Purpose: Follistatin (FST), an inhibitor of activin, regulates a variety of biological functions, including cell proliferation, differentiation, and apoptosis. However, the role of FST in cancer metastasis is still unknown. Previous research established a multiple-organ metastasis model of human small cell lung cancer in natural killer cell^depleted SCID mice. In this model, i.v. inoculated tumor cells produced metastatic colonies in multiple organs including the lung, liver, and bone. The purpose of this study is to determine the role of FST in multiple-organ metastasis using this model. Experimental Design: A human FST gene was transfected into the small cell lung cancer cell lines SBC-3 and SBC-5 and established transfectants secreting biologically active FST. The metastatic potential of the transfectants was evaluated using the metastasis model. Results: FST-gene transfection did not affect the cell proliferation, motility, invasion, or adhesion to endothelial cells in vitro. I.v. inoculated SBC-3 or SBC-5 cells produced metastatic colonies into multiple organs, including the lung, liver, and bone in the natural killer cell^depleted SCID mice. FST transfectants produced significantly fewer metastatic colonies in these organs when compared with their parental cells or vector control clones. Immunohistochemical analyses of the liver metastases revealed that the number of proliferating tumor cells and the tumor-associated microvessel density were significantly less in the lesions produced by FST transfectants. Conclusions: These results suggest that FST plays a critical role in the production of multipleorgan metastasis, predominantly by inhibiting the angiogenesis.This is the first report to show the role of FST in metastases.

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Purpose: Malignant pleural mesothelioma (MPM) is a biologically heterogeneous malignant disease with a poor prognosis. We reported previously that the anti-vascular endothelial growth factor (VEGF) antibody, bevacizumab, effectively inhibited the progression of VEGF-high-producing (but not VEGF-low-producing) MPM cells in orthotopic implantation models, indicating the need for novel therapeutic strategies to improve the poor prognosis of this disease. Therefore, we focused on the multityrosine kinase inhibitor E7080 and assessed its therapeutic efficacy against MPM cells with different proangiogenic cytokine production profiles. Experimental Design: The efficacy of E7080 was assayed in orthotopic implantation of severe combined immunodeficient mouse models with three human MPM cell lines (MSTO-211H, NCI-H290, and Y-MESO-14). Results: With regard to proangiogenic cytokine production profiles, MSTO-211H and Y-MESO-14 cells were MPM cells producing high levels of fibroblast growth factor-2 and VEGF, respectively. NCI-H290 cells produced low levels of fibroblast growth factor-2 and VEGF compared with the other two cell lines. E7080 potently suppressed the phosphorylation of VEGF receptor-2 and FGF receptor 1 and, thus, inhibited proliferation of endothelial cells, but not that of the MPM cell lines, in vitro. Orthotopically inoculated MSTO-211H cells produced only thoracic tumors, whereas NCI-H290 and Y-MESO-14 cells also developed pleural effusions. Treatment with E7080 potently inhibited the progression of these three MPM cell lines and markedly prolonged mouse survival, which was associated with decreased numbers of tumor-associated vessels and proliferating MPM cells in the tumor. Conclusions: These results strongly suggest broad-spectrum activity of E7080 against MPM with different proangiogenic cytokine production profiles in humans. (Clin Cancer Res 2009;15(23):7229-37) Malignant pleural mesothelioma (MPM) is an aggressively growing tumor, which disseminates into the thoracic cavity and frequently produces a malignant pleural effusion (1). More than 60% of patients with MPM present with a pleural effusion associated with breathlessness, often accompanied by chest wall pain, which compromises their quality of life (2). This type of tumor was once considered rare, but its incidence is increasing worldwide, due primarily to exposure to asbestos and possibly to the SV40 tumor virus. MPM is refractory to conventional chemotherapy and radiotherapy, and also has a poor prognosis, with a median survival time from onset of ∼1 year. Although the multitargeted antifolate agent, pemetrexed, in combination with cisplatin, was recently approved as first-line treatment of MPM (3), the overall prognosis of patients with MPM remains very poor, indicating the need for effective novel therapies.Angiogenesis, the formation of new blood vessels to deliver oxygen and nutrients to the expanding tumor mass, is crucial for the growth and metastasis of solid tumors (4). In many types of cancer, including MPM, there is an ...

The luteal phase in the normal human menstrual cycle is known to be about 14 days. The physiological mechanisms that regulate the corpus luteum remain to be clarified, although apoptosis is reported to be involved. This study was undertaken to investigate the regulation of luteal function by gonadotropins, cytokines, and PGs, concentrating attention on the incidence of apoptosis and its molecular mechanisms in cultured human luteinized granulosa cells collected at oocyte pick-up from patients undergoing in vitro fertilization and embryo transfer. Clusters of granulosa cells were pipetted in 0.1% hyaluronidase in phosphate-buffered saline. After cell separation by centrifugation using Ficoll-Paque, 1 x 104 viable cells/mL in RPMI 1640 medium with 10% FCS were used for experimentation. Substances added were FSH (100 ng/mL), hCG (100 ng/mL), LH (100 ng/mL), interleukin-1beta (IL-1beta; 10 ng/mL), transforming growth factor-beta1 (TGFbeta1; 10 ng/mL), macrophage colony-stimulating factor (MCSF; 10 ng/mL), tumor necrosis factor-alpha (TNFalpha; 10 ng/mL), and PGF2alpha (10 ng/mL). After 24-h culture at 37 C under 5% CO2 and air, cells were fixed with 4% neutral buffered formalin and stained with Hoechst 33258. Apoptotic bodies were counted under a fluorescence microscope, and immunostaining was performed using anti-Fas, Fas ligand, Bcl-2, Bax, and p53 antibodies. Incidences of apoptotic bodies in the group without substance addition were 0.7 +/- 0.2% (0 h), 5.9 +/-0.6% (24 h), and 7.9 +/- 1.2% (48 h); spontaneous increase was significant at the latter time points. Defining the incidence at 24 h as 100%, values after treatment were: FSH, 57%; LH, 84%; hCG, 44%; IL-1beta, 76%; TGFbeta1, 52%; M-CSF, 50%; TNFalpha, 177%; and PGF2alpha, 147%. Significant suppression was observed with FSH, hCG, TGFbeta1, and M-CSF (P < 0.01). On the other hand, significant induction occurred with TNFalpha and PGF2alpha (P < 0.01). On immunostaining, the incidence of stained cells with anti-Fas, Fas ligand, Bax, and p53 antibody was increased after 24-h incubation without addition. This was reduced by hCG, TGFbeta1, and M-CSF. No stained cells were observed with anti-Bcl-2 antibody before or after incubation. In conclusion, our results suggest that both gonadotropins (FSH and hCG) and cytokines (TGFbeta1 and M-CSF) may be involved in the support of luteal function via suppression of apoptosis, and that TNFalpha and PGF2alpha may contribute to ovarian dysfunction and/or luteal regression via its induction in human luteinized granulosa cells. Our results also suggest that Fas, Fas ligand, p53, and Bax may play roles in this apoptosis controlled by hCG, TGFbeta1, and M-CSF.

Lung cancer is currently the leading cause of cancer deaths worldwide. Primary lung cancers are classified into two main histological groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC constitutes approximately 85% of all lung cancers and often shows intrinsic multidrug resistance (MDR), whereas SCLC almost always responds well to various anticancer agents. 1) Cisplatin is an established antitumor agent for the treatment of advanced human NSCLC and is employed in cisplatin-based adjuvant chemotherapy. 2,3) Although the development of resistance to cisplatin is one of the major obstacles in the successful treatment of NSCLC, the molecular mechanisms involved remain poorly understood. In order to overcome the problems related to drug resistance and to improve the clinical outcome of patients with NSCLC, the mechanisms of cancer chemoresistance must be more clearly elucidated.Our previous in vitro study using three NSCLC cell lines has demonstrated that cisplatin has triggered apoptosis more easily in the chemosensitive human NSCLC cell line than the chemoresistant cell line, based on biochemical and morphological findings. 4) We have also reported that an increased transcriptional level of constitutive signal transducer and activator of transcription (STAT) 3 may be related to the suppressive regulation of the apoptotic pathway in intrinsically chemoresistant NSCLC cells. 4)However, multiple factors are involved in the chemoresistance of cancer cells, suggesting that other mechanisms or factors may also contribute to the resistance of the NSCLC cell lines. Therefore, it is needed to further study other cellular mechanisms of chemoresistance, such as ATP-binding cassette transporters and lung resistance-related protein (LRP), important in drug disposition and in the development of MDR, together with measuring the concentration of anticancer agent in cancer cells.During the past decade, there have been many studies linking various transporters to MDR both in cell culture and in the clinical setting. Of these proteins, P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and LRP have attracted considerable attention in studies of cancer chemotherapy. In tumor cell lines, MDR is often associated with overexpression of ATP-dependent drug efflux proteins belonging to the superfamily of ATP-binding cassette transporters: the 170-kDa MDR1 and the 190-kDa MRP1.5) These proteins bind to and transport various structurally unrelated compounds to maintain their intracellular concentrations below cytotoxic levels. In addition to an overall decrease in intracellular drug concentration, redistribution of the drug from the nucleus to the cytoplasm has also been implicated in MDR of cancer cells. 6) Recently, another drug resistance-related protein, referred to as LRP, has been identified.7) LRP has been found to be identical to the human major vault protein, which is the major component of vaults.8) Vaults are mainly located in the cytoplasm, and approximately 5% of cellular va...