Luteinizing Hormone-Releasing Hormone (LHRH)-I Antagonist Cetrorelix Inhibits Myeloma Cell Growth in Vitro and in Vivo

Published OnlineFirst November 9, 2010; DOI: 10.1158/1535-7163.MCT-10-0829

Molecular Cancer Preclinical Development Therapeutics

Luteinizing Hormone-Releasing Hormone (LHRH)-I Antagonist Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

Jianguo Wen1, Yongdong Feng2, Chad C. Bjorklund3, Michael Wang3, Robert Z. Orlowski3,4, Zheng-Zheng Shi5, Bing Liao6, Jacqueline O'Hare1, Youli Zu1,7, Andrew V. Schally8,9, and Chung-Che Chang1,7

Abstract The objective of this study was to determine the effects of an luteinizing hormone-releasing hormone (LHRH)-I antagonist, Cetrorelix, on human multiple myeloma (MM) cells and to elucidate the mechanisms of action. We showed that LHRH-I and LHRHR-I genes were expressed in MM cell lines and primary MM cells. Treatment with Cetrorelix inhibited growth and colony-forming ability of myeloma cells, including cell lines resistant to arsenic trioxide, bortezomib, or lenalidomide. Cetrorelix induced apoptosis in myeloma cells including primary myeloma cells. In addition, Cetrorelix inhibited the growth of human myeloma cells xenografted into mice without any apparent side effects. Cetrorelix downregulated the nuclear factor-kappa B (NF-kB) pathway activity and the expression of cytokines, including interleukin 6, insulin-like growth factor 1, VEGF-A, and stromal-derived factor 1, important for myeloma cell growth and survival in myeloma cells and/or marrow stromal cells from myeloma patients. Cetrorelix decreased the phosphorylation of extracellular signal regulated kinase 1/2 and STAT3 in myeloma cells, two crucial pathways for myeloma cells growth and survival. Moreover, the expression of p21 and p53 was increased, whereas that of antiapoptotic

proteins Bcl-2 and Bcl-xL was reduced by Cetrorelix. Our findings indicate that Cetrorelix induces cytotoxicity in myeloma cells through various mechanisms and provide a rationale for investigating Cetrorelix for the treatment of MM. Mol Cancer Ther; 10(1); 148–58. 2010 AACR.

Introduction refractory or relapsed disease show resistance to these therapies (4, 5). Thus, the development of novel agents to Multiple myeloma (MM) is the second most common treat MM remains an important task. hematologic cancer in the United States, representing We observed in our gene array experiments that RPMI 10% of all hematopoietic malignancies (1). It is incurable, 8226 myeloma cells expressed the luteinizing hormone- although novel approaches such as the use of proteasome releasing hormone (LHRH)-I and LHRHR-I, a finding inhibitor bortezomib, have improved the treatment out- that has not been reported previously (data not shown). come (2, 3). However, the majority of patients with Hypothalamic LHRH is the primary link between the brain and the pituitary in the regulation of gonadal functions and plays a key role in control of vertebrate Authors' Affiliations: 1Department of Pathology, The Methodist Hospital reproduction (6). It has been shown that most vertebrate and The Methodist Hospital Research Institute, Houston, Texas; 2Depart- species express at least 2 forms of LHRH (7). LHRH-I and ment of General Surgery, Tongji Hospital, Tongji Medical College in its cognate receptor, LHRHR-I, have been found in the Huazhong University of Science and Technology, Wuhan, China; Depart- ments of 3Lymphoma and Myeloma, and 4Experimental Therapeutics, The human endometrium, placenta, breast, ovary, testis, and University of Texas M. D. Anderson Cancer Center; Departments of prostate (8–12), as well as several malignant tumors and 5Radiology and 6Neurology, The Methodist Hospital and Research Insti- tute, Houston, Texas; 7Department of Pathology, Weill Medical College of cell lines (10). Cornell University, New York; 8Veterans Affairs Medical Center and South In a number of human cancers, including endometrial, Florida Vetarans Affairs Foundation for Research and Education; and prostatic, colorectal, lung, and ovarian tumors, the pro- 9Department of Pathology and Medicine, Division of Hematology/Oncol- ogy, University of Miami Miller School of Medicine, Miami, Florida liferation of cancer cells can be inhibited by agonistic or antagonistic analogues of LHRH (13–24). Cetrorelix espe- J. Wen and Y. Feng contributed equally to this work. cially, a third-generation LHRH antagonist, has been Corresponding Author: Chung-Che Chang, Department of Pathology, The Methodist Hospital and The Methodist Hospital Research Institute, shown to induce apoptosis, growth inhibition, and cell- 6565 Fannin MS205, Houston, TX 77030. Phone: 7134414458; Fax: cycle arrest of cancer cells (13, 16, 22). Thus, Cetrorelix 7137902681. E-mail: [email protected] decreased the levels of cyclin A and Cdk2 (cyclin-depen- doi: 10.1158/1535-7163.MCT-10-0829 dent kinase) but increased the level of p21 and p53 in 2010 American Association for Cancer Research. epithelial ovarian cancer cells (22). In leiomyoma cells

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Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

treated with Cetrorelix, an augmented expression of Fas, lines were cultured as reported (26). No authentication Fas ligand, Bcl-xL, Bax, and caspase 3 and reduced was done by the authors. Bone marrow aspirates were expression of Bcl-2 have been reported (19). collected from myeloma patients or healthy donors under These findings prompted us to examine cultured protocols approved by the institutional review boards of human MM cells and primary myeloma cells for the The University of Texas M. D. Anderson Cancer Center presence of LHRH-I and LHRHR-I and to determine and The Methodist Hospital Research Institute and the effects of the LHRH antagonist Cetrorelix on cell informed consent was obtained in compliance with the growth. Our work shows that Cetrorelix induces apop- Declaration of Helsinki. Bone marrow mononuclear cells tosis and inhibits the growth of myeloma cells, including were isolated by density gradient centrifugation with some cell lines resistant to arsenic trioxide (ATO), borte- Lymphocyte Separation Medium (MP Biomedicals). þ zomib (BZM), or lenalidomide, in vitro and in vivo. This CD138 plasma cells were isolated with AutoMACS, effect is likely exerted through the suppression of NF-kB using CD138 antibodies conjugated with magnetic beads pathway and expression of several key growth/survival (Miltenyi Biotec). BMSC layers were established from factors of myeloma cells, including IL-6 (interleukin 6), MM patients as described previously (27). Samples were IGF-1 (insulin like growth factor 1), VEGF-A, SDF1-a provided from The Methodist Hospital and the M. D. (stromal-derived factor 1-a), expressed by myeloma cells Anderson Cancer Center Department of Lymphoma and and/or myeloma bone marrow stromal cells (BMSC) Myeloma Tissue Bank. Usage of these samples was from myeloma patients; inhibition of the activation of approved by the institutional review board of The Metho- ERK (extracellular signal regulated kinase) and STAT3 dist Hospital Research Institute. pathways; upregulation of p53 and p21 expression; and k downregulation of Bcl-2 and Bcl-xL expression. Alto- Flow cytometric analysis for phosphorylaiton NF- B gether, these findings support the merit of further eva- p65 (RelA) and quantitative RT-PCR in cocultures luation of LHRH-I antagonists in the treatment of MM. MM BMSCs were harvested and seeded into 12-well plates and incubated overnight. The medium was Materials and Methods exchanged and RPMI 8226 cells were seeded at the top of confluent, nonproliferative monolayer of BMSCs and Reagents and antibodies allowed to adhere for 12 hours. The cultures were then 1 The LHRH-I antagonist Cetrorelix [Ac-D-Nal(2) , D-Phe treated with 5 mmol/L Cetrorelix for 3, 6, or 12 hours. The 2 3 6 10 (4Cl) , D-Pal(3) , D-Cit , D-Ala ]LH-RH [Nal(2) is 3-(2- MM cells were collected with gentle pipetting up and naphthyl)alanine, Pal(3) is 3-(3-pyridyl)alanine, and Cit is down without touching the monolayer and then BMSCs citrulline], was originally synthesized in our laboratory were collected with trypsinization. Phospho-NF-kB p65 (25). Cetrorelix was dissolved in distilled water contain- was tested using methods previously described (26) and ing 5% mannitol (vehicle solution for injection). Antibo- cytokine quantification by quantitative reverse transcrip- dies against phospho-ERK1/2, ERK1/2, phospho- tion PCR (qRT-PCR) was described in the following text. STAT3, STAT3, p21, p27, p53, Mcl-1, Bcl-2, Bcl-xL (Cell Signaling Technology), CD138, LHRHR-I (Abcam), RNA extraction, reverse transcription, and PCR b-actin (Sigma-Aldrich), and Alexa 647–conjugated phos- Total cellular RNA was extracted and cDNA was pho-NF-kB p65 (RelA; Becton Dickinson) were pur- synthesized as previously described (26). For qRT-PCR, chased from the indicated vendors. amplification was conducted using the LightCycler, using FastStart DNA Master SYBR Green (Roche Applied Myeloma cell lines and primary cell cultures Science), and 18S RNA was used as a control to evaluate Dexamethasone-sensitive (MM.1S) and -resistant the expression of the genes listed in text, using the (MM.1R) human MM cell lines were kindly provided following primers: 50-ATCACCAGCCACAGAGATCC- by Dr. Steven Rosen (Northwestern University, Chicago, 30 and 50-CAAGGGGGCCTCTAATTTTC-30 for human IL), whereas RPMI 8226 and doxorubicin-resistant RPMI LHRH-I, 50-CAAGGCTTGAAGCTCTGTCC-30 and 50- 8226-Dox40 cells were gifts from Dr. William Dalton AAGGTCAGAGTGGGGAGGTT-3 for human LHR- (Moffitt Cancer Center, Tampa, FL). ATO-resistant cells HR-I, 50-CTCTGCTGACCCAACAAACAG-30 and 50- (RPMI 8226-ATOR, stably maintained at 0.5 mmol/L TTTTCCGGGATCTCTCCCCAT-30 for human APRIL, ATO), and BZM-resistant cells (RPMI 8226-BZMR, stably 50-GCTACCTGGAGCACCAACTC-30 and 50-GCCACG- maintained at 5 nmol/L BZM) were generated from CAAGTAACACAGAA-30 for human BSF3, 5-TTTATTT- RPMI 8226 in our laboratory by slowly increasing con- CAACAAGCCCACAGG-30 and 50-GCAATACATCT- centrations of ATO or BZM for selection (26). KAS-6/1 IL- CCAGCCTCCTTA-30 for human IGF-1, 50-AGAGC- 6–dependent cells were kindly provided by Dr. Diane CAACGTCAAGCATCT-30 and 50-CTTTAGCTTCGGG- Jelinek (Department of Immunology, Mayo Clinic/Foun- TCAATGC-30 for human SDF1-a,50-CGACTACTACGC- dation, Rochester, MN). Lenalidomide-resistant KAS-6/ CAAGGAGG-30 and 50-CGGAGCTCTGATGTGTTGAA- R10R cells were generated from KAS-6/1 cells by expos- 30 for human TGF-b,50-CCTTGCCTTGCTGCTCT- ing them to slowly increasing drug concentrations and ACCTC-30 and 5-CCACCAGGGTCTCGATTGGAT-30 were propagated in 10 mmol/L lenalidomide. All cell for human VEGF, 50-TACCCCCAGGAGAAGATTCC-30

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and 50-TTTTCTGCCAGTGCCTCTTT-30 for human IL-6, duced cells were purified on the basis of GFP expression and 50-TTCGGAACTGAGGCCATGAT-30 and 50-TTT- by an Aria flow cytometer (BD Biosciences). Then, cells CGCTCTGGTCCGTCTTG-30 for human 18SrRNA. Each were injected intravenously into sixteen 8- to 10-week-old sample was measure in triplicate, and the results were female NOD/SCID Il2rg / mice (Jackson Laboratory). analyzed as previously reported (28). For imaging, each mouse was anesthetized and imaged in a Xenogen/Caliper IVIS 200 optical scanner (Wave- Sequencing and BLAST analysis metrics) at approximately 10 minutes following the i.p. PCR products of LHRH-I and LHRHR-I genes, purified administration of D-luciferin (150 mg/kg). Semiquantita- using QIAquick Gel Extraction Kit (Qiagen), were tive region of interest analysis was conducted with the sequenced at Baylor College of Medicine Sequencing dedicated software Living Image v3.1. The mice were Core Laboratory. These were then submitted through subjected to imaging weekly after injection. As soon as the NCBI server for BLAST analysis to confirm the the imaging study showed tumor growth, the mice were expression of these genes. injected i.p. daily with vehicle (5% mannitol; n ¼ 8 mice) or Cetrorelix (75 mg per mouse per day; n ¼ 8 mice) and Immunohistochemistry imaged every week for 8 weeks to monitor the tumor size. The immunohistochemistry using antibody against The mice were sacrificed when signs of discomfort CD138 and LHRHR-I were done on bone marrow biop- emerged or when the tumor volume exceeded 2.0 cm. sies from a MM patient with previously reported meth- All experiments were conducted in accord with NIH ods (29). guidelines and with approval of The Methodist Hospital Research Institute Committee for the Protection of Ani- Western blotting analysis mals in Research. Cells were treated as specified in the figure legends, collected, and immunoblotted with the antibodies listed Statistical analysis earlier using previously described methods (26). Statistical analysis was conducted with the SPSS 11.5, using t test or 1-way ANOVA. Growth inhibition assay, colony assay, and evaluation of apoptosis Myeloma cells were treated as specified in each figure Results legend. The growth inhibitory effect on MM cell lines was then assessed by MTT assays (Chemicon International) as Expression of LHRH and LHRHR in MM cells lines previously described (26). Colony formation was assayed and plasma cells from myeloma patients or healthy by soft-agar method as shown (26). The induction of donors apoptosis was evaluated using the Annexin V assay Because there is no information on the expression of (BD Pharmingen) as reported previously (26). LHRH and receptor in MM cells, we first determined the expression of LHRH-I and LHRHR-I by RT-PCR in 3 Flow cytometric evaluation of caspase 3 activity myeloma cell lines and samples from myeloma patients. Cells were treated as specified in the figure legends, As presented in Figure 1A, amplified products with the collected, and caspase 3 activity was evaluated by flow predicted size of 139 bp (LHRH-I) and 128 bp (LHRHR-I) cytometry using previously described methods (26). were observed in human MM cell lines RPMI 8226, þ MM.1S, and U266 and in CD138 plasma cells from bone Transient transfection of siRNA marrow samples of healthy donors (NM1–5) and mye- The scrambled siRNA (siSCR; catalogue no. sc-37007) loma patients (MM1–5). Products from RPMI 8226 cDNA and siRNA to knock down LHRHR-I (catalogue no. sc- were sequenced and confirmed by BLAST analysis as a 4002) were obtained from Santa Cruz Biotechnology, part of human LHRH-I and LHRHR-I genes. MM cell lines Inc. RPMI 8226 cells were transfected with siRNA, using Dox40, MM.1R, ATOR, BZMR, KAS-6/1, and KAS-6/ the RNAiFect transfection system (Qiagen) as reported R10R all have LHRH-I and LHRHR-I expression, verified (26). Cells were incubated at 37C for 40 hours before by PCR (data not shown). Western blot also revealed that replating onto 12-well plates. Cells were allowed to the LHRHR-I is expressed in MM cells lines and primary attach overnight (about 8 hours), then treated with myeloma cells (Fig. 1B). The immunohistochemical stain- Cetrorelix for 24 hours, followed by apoptosis and ing showed that LHRHR-I was expressed by the neo- þ Western blot. plastic CD138 plasma cells (Fig. 1C). We next studied by þ qRT-PCR the expression levels of LHRHR-I in CD138 Animal study plasma cells and CD138 cells from bone marrow sam- The pLenti7.3/V5-GFP/luciferase plasmid developed ples of control individuals and myeloma patients. As at our laboratory, along with packaging mix, was trans- shown in Figure 1D, the neoplastic plasma cells showed fected into 293FT with Lipofetamin 2000 according to the the highest level of expression of LHRHR-I, whereas manufacturer’s procedure. RPMI 8226 cells were trans- there were no significant differences among normal þ duced with the collected virus particles and stably trans- CD138 cells and CD138 cells from controls or myeloma

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Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

+ + + + + + + + + + + 8 8 8 8 + + + + 8 8 8 8 8 8 8 3 3 3 3 8 8 8 8 6 3 3 3 3 3 3 3 1 1 1 1 6 3 3 3 3 2 1 1 1 1 1 1 1 D D D 2 1 1 1 1 2 D D D D 2 D D D D D D D 8 C C C CD A ive B S C 8 C C C S C C C C C C C t I 1 I a 1 2 3 4 1 6 1 2 3 4 5 . 1 2 3 4 5 g M M 6 M. M M M M M5 M M M M M M e P P 2 M M M M M M N N N N M M M M M N R M M M M M R M U N LHRHR-I LHRHR-I Actin LHRH-I

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Figure 1. The presence of LHRH receptor mRNA and LHRH receptor proteins in MM cell lines and primary samples from myeloma patients. A, RNA was extracted from RPMI 8226, MM.1S, U266, primary normal CD138þ cells (NM1–5), and MM CD138þ cells (MM1–5) and then LHRH-I and LHRHR-I gene expression was assayed with RT-PCR. PCR products were electrophoresed on a 2% agarose gel. Results are from 1 experiment representative of 3. B, total cell lysates from RPMI 8226, MM.1S, and primary MM CD138þ cells (MM1–5) were immunoblotted with antibodies against the LHRHR-I. C, immunohistochemical staining shows the expression of LHRHR-I in the neoplastic CD138þ cells. D, mRNA levels of LHRHR-I in primary normal CD138þ/ and MM CD138þ/ cells were assayed by qRT-PCR. The results of all samples are shown as individual bars of relative mRNA levels normalized to primary normal 1 (NM1) CD138/ cells. Groups with the same letter indicate no significant differences (P > 0.05).

patients. This raises the possibility of targeting LHRHR-I (1 mmol/L) reduced colony-forming ability of the two for the treatment of myeloma patients. MM cell lines tested.

Cetrorelix decreases MM cell growth and colony- Cetrorelix induces apoptosis in MM cells forming ability The changes of cell growth and viability indicated To investigate the effect of LHRH-I antagonist on earlier suggested the induction of cell death by Cetrorelix myeloma cell growth, we used the Cetrorelix to treat in MM cells. Therefore, we investigated the Cetrorelix- 8 MM cells lines, including the RPMI 8226–derived induced apoptosis with flow cytometry. As shown ATO- or BZM-resistant cells, and KAS-6/1–derived, in Figure 3A, as compared with controls, treatment lenalidomide-resistant myeloma cells generated in our with Cetrorelix for 24 hours significantly increased apop- laboratories. Cetrorelix treatment reduced cell growth tosis in MM cells in a dose-dependent manner. We also after 48 hours in all cell lines. At the concentration of 1 to investigated the effect of Cetrorelix on caspase-mediated 2 mmol/L (a concentration comparable with the pre- apoptosis. Results showed that Cetrorelix significantly viousstudies;ref.16),a20%to50%decreaseofcell induced the activities of caspase 3 in RPMI 8226, MM.1S, growth as compared with the vehicle was seen and a and U266 cells (Fig. 3A). Furthermore, Cetrorelix showed þ maximal response was reached at 4 mmol/L (Fig. 2A). a similar cytotoxic effect in all primary MM CD138 cells Importantly, although RPMI 8226-ATOR cells, RPMI (MM1–5). Of note, there were no significant toxicities to 8226-BZMR, and KAS-6/R10R cells show resistance to CD138 cells (Fig. 3B), suggesting that the toxicity of ATO, BZM, and lenalidomide, respectively (Fig. 2A Cetrorelix is specific to myeloma cells. We next explored inset), they remained sensitive to Cetrorelix treatment. whether this Cetrorelix-induced apoptosis is mediated The growth inhibition was further confirmed by colony through LHRHR-I. Compared with siSCR-transfected formation assays (Fig. 2B), showing that Cetrorelix cells, the apoptosis was largely abrogated by the

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Dox40 MM.1R Downloaded from RPMI 8226 MM.1S A 100 100 100 100 * ** ** 80 80 ** 80 ** 80 ** ** ** ** ** 60 60 ** ** 60 60 ** ** Published OnlineFirstNovember9,2010;DOI:10.1158/1535-7163.MCT-10-0829 40 40 40 40

20 20 20 20 mct.aacrjournals.org Fold change of cell viability change (%) vehicle Fold to Fold change of cell viability to vehicle (%) 0 0 0 0 cell(%) viabilityof vehicle to change Fold 0 1 2 4 8 0 1 2 4 8 of cellviability vehicle (%) change to Fold 0 1 2 4 8 0 1 2 4 8 μ Cetrorelix (μmol/L) μ Cetrorelix (μmol/L) Cetrorelix ( mol/L) 120 Cetrorelix ( mol/L) RPMI 8226 120 KAS-6wt RPMI 8226 BZMR 120 KAS-6/R10R ATOR 100 100 100 80 80 ATOR 80 BZMR 60 KAS-6/1 60 100 100 100 60 40 KAS-6/R10R 40 ** 40 20 20 ** 80 100 20

80 (%) vehicle to viability cell of change Fold ** 80 (%) vehicle to viability cell of change Fold 0 0 0 0.5 1.0 2.0 4.0 8.0 16 0 1.25 2.5 5.0 10 20 *Fold changeof cell viability to vehicle (%) 0 ATO (uM) BZM (nM) 0 0.1* 1 10 100 ** ** ** Lenalidomide (μM) 60 80 ** 60 60 ** ** on September 30, 2021.©2011AmericanAssociation forCancer ** **

** ** ** 40 60 40 40

20 40 20 20 Research.

0 20 Fold change of cell of viabilityvehicle change (%)Fold to 0 0 Fold change of cell viability to vehicle (%) viability to vehicle of cell change Fold Fold change ofFold cell viability to vehicle (%) 0 1 2 4 8 0 1 2 4 8 0 1 2 4 8 μ Cetrorelix (μmol/L) Cetrorelix (μmol/L) Cetrorelix ( mol/L) 0 6/1 cell viabilityFold change (%) of to vehicle 0 1 2 4 8 ATO RPMI8226 BZMR Dox 40 KAS MM.1S MM.1R KAS-6/R10R Cetrorelix (μmol/L) Reduction in cell viability 0.6 0.56 0.5 0.47 0.41 0.35 0.31 0.3 ** 35 Vehicle B Cetrorelix 1 μmol/L 30 ** 25

15 oeua acrTherapeutics Cancer Molecular

Colony number 10

0 Vehicle Cetrorelix 1µmol/L RPMI 8226 MM1.S RPMI 8226

Figure 2. Effects of Cetrorelix on the growth and colony-forming ability of MM cells. A, RPMI 8226, RPMI 8226-Dox40, MM.1S, MM.1R, ATOR, BZMR, KAS-6/1, and KAS-6/R10R cells were cultured in 96-well plate and treated with Cetrorelix (1, 2, 4, and 8 mmol/L). The MTT analysis was done after 48 hours of culture. Inset, RPMI 8226 and ATOR were cultured and treated with ATO (0, 1, 2, 4, 8, and 16 mmol/L); RPMI 8226 and BZMR were cultured and treated with BZM (0, 1.25, 2.5, 5, 10, and 20 nmol/L) to show the resistance to ATO and BZM, respectively; KAS-6 and KAS-6/R10R were cultured and treated with lenalidomide (0, 0.1, 1, 10, and 100 mmol/L) to show the resistance. The MTT analysis was done after 48 hours of culture. Data are mean SD of 3 independent experiments. *, P < 0.05; **, P < 0.01 versus vehicle. The reduction in cell viability (¼ 1 relative viability normalized to vehicle) caused by Cetrorelix (4 mmol/L) in different MM cell lines are shown in the table. B, RPMI 8226 and MM.1S cells were cultured in agar with 1 mmol/L Cetrorelix in a 6-well plate. After 2 weeks, the dishes were stained with methylene blue and colonies were photographed and counted. Data are mean SD of 3 independent experiments. **, P < 0.01 versus vehicle. Representative images of colonies are presented. Published OnlineFirst November 9, 2010; DOI: 10.1158/1535-7163.MCT-10-0829

Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

siSCR LHRHR-I ** A 4 C Vehicle 4 LHRHR-I 7 ** Cetrorelix 5 μmol/L ** Vehicle Actin Cetrorelix 1 μmol/L 6 μ 3 Cetrorelix 5 mol/L ** 3 Cetrorelix 10 μmol/L ** ** 5 siSCR siRNA LHRHR-I 4 ** 2 ** 2 3 ** ** 2 1 1

0 0 Fold change of apoptosis to vehicle 0 Fold change of apoptosis to vehicle

RPMI 8226 MM.1S U266 Caspase 3 activity normalized to vehicle RPMI8226 MM.1S U266 Vehicle Cetrorelix

B 4.0 4.0 Vehicle Cetrorelix 5 μmol/L 4.0 Vehicle 3.5 Vehicle 3.5 μ Cetrorelix 10 μmol/L Cetrorelix 5 mol/L 3.5 Cetrorelix 5 μmol/L μ Cetrorelix 10 mol/L 3.0 Cetrorelix 10 μmol/L 3.0 Patient 2 3.0 2.5 2.5 Patient 1 2.5 Patient 3 2.0 2.0 2.0 1.5 1.5 1.5 1.0 1.0 1.0

0.5 0.5 0.5 Fold change of apoptosis to vehicle of apoptosis Fold change Fold changeapoptosis of tovehicle Fold change of apoptosis to vehicle 0.0 0.0 0.0 + – CD138+ CD138– CD138+ CD138– CD138 CD138 4.0 4.0 Vehicle Vehicle 3.5 μ Cetrorelix 5 mol/L 3.5 Cetrorelix 5 μmol/L μ 3.0 Cetrorelix 10 mol/L μ 3.0 Cetrorelix 10 mol/L 2.5 Patient 4 2.5 Patient 5 2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

Fold change of apo ptosis to vehicle 0.0

Fold change of apoptosis to vehicle 0.0 + – CD138 CD138 CD138+ CD138–

Figure 3. Effects of Cetrorelix on the apoptosis of MM cells. A, left, RPMI 8226, MM.1S, and U226 cells were cultured in 6-well plates and treated with Cetrorelix (1, 5, and 10 mmol/L) for 24 hours and the analysis for apoptosis was done. Right, RPMI 8226, MM.1S, and U226 cells were cultured in 6-well plates and treated with Cetrorelix (5 mmol/L) for 24 hours and the analysis for caspase 3 was done. Data are mean SD of 3 independent experiments. **, P <0.01 versus vehicle. B, CD138þ and CD138 cells from primary MM samples (MM1–5) were cultured in 6-well plate and treated with Cetrorelix (5 and 10 mmol/L) for 24 hours and the analysis for apoptosis was done. C, RPMI 8226 cells were transfected with siSCR or siRNA to knockdown LHRHR-I. Forty hours after transfection, cells were replated and cultured overnight, allowed to attach, and then treated with Cetrorelix for another 24 hours. Cells were collected for apoptosis as well as Western blot to determine the knockdown efficiency. Data are mean SD of 3 independent experiments, normalized to vehicle treatment. **, P < 0.01 versus siSCR control.

LHRHR-I knockdown with siRNA transfection. This the inhibition of tumor growth continued for the 8-week result suggested that the LHRHR-I is necessary for duration of the experiment. The Kaplan–Meier survival Cetrorelix to function in myeloma cells. The remaining curve revealed that there was a significant improvement apoptosis effect may come from the residual LHRHR-I or in overall survival of mice treated with Cetrorelix com- another unknown target receptor (Fig. 3C). pared with vehicle-treated mice (Fig. 4C; P < 0.05).

Cetrorelix inhibits myeloma cell growth in vivo Cetrorelix regulates the expression of cytokines and To show the in vivo activity of Cetrorelix, we next NF-kB pathway treated NOD/SCID Il2rg / mice bearing human mye- To understand the mechanisms involved in the induc- loma RPMI 8226 tumors with Cetrorelix. At a dose of 75 tion of cytotoxicity by Cetrorelix in myeloma cells, we mg Cetrorelix per animal per day, a dose lower than the conducted several studies. First, because several cyto- previous studies (30, 31), bioluminescence imaging began kines expressed by myeloma cells and/or the microen- to show significantly decreased tumor volume with vironment have been shown to be important for myeloma Cetrorelix treatment at 4 weeks (Fig. 4A and B) and cell growth and survival through autocrine and/or

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A 1.5 Vehicle

1.0

Day 0 Day 28 Day 56 x 106

0.5

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photons/cm2/sr B C 400 ** 1.2 6

350 Vehicle Cetrorelix 1.0 300 / sr) 2 250 0.8

200 ** 0.6 150

100 ** 0.4 photons/cm Vehicle 50 ** Survival function ** Cetrorelix Bioluminescence (x 10 0.2 0

-50 0.0 0 102030405060 0 1020304050607080 Days after treatment Days after treatment

Figure 4. Cetrorelix is active in an MM xenograft model. A, NOD/SCID Il2rg/ mice bearing RPMI 8226 tumors were injected i.p. daily with vehicle (n ¼ 8) or Cetrorelix (75 mg/mouse/day; n ¼ 8) for 8 weeks. Tumor volume was monitored with bioluminescence imaging and a representative example from each group is shown. Color gradation scale ranges from purple (low signal; low tumor burden) to red (high signal; high tumor burden). Units are 106 photons/s/cm2/ sr. B, mice receiving Cetrorelix or vehicle were imaged weekly. The images were analyzed using Living Image software, and a region of interest tool was used to measure the fluorescence efficiency. Data are mean of 8 independent experiments. **, P < 0.01 versus vehicle. C, the Kaplan–Meier survival curve for mouse groups that received Cetrorelix (n ¼ 8) or vehicle (n ¼ 8).

paracrine signaling, we evaluated the effects of Cetror- has been shown to induce activation of NF-kB (32), and elix on these cytokines and growth factors. Thus, we many of these cytokines expression are regulated by used a qRT-PCR to assess the effect of Cetrorelix on NF-kB pathway (33, 34), we further examined the effect mRNA expression of IL-6, IGF-1, VEGF-A, A prolifera- of Cetrorelix on NF-kB activity in the MM coculture tion-inducing ligand (APRIL), B-cell–stimulating factor- system. The phosphorylation of p65 (RelA), an indicator 3 (BSF3), SDF1-a,andTGF-b in RPMI 8226 cells and for NF-kB activation, was significantly decreased after 6 BMSCs established from myeloma patients when they hours of Cetrorelix treatment in both MM cells and were cocultured. As shown in Figure 5A, Cetrorelix BMSCs (left and right panels, respectively, Fig. 5C). significantly decreased the mRNA expression for IL-6, This suggests that the decreased NF-kB activity and IGF-1, VEFG-A, APRIL, BSF3, SDF1-a,andTGF-b in the decreased expression of these cytokines. which RPMI 8226 cells. In addition, Cetrorelix also signifi- are important for myeloma cell growth and survival. cantly decreased the expression of mRNA for IL-6, may play a role in Cetrorelix-induced cytotoxicity and VEGF-A, and BSF3 in BMSCs. Because LHRH pathway growth inhibition.

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A B Vehicle RPMI 8226 Vehicle 125 Cetrorelix 125 Cetrorelix BMSCs * ** ** ** ** ** ** * ** *

100 100

75 75

50 50

25 25

0 0 Fold change of gene expression to vehicle (%) vehicle to expression of gene change Fold α β IL-6 IGF-1 VEGF APRIL BSF-3 SDF1α TGFβ expressionFold change of gene to vehicle (%) IL-6 IGF-1 VEGF APRIL BSF-3 SDF1 TGF

C RPMI 8226 BMSCs

Isotype Vehicle Cetrorelix 3 h Counts Cetrorelix 6 h

Phospho-p65 Phosphop-p65

Figure 5. Cetrorelix regulates the expression of cytokines and NF-kB pathway. RPMI 8226 cells cocultured with BMSCs were starved overnight and cultured without cytokine. A, cells were then treated with Cetrorelix (5 mmol/L) for 12 hours. RNA was extracted from RPMI 8226 and BMSCs separately and qRT-PCR was done. Data are mean SD of 3 independent experiments after normalizing the data to the vehicle. *, P <0.05; **, P <0.01 versus vehicle. B, cells were treated with Cetrorelix (5 mmol/L) for 3 or 6 hours and then collected, fixed, permeabilized, and labeled with phycoerythrin-conjugated antibody against phosphorylated form of p65 followed by flow cytometric analysis. The x-axis shows the fluorescence intensity of PE, and the y-axis represents the cell counts. Data are from 3 independent experiments.

Cetrorelix regulates the phosphorylation of STAT3 various pathways important for the survival and growth and ERK: expression of cell-cycle–related and of myeloma cells. Of note, JNK and AKT did not show apoptosis-related proteins significant changes with Cetrorelix treatment (data not We next examined whether Cetrorelix inhibited signal- shown). ing pathways important for myeloma growth and survival, including STAT3, ERK, JNK (c-jun NH, kinase), and Discussion AKT pathways. As shown in Figure 5B, Cetrorelix mark- edly inhibited phosphorylation of STAT3 and ERK. We Various investigators showed that Cetrorelix inhibits in then studied the influence of Cetrorelix on cell-cycle– vivo and in vitro growth of human ovarian, endometrial, related and apoptosis-related protein expression in mye- mammary, and prostatic cancers (13, 15–18, 21–24). Here, loma cells (Fig. 6). Results showed that Cetrorelix we report that Cetrorelix induced cell growth inhibition increased the expression of genes inhibiting cell cycling, and apoptosis in multiple myeloma cells. To the best of p21 and p53, but decreased the expression of antiapopto- our knowledge, our study is the first to show the anti- tic genes, including Bcl-2 and Bcl-xL. These results suggest tumor properties of Cetrorelix in a hematologic malig- that Cetrorelix generates antimyeloma effects through nancy.

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A B p21

p-ERK p27

p53 ERK Bcl-2a p-STAT3 - Bcl xL STAT3 Mcl-1 Actin Actin 0 1 6 12 24 - - Cetrorelix time (h) Cetrorelix + + RPMI 8226 MM.1S

Figure 6. Cetrorelix regulates the phosphorylation of ERK and STAT3; expression of cell-cycle–related and apoptosis-related protein. A, RPMI 8226 cells were starved overnight and cultured without cytokine. Cells were then exposed to Cetrorelix (5 mmol/L) for 0, 1, 6, and 12 hours. Lysates from unstimulated or stimulated myeloma cells were immunoblotted with either anti-phospho–specific ERK antibody and then reprobed with anti-ERK antibody or with anti- phospho–specific STAT3 antibody and reprobed with anti-STAT3 antibody. The membrane was reprobed again with anti-actin antibody as a loading control. Data are from 3 independent experiments. B, RPMI 8226 and MM.1S cells were starved overnight and cultured without cytokines. Cells were then exposed to Cetrorelix (5 mmol/L) for 12 hours. Lysates from unstimulated or stimulated myeloma cells were immunoblotted with anti-p21, anti-p27, anti-p53, anti-Bcl-2, or

anti-Bcl-xL antibody. The membrane was reprobed again with anti-actin antibody as a loading control. Data are from 3 independent experiments.

Our results indicate that the antimyeloma effects of IL-6-induced JAK/STAT3 signaling promotes MM cell Cetrorelix are likely to be mediated at several levels. survival by modulating Bcl-xL and/or Mcl-1 protein First, Cetrorelix is capable of regulating the NF-kB levels (38–42). Of note, it has been shown that engage- activity and expression of several key cytokines impor- ment of the LHRHR by LHRH initiates a complex series tant for survival and growth of myeloma cells and of signaling events that include the activation of ERK1/ in BMSCs. It has been shown that adhesion of 2 (43). Therefore, the abrogation of ERK and STAT3 multiple myeloma cells to BMSCs triggers the NF- activation may contribute to the growth inhibition and kB–dependent transcription and secretion of cytokines apoptosis induced by Cetrorelix in myeloma cells. This such as IL-6, VEGF, IGF-1, SDF1-a, BAFF, APRIL, HGF, phenomenon is further supported by our finding that and TNF-a (tumor necrosis factor a) in BMSCs (33, 34). Cetrorelix decreased the expression of STAT3-regulated These cytokines then enhance the growth, survival, antiapoptoic proteins, Bcl-2 and Bcl-xL,consistentwith drug resistance, and migration of myeloma cells (33, the results by Kwon et al. (19). However, effects of 34). In addition, myeloma cells localized in the bone Cetrorelix on STAT3 activation have not been pre- marrow milieu can secrete cytokines such as TNF-a, viously evaluated. TGF-b, and VEGF, which further augment secretion of Our findings suggest that the cell-cycle regulatory IL-6 from BMSCs, forming a positive feedback loop genes p53 and p21 play significant roles in Cetrorelix- between myeloma cells and BMSCs to support their induced growth inhibition. p53 is known to induce cell- survival and growth (34–37). IL-6 has been shown to be cycle arrest and apoptosis. p21 is an inhibitor of cyclin- one of the most important factors for myeloma cell dependent kinases and plays a critical role in controlling growth and survival. In the current study, we showed the cell cycle. It is known that p21 can be upregulated by that Cetrorelix decreased the mRNA expression of IL-6, both p53-dependent and p53-independent pathways (22). VEGF, and BSF3 in both myeloma cells and BMSCs. In our study, the expression of p53 protein was increased Cetrorelix also downregulated the mRNA expression in association with the increase of p21 protein, suggesting of IGF-1, APRIL, SDF1-a,andTGF-b in myeloma that Cetrorelix-induced upregulation of p21 may be cells. Previous studies on the anticancer activities of mediated by p53. Although RPMI 8226 is regarded as Cetrorelix have not evaluated the effect of Cetrorelix in a mutant p53-expressing cell line, RPMI 8226 cells still regulating expression of these cytokines, except for produce small amounts of wild-type p53 with correct IGF-1. conformation (44, 45). It has been reported that IL-6 induces proliferation of Our results revealed that the LHRH-I and LHRHR-I MM cells through the activation of the Ras/Raf/MEK were expressed in myeloma cells lines and the expression (MAP/ERK kinase)/ERK signaling pathway whereas levels of LHRHR-I were upregulated in the neoplastic

156 Mol Cancer Ther; 10(1) January 2011 Molecular Cancer Therapeutics

Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

myeloma cells from patients compared with normal trials, are warranted to evaluate the possible benefits plasma cells from controls (Fig. 1). These results agree of using Cetrorelix in the treatment of myeloma with those of gene expression profiling study database patients. from Multiple Myeloma Genomics Portal (http://www. broadinstitute.org/mmgp/home). In that database, LHRH-I and LHRHR-I are expressed in neoplastic Disclosure of Potential Conflicts of Interest plasma cells isolated from all patients of MGUS (mono- Dr. Andrew V. Schally is a co-inventor on the patent for LHRH clonal gammopathy of undetermined significance), smol- antagonist Cetrorelix, which is assigned to Tulane University School of dering myeloma, and MM of different prognostic Medicine. subtypes. Although we do not observe significantly different expressions of these genes among these groups of Acknowledgments plasma cell dyscrasias, the wide expression of LHRHR-I We thank the Multiple Myeloma Tissue Bank at the Department of in myeloma patients suggests that targeting these mole- Lymphoma and Myeloma of The M. D. Anderson Cancer Center for cules may represent a treatment option for most of providing the myeloma samples for this study. myeloma patients. In conclusion, our results show for the first time that Grant Support Cetrorelix significantly suppressed growth of multiple myeloma cells in vitro and in vivo through various This work was supported by the Medical Research Service of the Veterans mechanisms. In addition, we found that RPMI 8226– Affairs Department and South Florida Veterans Affairs Foundation for Research and Education and the University of Miami Miller School of Medicine Depart- derived, ATO- and BZM-resistant cells are sensitive to ments of Pathology and Medicine, Division of Hematology/Oncology (A.V. Cetrorelix treatment. The proteasome inhibitor BZM Schally) and The Methodist Hospital Research Institute Scholar Grant (C.-C. Chang). and ATO are recent additions to the MM treatment The costs of publication of this article were defrayed in part by the armamentarium, and both drugs show significant payment of page charges. This article must therefore be hereby marked beneficial effects in myeloma treatment (46, 47). How- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ever, more than 50% of patients with refractory or relapsed diseases show resistance to BZM or ATO Received September 2, 2010; revised October 15, 2010; accepted treatment (4, 5). Future studies, including clinical November 1, 2010; published OnlineFirst November 9, 2010

References 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 12. Palmon A, Ben Aroya N, Tel-Or S, Burstein Y, Fridkin M, Koch Y. The 2007. CA Cancer J Clin 2007;57:43–66. gene for the neuropeptide gonadotropin-releasing hormone is 2. Cavo M. Proteasome inhibitor bortezomib for the treatment of multiple expressed in the mammary gland of lactating rats. Proc Natl Acad myeloma. Leukemia 2006;20:1341–52. Sci USA 1994;91:4994–6. 3. Nencioni A, Grunebach F, Patrone F, Ballestrero A, Brossart P. 13. Chen W, Yoshida S, Ohara N, Matsuo H, Morizane M, Maruo T. Proteasome inhibitors: antitumor effects and beyond. Leukemia Gonadotropin-releasing hormone antagonist Cetrorelix down-regu- 2007;21:30–6. lates proliferating cell nuclear antigen and epidermal growth factor 4. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of expression and up-regulates apoptosis in association with enhanced bortezomib in relapsed, refractory myeloma. N Engl J Med 2003;348: poly(adenosine 5’-diphosphate-ribose) polymerase expression in cul- 2609–17. tured human leiomyoma cells. J Clin Endocrinol Metab 2005;90:884– 5. Munshi NC, Tricot G, Desikan R, et al. Clinical activity of arsenic 92. trioxide for the treatment of multiple myeloma. Leukemia 2002;16: 14. Emons G, Ortmann O, Becker M, et al. High affinity binding and direct 1835–7. antiproliferative effects of LHRH analogues in human ovarian cancer 6. Stojilkovic SS, Catt KJ. Expression and signal transduction pathways cell lines. Cancer Res 1993;53:5439–46. of gonadotropin-releasing hormone receptors. Recent Prog Horm Res 15. Emons G, Schally AV. The use of luteinizing hormone releasing 1995;50:161–205. hormone agonists and antagonists in gynaecological cancers. Hum 7. Sherwood NM, Lovejoy DA, Coe IR. Origin of mammalian gonado- Reprod 1994;9:1364–79. tropin-releasing hormones. Endocr Rev 1993;14:241–54. 16. Grundker C, Schlotawa L, Viereck V, et al. Antiproliferative effects of 8. Clayton RN, Catt KJ. Gonadotropin-releasing hormone receptors: the GnRH antagonist Cetrorelix and of GnRH-II on human endometrial characterization, physiological regulation, and relationship to repro- and ovarian cancer cells are not mediated through the GnRH type I ductive function. Endocr Rev 1981;2:186–209. receptor. Eur J Endocrinol 2004;151:141–9. 9. Adelman JP, Mason AJ, Hayflick JS, Seeburg PH. Isolation of the gene 17. Halmos G, Schally AV, Kahan Z. Down-regulation and change in and hypothalamic cDNA for the common precursor of gonadotropin- subcellular distribution of receptors for luteinizing hormone-releasing releasing hormone and prolactin release-inhibiting factor in human hormone in OV-1063 human epithelial ovarian cancers during therapy and rat. Proc Natl Acad Sci U S A 1986;83:179–83. with LH-RH antagonist Cetrorelix. Int J Oncol 2000;17:367–73. 10. Limonta P, Moretti RM, Marelli MM, Motta M. The biology of 18. Kleinman D, Douvdevani A, Schally AV, Levy J, Sharoni Y. Direct gonadotropin hormone-releasing hormone: role in the control of growth inhibition of human endometrial cancer cells by the gonado- tumor growth and progression in humans. Front Neuroendocrinol tropin-releasing hormone antagonist SB-75: role of apoptosis. Am J 2003;24:279–95. Obstet Gynecol 1994 Jan;170:96–102. 11. Oikawa M, Dargan C, Ny T, Hsueh AJ. Expression of gonadotropin- 19. Kwon JY, Park KH, Park YN, Cho NH. Effect of Cetrorelix acetate on releasing hormone and prothymosin-alpha messenger ribonucleic apoptosis and apoptosis regulatory factors in cultured uterine leio- acid in the ovary. Endocrinology 1990;127:2350–6. myoma cells. Fertil Steril 2005;84:1526–8.

www.aacrjournals.org Mol Cancer Ther; 10(1) January 2011 157

Wen et al.

20. Noci I, Coronnello M, Borri P, et al. Inhibitory effect of luteinising 34. Hideshima T, Chauhan D, Schlossman R, Richardson P, Anderson hormone-releasing hormone analogues on human endometrial cancer KC. The role of tumor necrosis factor alpha in the pathophysiology of in vitro. Cancer Lett 2000;150:71–8. human multiple myeloma: therapeutic applications. Oncogene 2001; 21. Schally AV. Luteinizing hormone-releasing hormone analogs: their 20:4519–27. impact on the control of tumorigenesis. Peptides 1999;20:1247–62. 35. Urashima M, Ogata A, Chauhan D, , et al. Transforming growth factor- 22. Tang X, Yano T, Osuga Y, et al. Cellular mechanisms of growth beta1: differential effects on multiple myeloma versus normal B cells. inhibition of human epithelial ovarian cancer cell line by LH-releasing Blood 1996;87:1928–38. hormone antagonist Cetrorelix. J Clin Endocrinol Metab 2002;87: 36. Dankbar B, Padro T, Leo R, et al. Vascular endothelial growth factor 3721–7. and interleukin-6 in paracrine tumor-stromal cell interactions in multi- 23. Yano T, Pinski J, Radulovic S, Schally AV. Inhibition of human ple myeloma. Blood 2000;95:2630–6. epithelial ovarian cancer cell growth in vitro by agonistic and antag- 37. Gupta D, Treon SP, Shima Y, et al. Adherence of multiple myeloma onistic analogues of luteinizing hormone-releasing hormone. Proc cells to bone marrow stromal cells upregulates vascular endothelial Natl Acad Sci USA 1994 Mar 1;91:1701–5. growth factor secretion: therapeutic applications. Leukemia 2001;15: 24. Engel JB, Schally AV. Drug Insight: clinical use of agonists and 1950–61. antagonists of luteinizing-hormone-releasing hormone. Nat Clin Pract 38. Hideshima T, Chauhan D, Hayashi T, et al. Proteasome inhibitor PS- Endocrinol Metab 2007;3:157–67. 341 abrogates IL-6 triggered signaling cascades via caspase-depen- 25. Bajusz S, Csernus VJ, Janaky T, Bokser L, Fekete M, Schally AV. New dent downregulation of gp130 in multiple myeloma. Oncogene antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely 2003;22:8386–93. related analogues. Int J Pept Protein Res 1988;32:425–35. 39. Hideshima T, Nakamura N, Chauhan D, Anderson KC. Biologic 26. Wen J, Cheng HY, Feng Y, et al. P38 MAPK inhibition enhancing ATO- sequelae of interleukin-6 induced PI3-K/Akt signaling in multiple induced cytotoxicity against multiple myeloma cells. Br J Haematol myeloma. Oncogene 2001;20:5991–6000. 2008;140:169–80. 40. Mitsiades CS, Mitsiades N, Poulaki V, et al. Activation of NF-kappaB 27. Stromberg T, Ekman S, Girnita L, et al. IGF-1 receptor tyrosine kinase and upregulation of intracellular anti-apoptotic proteins via the IGF-1/

inhibition by the cyclolignan PPP induces G2/M-phase accumulation Akt signaling in human multiple myeloma cells: therapeutic implica- and apoptosis in multiple myeloma cells. Blood 2006 15;107:669–78. tions. Oncogene 2002;21:5673–83. 28. Pfaffl MW. A new mathematical model for relative quantification in 41. Podar K, Tai YT, Cole CE, et al. Essential role of caveolae in inter- real-time RT-PCR. Nucleic Acids Res 2001;29:e45. leukin-6- and insulin-like growth factor I-triggered Akt-1-mediated 29. Shahjahan M, Dunphy CH, Ewton A, et al. p38 mitogen-activated survival of multiple myeloma cells. J Biol Chem 2003;278:5794– protein kinase has different degrees of activation in myeloproliferative 801. disorders and myelodysplastic syndromes. Am J Clin Pathol 42. Puthier D, Bataille R, Amiot M. IL-6 up-regulates mcl-1 in human 2008;130:635–41. myeloma cells through JAK / STAT rather than ras / MAP kinase 30. Yano T, Pinski J, Halmos G, Szepeshazi K, Groot K, Schally AV. pathway. Eur J Immunol 1999;29:3945–50. Inhibition of growth of OV-1063 human epithelial ovarian cancer 43. Caunt CJ, Finch AR, Sedgley KR, McArdle CA. GnRH receptor xenografts in nude mice by treatment with luteinizing hormone- signalling to ERK: kinetics and compartmentalization. Trends Endo- releasing hormone antagonist SB-75. Proc Natl Acad Sci USA crinol Metab 2006;17:308–13. 1994;91:7090–4. 44. Tai YT, Podar K, Gupta D, et al. CD40 activation induces p53- 31. Jungwirth A, Pinski J, Galvan G, et al. Inhibition of growth of androgen- dependent vascular endothelial growth factor secretion in human independent DU-145 prostate cancer in vivo by luteinising hormone- multiple myeloma cells. Blood 2002;99:1419–27. releasing hormone antagonist Cetrorelix and bombesin antagonists 45. Teoh G, Tai YT, Urashima M, et al. CD40 activation mediates p53- RC-3940-II and RC-3950-II. Eur J Cancer 1997;33:1141–8. dependent cell cycle regulation in human multiple myeloma cell lines. 32. Grundker C, Schulz K, Gunthert AR, Emons G. Luteinizing hormone- Blood 2000;95:1039–46. releasing hormone induces nuclear factor kappaB-activation and 46. Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high- inhibits apoptosis in ovarian cancer cells. J Clin Endocrinol Metab dose dexamethasone for relapsed multiple myeloma. N Engl J Med 2000;85:3815–20. 2005;352:2487–98. 33. Chauhan D, Uchiyama H, Akbarali Y, et al. Multiple myeloma cell 47. Hussein MA, Saleh M, Ravandi F, Mason J, Rifkin RM, Ellison R. Phase adhesion-induced interleukin-6 expression in bone marrow stromal 2 study of arsenic trioxide in patients with relapsed or refractory cells involves activation of NF-kappa B. Blood 1996;87:1104–12. multiple myeloma. Br J Haematol 2004;125:470–6.

158 Mol Cancer Ther; 10(1) January 2011 Molecular Cancer Therapeutics

Luteinizing Hormone-Releasing Hormone (LHRH)-I Antagonist Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

Jianguo Wen, Yongdong Feng, Chad C. Bjorklund, et al.

Mol Cancer Ther 2011;10:148-158. Published OnlineFirst November 9, 2010.

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