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Treatment for Malignant Pleural Effusion of Human Lung Adenocarcinoma by Inhibition of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Phosphorylation1

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Treatment for Malignant Pleural Effusion of Human Lung Adenocarcinoma by Inhibition of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Phosphorylation1 Vol. 6, 957–965, March 2000 Clinical Cancer Research 957 Treatment for Malignant Pleural Effusion of Human Lung Adenocarcinoma by Inhibition of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Phosphorylation1 Seiji Yano, Roy S. Herbst, Hisashi Shinohara, expression of VEGF/VPF protein and mRNA in the lung Barbara Knighton, Corazon D. Bucana, tumors of PC14PE6 cells in vivo. These findings indicate that Jerald J. Killion, Jeanette Wood, and PTK 787 reduced PE formation mainly by inhibiting vascu- 2 lar permeability, suggesting that this VEGF/VPF receptor Isaiah J. Fidler tyrosine kinase inhibitor could be useful for the control of Department of Cancer Biology, The University of Texas M. D. malignant PE. Anderson Cancer Center, Houston, Texas 77030 [S. Y., R. S. H., H. S., B. K., C. D. B., J. J. K., I. J. F.], and Oncology Research , Novartis, Ltd., CH-4002 Basel, Switzerland [J. W.] INTRODUCTION Malignant PE3 is associated with highly symptomatic, ad- vanced-stage lung cancer. Most patients with PE present with ABSTRACT progressive dyspnea, cough, or chest pain that compromises Malignant pleural effusion (PE) is associated with ad- their quality of life (1). Malignant PE is most often caused by vanced human lung cancer. We found recently, using a nude lung adenocarcinoma, because this type often forms a primary mouse model, that vascular endothelial growth factor/vas- tumor in the periphery of the lung and invades the pleural cavity cular permeability factor (VEGF/VPF) is responsible for PE (1). Malignant PE has consistently been shown to indicate a induced by non-small cell human lung carcinoma cells. The poor prognosis in advanced lung cancer patients, being associ- purpose of this study was to determine the therapeutic ated with high morbidity and mortality (2, 3). Previous reports potential of a VEGF/VPF receptor tyrosine kinase phospho- demonstrate that drainage followed by instillation of sclerosing rylation inhibitor, PTK 787, against PE formed by human agents is useful for controlling PE and improving the quality of lung adenocarcinoma (PC14PE6) cells. PTK 787 did not life of patients. However, the efficacy of this treatment is affect the in vitro proliferation of PC14PE6 cells, whereas it variable and does not extend the survival of lung cancer patients specifically inhibited proliferation of human dermal micro- (1, 4). Clearly, a more effective therapy for malignant PE is vascular endothelial cells stimulated by VEGF/VPF. A spe- needed. cific platelet-derived growth factor receptor tyrosine kinase Among the possible targets for PE treatment is VEGF, also inhibitor, CGP57148 (used as a control because PTK 787 called VPF (5), an important multifunctional cytokine that pro- also inhibits platelet-derived growth factor receptor tyrosine motes developmental, physiological, and pathological neovas- kinases), had no effect on proliferation of PC14PE6 or hu- cularization (6–8). VEGF/VPF consists of at least four isoforms man dermal microvascular endothelial cells. i.v. injection of (VEGF121, VEGF165, VEGF189, and VEGF206), arising PC14PE6 cells into nude mice produced lung lesions and a through alternate splicing of mRNA from a single gene (6). It large volume of PE containing a high level of VEGF/VPF. can be produced by various cell types, including many tumor Oral treatment with CGP57148 had no effect on PE or lung cells and activated macrophages (6). VEGF/VPF has been metastasis. In contrast, oral treatment with PTK 787 signif- shown to stimulate the proliferation and migration of endothelial icantly reduced the formation of PE but not the number of cells and to induce the expression of metalloproteinases and lung lesions. Furthermore, treatment with PTK 787 signifi- plasminogen activity by these cells (6, 9–12). The cytokine is cantly suppressed vascular hyperpermeability of PE-bear- also a powerful inducer of vascular hyperpermeability. Through ing mice but did not affect the VEGF/VPF level in PE or this property, the molecule plays a central role in ascites fluid formation by murine tumors and human ovarian cancer cell lines in animal models (13–17). Received 10/7/99; revised 12/9/99; accepted 12/13/99. 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 3 The abbreviations used are: PE, pleural effusion; VEGF/VPF, vascular indicate this fact. endothelial growth factor/vascular permeability factor; Flt-1, fms-like 1 Supported in part by Cancer Center Support Core Grant CA16672 and tyrosine kinase; Flk-1, fetal liver kinase; KDR, kinase insert domain- Grant R35-CA42107 (to I. J. F.) from the National Cancer Institute, containing receptor; FBS, fetal bovine serum; bFGF, basic fibroblast NIH. growth factor; PDGF, platelet-derived growth factor; IL, interleukin; rh, 2 To whom requests for reprints should be addressed, at the Department of recombinant human; ISH, in situ hybridization; IHC, immunohisto- Cancer Biology, Box 173, The University of Texas M. D. Anderson Cancer chemistry; HDMEC, human dermal microvascular endothelial cell; PTK Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792- 787/ZK232394, 1-[4-chloroanilino]-4-[pyridylmethyl] phthalazine di- 8577; Fax: (713) 792-8747; E-mail: [email protected]. hydrochloride. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 958 VEGF Receptor Inhibitor Therapy against PE Recently, we developed a model for PE by human lung supplemented with 10% FBS, sodium pyruvate, nonessential adenocarcinoma cells (PC14 and its highly metastatic variant, amino acids, L-glutamine, 2-fold vitamin solution, and penicil- PC14PE6) in nude and severe combined immunodeficiency lin-streptomycin (Flow Laboratories, Rockville, MD) in a mice (18) and clarified the role of VEGF/VPF in PE formation. 10-cm dish and incubated in 5% CO2-95% air at 37°C. The i.v. injection of PC14 and PC14PE6 cells, expressing high PC14PE6 cells were free of Mycoplasma and pathogenic murine levels of VEGF/VPF, produced lung lesions that in turn pro- viruses (assayed by Microbiological Associates, Bethesda, MD). duced large-volume PE in mice. On the other hand, i.v. or Cultures were maintained for no longer than 6 weeks after intrathoracic injection of H226 cells (VEGF/VPF low-express- recovery from frozen stocks. HDMECs (Cascade Biologicals, ing human lung squamous cell carcinoma) produced lung le- Portland, OR) were cultured in Medium 131 with Microvascular sions without detectable PE. Interestingly, VEGF/VPF gene Growth Supplement (Cascade Biologicals). For proliferation transfection into H226 cells resulted in induction of PE when assays, HDMECs were used at passages 2–5. tumor cells were injected intrathoracicly.4 This evidence sug- Reagents. rh VEGF165, rhbFGF, and antihuman VEGF gests that VEGF/VPF plays a crucial role in PE formation by polyclonal antibody were purchased from R&D Systems (Min- human non-small cell carcinoma cells. neapolis, MN). PTK 787/ZK232394 was discovered and syn- Two VEGF/VPF receptors have been identified: Flt-1 and thesized in the Department of Oncology Research, Novartis the Flk-1/kinase insert domain-containing receptor (KDR), are Pharmaceuticals (Basel, Switzerland) and was profiled in col- high-affinity VEGF/VPF receptors with an extracellular domain laboration with the Institute of Molecular Medicine (Tumor containing seven immunoglobulin-like domains and a split ty- Biology Center, Freiburg, Germany), as well as the Oncology rosine kinase intracellular domain (6). Flk-1 has 85% homology Research Laboratories of Schering AG (Berlin, Germany). The with the human homologue, KDR. Both Flt-1 and Flk-1/KDR studies described in this report were performed with either a have been shown to be important regulatory systems for vascu- dihydrochloride or succinate salt. For in vitro assays, a stock logenesis and physiological angiogenesis (19–24). However, solution of 10 mM of PTK787/ZK 222584 was prepared in the interaction of VEGF/VPF with Flk-1/KDR is thought to be DMSO. This was diluted further in buffer or medium so that the the more important interaction for tumor angiogenesis because it concentration of DMSO in assay systems did not exceed 0.1%. is essential for induction of the full spectrum of VEGF/VPF For in vivo studies, the vehicle for the dihydrochloride salt was functions (6). In fact, many compounds and molecules devel- distilled water. The succinate salt was suspended in vehicle oped to block VEGF/VPF activities mediated by Flk-1/KDR containing 5% DMSO and 0.5% Tween 80 in distilled water have been shown to have antiangiogenic activity in animal (28). The PDGF receptor tyrosine kinase phosphorylation inhib- models (25–27). itor CGP57148 (28, 29) was from Novartis. One such molecule is an inhibitor of tyrosine kinase phos- Cell Proliferation Assay. HDMECs (5 ϫ 103/well) phorylation of Flk-1/KDR and Flt-1, called PTK 787 (28). PTK plated in triplicate in 96-well plates precoated with 1.5% gelatin 787 directly inhibits phosphorylation of the VEGF/VPF receptor were incubated overnight in supplemented M131 medium. tyrosine kinases and suppresses angiogenesis induced by VEGF/ PC14PE6 cells (2 ϫ 103/well) plated in triplicate in 96-well VPF. At slightly higher doses, it also inhibits PDGF receptor plates were incubated in MEM containing 5% FBS. The cells tyrosine kinase phosphorylation (28). It can be given p.o., is were then washed and incubated for 72 h with test samples in well tolerated, and has been demonstrated to inhibit the growth fresh MEM containing 5% FBS. The proliferative activity was of several carcinomas in nude mice (28). determined by the 3-(4,4-dimethylthiazol-2-yl)-2.5-diphenyl- In this study, we examined the therapeutic efficiency of tetrazolium bromide assay using an MR-5000 96-well microtiter PTK 787 against malignant PE caused by human lung adeno- plate reader set at 570 nm (12).
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