CD36-mediated activation of endothelial cell apoptosis by an N-terminal recombinant fragment of thrombospondin-2 inhibits breast cancer growth and metastasis in vivo

Thus far the clinical benefits seen in breast cancer patients treated with drugs targeting the vascular endothelial growth factor (VEGF) pathway are only modest. Consequently, additional antiangiogenic approaches for treatment of breast cancer need to be investigated. Thrombospondin-2 (TSP-2) has been shown to inhibit tumor growth and angiogenesis with a greater potency than the related molecule TSP-1. The systemic effects of TSP-2 on tumor metastasis and the underlying molecular mechanisms of the antiangiogenic activity of TSP-2 have remained poorly understood. We generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and the IgG-Fc1 fragment (N-TSP2-Fc) and could demonstrate that the antiangiogenic activity of N-TSP2-Fc is dependent on the CD36 receptor. We found that N-TSP2-Fc inhibited VEGF-induced tube formation of human dermal microvascular endothelial cells (HDMEC) on matrigel in vitro and that concurrent incubation of anti-CD36 antibody with N-TSP2-Fc resulted in tube formation that was comparable to untreated control. N-TSP2-Fc potently induced apoptosis of HDMEC in vitro in a CD36-dependent manner. Moreover, we could demonstrate a CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3 in HDMEC in vitro. Daily intraperitoneal injections of N-TSP2-Fc resulted in a significant inhibition of the growth of human MDA-MB-435 and MDA-MB-231 tumor cells grown in the mammary gland of immunodeficient nude mice and in reduced tumor vascularization. Finally, increased serum concentrations of N-TSP2-Fc significantly inhibited regional metastasis to lymph nodes and distant metastasis to lung as shown by quantitative real-time alu PCR. These results identify N-TSP2-Fc as a potent systemic inhibitor of tumor metastasis and provide strong evidence for an important role of the CD36 receptor in mediating the antiangiogenic activity of TSP-2.     

Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.     

The effect of thrombospondin-1 on breast cancer metastasis

Thrombospondin-1 (TSP-1) has been proposed to have both pro-metastatic and anti-metastatic properties. To elucidate its role in breast cancer metastasis, we compared tumor progression in the polyomavirus middle T antigen (Pyt) transgenic mouse and the TSP-1-null Pyt transgenic mouse. We characterized the tumors in these mice at 45, 60 and 90 days of age. Tumor size, areas of necrosis, macrophage infiltration, levels of active and total TGF-β, vessel morphology, and lung and blood metastasis were measured in these mice. Mammary tumors were larger in the TSP-1-null mouse, and vessels were larger, but fewer in number in these tumors. The level of total TGF-β was significantly higher in the Pyt tumors at 90 days of age. Importantly, significantly fewer metastases were observed in the lungs of the TSP-1-null/Pyt mouse. Primary Pyt tumor cells were more migratory than TSP-1-null Pyt tumor cells on collagen. Treatment of Pyt mice with recombinant proteins that contain the type-1 repeats of TSP-1 resulted in decreased primary tumor growth and metastasis. Sequences that are involved in CD36 binding and those required for TGF-β activation mediated the inhibition of primary tumor growth. Thus, TSP-1 in the mammary tumor microenvironment inhibits angiogenesis and tumor growth, but promotes metastasis to the lung in the Pyt transgenic mouse. The ability of TSP-1 to support metastasis correlates with its ability to promote tumor cell migration.     

Mediation of glucocorticoid receptor function by the activation of latent transforming growth factor beta 1 in MG-63 human osteosarcoma cells

We analyzed glucocortcoid receptor function using ligand binding assays, DNA band-shift analysis and trans-activation of the murine mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase reporter gene in transiently transfected MG-63 human osteosarcoma cells. Dexamethasone increased the distribution of MG-63 cells in the G₁/G₀ phase of the cell cycle, thus decreasing the rate of DNA synthesis and cell growth. Its effect on MG-63 cell growth was neutralized by RU486 and anti-transforming growth factor beta 1 (TGFβ1) antibody. In addition, (i) dexamethasone increased the levels of active TGFβ1 in MG-63-conditioned media without significantly altering the expression of TGFβ1 mRNA in MG-63 cells and (ii) TGFβ1 inhibited proliferation of MG-63 cells. Therefore, we conclude that glucocorticoid receptor function is mediated by the activation of latent-TGFβ1 in MG-63 osteosarcoma cells. © 1995 Wiley-Liss, Inc.     

腺病毒介导VEGF-siRNA对荷人骨肉瘤裸鼠移植瘤生长的影响

目的:观察腺病毒介导的VEGF-siRNA对荷人骨肉瘤细胞株MG63裸鼠移植瘤生长的影响。方法:将Ad-VEGF-siRNA感染MG63,用噻唑蓝(thiazolyl blue,MTT)法检测细胞体外增殖活力,反转录-聚合酶链反应(reverse transeription-poly-merase chain reaction,RT-PCR)法检测VEGF抑制效果。建立裸鼠移植瘤模型,定期瘤内注射Ad-VEGF-siRNA,观察裸鼠致瘤率、肿瘤体积、抑瘤率及免疫组化法检测肿瘤组织中bcl-2的表达。结果:感染Ad-VEGF-siRNA能明显抑制MG63细胞的生长,瘤灶内注射Ad-VEGF-siRNA后裸鼠移植瘤的质量和体积均显著低于对照组(P<0.01)。TUNEL染色显示肿瘤细胞凋亡增加,免疫组化结果提示肿瘤组织中bcl-2表达明显减少(P<0.01)。结论:Ad-VEGF-siRNA可有效而特异地阻断VEGF基因表达,抑制裸鼠移植瘤生长,促进肿瘤细胞的凋亡。     

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.     

Recombinant antisense C-myc adenovirus increase in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin

C-myc is an oncogene with the important role of cell proliferation controller. It has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation and induce metastatic features. Some studies showed that overexpression of c-myc could induce resistance in response to antineoplastic agents. Currently, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin(CDDP). The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Western Blot, MTT assay, RT-PCR, flow cytometry (FCM), and transmission electron microscopy (TEM) were used to study expression of c-myc and caspase-3 protein, tumor cell proliferation in vitro, cell apoptotic morphology and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2.0 x 10(9) pfu/ml. Ad-Asc-myc downregulated the expression of c-myc protein after transfected MG-63 cells for 48 hours, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 hours can inhibited tumor cells proliferation in vitro by 33.4 and 54.2 percent, respectively, which had significant difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). RT-PCR revealed that Ad-Asc-myc downregulated expression of bcl-2 and upregulated expression of Bax, and no appreciable changes were observed in the expression of E2F-1. Detection of caspase-3 protein TEM, and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin. Cell cycle analysis showed that obvious G(2)/M phase arrested in transfected cells. In conclusion, Ad-Asc-myc increased the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.     

TSP-1及TP的表达与骨肉瘤新生血管生成的相关性

目的研究血小板反应蛋白-1(TSP-1)和胸苷磷酸化酶(TP)在骨肉瘤组织中的表达情况,探讨两者的平衡状态在新生血管生成中的作用。方法收集54例骨肉瘤手术切除标本,应用免疫组织化学法检测骨肉瘤组织中TSP-1及TP的表达,CD34单克隆抗体标记血管内皮细胞,计数骨肉瘤微血管密度(microvessel density,MVD)。结果骨肉瘤TSP-1的表达程度与MVD呈负相关(Spearman秩和检验:rs=-0.241,P=0.000);TP的表达与MVD计数呈正相关(Spearman秩和检验:rs=0.511,P=0.000);TSP-1、TP的表达与骨肉瘤临床病理因素无统计学意义(均P0.05),但都与肺转移显著相关(P0.05)。结论 TSP-1及TP在骨肉瘤发生发展中起重要作用,并影响血管形成及肿瘤侵袭性,两者表达的不平衡是骨肉瘤组织中新生血管生成的关键因素之一。     

Halofuginone inhibits tumor growth in the polyoma middle T antigen mouse via a thrombospondin-1 independent mechanism

Halofuginone inhibits fibrosis by decreasing type I collagen synthesis and tumor growth through an anti-angiogenic mechanism. In vitro data suggested that halofuginone inhibits angiogenesis through upregulating thrombospondin-1 (TSP-1) expression and by inhibiting cell proliferation. To determine whether thrombospondin-1 (TSP-1) is necessary for inhibition of tumor growth and angiogenesis by halofuginone, we tested the effect of halofuginone on mammary tumor growth in polyoma middle T antigen, TSP-1 null (TSP-1-/-PyT) transgenic mice. After 30 days of treatment, we found a significant decrease in tumor weight in these mice and the extent of tumor growth inhibition was comparable to that found in TSP-1 expressing PyT mice (TSP-1+/+PyT). However, no significant difference in tumor weight was observed after 60 days of halofuginone treatment between control and treated mice in both genotypes. Interestingly, type I collagen level was lower in the halofuginone treated TSP-1+/+PyT tumors at 30 days, but this was not observed in the TSP-1-/-PyT mice. Levels of type I collagen did not correlate with blood vessel number as a decrease in the number of vessels was observed in the halofuginone treated tumors from both the TSP-1+/+PyT and TSP-1-/-PyT mice as compared to control tumors. Because halofuginone has been shown to inhibit type I collagen synthesis by inhibiting the TGF-beta signaling pathway, we measured Smad 2/3 phosphorylation levels and found that halofuginone inhibited Smad 2/3 phosphorylation in cells derived from TSP-1+/+PyT tumors. We also found that it inhibited Smad 2/3 phosphorylation in cells treated with the TGF-beta activating sequence of TSP-1, TSR2+RFK. Our data demonstrate that halofuginone inhibits mammary tumor growth in a transgenic mouse model via a TSP-1 independent pathway, by decreasing tumor angiogenesis and by inhibiting TGF-beta signaling.     

Upregulation of CENPF is linked to aggressive features of osteosarcoma

Centromere protein F (CENPF) plays a key role in the regulation of the cell cycle. The present study revealed that CENPF was overexpressed in a variety of tumors and associated with the poor prognosis of osteosarcoma. The mRNA expression levels of CENPF were analyzed using the Gene Expression Profiling Interactive Analysis database and the protein levels of CENPF were detected in the specimens from patients with osteosarcoma using immunohistochemistry. Cell proliferation, cell cycle and flow cytometry assays were performed after the transfection of control or CENPF plasmids into osteosarcoma cells. A xenografts assay was used to determine the effects of CENPF on tumor growth in vivo. The results showed that CENPF was upregulated in osteosarcoma tissues and associated with high-grade tumor stage (P=0.023) and intraglandular dissemination (P=0.046). The transfection-induced depletion of CENPF in human osteosarcoma MG-63 and U-2 OS cell lines inhibited cell proliferation, stimulated apoptosis and induced cell cycle arrest. Induced CENPF depletion in MG-63 cells inhibited tumor growth of osteosarcoma cells in mice. These findings suggested that elevated CENPF levels contributed to increased cell proliferation by mediating apoptosis and cell cycle in osteosarcoma. Therefore, CENPF might be a potential biomarker for poor prognosis of osteosarcoma.     

Small interfering RNA-directed knockdown of S100A4 decreases proliferation and invasiveness of osteosarcoma cells

Osteosarcoma is the most common osteogenic malignant tumor characterized by a high level of malignancy, relapse, metastasis and poor prognosis. S100A4 has been implicated in the proliferation, cell cycle progression, and metastasis of many malignant tumors, although the roles of S100A4 in osteosarcoma have not been documented. This study showed that the expression of S100A4 was found in two osteosarcoma cell lines MG-63 and U-2OS, and in 70.7% of osteosarcoma clinical tissues, and the expression was correlated with the expression of CD44V6. In addition, transfection with S100A4 siRNA significantly reduced the proliferation and the invasiveness of MG-63 cells. Furthermore, S100A4 siRNA down-regulated the expression of CD44 and MMP2, suggesting that S100A4 may promote the proliferation, invasion and metastasis of osteosarcoma cells by regulating the expression of other proteins that are crucial in modulating cell–ECM adhesion and facilitating ECM degradation. Therefore, siRNA-directed knockdown of S100A4 may represent a viable clinical therapy for osteosarcoma.     

Meloxicam inhibits osteosarcoma growth, invasiveness and metastasis by COX-2-dependent and independent routes

Cyclooxygenase-2 (COX-2) inhibitors exert antitumor activity via COX-2-dependent and independent pathways. We wished to evaluate the antitumor activity of meloxicam, a preferential COX-2 inhibitor, in osteosarcoma, the most common primary malignant bone tumor, and determine whether its antitumor effect is COX-2-dependent. COX-2 expression in the osteosarcoma cell lines MG-63, HOS and U2-OS was determined by real-time RT-PCR and western blotting. Subsequently, the inhibitory effects of meloxicam on osteosarcoma cell growth and invasiveness were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and matrigel invasion assays, respectively. Apoptotic activity was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining and semi-quantification of Bax and Bcl-2 expression by real time RT-PCR and western blotting. Prostaglandin-E(2) (PGE(2)) production in the presence and absence of meloxicam was analyzed by enzyme immunoassay, and to determine whether the effects of meloxicam are COX-2-dependent or independent, PGE(2) was added to see if it reversed the effects of meloxicam. In addition, the effects of meloxicam on tumor growth and metastasis were evaluated in an in vivo mouse model using grafted LM-8 mouse osteosarcoma cells, together with immunohistochemical analysis for vascular endothelial growth factor in lung metastatic lesion. Meloxicam inhibited PGE(2) production, proliferation and invasiveness especially in MG-63 cells, which express relatively high levels of COX-2. Only high concentrations of meloxicam caused apoptosis and upregulated Bax mRNA and protein in MG-63 cell culture. In contrast, meloxicam did not induce apoptosis in HOS and U2-OS cells, expressing relatively low levels of COX-2. Exogenous PGE(2) reduced the effects of meloxicam on cell viability and invasiveness, but not its effect on Bax mRNA. In vivo, high doses of meloxicam suppressed LM-8 tumor growth and lung metastasis. Meloxicam, may have both COX-2-dependent and independent inhibitory actions on osteosarcoma. Its effects are more prominent in osteosarcoma cells that have relatively high levels of COX-2.     

The application of an anti-angiogenic gene (thrombospondin-1) in the treatment of human prostate cancer xenografts

Angiogenesis is a critical event for solid tumor growth and metastasis. Within a given microenvironment, the angiogenic response is determined in part by the balance between angiogenesis inducers and inhibitors. The aim of this study was to establish a thrombospondin-1 (TSP-1) ( an antiangiogenic gene) expression vector, and to determine the feasibility for use of TSP-1 in prostate cancer gene therapy. The results of this study showed that pCR-TSP-1, the cloned TSP-1 expression plasmid vector, expressed the TSP-1 gene efficiently in DU145, a human prostate cancer cell line. pCR -TSP-1 did not exert any significant growth inhibitory activity on the tested cell line in vitro. However, TSP-1 overexpression inhibited the growth of DU-145 xenografts in Balb/c nude mice when directly transfected with pCR-TSP-1 in combination with a liposomal agent (DOSPER). Histological analysis showed that there were extensive areas of necrosis in the TSP-1 overexpressing tumors, whereas no necrotic foci were observed in the control tumors. Furthermore, the microvessel density was lower in the TSP-1 overexpressing tumors compared to the control tumors. These results suggest that TSP-1 may be a potentially useful gene for prostate cancer gene therapy.     

Kv1.3钾通道在骨肉瘤中的表达及作用研究

目的 探讨特异性短发卡RNA（shRNA）抑制Kv1.3钾通道对骨肉瘤MG-63细胞增殖和凋亡的影响。方法 采用实时荧光定量聚合酶链反应、Western blotting和免疫组化检测骨肉瘤MG-63细胞中Kv1.3钾通道的表达,并分别采用CCK-8法、裸鼠骨肉瘤异种移植模型和流式细胞仪检测MG-63细胞增殖、生长和凋亡的变化,采用Western blotting检测MG-63细胞中多聚ADP核糖聚合酶（PARP）和caspase-3的表达水平。结果Kv1.3钾通道在骨肉瘤细胞中异常高表达。沉默Kv1.3钾通道的Ad5-Kv1.3-shRNA能从体内外有效地抑制MG-63细胞增殖并促进其早期凋亡。沉默Kv1.3钾通道可明显诱导细胞凋亡相关蛋白caspase-3和PARP的裂解。结论Kv1.3钾通道参与骨肉瘤细胞增殖和凋亡的过程,有望成为骨肉瘤治疗及诊断的新靶点基因。     

c-Jun N-terminal kinase activation is required for the inhibition of neovascularization by thrombospondin-1.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.     

仿血管通道结构在大鼠和裸鼠骨肉瘤组织微血管形成中的作用

目的:研究肿瘤仿血管通道在骨肉瘤裸鼠移植瘤组织中的结构特点及其在肿瘤微血管形成中的作用。方法:建立荷人骨肉瘤MG-63细胞裸鼠移植瘤和荷骨肉瘤UMR106细胞大鼠移植瘤模型,以CD34、CD147免疫荧光和GFP荧光法观察移植瘤组织中肿瘤仿血管通道的结构特点及其与肿瘤微血管的关系。结果:骨肉瘤MG-63细胞移植瘤和UMR106细胞移植瘤组织CD147染色均呈阳性,血管内皮细胞CD34染色均阳性。肿瘤仿血管通道主要位于移植瘤组织中心区域,主要由骨肉瘤细胞构成,是具有功能的类血管通道;肿瘤微血管主要位于移植瘤周边区域,主要由宿主组织的血管内皮细胞组成,是与肿瘤仿血管通道存在直接相通的管道样结构。结论:大鼠和裸鼠骨肉瘤细胞移植瘤组织中存在的肿瘤仿血管通道具有类血管功能,并与来源于宿主组织的肿瘤微血管相互联通,诱导肿瘤微血管侵入肿瘤内部生长。     

Thrombospondin binds to the surface of human osteosarcoma cells and mediates platelet-osteosarcoma cell interaction.

We have previously shown that thrombospondin (TSP) is synthesized and secreted by human MG-63 osteosarcoma cells. In this study, the secretion and cell surface expression of TSP by two different human osteosarcoma cell lines (MG-63 and TE-85) as well as the involvement of TSP in the platelet-aggregating activity of these tumor cells were studied. Using a sandwich enzyme-linked immunosorbent assay, MG-63 cells secreted 3-fold as much TSP as TE-85 cells at 48 h (0.17 +/- 0.01 (SD) versus 0.06 +/- 0.006 micrograms/10(6) cells, P = 0.007). Binding of exogenous 125I-TSP to MG-63 and TE-85 cells in monolayer indicated that binding was time and concentration dependent, saturable, and inhibited by excess cold TSP. However, despite a similar affinity, MG-63 cells had 10-fold more TSP-binding sites than TE-85 cells (402,394 +/- 130,346 versus 36,748 +/- 7,708 TSP-binding sites/cell; P = 0.002). Similar binding differences of 125I-TSP were observed with both osteosarcoma cell lines in suspension. A fluorescence-activated cell-sorting analysis was used in conjunction with an anti-TSP polyclonal antibody, and binding of endogenous TSP to MG-63 and TE-85 cells in suspension was investigated. Addition of an anti-TSP antibody to MG-63 and TE-85 cells in suspension increased the mean fluorescence intensity 50-fold when compared to an irrelevant antibody. Moreover, the fluorescence intensity of MG-63 cells with an anti-TSP polyclonal antibody was increased by 40% when compared to TE-85 cells. Since TSP was expressed on the surface of osteosarcoma cells, the involvement of this glycoprotein in the platelet-aggregating activity of MG-63 and TE-85 cells was therefore investigated using an anti-TSP polyclonal antibody and two monoclonal antibodies (P10 and MA-II), the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment and the Mr 25,000 heparin-binding fragment of TSP, respectively. Preincubation of MG-63 cells (1 x 10(6) cells/ml) with either an anti-TSP polyclonal antibody (100 micrograms/ml) or monoclonal antibody P10 (15 micrograms/ml) inhibited by 80% other platelet-aggregating activity of these tumor cells, while anti-TSP monoclonal antibody MA-II (15 micrograms/ml) had no effect. In sharp contrast, the anti-TSP polyclonal antibody (100 micrograms/ml) only exhibited a slight inhibitory effect on platelet aggregation induced by TE-85 cells when using a low concentration of tumor cells (0.6 x 10(6) cells/ml).(ABSTRACT TRUNCATED AT 400 WORDS)     

Antitumor activity of histone deacetylase inhibitor trichostatin A in osteosarcoma cells

Background: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest,apoptosis and differentiation of tumor cells. The present study aimed to examine the effects of trichostatin A(TSA), one such inhibitor, on the cell cycle, apoptosis and invasiveness of osteosarcoma cells. Methods: MG-63 cells were treated with TSA at various concentrations. Then, cell growth and apoptosis were determinedby 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively; cell cyclingwas assessed by flow cytometry; invasion assays were performed with the transwell Boyden Chamber system.Results: MTT assays revealed that TSA significantly inhibited the growth of MG-63 cells in a concentrationand time dependent manner. TSA treated cells demonstrated morphological changes indicative of apoptosisand TUNEL assays revealed increased apoptosis of MG-63 cells after TSA treatment. Flow cytometry showedthat TSA arrested the cell cycle in G1/G2 phase and annexin V positive apoptotic cells increased markedly. Inaddition, the invasiveness of MG-63 cells was inhibited by TSA in a concentration dependent manner. Conclusion:Our findings demonstrate that TSA inhibits the proliferation, induces apoptosis and inhibits invasiveness ofosteosarcoma cells in vitro. HDAC inhibitors may thus have promise to become new therapeutic agents againstosteosarcoma.     

c-Ski overexpression promotes tumor growth and angiogenesis through inhibition of transforming growth factor-β signaling in diffuse-type gastric carcinoma

c-Ski, originally identified as a proto-oncogene product, is an important negative regulator of transforming growth factor (TGF)-β family signaling through interaction with Smad2, Smad3, and Smad4. High expression of c-Ski has been found in some cancers, including gastric cancer. We previously showed that disruption of TGF-β signaling by dominant-negative TGF-β type II receptor in a diffuse-type gastric carcinoma model accelerated tumor growth through induction of tumor angiogenesis by decreased expression of the anti-angiogenic factor thrombospondin (TSP)-1. Here, we examined the function of c-Ski in human diffuse-type gastric carcinoma OCUM-2MLN cells. Overexpression of c-Ski inhibited TGF-β signaling in OCUM-2MLN cells. Interestingly, c-Ski overexpression resulted in extensive acceleration of the growth of subcutaneous xenografts in BALB/c nu/nu female mice (6 weeks of age). Similar to tumors expressing dominant-negative TGF-β type II receptor, histochemical studies revealed less fibrosis and increased angiogenesis in xenografted tumors expressing c-Ski compared to control tumors. Induction of TSP-1 mRNA by TGF-β was attenuated by c-Ski in vitro , and expression of TSP-1 mRNA was decreased in tumors expressing c-Ski in vivo . These findings suggest that c-Ski overexpression promotes the growth of diffuse-type gastric carcinoma through induction of angiogenesis. ( Cancer Sci  2009; 100: 1809–1816)