Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.     

Addition of integrin binding sequence to a mutant human endostatin improves inhibition of tumor growth

Tumor vasculatures express high levels of alphaVbeta3/alphaVbeta5 and alpha5beta1 integrins. Consequently, peptides containing the RGD (Arg-Gly-Asp) sequence, which is present in ligands of integrins, is effective in targeting therapeutic reagents to tumor vascular endothelium. In our study, we investigated whether the biologic activity of endostatin can be enhanced by the addition of an integrin targeting sequence. RGD sequence was added to either the amino or carboxyl terminus of endostatin containing a point mutation, P125A-endostatin. Earlier we have shown that the P125A mutation did not affect the biologic activity of endostatin but in fact had better antiangiogenic activity when compared to the native molecule. Further modification of P125A-endostatin with the RGD motif showed specific and increased binding to endothelial cells, and the increased binding coincided with improved antiangiogenic properties. Both amino and carboxyl terminal RGD-modification of P125A-endostatin resulted in greater inhibition of endothelial cell migration and proliferation. RGD modification increased tumor localization without affecting the circulatory half-life of P125A-endostatin, and RGD-modified P125A-endostatin was found to be more effective when compared to the P125A-endostatin in inhibiting ovarian and colon cancer growth in athymic mice. Complete inhibition of ovarian tumor growth was observed when P125A-endostatin-RGD was encapsulated into alginate beads. These studies demonstrate that addition of a vascular targeting sequence can enhance the biologic activity of an antiangiogenic molecule.     

Improved biological activity of a mutant endostatin containing a single amino-acid substitution

Human endostatin has an internal Asn-Gly-Arg (NGR) motif at position 126-128 following a proline at position 125. Asn-Gly-Arg-containing peptides have been shown to target tumour vasculature and inhibit aminopeptidase N activity. We previously compared the in vitro and in vivo biological activities of native endostatin and endostatin with a proline to alanine mutation (P125A-endostatin). P125A-endostatin exhibited greater inhibition of both endothelial cell proliferation and human ovarian cancer growth compared to native endostatin. Here we explore further the effects on biological activity of the P125A mutation, and show that aminopeptidase N is not involved. To determine whether the increased biological activity of the mutant was due to unmasking of downstream NGR-sequence, effect of endostatin on aminopeptidase N activity was investigated. Neither the native nor the P125A-endostatin inhibited aminopeptidase N. However, synthetic peptides consisting of the S118-T131 region of endostatin inhibited aminopeptidase N. These results suggest that the internal NGR site in native or mutant endostatin is not accessible to aminopeptidase N, and that this activity is not involved in the enhanced biological activity of the P125A form. P125A-endostatin bound to endothelial cells more efficiently than native endostatin and exhibited greater inhibition of not only proliferation but also migration of endothelial cells. P125A-endostatin also localised into tumour tissue to a higher degree than the native protein, and displayed greater inhibition of growth of colon cancer in athymic mice. Both proteins inhibited tumour cell-induced angiogenesis effectively. Real-time PCR analysis showed that both native and P125A-endostatin decreased expression of key proangiogenic growth factors. Vascular endothelial growth factor and angiopoietin 1 were downregulated more by the mutant. These studies suggest that the region around P125 can be modified to improve the biological activity of endostatin.     

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.     

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.     

Intratumoral administration of endostatin plasmid inhibits vascular growth and perfusion in MCa-4 murine mammary carcinomas

Endostatin, a fragment of the COOH-terminal domain of mouse collagen XVIII is a recently demonstrated endogenous inhibitor of tumor angiogenesis and endothelial cell growth. Antiangiogenic therapy with endostatin in animals requires multiple and prolonged administration of the protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenic factor in vivo. Established MCa-4 murine mammary carcinomas, grown in immunodeficient mice, were treated with intratumoral injection of endostatin plasmid at 7-day intervals. At the time of sacrifice, 14 days after the first injection, endostatin-treated tumor weights were 51% of controls (P < 0.01). Tumor growth inhibition was accompanied by a marked reduction in total vascular density. Specifically, computerized image analysis showed a 18-21% increase in the median distances between tumor cells and both the nearest anatomical (CD31-stained) vessel [48.1 +/- 3.8 versus 38.3 +/- 1.6 microm (P < 0.05)] and the nearest tumor-specific (CD105-stained) vessel [48.5 +/- 1.5 versus 39.8 +/- 1.5 microm (P < 0.01)]. An increased apoptotic index of tumor cells in endostatin-treated tumors [3.2 +/- 0.5% versus 1.9 +/- 0.3% (P < 0.05)] was observed in conjunction with a significant decrease in tumor perfused vessels (DiOC7 staining), and an increase in tumor cell hypoxia (EF5 staining). Hypoxia resulting from endostatin therapy most likely caused a compensatory increase of in situ vascular endothelial growth factor (VEGF) and VEGF receptor mRNA expression. Increased immunoreactivity of endostatin staining in endostatin-treated tumors was also associated with an increased thrombospondin-1 staining [1.12 +/- 0.16 versus 2.44 +/- 0.35]. Our data suggest that intratumoral delivery of the endostatin gene efficiently suppresses murine mammary carcinoma growth and support the potential utility of the endostatin gene for cancer therapy.     

Localization of endostatin in rat and human gliomas

BACKGROUND: Endostatin is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. Its occurrence and localization has not yet been examined in human brain tumors. The authors report the production of a monoclonal antibody and detection of endostatin in rat and human gliomas by immunohistochemistry. METHODS: The authors analyzed localization and tissue distribution of endostatin in 41 paraffin embedded glioma samples (18 glioblastoma multiforme, 7 WHO Grade III astrocytomas, 13 fibrillary, and 3 protoplasmic WHO Grade II astrocytomas) of human origin and 21 rat C6 gliomas by immunohistochemistry. Double labeling experiments confirmed the origin of endostatin-labeled cells. RESULTS: Endostatin immunoreactivity was detected in tumor cells, endothelial cells, macrophages, and lymphocytes of both rat and human gliomas. The percentage of cells labeled with the endostatin antibody was significantly lower (P = 0.0126) in the tumor parenchyma of human glioblastomas than in WHO Grade II astrocytomas. CONCLUSIONS: Endostatin was present in various cell types in rat and human gliomas in vivo. Lower levels in glioblastomas than in WHO Grade II astrocytomas might have reflected the shift of a probable regulatory balance between promoters and inhibitors of angiogenesis towards facilitation of neovascularization.     

Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth

Ovarian cancer is the leading cause of fatality among gynecological malignancies. Ovarian cancer growth is angiogenesis-dependent, and an increased production of angiogenic growth factors such as vascular endothelial growth factor is prognostically significant even during early stages of the disease. Therefore, we investigated whether antiangiogenic treatment can be used to inhibit the growth of ovarian cancer in an experimental model system. Mouse angiostatin (kringle 1-4) and endostatin were expressed in yeast. Purified angiostatin and endostatin were then used to treat established ovarian cancers in athymic mice. These studies showed that both angiostatin and endostatin inhibited tumor growth. However, angiostatin treatment was more effective in inhibiting ovarian cancer growth when compared with endostatin in parallel experiments. Residual tumors obtained from angiostatin- and endostatin-treated animals showed decreased number of blood vessels and, as a consequence, increased apoptosis of tumor cells. Subsequently, the efficacy of a combined treatment with angiostatin and endostatin was investigated. In the presence of both angiostatic proteins, endothelial cell proliferation was synergistically inhibited. Similarly, a combination regimen using equal amounts of angiostatin and endostatin showed more than additive effect in tumor growth inhibition when compared with treatment with individual angiostatic protein. These studies demonstrate synergism between two angiostatic molecules and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.     

Angiosuppressive and angiostimulatory effects exerted by synthetic partial sequences of endostatin.

PURPOSE: Endostatin, a peptide derived from proteolysis of collagen XVIII, is an endogenous inhibitor of angiogenesis and tumor growth. We have synthesized five peptide fragments designed to cover the whole length of the endostatin molecule (containing 40-50 amino acids each) with the aim of exploring the possibility that a specific sequence within the molecule might be responsible for its antiangiogenic effects. EXPERIMENTAL DESIGN: The five peptide sequences, termed fragments I, II, III, IV, and IVox, the latter bearing the original disulfide bond Cys(135)-Cys(165), were investigated for their effects on cultured endothelial cells, on enzyme activities related to angiogenesis, on tube formation in three-dimensional gel matrices, in vivo in the avascular rabbit cornea assay, and in an experimental tumor burden paradigm. RESULTS: Both the fragment covering the COOH-terminal endostatin region, fragment IV, and particularly fragment IVox, retained the angioinhibitory effects of endostatin. Fragment IVox strongly inhibited endothelial cell migration and proliferation, in vitro tube formation, and in vivo angiogenesis, displaying a potency comparable with that of endostatin. When tested in vivo on tumor growth, fragment IVox demonstrated to be more effective than full-length endostatin. Unexpectedly, fragment III exhibited proangiogenic activity, increasing endothelial cell migration, producing neovascularization to an extent similar to that of vascular endothelial growth factor, and enhancing the angiogenic response to vascular endothelial growth factor in the cornea assay. Peptides encompassing the NH(2)-terminal region of endostatin (fragments I and II) had negligible effects on angiogenesis. CONCLUSIONS: In view of these results, which show strikingly distinct profiles of endostatin fragments, we propose that the amino acid sequence of endostatin contains both angiosuppressive and angiostimulatory domains.     

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.     

Inhibition of ovarian cancer by RGD-P125A-endostatin-Fc fusion proteins

Previous studies have shown that a single point mutation in endostatin at position 125 (P125A) can improve the biological activity of endostatin. Addition of an integrin-targeting moiety, R-G-D, resulted in better localization to tumor vasculature and improved the antiangiogenic activity of endostatin. Because endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In our study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its antiangiogenic and antitumor effects in athymic mice. Two genetic constructs were made, RGD-P125A-endostatin-Fc (RE-Fc) and P125A-endostatin-RGD-Fc (ER-Fc). Both constructs were cloned and expressed in mammalian cells. Purified fusion proteins inhibited endothelial cell migration and proliferation better than yeast-derived P125A-endostatin. Both RE-Fc and ER-Fc inhibited ovarian cancer growth and were found to be as effective as Bevacizumab treatment. Fusion protein showed marked increased half-life. Combination treatment with Bevacizumab and ER-Fc showed additive inhibition of ovarian cancer growth. These studies demonstrate that genetic fusion with human IgG4-Fc increases the half-life of P125A-endostatin and can be used along with Bevacizumab to improve antiangiogenic and antitumor activities.     

Effect of retroviral endostatin gene transfer on subcutaneous and intraperitoneal growth of murine tumors.

BACKGROUND: Inhibiting tumor angiogenesis is a promising new strategy for treating cancer. Difficulties with the stability, manufacture, and long-term administration of recombinant antiangiogenic proteins have prompted investigators to use gene therapy to generate these proteins in vivo. We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the murine liver cell line NMuLi could inhibit tumor growth in vivo. METHODS: NMuLi cells were transduced with retroviral vectors containing the murine endostatin gene. The presence and function of endostatin in transduced cell supernatants were confirmed by competitive enzyme immunoassay and endothelial cell proliferation assays. Nude mice were given a subcutaneous or intraperitoneal injection with NMuLi cells, control transduced cells (NEF-null), or endostatin-transduced clones (NEF-Endo1 to 4) and were monitored for tumor growth. All statistical tests were two-sided. RESULTS: Supernatants from the clone secreting the lowest amount of endostatin (NEF-Endo4, 28 ng/mL) inhibited endothelial cell proliferation by 6% (95% confidence interval [CI] = 0% to 12%), and those from the clone secreting the highest amount (NEF-Endo1, 223 ng/mL) inhibited endothelial cell proliferation by 20% (95% CI = 13% to 27%). Increased levels of endostatin were detected in tumor lysates, but not serum, of mice given a subcutaneous injection of NEF-Endo1 cells. After 63 days, mice given a subcutaneous injection of parental NMuLi or NEF-null cells had tumor volumes of 2400 mm(3) (95% CI = 1478 mm(3) to 3300 mm(3)) and 2700 mm(3) (95% CI = 2241 mm(3) to 3144 mm(3)), respectively, compared with mean tumor volumes of less than 30 mm(3) in mice given an injection of NEF-Endo clones, a statistically significant difference (P<.001). After 123 days, all 16 mice given an intraperitoneal injection of parental NMuLi or NEF-null cells had died, compared with only three (9%) of 32 mice given an injection of NEF-Endo clones. CONCLUSIONS: Retroviral endostatin gene transfer leads to secretion of functional endostatin that is sufficiently active to inhibit tumor growth. Further studies of retroviral endostatin gene transfer for the treatment of cancer are warranted.     

Serum endostatin predicts tumor vascularity in hepatocellular carcinoma

BACKGROUND: Tumor angiogenesis is a strong prognostic factor in patients with hepatocellular carcinoma (HCC). However, to the authors' knowledge, details regarding the serum levels of proangiogenic and antiangiogenic growth factors controlling this process are not yet known. METHODS: Serum endostatin, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) levels were measured by the enzyme immunoassay method in prospectively collected samples from 33 HCC patients who had received no preoperative therapy. The angiogenic score (AS) and endostatin localization were evaluated using immunohistochemistry. RESULTS: Significant decreases in serum endostatin (P = 0.0007) and bFGF (P = 0.0004) were observed in postoperative samples compared with the preoperative values. A very strong direct correlation was noted between VEGF and endostatin (P < 0.0001). Only the preoperative serum endostatin was found to have a significant (P = 0.0025) inverse correlation with the AS. Furthermore, the combined positivity for bFGF and VEGF and negativity for endostatin was found to have a significantly (P = 0.0069) positive correlation with AS. Significantly high levels of endostatin were noted in patients with trabecular-type tumors (P = 0.0446) and in patients with hepatitis B infection (P = 0.0183). The serum endostatin level was found to be significantly (P = 0.0166) higher in living patients and patients with high serum endostatin levels had a tendency (P = 0.0871) toward long survival. Tissue endostatin expression was found to have a direct correlation with the serum endostatin level (P = 0.0117). CONCLUSIONS: The measurement of serum endostatin can predict tumor vascularity and may serve as a promising tool in the antiangiogenic therapy for patients with HCC.     

Endostatin improves radioresponse and blocks tumor revascularization after radiation therapy for A431 xenografts in mice

PURPOSE: Clinical trials of antiangiogenic agents used alone for advanced malignancy have been disappointing but preclinical studies suggest that the addition of radiation therapy could improve antitumor efficacy. To test the hypothesis that antiangiogenic therapy combined with radiation therapy can overcome the limitations of antiangiogenic monotherapy, we studied the effects of endostatin combined with radiation on the growth and vascularization of A431 human epidermoid carcinomas growing intramuscularly in the legs of mice. METHODS AND MATERIALS: Mice with established A431 human epidermoid leg tumors were treated with radiation, endostatin, both radiation and endostatin, or vehicle control. The experiment was repeated and mice from each group were killed at 2, 7, and 10 days after irradiation so that tumor tissue could be obtained to further analyze the kinetics of the antitumor, antivascular, and antiangiogenic response to therapy. RESULTS: Endostatin enhanced the antitumor effects of radiation, and prolonged disease-free survival was observed in the combined treatment group. Endothelial cell proliferation was increased in tumors after irradiation but was blocked by the concurrent administration of endostatin, and the combination of endostatin with radiation enhanced endothelial cell apoptosis within 48 h after irradiation. Expression of vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 were increased in tumors after irradiation, and this increase was blocked by concurrent administration of endostatin. CONCLUSION: These data indicate that endostatin can block tumor revascularization after radiation therapy and thereby augment radioresponse.     

Endostatin overexpression inhibits lymphangiogenesis and lymph node metastasis in mice

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and control mice, nor in the rate of conversion of benign papillomas to malignant squamous cell carcinomas (SCC). We did find, however, that endostatin regulates the terminal differentiation of keratinocytes because the SCCs in the J4 mice were less aggressive and more often well differentiated than those in the control mice. We observed an inhibition of tumor angiogenesis by endostatin at an early stage in skin tumor development, but more strikingly, there was a significant reduction in lymphatic vessels in the papillomas and SCCs in association with elevated endostatin levels and also a significant inhibition of lymph node metastasis in the J4 mice. We showed that tumor-infiltrating mast cells strongly expressed vascular endothelial growth factor-C (VEGF-C), and that the accumulation of these cells was markedly decreased in the tumors of the J4 mice. Moreover, endostatin inhibited the adhesion and migration of murine MC/9 mast cells on fibronectin in vitro. Our data suggest that endostatin can inhibit tumor lymphangiogenesis by decreasing the VEGF-C levels in the tumors, apparently via inhibition of mast cell migration and adhesion, and support the view that the biological effects of endostatin are not restricted to endothelial cells because endostatin also regulates tumor-associated inflammation and differentiation, and the phenotype of epithelial tumors.     

Enhancement of radiation effects by pXLG-mEndo in a lung carcinoma model

PURPOSE: Endostatin is a potent antiangiogenesis protein with little or no toxicity that has potential to enhance radiotherapy. The major goal of this study was to evaluate the combination of radiation and endostatin gene therapy in a preclinical lung cancer model. METHODS: Plasmid pXLG-mEndo, constructed in our laboratory, includes the mouse endostatin gene cloned into the pWS4 vector. The kinetics of endostatin expression and efficacy of the pXLG-mEndo and radiation ((60)Co gamma-rays) combination was evaluated in the C57BL/6 mouse-Lewis lung carcinoma (LLC) model. The LLC cells were implanted s.c. and pXLG-mEndo was injected intratumorally 12-14 days later without any transfection agent; a dose of 10 Gy radiation was applied approximately 16 h thereafter. Some groups received each modality twice. Endostatin, vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were quantified in plasma and tumors, and tumor vasculature was examined. RESULTS: Endostatin expression within LLC tumors peaked on Day 7 after pXLG-mEndo injection. Addition of radiation to pXLG-mEndo significantly enhanced the level of tumor endostatin compared with plasmid alone (p < 0.05). Tumor growth was significantly delayed in mice receiving pXLG-mEndo plus radiation compared with no treatment (p < 0.005), radiation (p < 0.05), and control plasmid (p < 0.05). The number of LLC tumor vessels was reduced after combined treatment (p < 0.05), and significant treatment-related changes were observed in both VEGF and TGF-beta1. CONCLUSIONS: The data demonstrate that delivery of endostatin by pXLG-mEndo as an adjuvant to radiation can significantly enhance the antitumor efficacy of radiotherapy in the LLC mouse tumor model and support further investigation of this unique combination therapy.     

重组内皮抑素对肿瘤血管结构和乏氧改善作用的实验观察

目的 观察重组内皮抑素作用下小鼠Lewis肺癌移植瘤在血管退化前是否存在血管正常化时间窗,在此时间窗内肿瘤乏氧是否改善.方法 激光共聚焦显微镜动态观察重组内皮抑素作用下肿瘤血管的形态学变化,免疫组化染色检测不同时间段肿瘤乏氧细胞比例,观测肿瘤生长情况并绘制肿瘤生长曲线.结果 重组内皮抑素处理后肿瘤血管密度逐渐下降,连续治疗9 d较对照组下降最为明显.重组内皮抑素处理后周细胞覆盖内皮细胞比例逐渐增加,第3天开始明显增加,第5天增加最明显,第7天后明显下降.对照组基底膜与内皮细胞连接松散,厚度增加,而重组内皮抑素处理后,基底膜与内皮细胞连接紧密,厚度下降.在重组内皮抑素处理后第5天肿瘤乏氧细胞比例下降最明显,连续5 d用重组内皮抑素,肿瘤生长未见加快.结论 重组内皮抑素能使肿瘤血管正常化,其时间窗为治疗后3～7 d;此时肿瘤氧供明显改善,为临床联合应用放疗和重组内皮抑素提供了实验依据.

Abstract：

Objective To investigate whether recombinant human endostatin can create a time window of vascular normalization prior to vascular pruning to alleviate hypoxia in Lewis lung carcinoma in mice. Methods Kinetic changes in morphology of tumor vasculature in response to recombinant human endostatin were detected under a confocal microscope with immunofluorescent staining in Lewis lung carcinomas in mice. The hypoxic cell fraction of different time was assessed with immunohistochemical staining . Effects on tumor growth were monitored as indicated in the growth curve of tumors . Results Compared with the control group vascularity of the tumors was reduced over time by recombinant human endostatin treatment and significantly regressed for 9 days. During the treatment, pericyte coverage increased at day 3, increased markedly at day 5, and fell again at day 7. The vascular basement membrane was thin and closely associated with endothelial cells after recombinant human endostatin treatment, but appeared thickened, loosely associated with endothelial cells in control tumors. The decrease in hypoxic cell fraction at day 5 after treatment was also found. Tumor growth was not accelerated 5 days after recombinant human endostatin treatment. Conclusions Recombinant human endostatin can normalize tumor vasculature within day 3 to 7, leading to improved tumor oxygenation. The results provide important experimental basis for combining recombinant human endostatin with radiation therapy in human tumors.     

脂质体介导的鼠内皮细胞抑制素基因治疗人肝癌的研究

目的：利用内皮细胞抑制素进行肿瘤抗血管基因治疗的尝试。方法：用ＲＴ－ＰＣＲ从鼠地脏扩增出内皮细胞抑制素ｃＤＮＡ,经测序证实后,装入分泌表达载体ｐＳＥＣ－ｈｙｇｒｏｍｙｃｉｎ,用ＤＥＡＥ－葡聚糖将其转染ＣＯＳ－７细胞,从ｍＲＮＡ水平及蛋白水平检测内皮细胞抑制素的表达,并观察分泌上肖是否具有特异性抑制ｂＦＧＦ刺激的内皮细胞增殖作用,并通过ＴＤＴ介导的ｄＵＴＰ缺口末端标记法和琼脂糖电泳来探讨其作用机制。     

Enhanced antiangiogenic therapy of squamous cell carcinoma by combined endostatin and epidermal growth factor receptor-antisense therapy.

PURPOSE: We tested the combined effects of antiangiogenic endostatin and epidermal growth factor receptor (EGFR) antisense gene therapy on squamous cell carcinoma (SCC). EXPERIMENTAL DESIGN and Results: The 1483 cell line of human head and neck SCC (HNSCC) and SCC-VII/SF murine SCC cells was used to establish tumors in nude mice and immunocompetent C3H mice, respectively. Tumor-bearing mice were treated with endostatin (20 mg/kg/day, s.c.), liposomal EGFR-antisense expression plasmid (25 microg/mouse, three times/week, intratumoral), a combination of both agents, or liposomal EGFR-sense plasmid as a control. Endostatin or EGFR-antisense alone significantly, yet partially, inhibited the growth of 1483 and SCC-VII/SF tumors, and a combination of both treatments completely blocked tumor growth. Immunohistochemistry analysis demonstrated that a complete suppression of tumor angiogenesis was achieved by the combination treatment. Down-regulation of vascular endothelial growth factor was shown in EGFR-antisense-treated tumors. These results suggest that the EGFR-antisense treatment, in addition to its inhibitory activity on tumor cell proliferation, might have a synergistic effect with endostatin on SCC-induced angiogenesis. In vitro studies demonstrated that EGFR inhibition by antisense oligonucleotides or EGFR-specific tyrosine kinase inhibitor down-regulated the production of VEGF in HNSCC cells. Additional experiments demonstrated that these EGFR inhibition approaches also directly suppressed the growth of endothelial cells. CONCLUSION: A combination of endostatin and EGFR targeting strategies profoundly inhibited the angiogenesis and growth of SCC in vivo. EGFR-antisense therapy might have multiple inhibitory effects against both tumor cells and endothelial cells, leading to enhanced antitumor efficacy. Such a combination strategy might represent a novel and promising approach for HNSCC therapy.     

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.