, whereas shuIL-6R continued to increase in mice treated with vehicle control. We also observed a trend toward prolonged survival in animals treated versus control. Mice of the control group have been sacrificed 6 days before the treated group for the occurrence of deaths and paralysis (data not shown). These results confirm activity of MLN120B as an antitumor agent in vivo in this SCID-hu model of human multiple myeloma. Evaluation of terminal bleeds did not reveal any differences in hematologic variables in drug-treated mice versus control mice (data not shown).


Figure 6
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 6. MLN120B inhibits human multiple myeloma cell growth in vivo. CB-17 SCID-hu mice were engrafted with INA6 cells, and antitumor effects of MLN120B (50 mg/kg, twice daily; {circ}) versus vehicle control (bullet) were assessed by weekly measurement of shuIL-6R in mouse sera.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we characterize the biological sequelae of NF-{kappa}B inhibition in multiple myeloma cells in the context of the bone marrow microenvironment. Specific NF-{kappa}B blockade using the IKKß inhibitor MLN120B confirms the role of NF-{kappa}B in growth, survival, and drug resistance in multiple myeloma cells within the bone marrow milieu and suggests the therapeutic benefit of targeting NF-{kappa}B in multiple myeloma. We first determine baseline NF-{kappa}B blocking activity by EMSA in multiple myeloma cell lines, including MM.1S, MM.1R, RPMI 8226, RPMI-LR5, RPMI-Dox40, U266, and INA6 cells. Although some variation is recognized, all multiple myeloma cell lines show baseline NF-{kappa}B activity. For example, the highest and lowest NF-{kappa}B activity is observed in MM.1R and RPMI-LR5 cells, respectively. We next show the inhibitory effect of MLN120B on phosphorylation and degradation of I{kappa}B{alpha} in MM.1S, RPMI 8226, and INA6 cells. Although MLN120 inhibits baseline phosphorylation of I{kappa}B{alpha} and NF-{kappa}B activity in RPMI 8226 and INA6, inhibition of neither I{kappa}B{alpha} phosphorylation nor NF-{kappa}B activity by MLN120B is recognized in MM.1S cells. Importantly, TNF-{alpha}-induced I{kappa}B{alpha} phosphorylation and NF-{kappa}B activation are completely blocked by MLN120B in MM.1S cells. Our results suggest that baseline NF-{kappa}B activity in MM.1S cells may not be mediated via IKKß, whereas, inducible (i.e., TNF-{alpha} stimulation) NF-{kappa}B activity is triggered by IKKß. Based on NF-{kappa}B inhibitory effect, we next examined whether MLN120B inhibits multiple myeloma cell proliferation. MLN120B (20 µmol/L) induced up to 35% and 75% inhibition, assessed by MTT assay and [3H]thymidine uptake, respectively. There is no relationship between baseline level NF-{kappa}B activity and susceptibility to MLN120B in these multiple myeloma cell lines.

Both IL-6 and IGF-I are abundant in the bone marrow microenvironment and promote multiple myeloma cell growth and survival (9, 33); importantly, MLN120B almost completely abrogates the growth-stimulatory effect triggered by these cytokines. Because TNF-{alpha} induces both NF-{kappa}B activation and cytotoxic signaling cascades, we hypothesized that NF-{kappa}B blockade by MNL120B would augment TNF-{alpha}-induced multiple myeloma cell cytotoxicity. As expected, MLN120B significantly augments TNF-{alpha}-induced cytotoxicity, suggesting that NF-{kappa}B mediates protection against TNF-{alpha}-induced apoptosis in multiple myeloma cells. Because NF-{kappa}B also regulates transcription of cell cycle– and apoptosis-regulating proteins (34, 35), we further examined whether MLN120B augments cytotoxicity of conventional anti-multiple myeloma agents. Importantly, MLN120B additively augments doxorubicin-, melphalan-, and dexamethasone-induced cytotoxicity in multiple myeloma cell lines, as well as in freshly isolated tumor cells from multiple myeloma patients, suggesting potential clinical utility of IKKß or NF-{kappa}B inhibitors with other chemotherapeutics in multiple myeloma.

The bone marrow microenvironment plays a crucial role in multiple myeloma cell proliferation, survival, migration, and drug resistance, due to both multiple myeloma cell adherence to BMSCs and the cytokine circuit between multiple myeloma cells and BMSCs (9, 33). For example, IL-6 secretion from BMSCs is significantly enhanced both by multiple myeloma cell adhesion (15, 36) and by cytokines secreted from multiple myeloma cells [i.e., vascular endothelial growth factor (37), TNF-{alpha} (12, 14), and transforming growth factor-ß (20, 38)]. Specifically, IL-6 transcription and secretion from BMSCs is regulated by NF-{kappa}B. Indeed, baseline, TNF-{alpha}-induced, and multiple myeloma cell adhesion–induced IL-6 secretion from BMSCs is significantly blocked by MLN120B. Most importantly, multiple myeloma cell growth stimulation by BMSCs is markedly inhibited by MLN120B, suggesting that multiple myeloma cell growth stimulation by BMSCs is, at least in part, mediated by IKKß activity.

We have shown that dexamethasone-induced apoptosis in multiple myeloma cells is mediated via related adhesion focal tyrosine kinase activation; second mitochondria-derived activator of caspases, but not cytochrome c release from mitochondria; and caspase-9 activation (39–41). Conversely, IL-6 protects against dexamethasone-induced apoptosis via phosphatidylinositol 3-kinase/Akt signaling (6–8) and activation of SH2 domain containing protein tyrosine phosphatase SHP2, thereby blocking related adhesion focal tyrosine kinase activation (39). We therefore examined whether MLN120B could overcome protective effect of BMSCs against dexamethasone-induced apoptosis. Dexamethasone significantly inhibits [3H]thymidine uptake of MM.1S cells in a dose-dependent fashion, which is abrogated in the presence of BMSCs. Importantly MLN120B almost completely abrogates this protective effect of BMSCs against dexamethasone-induced apoptosis, suggesting its utility to either sensitize or overcome resistance to dexamethasone therapy.

Finally, we evaluated the in vivo effects of MLN120B on multiple myeloma cell growth within the human bone marrow milieu using SCID mice implanted with a human fetal bone chip, into which human IL-6-dependent INA6 cells were injected. In our models, tumor growth has been monitored by measurement of serum shuIL-6R levels, released from INA6, progressive increased during the progression of the disease, and predictably detected after 4 weeks from cell injection. Whereas the detection of human {kappa} chain, which is produced in very small amounts when cells are cultured in vitro (42), is associated only with the presence of a high burden of disease and does not represent a sensitive marker of early tumor growth. In this model, we observed a reduction of shuIL-6R levels released from INA6 in mice treated with MLN120B. These results therefore suggest that MLN120B can target both multiple myeloma cells and the bone marrow microenvironment in vivo. In summary, our data show that NF-{kappa}B activation promotes growth, survival, and drug resistance of multiple myeloma cells in the bone marrow microenvironment and provide the framework for clinical trials of novel agents, such as MLN120B, specifically targeting IKKß in multiple myeloma.


   Footnotes
 
Grant support: NIH Specialized Programs of Research Excellence grants IP50 CA10070, PO-1 78378, and RO-1 CA 50947; Doris Duke Distinguished Clinical Research Scientist Award (K.C. Anderson); Multiple Myeloma Research Foundation (T. Hideshima, D. Chauhan, and C. Mitsiades); and Cure for Myeloma Research Fund (K.C. Anderson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: T. Hideshima and P. Neri equally contribute to this study.

Received 11/15/05; revised 7/14/06; accepted 8/ 2/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sonneveld P, Schoester M, de Leeuw K. Clinical modulation of multidrug resistance in multiple myeloma: effects of cyclosporine on resistant tumor cells. J Clin Oncol 1994;12:1584–91.[Abstract/Free Full Text]
  2. Covelli A. Modulation of multidrug resistance (MDR) in hematological malignancies. Ann Oncol 1999;10:53–9.[Abstract/Free Full Text]
  3. Schwarzenbach H. Expression of MDR1/P-glycoprotein, the multidrug resistance protein MRP, the lung-resistance protein LRP in multiple myeloma. Med Oncol 2002;19:87–104.[CrossRef][Medline]
  4. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 1999;93:1658–67.[Abstract/Free Full Text]
  5. Hazlehurst LA, Damiano JS, Buyuksal I, Pledger WJ, Dalton WS. Adhesion to fibronectin via ß1 integrins regulates p27kip1 levels and contributes to cell adhesion mediated drug resistance (CAM-DR). Oncogene 2000;19:4319–27.[CrossRef][Medline]
  6. Tu Y, Gardner A, Lichtenstein A. The phosphatidylinositol 3-kinase/AKT kinase pathway in multiple myeloma plasma cells: roles in cytokine-dependent survival and proliferative responses. Cancer Res 2000;60:6763–70.[Abstract/Free Full Text]
  7. Hideshima T, Nakamura N, Chauhan D, Anderson KC. Biologic sequelae of interleukin-6 induced PI3-K/Akt signaling in multiple myeloma. Oncogene 2001;20:5991–6000.[CrossRef][Medline]
  8. Mitsiades CS, Mitsiades N, Poulaki V, et al. Activation of NF-{kappa}B and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications. Oncogene 2002;21:5673–83.[CrossRef][Medline]
  9. Hideshima T, Anderson KC. Molecular mechanisms of novel therapeutic approaches for multiple myeloma. Nat Rev Cancer 2002;2:927–37.[CrossRef][Medline]
  10. Hideshima T, Bergsagel PL, Kuehl WM, Anderson KC. Advances in biology of multiple myeloma: clinical applications. Blood 2004;104:607–18.[Abstract/Free Full Text]
  11. Baldwin AS, Jr. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 1996;14:649–83.[CrossRef][Medline]
  12. Hideshima T, Chauhan D, Schlossman RL, Richardson PR, Anderson KC. Role of TNF-{alpha} in the pathophysiology of human multiple myeloma: therapeutic applications. Oncogene 2001;20:4519–27.[CrossRef][Medline]
  13. Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res 2001;61:3071–6.[Abstract/Free Full Text]
  14. Hideshima T, Chauhan D, Richardson P, et al. NF-{kappa}B as a therapeutic target in multiple myeloma. J Biol Chem 2002;277:16639–47.[Abstract/Free Full Text]
  15. Chauhan D, Uchiyama H, Akbarali Y, et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-{kappa}B. Blood 1996;87:1104–12.[Abstract/Free Full Text]
  16. Klein B, Zhang XG, Lu XY, Bataille R. Interleukin-6 in human multiple myeloma. Blood 1995;85:863–72.[Free Full Text]
  17. Lichtenstein A, Tu Y, Fady C, Vescio R, Berenson J. Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol 1995;162:248–55.[CrossRef][Medline]
  18. Mitsiades N, Mitsiades CS, Poulaki V, et al. Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma cells: therapeutic implications. Blood 2002;99:4525–30.[Abstract/Free Full Text]
  19. Catley L, Weisberg E, Tai YT, et al. NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma. Blood 2003;102:2615–22.[Abstract/Free Full Text]
  20. Hayashi T, Hideshima T, Nguyen AN, et al. TGF-ß receptor I kinase inhibitor downregulates cytokine secretion and multiple myeloma cell growth in the bone marrow microenvironment. Clin Cancer Res 2004;10:7540–6.[Abstract/Free Full Text]
  21. Hideshima T, Chauhan D, Hayashi T, et al. Anti-tumor activity of lysophosphatidic acid acyltransferase (LPAAT)-ß inhibitors, a novel class of agents, in multiple myeloma. Cancer Res 2003;63:8428–36.[Abstract/Free Full Text]
  22. Ito K, Nakazato T, Xian MJ, et al. 1'-acetoxychavicol acetate is a novel nuclear factor {kappa}B inhibitor with significant activity against multiple myeloma in vitro and in vivo. Cancer Res 2005;65:4417–24.[Abstract/Free Full Text]
  23. Nagashima K, Sasseville VG, Wen D, et al. Rapid TNFR1 dependent lymphocyte depletion in vivo with a selective chemical inhibitor of IKKß. Blood 2006;107:4266–73.[Abstract/Free Full Text]
  24. Urashima M, Chen BP, Chen S, et al. The development of a model for the homing of multiple myeloma cells to human bone marrow. Blood 1997;90:754–65.[Abstract/Free Full Text]
  25. Tassone P, Goldmacher VS, Neri P, et al. Cytotoxic activity of the maytansinoid immunoconjugate B-B4-1 against CD138+ multiple myeloma cells. Blood 2004;104:3688–96.[Abstract/Free Full Text]
  26. Tassone P, Neri P, Carrasco DR, et al. A clinically relevant SCID-hu in vivo model of human multiple myeloma. Blood 2005;106:713–6.[Abstract/Free Full Text]
  27. Chauhan D, Kharbanda S, Ogata A, et al. Interleukin-6 inhibits Fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells. Blood 1997;89:227–34.[Abstract/Free Full Text]
  28. Puthier D, Bataille R, Amiot M. IL-6 up-regulates mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase pathway. Eur J Immunol 1999;29:3945–50.[CrossRef][Medline]
  29. Jourdan M, De Vos J, Mechti N, Klein B. Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-{alpha} and insulin-like growth factor 1. Cell Death Differ 2000;7:1244–52.[CrossRef][Medline]
  30. Ogawa M, Nishiura T, Oritani K, et al. Cytokines prevent dexamethasone-induced apoptosis via the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in a new multiple myeloma cell line. Cancer Res 2000;60:4262–9.[Abstract/Free Full Text]
  31. Mitsiades CS, Mitsiades NS, McMullan CJ, et al. Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer Cell 2004;5:221–30.[CrossRef][Medline]
  32. Tassone P, Neri P, Burger R, et al. Combination therapy with IL-6 receptor super-antagonist Sant7 and dexamethasone induces antitumor effects in a novel SCID-hu in vivo model of human multiple myeloma. Clin Cancer Res 2005;11:4251–8.[Abstract/Free Full Text]
  33. Mitsiades CS, Mitsiades N, Munshi NC, Anderson KC. Focus on multiple myeloma. Cancer Cell 2004;6:439–44.[CrossRef][Medline]
  34. Mitsiades N, Mitsiades CS, Poulaki V, et al. Biologic sequelae of nuclear factor-{kappa}B blockade in multiple myeloma: therapeutic applications. Blood 2002;99:4079–86.[Abstract/Free Full Text]
  35. Nakanishi C, Toi M. Nuclear factor-{kappa}B inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 2005;5:297–309.[CrossRef][Medline]
  36. Uchiyama H, Barut BA, Mohrbacher AF, Chauhan D, Anderson KC. Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates IL-6 secretion. Blood 1993;82:3712–20.[Abstract/Free Full Text]
  37. Dankbar B, Padro T, Leo R, et al. Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma. Blood 2000;95:2630–6.[Abstract/Free Full Text]
  38. Urashima M, Ogata A, Chauhan D, et al. Transforming growth factor b1: Differential effects on multiple myeloma versus normal B cells. Blood 1996;87:1928–38.[Abstract/Free Full Text]
  39. Chauhan D, Hideshima T, Pandey P, et al. RAFTK/PYK2-dependent and independent apoptosis in multiple myeloma cells. Oncogene 1999;18:6733–40.[CrossRef][Medline]
  40. Chauhan D, Hideshima T, Rosen S, Reed JC, Kharbanda S, Anderson KC. Apaf-1/cytochrome c independent and Smac dependent induction of apoptosis in multiple myeloma cells. J Biol Chem 2001;276:24453–6.[Abstract/Free Full Text]
  41. Chauhan D, Anderson KC. Apoptosis in multiple myeloma: therapeutic implications. Apoptosis 2001;6:47–55.[CrossRef][Medline]
  42. Burger R, Guenther A, Bakker F, et al. Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma. Hematol J 2001;2:42–53.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. K. Pandey, B. Sung, A. B. Kunnumakkara, G. Sethi, M. M. Chaturvedi, and B. B. Aggarwal
Berberine Modifies Cysteine 179 of I{kappa}B{alpha} Kinase, Suppresses Nuclear Factor-{kappa}B-Regulated Antiapoptotic Gene Products, and Potentiates Apoptosis
Cancer Res., July 1, 2008; 68(13): 5370 - 5379.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hideshima, T.
Right arrow Articles by Anderson, K. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hideshima, T.
Right arrow Articles by Anderson, K. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation