Tumor escape from endogenous, extracellular matrix-associated angiogenesis inhibitors by up-regulation of multiple proangiogenic factors.

PURPOSE: Thrombospondin-1 (Tsp1), endostatin, and tumstatin are extracellular matrix-associated proteins that inhibit angiogenesis. We examined the mechanisms by which tumor cells may bypass the antiangiogenic effects of these endogenous regulators. EXPERIMENTAL DESIGN: CT26 colon and RenCa renal carcinoma cells were stably transfected with Tsp1, endostatin, or tumstatin cDNA. Subcutaneous and metastatic tumor growth in syngeneic mice was analyzed. Expression of proangiogenic factors in resulting tumors was measured by quantitative real-time PCR. The combination of Tsp1 and vascular endothelial growth factor (VEGF) receptor-2 inhibition was also examined. RESULTS: There was significant suppression of angiogenesis in flank tumors and liver metastases formed from cells overexpressing Tsp1, endostatin, or tumstatin. However, all tumors ultimately escaped angiogenesis inhibition. The combination of all three angiogenesis inhibitors had no additive effect beyond overexpression of a single inhibitor. Using quantitative real-time PCR, we found that VEGF and platelet-derived growth factor (PDGF)-A levels were routinely up-regulated at least 5-fold in all CT26 tumors overexpressing any antiangiogenic protein, and there were variable increases in angiopoietin 2 (Ang2), basic fibroblast growth factor, and PDGF-B. In contrast, RenCa tumors, which have high baseline levels of VEGF and PDGF-B, relied on basic fibroblast growth factor, Ang1, and PDGF-A up-regulation to counteract Tsp1 overexpression. Growth of CT26 cells with Tsp1 overexpression was suppressed when anti-VEGFR-2 treatment was added. CONCLUSIONS: Cancer cells with overexpression of three different endogenous angiogenesis inhibitor eventually escape angiogenesis inhibition by up-regulation of various proangiogenic factors. Tsp1, endostatin, and tumstatin may be functionally redundant in this system. These endogenous angiogenesis inhibitors are likely best used in combination with the blockade of proangiogenic pathways or with traditional chemotherapy or radiation therapy.     

肿瘤抑素的应用及研究进展

肿瘤的生长需要新生血管来为其提供充足的营养物质和氧气,因此通过抑制血管的生成能够抑制肿瘤的生长。在所有来源于Ⅳ型胶原的内源性血管生成抑制剂中,肿瘤抑素的研究最广泛。肿瘤抑素具有抑制血管内皮细胞增殖、诱导内皮细胞凋亡、直接抑制肿瘤细胞增殖,促进肿瘤细胞凋亡等多种功能。本文就肿瘤抑素的研究现状进行综述。     

内皮抑素的作用机制以及在肿瘤治疗中的研究现状

内皮抑素是胶原ⅩⅧ的20kD水解片断，它是血管生成最有效的天然抑制剂之一，特异性抑制内皮细胞的增殖，通过抑制细胞周期素D1而诱导凋亡。在内皮细胞表面，内皮抑素与整合素α5β1结合从而激活Src激酶，此外也下调RhoAGTP酶的活性，抑制Ras和Raf激酶家族介导的信号传导。所有的事件均导致激动蛋白细胞骨架的解体，细胞-基质相互作用的紊乱，内皮细胞迁移活性的下降等，抑制血管生成。本文概述内皮抑素的生物学活性及作用机制。     

Endostatin expression by MDA-MB-435 breast cancer cells effectively inhibits tumor growth

Tumors must induce the formation of new blood vessels in order to grow and metastasize. Endostatin, a cleaved product of collagen XVIII, inhibits endothelial cell proliferation and suppresses tumor growth and metastases. Several recent reports have questioned the efficacy of endostatin as a tumor suppressor in experimental animals. Our objective was to determine whether endostatin expression in breast cancer cells inhibits neovascularization and tumor growth in nude mice. MDA-MB-435 cells were transfected with an endostatin expression vector while control cells were transfected with an empty vector. Endostatin expression and secretion were confirmed by RT-PCR and a dot blot assay. No differences were observed in the growth rates of the endostatin-expressing and control clones in vitro. When injected into male and female nude mice, tumors from the control clones increased in size 10-15 fold over 8-10 weeks. In contrast, the endostatin clones formed small tumors which did not increase in size after the first 3 weeks. The endostatinderived tumors had a significantly higher apoptotic index (5.6%) compared to controls (2.0%) and showed a marked reduction in vascularization. In conclusion, expression of endostatin in MDA-MB-435 breast cancer cells effectively suppressed breast tumor growth by inhibiting angiogenesis and increasing apoptosis.     

The PTEN/Akt pathway dictates the direct alphaVbeta3-dependent growth-inhibitory action of an active fragment of tumstatin in glioma cells in vitro and in vivo

The collagen type IV cleavage fragment tumstatin and its active subfragments bind to integrin alpha(V)beta(3) and inhibit activation of focal adhesion kinase, phophoinositol-3 kinase, Akt, and mammalian target of rapamycin (mTOR) in what is thought to be an endothelial cell-specific manner. The resultant endothelial cell apoptosis accounts for the ability of tumstatin to function as an endogenous inhibitor of angiogenesis and an indirect suppressor of tumor growth. We hypothesized that the inability of tumstatin to directly suppress tumor cell growth might be the result of the constitutive activation of the Akt/mTOR pathway commonly seen in tumors. Consistent with this idea, several integrin alpha(V)beta(3)-expressing glioma cell lines with PTEN mutations and high levels of phospho-Akt (pAkt) were unaffected by exposure to an active fragment of tumstatin (T3), whereas alpha(V)beta(3)-expressing glioma cell lines with a functional PTEN/low levels of pAkt exhibited T3-induced growth suppression that could be bypassed by small interfering RNA-mediated suppression of PTEN, introduction of a constitutively expressed Akt, or introduction of the Akt and mTOR target eukaryotic translation initiation factor 4E. The direct tumor-suppressive actions of T3 were further shown in an alpha(V)beta(3)-deficient in vivo mouse model in which T3, while unable to alter the tumstatin-insensitive vasculature contributed by the alpha(V)beta(3)-deficient host, nonetheless suppressed the growth and proliferative index of i.c. implanted alpha(V)beta(3)-expressing PTEN-proficient glioma cells. These results show that tumstatin, previously considered to be only an endogenous inhibitor of angiogenesis, also directly inhibits the growth of tumors in a manner dependent on Akt/mTOR activation.     

Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.     

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.     

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.     

Targeted expression of maspin in tumor vasculatures induces endothelial cell apoptosis

Angiogenesis, the formation of new blood vessels, is required for normal tissue development and pathological conditions such as tumorigenesis. Most solid tumors can not grow beyond a few millimeters without the recruitment of neovessels since cancer cells require access to blood vessels for nutrients and to escape the local environment and metastasize to other tissue and organ sites. Targeting tumor vessel endothelium therefore should serve as an effective therapy for cancers. Maspin is a serpin that exhibits antiangiogenic properties. In this report, we show that when maspin overexpression is targeted in vivo to endothelial cells, it actively induces endothelial cell apoptosis. Intravascular administration of adenovirus-maspin to mice bearing mammary tumors disrupts tumor-induced angiogenesis. Interestingly, tumor neovessels become leaky after maspin treatment, whereas normal mature vessels are not affected by maspin treatment. We further demonstrate that maspin directly induces endothelial cell apoptosis in vitro, and this effect is maspin specific. The induction of apoptosis is accompanied by changes in the expression of Bcl-2 family genes and is blocked by caspase inhibitors. In addition, the apoptotic effect is mediated by intracellular maspin and is dependent on the RSL region of maspin. Furthermore, we have shown that transient overexpression of Bcl-2 protected the HUVECs from maspin-mediated apoptosis, and the presence of both maspin and Bax accelerated the apoptosis process. These findings demonstrate that neovascular endothelial cells are highly sensitive to maspin level inside the cells. This property can be used for targeted therapy against tumor angiogenesis and metastasis.     

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.     

Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis in an orthotopic oral squamous cell carcinoma model

Tumstatin – non-collagenous (NC1) domain of the alpha 3 chain of type IV collagen – is a potent inhibitor of tumor angiogenesis. Successful tumor inhibition has been reported in glioma, bronchopulmonary cancer and melanoma experimental model. In this study, the effects of tumstatin, in vitro and in vivo, were investigated in an oral cancer model. Recombinant human tumstatin proteins were obtained by the transformation of Tn 5B1-4 cells, transfected with a plasmid containing tumstatin cDNA using the lipofection method, as previously described. Tumstatin inhibited the proliferation of human umbilical vascular endothelial cells in a dose dependent manner in a proliferation assay. For the in vivo analysis, we established an orthotopic oral squamous cell carcinoma (AT-84 cells) animal (C3H/He) model. In this animal model, the in vivo inhibitory effects of tumstatin on the tumor growth and on the metastasis of tumors were demonstrated. However, the tumors did not show complete remission. Immunostaining of the tumor microvessels (CD-31/PECAM) revealed that the density of tumor microvessels was significantly decreased in the tumstatin treated primary tumors. The results demonstrated that tumstatin delayed the tumor growth and the metastasis of oral squamous cell carcinomas. However, tumstatin alone failed to achieve tumor regression. Therefore, tumstatin might have an adjuvant role in the treatment of oral cancers, in combination with the conventional therapy.     

Thrombospondin-1 treatment prevents growth of dormant lung micrometastases after surgical resection and curative radiation therapy of the primary tumor in human melanoma xenografts

PURPOSE: Intradermal D-12 human melanoma xenografts develop pulmonary micrometastases in BALB/c nu/nu mice, and these metastases are kept dormant for prolonged times, because the primary tumor secretes thrombospondin-1 (TSP-1) into the blood circulation of the host. In this study, we report on the development of macroscopic metastases after surgical resection and curative radiation treatment of the primary tumor, the mechanisms involved, and the effects of treating the host with exogenous TSP-1 after the eradication of the primary tumor. METHODS AND MATERIALS: Xenografted tumors of the D-12 human melanoma were used as tumor model. Macroscopic metastases were scored by using a stereomicroscope. Micrometastases were detected by histologic examinations. Angiogenesis was studied by using an intradermal angiogenesis assay. Apoptotic endothelial cells were detected by immunohistochemistry by using an in situ apoptosis detection kit. RESULTS: Surgical resection as well as curative radiation treatment of the primary tumor resulted in accelerated growth of dormant micrometastases. This growth could be prevented by treating the host with exogenous TSP-1 after the surgery or the irradiation. Endogenous and exogenous TSP-1 prevented metastatic growth by suppressing angiogenesis, i.e., by inducing apoptosis in activated endothelial cells adjacent to dormant micrometastases. CONCLUSIONS: TSP-1 has antiangiogenic and antimetastatic effects that should be investigated further in clinical studies. Cancer patients with TSP-1-producing primary tumors may benefit from combined local treatment and antiangiogenic/antimetastatic treatment with TSP-1.     

Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor tyrosine kinase c-Met have emerged as key determinants of brain tumor growth and angiogenesis. SF/HGF and c-Met are expressed in brain tumors, the expression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of SF/HGF and/or c-Met in brain tumor cells enhances their tumorigenicity, tumor growth, and tumor-associated angiogenesis. Conversely, inhibition of SF/HGF and c-Met in experimental tumor xenografts leads to inhibition of tumor growth and tumor angiogenesis. SF/HGF is expressed and secreted mainly by tumor cells and acts on c-Met receptors that are expressed in tumor cells and vascular endothelial cells. Activation of c-Met leads to induction of proliferation, migration, and invasion and to inhibition of apoptosis in tumor cells as well as in tumor vascular endothelial cells. Activation of tumor endothelial c-Met also induces extracellular matrix degradation, tubule formation, and angiogenesis in vivo. SF/HGF induces brain tumor angiogenesis directly through only partly known mechanisms and indirectly by regulating other angiogenic pathways such as VEGF. Different approaches to inhibiting SF/HGF and c-Met have been recently developed. These include receptor antagonism with SF/HGF fragments such as NK4, SF/HGF, and c-Met expression inhibition with U1snRNA/ribozymes; competitive ligand binding with soluble Met receptors; neutralizing antibodies to SF/HGF; and small molecular tyrosine kinase inhibitors. Use of these inhibitors in experimental tumor models leads to inhibition of tumor growth and angiogenesis. In this review, we summarize current knowledge of how the SF/HGF:c-Met pathway contributes to brain tumor malignancy with a focus on glioma angiogenesis.     

Prediction of in vivo synergistic activity of antiangiogenic compounds by gene expression profiling

Angiogenesis, an essential phenotype for tumor formation, requires the interaction of many cells within the tumor microenvironment. Therefore, successful antiangiogenic therapies must be able to block all of the different mechanisms tumors use to induce neovascularization. A major challenge for developing such protocols is determining which agents are likely to have the highest degree of synergistic activity in vivo. We treated human microvascular endothelial cells with six inhibitors of angiogenesis and used microarrays to seek divergent patterns of gene expression suggestive of potential synergies. The expression profiles of a thrombospondin-mimetic peptide (DI-TSPa) and TNP-470 (TNP) were very similar, whereas endostatin had a dramatically different profile. In vitro, endostatin was synergistically antiangiogenic with either TNP-470 or DI-TSPa. In vivo, mice bearing Lewis lung carcinoma cells treated with a combination of endostatin and either DI-TSPa or TNP-470, at doses that were ineffective when used alone, resulted in a marked inhibition of tumor growth and decreased tumor angiogenesis. Conversely, animals treated with both DI-TSPa and TNP-470 demonstrated a modest effect on both tumor growth and angiogenesis. These results suggest that even in the absence of a complete mechanistic understanding of how these inhibitors work, gene expression profiling may be used to predict synergistic antiangiogenic activity and thus maximize their antitumor efficacy.     

Inhibition of orthotopic human bladder tumor growth by lentiviral gene transfer of endostatin.

PURPOSE: Inhibitors of endothelial cell proliferation, such as endostatin, result in suppression of tumor-associated angiogenesis and can achieve growth-inhibitory effects depending on the type of tumor treated. The purpose of this study was to investigate whether local overexpression of endostatin could serve to diminish tumor growth of bladder cancer in vivo. EXPERIMENTAL DESIGN: We examined the capability of lentiviral-mediated gene transfer in vitro and therapeutic effects of lentivirus-based vectors expressing endostatin on tumor growth using an orthotopic human bladder tumor model. RESULTS: We found that self-inactivating lentivirus vectors containing green fluorescent protein, alone or in combination with endostatin, were capable of efficient and stable gene transfer to a variety of human bladder tumor cell lines. The production and secretion of endostatin from lentivirus-transduced KU-7 human bladder cancer cells was confirmed by Western blot and competitive enzyme immunoassay. Intravesical instillation of untransduced, green fluorescent protein control lentivirus-transduced, and endostatin-transduced KU-7 cells was performed in murine models to establish orthotopic tumors. Sustained long-term expression of endostatin was achieved in lentivirus-transduced orthotopic bladder tumors, and it was associated with decreased vascularization and inhibition of tumor growth. Lentivirus vector-mediated overexpression of endostatin did not affect the intrinsic production of basic fibroblast growth factor and vascular endothelial growth factor. CONCLUSIONS: These findings suggest that lentivirus-mediated gene transfer might represent an effective strategy for expression of angioinhibitory peptides to achieve inhibition of human bladder cancer proliferation and tumor progression.     

血管内皮抑素与肿瘤关系研究进展

 新生血管的生成在肿瘤的生长和转移中起重要作用,抗血管生成治疗已成为当今肿瘤治疗领域的热点,血管内皮抑素作为一种新型的内源性血管生成抑制剂,能特异性抑制内皮细胞的增殖和迁移,诱导细胞凋亡,并能抑制动物模型中新生血管生成,文章就内皮抑素的抗血管生成机制,诱导细胞凋亡及临床应用等方面进行综述。     

Antiangiogenic treatment with thrombospondin-1 enhances primary tumor radiation response and prevents growth of dormant pulmonary micrometastases after curative radiation therapy in human melanoma xenografts

Thrombospondin-1 (TSP-1) is a potent antiangiogenic factor that has been shown to inhibit tumor growth by preventing endothelial cells from responding to a wide variety of angiogenic stimulators. We have demonstrated previously that D-12 primary tumors (human melanoma xenografts) suppress the growth of their spontaneous pulmonary micrometastases by secreting TSP-1 into the blood circulation. The same tumor model was used in the present work to study antitumor effects of combined radiation therapy and antiangiogenic treatment with TSP-1. Curative radiation treatment of D-12 primary tumors resulted in rapid growth of previously dormant micrometastases. Growth of dormant micrometastases could be prevented by treating the host mice with exogenous TSP-1 after the radiation treatment. Treatment with exogenous TSP-1 after subcurative radiation treatment reduced the growth rate of recurrent primary tumors in addition to suppressing metastatic growth. TSP-1 suppressed tumor growth at both primary and metastatic sites by inducing apoptosis in tumor-associated microvascular endothelial cells. Treatment with exogenous TSP-1 before radiation treatment enhanced the antitumor effect of the radiation treatment. The radiopotentiation by TSP-1 involved at least two distinctly different mechanisms, i.e., TSP-1 reduced the fraction of radiobiologically hypoxic parenchymal tumor cells and increased the radiation sensitivity of the tumor microvasculature by promoting radiation-induced endothelial cell apoptosis. In conclusion, the present preclinical study showed that TSP-1 has antiangiogenic, antimetastatic, and radiopotentiating properties that merit additional investigation in clinical studies.     

Thrombospondin 1 Triggers Osteosarcoma Cell Metastasis and Tumor Angiogenesis

Osteosarcomas, especially those with metastatic or unresectable disease, have limited treatment options. The antitumor effects of pharmacologic inhibitors of angiogenesis in osteosarcomas are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here we demonstrated that thrombospondin 1 (TSP-1) is a potent inhibitor of the growth and metastasis of the osteosarcoma cell line MG-63. Moreover, we demonstrate that upregulation of TSP-1 facilitated expression of vasculostatin in MG-63 cells. In angiogenesis assays, overexpression of TSP-1 inhibited MG-63 cells and induced tube formation of human umbilical vein endothelial cells (HUVECs) in a CD36-dependent fashion. Finally, in xenografted tumors, we observed that TSP-1 overexpression inhibited angiogenesis and tumor growth. These results provided strong evidence for an important role of the TSP-1/CD36/vasculostatin signaling axis in mediating the antiangiogenic activity of osteosarcoma.     

Gene transfer of endostatin enhances the efficacy of doxorubicin to suppress human hepatocellular carcinomas in mice

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death, and is chemoresistant to anticancer drugs. Anti-angiogenic therapy has been shown to enhance the efficacy of chemotherapy to treat solid tumors. The aim of the present study was to determine whether endostatin, a potent antiangiogenic agent, could enhance the efficacy of doxorubicin to combat HCC. An endostatin expression plasmid was constructed and its expression in vitro and in vivo was detected after gene transfer. Recombinant endostatin inhibited angiogenesis in the chorioallantoic membrane assay, and showed synergistic effects with doxorubicin in inhibiting the in vitro proliferation of endothelial cells, but not that of tumor cells. Both endostatin gene therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, and tumor angiogenesis. Combination therapy with endostatin gene therapy and doxorubicin showed a stronger effect in suppressing tumor growth, and tumor angiogenesis, than the respective monotherapies. Gene transfer of endostatin down-regulated the expression of both hypoxia-inducible factor-1alpha and vascular endothelial growth factor (VEGF), whereas doxorubicin only down-regulated VEGF expression. Endostatin and doxorubicin synergized to down-regulate VEGF expression. Endostatin and doxorubicin combination therapy warrants investigation as a therapeutic strategy to combat HCC.     

Anti-endostatin monoclonal antibody enhances growth of human hepatocellular carcinoma cells by inhibiting activity of endostatin secreted by the transplanted cells in nude mice

Endostatin, a fragment of collagen XVIII, inhibits angiogenesis in tumors, and is expected to become a new anticancer drug. However, its effectiveness is still controversial, because some researchers failed to reproduce the same marked regression of tumors by the peptide. We gave anti-endostatin monoclonal antibody, designated as CH18B, to nude mice transplanted with human hepatocellular carcinoma cells (JHH-1 line) that endogenously produced endostatin from collagen XVIII secreted by the cells themselves. As a result, CH18B promoted tumor angiogenesis by inhibiting endostatin activity in the tumor and subsequently increased tumor mass by preventing cancer cells from undergoing apoptosis. But the antibody itself did not stimulate proliferation of the tumor cells. Our present experimental procedure, the use of anti-endostatin antibody, definitely solved the question whether endostatin might exert its anticancer activity.