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Antitumor Effects of Thalidomide Analogs in Human Prostate Cancer Xenografts Implanted in Immunodeficient Mice

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Antitumor Effects of Thalidomide Analogs in Human Prostate Cancer Xenografts Implanted in Immunodeficient Mice 4192 Vol. 10, 4192–4197, June 15, 2004 Clinical Cancer Research Antitumor Effects of Thalidomide Analogs in Human Prostate Cancer Xenografts Implanted in Immunodeficient Mice Sylvia S. W. Ng,1 Gordon R. MacPherson,1 xenografts was significantly decreased by CPS45 and Michael Gu¨tschow,2 Kurt Eger,3 and CPS49. CPS49 also reduced MVD in PC3 xenografts. 1 Conclusions: Thalidomide analogs CPS11 and 49 are William D. Figg promising anti-cancer agents. PDGF signaling pathway may 1 Molecular Pharmacology Section, Cancer Therapeutics Branch, be a potential target for these thalidomide analogs. Detailed Center for Cancer Research, National Cancer Institute, NIH, microarray and functional analyses are under way with the Bethesda, Maryland; 2Pharmaceutical Institute, Poppelsdorf, University of Bonn, Bonn, Germany; and 3Institute of Pharmacy, aim of elucidating the molecular mechanism(s) of action of Pharmaceutical Chemistry, University of Leipzig, Leipzig, Germany these thalidomide analogs. INTRODUCTION ABSTRACT Prostate carcinoma is the most common cancer and the Purpose: Thalidomide has demonstrated clinical activ- second leading cause of cancer death in American men. Current ity in various malignancies including androgen-independent standard therapy for advanced prostate cancer involves andro- prostate cancer. The development of novel thalidomide an- gen ablation using luteinizing hormone-releasing hormone ago- alogs with better activity/toxicity profiles is an ongoing re- nists with or without an androgen antagonist such as flutamide search effort. Our laboratory previously reported the in or bicalutamide. However, the disease eventually becomes an- vitro antiangiogenic activity of the N-substituted thalidomide drogen independent, at which point therapeutic options are analog CPS11 and the tetrafluorinated analogs CPS45 and limited. Novel treatment modalities are required to improve CPS49. The current study evaluated the therapeutic poten- clinical outcome. tial of these analogs in the treatment of prostate cancer in Angiogenesis, the formation of new blood vessels from vivo. preexisting vessels, plays a significant role in solid tumor Experimental Design: Severely combined immunodefi- growth and metastasis (1). In particular, using the transgenic cient mice bearing s.c. human prostate cancer (PC3 or adenocarcinoma of the mouse prostate model, Huss et al. (2) 22Rv1) xenografts were treated with the analogs at their have identified two distinct angiogenic switches in prostate maximum tolerated doses. Tumors were then excised and cancer progression. It seems logical to speculate that inhibition processed for ELISA and CD31 immunostaining to deter- of angiogenesis would represent an effective treatment strategy mine the levels of various angiogenic factors and microvessel for prostate cancer. Thalidomide has been shown to inhibit basic density (MVD), respectively. fibroblast growth factor (bFGF)- and vascular endothelial Results: CPS11, CPS45, and CPS49 induced prominent growth factor (VEGF)-induced angiogenesis (3, 4). The antitu- and modest growth inhibition in PC3 and 22Rv1 tumors, mor activity of thalidomide has also been reported in numerous respectively. Thalidomide had no effect on tumor growth in clinical trials (5–8). In androgen-independent prostate cancer, either xenograft. Vascular endothelial growth factor and thalidomide caused a decrease in serum prostate-specific anti- basic fibroblast growth factor levels were not significantly gen and an improvement of clinical symptoms in 27% of pa- altered by any of the thalidomide analogs or thalidomide in tients (9). The common side effects of thalidomide, including both PC3 and 22Rv1 tumors. CPS45, CPS49, and thalido- dose-dependent somnolence and dizziness as well as peripheral mide significantly reduced PC3 tumor platelet-derived neuropathy (10), have prompted the development of thalidomide growth factor (PDGF)-AA levels by 58–82% (P < 0.05). analogs with better pharmacological profiles. Our laboratory Interestingly, treatment with the analogs and thalidomide previously demonstrated the superior antiangiogenic activity of resulted in differential down-regulation (>1.5-fold) of genes the N-substituted analog CPS11 and the tetrafluorinated analogs encoding PDGF and PDGF receptor isoforms as determined CPS45 and CPS49 in comparison with thalidomide in multiple by DNA microarray analysis. Intratumoral MVD of 22Rv1 in vitro assays (11). The current study investigated the in vivo therapeutic efficacy of these analogs in the treatment of prostate cancer. MATERIALS AND METHODS Received 12/10/03; revised 3/19/04; accepted 3/30/04. The costs of publication of this article were defrayed in part by the Drugs. Thalidomide analogs CPS11, CPS45, and CPS49 payment of page charges. This article must therefore be hereby marked were synthesized by Dr. Kurt Eger and his group at the Univer- advertisement in accordance with 18 U.S.C. Section 1734 solely to sity of Leipzig (Leipzig, Germany). Worldwide patents of these indicate this fact. analogs have been filed by the NIH and licensed to Celgene Requests for reprints: William D. Figg, Molecular Pharmacology Section, National Cancer Institute, NIH, Building 10, Room 5A01, MSC Corp. (Warren, NJ). Their chemical structures have been pub- 1910, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402- lished previously (11). Thalidomide was obtained from Celgene 3622; Fax: (301) 402-8606; E-mail: [email protected]. Corp. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 4193 Fig. 1 Effects of thalidomide and thalidomide analogs on PC3 (A) and 22Rv1 (B) tumor growth and on the body weight of PC3 (C) and 22Rv1 (D) tumor-bearing mice. f, vehicle control; F, CPS11; Œ, CPS45; ࡗ, CPS49; ƒ, thalidomide. Cell Lines. Human prostate cancer cell lines PC3 and and b is the width. Before euthanasia, the mice were anesthe- 22Rv1 and Lewis lung cancer cell line LLC1 were obtained tized with 2% isoflurane. Approximately 800-1000 ␮l of blood from the American Type Culture Collection (Manassas, VA). were drawn by cardiac puncture and collected in EDTA-con- Prostate cancer cells and Lewis lung cancer cells were main- taining microtainer tubes (Becton Dickinson, Franklin Lakes, tained at 37°C and 5% CO2 in RPMI 1640 and DMEM, respec- NJ). Plasma samples were separated by centrifugation and then tively, supplemented with 10% fetal bovine serum and antibi- stored at –80°C for subsequent detection of angiogenic factors. otics (100 units/ml penicillin, 100 ␮g/ml streptomycin, and 0.25 Harvested tumors were snap frozen in OCT (Miles Inc., Elkhart, ␮g/ml amphotericin B). IN) in liquid nitrogen and subsequently processed for immuno- Human Prostate Cancer Xenograft Model. All animal histochemistry. experiments were done in accordance with institutional guide- Measurement of Angiogenic Factors. The Quantikine lines for animal welfare. PC3 (5 ϫ 106) and 22Rv1 (3 ϫ 106) human VEGF, platelet-derived growth factor (PDGF)-AA, and cells were injected s.c. into 5–6-week-old male severely com- bFGF ELISA kits (R&D Systems, Minneapolis, MN) were used bined immunodeficient mice. When tumor volume reached to determine plasma levels of human VEGF, PDGF-AA, and ϳ150–200 mm3, animals were randomized into five groups bFGF, respectively, according to the manufacturer’s instruc- (n ϭ 5 each). Each group was treated with i.p. bolus injections tions. of either the drug vehicle (0.5% carboxymethylcellulose), Immunohistochemistry. Five-␮m-thick sections ob- CPS11 (100 mg/kg), CPS45 (100 mg/kg), CPS49 (12.5 mg/kg), tained from each frozen tumor were stained with H&E for or thalidomide (100 mg/kg) 5 days a week for 4 weeks. These histological examination. For detection of microvessels, sec- doses were the maximum tolerated doses determined in a pre- tions were stained with the polyclonal anti-CD31/PECAM-1 vious study (11). Tumors were measured with a caliper once a antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). week, and their volumes were calculated using the formula Antigens were visualized using the streptavidin-biotin-peroxi- ␲/6 ϫ a ϫ b2, where a is the longest dimension of the tumor, dase method. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. 4194 Thalidomide Analogs in Human Prostate Cancer Quantification of Intratumor Microvessel Density (MVD). Tumor sections stained with anti-CD31/PECAM-1 antibody were examined by light microscopy. Clusters of stained endothelial cells distinct from adjacent microvessels, tumor cells, or other stromal cells were counted as one mi- crovessel. The MVD for each tumor was expressed as the average count of the three most densely stained fields identified using a ϫ40 objective. Five different tumors per vehicle control or treatment group were analyzed. DNA Microarrays. RNA was extracted from PC3 and 22Rv1 tumor xenografts harvested from severely combined immunodeficient mice treated with the vehicle (0.5% carboxym- ethylcellulose), CPS11, CPS45, CPS49, or thalidomide using the RNeasy system (Qiagen, Valencia, CA). Messenger RNAs from each of the vehicle control reference tumors (n ϭ 5) and from each of the drug-treated tumors (n ϭ 5 tumors/treatment group) were converted to cDNA and labeled with cyanine 3-dUTP (Cy3) and cyanine 5-dUTP (Cy5), respectively, using the LabelStar system (Qiagen). Labeled cDNAs from each n ϭ 5 set were pooled, and aliquots were used to interrogate separate
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