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.     

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti‐angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti‐proliferative and anti‐angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO‐1 and cisplatin resistant, BRCA2 proficient PEO‐4 ovarian cancer cells. Both PEO‐1 and PEO‐4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF‐A and Glut‐1 in PEO‐1 and PEO‐4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR‐5 cells. Plumbagin challenge also restricts the VEGF induced pro‐angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR‐5 tumor‐bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti‐cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.     

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.     

Clodronate inhibits tumor angiogenesis in mouse models of ovarian cancer

Purpose Bisphosphonates have been shown to inhibit and deplete macrophages. The effects of bisphosphonates on other cell types in the tumor microenvironment have been insufficiently studied. Here, we sought to determine the effects of bisphosphonates on ovarian cancer angiogenesis and growth via their effect on the microenvironment, including macrophage, endothelial and tumor cell populations.Experimental Design Using in vitro and in vivo models, we examined the effects of clodronate on angiogenesis and macrophage density, and the overall effect of clodronate on tumor size and metastasis.Results Clodronate inhibited the secretion of pro-angiogenic cytokines by endothelial cells and macrophages, and decreased endothelial migration and capillary tube formation. In treated mice, clodronate significantly decreased tumor size, number of tumor nodules, number of tumor-associated macrophages and tumor capillary density.Conclusions Clodronate is a potent inhibitor of tumor angiogenesis. These results highlight clodronate as a potential therapeutic for cancer.     

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 peritoneal dissemination of ovarian cancer by tyrosine kinase receptor inhibitor SU6668 (TSU-68)

SU6668 (TSU-68) is a small-molecule synthetic inhibitor of the angiogenic related receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. Using a mouse model of peritoneally disseminated ovarian cancer, we investigated whether SU6668 inhibits peritoneal dissemination and prolongs survival time. BALB/c nude mice were intraperitoneally (i.p.) inoculated with SHIN-3 (VEGF-hypersecretory) or KOC-2S (PDGF-hypersecretory) ovarian serous adenocarcinoma cells with marked peritoneal dissemination ability. From the day after i.p. inoculation of tumor cells, SU6668 was orally administered 6 times weekly at a daily dose of 100 mg/kg or 400 mg/kg. The SU6668-administered group and the vehicle-administered control group were compared for the number of tumor vascular endothelial cells, weight of peritoneally disseminated tumors, amount of ascitic fluid and survival time. As a result, these 3 parameters were significantly smaller in the SHIN-3-inoculated, SU6668-administered mice than in the control group (p = 0.03, p = 0.002, and p = 0.02, respectively). The mean survival time was significantly longer, at 58.1 +/- 11.2 days, in the SU6668-administered mice than that (34.5 +/- 8.8 days) in the control group (p = 0.002). Similarly, in the KOC-2S-inoculated mice, the oral administration of SU6668 significantly reduced these 3 parameters (p = 0.04, p = 0.04, and p = 0.03, respectively), and significantly prolonged survival (16.6 +/- 1.7 days vs. 11.0 +/- 0.7 days, p = 0.008). Thus, the oral administration of SU6668 inhibited angiogenesis and peritoneal dissemination and prolonged survival in mice with peritoneally disseminated ovarian cancer. These effects were observed with both the VEGF- and PDGF-hypersecretory cell lines. Our results suggest that molecular targeting with oral SU6668 will become a new therapeutic strategy targeting peritoneally disseminated ovarian cancer.     

Ascites modulates cancer cell behavior,contributing to tumor heterogeneity in ovarian cancer

Malignant ascites constitute a unique tumor microenvironment providing a physical structure for the accumulation of cellular and acellular components. Ascites is initiated and maintained by physical and biological factors resulting from underlying disease and forms an ecosystem that contributes to disease progression. It has been demonstrated that the cellular contents and the molecular signatures of ascites change continuously during the course of a disease. Over the past decade, increasing attention has been given to the characterization of components of ascites and their role in the progression of ovarian cancer, the most malignant gynecologic cancer in women. This review will discuss the role of ascites in disease progression, in terms of modulating cancer cell behavior and contributing to tumor heterogeneity.     

Expression pattern and circulating levels of endostatin in patients with pancreas cancer

Endostatin is a potent inhibitor of angiogenesis that is cleaved from the basement membrane protein type XVIII collagen. Expression of endostatin has recently been shown by Western blot analysis of tissue lysates in normal pancreas and pancreas cancer tissue. We show here that the expression pattern of type XVIII collagen/endostatin is shifted from a general basement membrane staining and is mainly located in the vasculature during tumor progression. This shift in type XVIII collagen/endostatin expression pattern coincides with an up-regulation of MMPs involved in endostatin processing in the tumor microenvironment, such as MMP-3, MMP-9 and MMP-13. The circulating levels of endostatin was analyzed in patients with pancreas cancer and compared to that of healthy controls, as well as after surgical treatment or in a group of nonoperable patients after intraperitoneal fluorouracil (5-FU) chemotherapy. The results show that patients with pancreas cancer have increased circulating levels of endostatin and that these levels are normalized after surgery or intraperitoneal chemotherapy. These findings indicate that endostatin could be used as a biomarker for pancreas cancer progression.     

Polymorphism in endostatin,an angiogenesis inhibitor,and prostate cancer risk and survival: A prospective study

Endostatin inhibits endothelial cell proliferation and migration, prerequisites of angiogenesis. A functional missense mutation (D104N) in endostatin was associated with an increased prostate cancer risk in a small study. We undertook a larger, prospective study within the Physicians' Health Study to examine D104N and prostate cancer risk and progression among 544 incident prostate cancer cases (1982–1995) and 678 matched controls. The association between endostatin genotype and cancer risk was estimated using logistic regression models. Among cases, Cox models were used to assess D104N and lethal prostate cancer. Given the role of endostatin in neovascularization of adipose tissue, we cross classified individuals on D104N genotype and body mass index (BMI). The genotype frequency was 1.3% homozygous (NN), 14.5% heterozygous (DN) and 84.2% wildtype homozygous (DD). There was no overall association between carriage of the N allele and prostate cancer risk (RR = 1.2, 95% CI: 0.9–1.6) or cancer‐specific mortality (HR = 1.2, 0.7–1.8). Cases with the polymorphic allele were less likely to be overweight (BMI 25 kg/m² or greater, 26%) compared to men wildtype homozygous (48%), p < 0.0001. Being overweight was associated with a 60% greater prostate cancer risk among those who were wildtype homozygous. In contrast, being overweight was associated with a 50% lower risk of cancer among those with the N allele. We did not confirm an earlier observation between the D104N polymorphism and prostate cancer. However, our data indicate that prostate cancer cases who carry the variant N allele are more likely to be overweight, and may be more susceptible to the angiogenic influences of obesity in prostate cancer pathogenesis. © 2009 UICC     

Indoleamine 2,3‐dioxygenase promotes peritoneal metastasis of ovarian cancer by inducing an immunosuppressive environment

Indoleamine 2,3‐dioxygenase (IDO) is a tryptophan‐catabolizing enzyme that has immunoregulatory functions. Our prior study showed that tumoral IDO overexpression is involved in disease progression and impaired patient survival in human ovarian cancer, although its mechanism remains unclear. The purpose of the present study is to clarify the role of IDO during the process of peritoneal dissemination of ovarian cancer. Indoleamine 2,3‐dioxygenase cDNA was transfected into the murine ovarian carcinoma cell line OV2944‐HM‐1, establishing stable clones of IDO‐overexpressing cells (HM‐1‐IDO). Then HM‐1‐IDO or control vector‐transfected cells (HM‐1‐mock) were i.p. transplanted into syngeneic immunocompetent mice. The HM‐1‐IDO‐transplanted mice showed significantly shortened survival compared with HM‐1‐mock‐transplanted (control) mice. On days 11 and 14 following transplantation, the tumor weight of peritoneal dissemination and ascites volume were significantly increased in HM‐1‐IDO‐transplanted mice compared with those of control mice. This tumor‐progressive effect was coincident with significantly reduced numbers of CD8⁺ T cells and natural killer cells within tumors as well as increased levels of transforming growth factor‐β and interleukin‐10 in ascites. Finally, treatment with the IDO inhibitor 1‐methyl‐tryptophan significantly suppressed tumor dissemination and ascites with reduced transforming growth factor‐β secretion. These findings showed that tumor‐derived IDO promotes the peritoneal dissemination of ovarian cancer through suppression of tumor‐infiltrating effector T cell and natural killer cell recruitment and reciprocal enhancement of immunosuppressive cytokines in ascites, creating an immunotolerogenic environment within the peritoneal cavity. Therefore, IDO may be a promising molecular target for the therapeutic strategy of ovarian cancer.     

Human endostatin inhibits growth of human non-small-cell lung cancer in a murine xenotransplant model.

Overall prognosis in human NSCLC remains poor. Antiangiogenic treatment has become a promising concept for the treatment of solid malignancies. Our purpose was to evaluate the efficacy of recombinant HSENDO for the treatment of human NSCLC in an orthotopic murine xenotransplantation model. The efficacy of HSENDO was tested in vitro in cell-proliferation, cell-migration and tube-formation assays. In vivo, the effect of HSENDO on tumor growth was tested in s.c. xenotransplanted human NSCLC and on intrapulmonary induced human NSCLC. In vitro, HSENDO inhibited both human and rodent endothelial cell proliferation in a time- and dose-dependent fashion. Endothelial cell migration was inhibited by 97%. Tube formation of murine endothelial cells was inhibited and preexisting tubes degenerated after HSENDO exposure. In vivo, HSENDO delayed growth of s.c. xenotransplanted tumors. Immunohistochemic staining demonstrated no change in microvessel density but a significant reduction of proliferating tumor cells and an increase in bFGF and VEGF expression, reflecting the antiangiogenic effect of HSENDO. Intrapulmonary tumor induction caused death subsequent to metastatic disease. Systemic HSENDO application extended survival significantly. HSENDO was demonstrated to inhibit endothelial cell proliferation, migration and tube formation effectively. In vivo growth of s.c. transplanted tumors was delayed and survival extended by 32% and 69%, respectively, after intrapulmonary NSCLC induction.     

人卵巢癌细胞系干/祖细胞分离及其鉴定

目的:自人卵巢癌细胞系SK-OV-3中分离干/祖细胞并进行鉴定。方法:采用无血清球形体形成法从SKOV-3中分离培养卵巢癌干/祖细胞;采用实时定量PCR和蛋折质印迹法测定球形体细胞干/祖细胞相关标志ABCG2、Oct-4、Nanog基因和蛋白的表达;流式细胞仪检测其耐药性;双层软琼脂检测其克隆形成能力;NOD/SCID小鼠检测其体内致瘤性。结果:球形体细胞表达干/祖细胞相关标志Oct-4、ABCG2、Nanog;对顺铂高耐药;在双层软琼脂上克隆形成率达(13.67±1.48)%;1 000个球形体形成细胞就能在NOD/SCID鼠中成瘤。结论:采用无血清培养基中球形体形成法从SKOV-3细胞系中可以分离出具有干/祖特性的卵巢癌细胞,可为今后研究卵巢癌的发生、发展、复发及其化疗药物筛选提供简便实用的体外模型。     

Pazopanib in ovarian cancer

The majority of women with ovarian cancer present with advanced disease, and ultimately relapse following primary surgery and platinum-taxane chemotherapy. Despite recent advances in the development of targeted agents in ovarian cancer, survival rates remain poor. The promising activity of bevacizumab, a VEGF receptor inhibitor, has stimulated research on the use of additional anti-angiogenic agents in ovarian cancer. Pazopanib, an oral tyrosine kinase inhibitor, targets VEGF receptor-1, -2 and -3, platelet-derived growth factor receptor-α and -β and c-kit; resulting in the inhibition of angiogenesis and tumor proliferation. Early phase studies have demonstrated promising efficacy and tolerability. To date, there has been one Phase III trial of pazopanib in ovarian cancer, demonstrating a progression-free survival benefit in women treated with maintenance pazopanib following primary surgery and systemic therapy. This article summarizes the preclinical and clinical data of pazopanib in ovarian cancer, highlighting future research options for this agent.     

Survivin与卵巢癌关系的Meta分析

目的:应用Meta分析方法评估国内外有关卵巢癌组织中Survivin的表达及其临床意义。方法:通过计算机检索PubMed、CNKI等数据库全面检索国内外已发表的相关文献,最终进入系统评价的有13篇病例对照研究。应用Stata 11.0进行Meta分析,计算OR及95%CI。结果:表明Survivin的表达在卵巢癌组与卵巢良性肿瘤组,卵巢癌组与卵巢正常组织组,临床Ⅰ-Ⅱ组与临床Ⅲ-Ⅳ组,中、高分化组与低、未分化组的差异有统计学意义(P<0.05);Survivin淋巴结转移组与淋巴结未转移组的表达差异无统计学意义(P>0.05)。结论:Survivin的高表达可能在卵巢癌的发生发展中起重要作用。     

The importance of angiogenesis in ovarian cancer

Advanced stage ovarian cancer has a high rate of recurrence even after surgery followed by chemotherapy combining carboplatin and a taxane. New strategies are currently under way to combat this situation and one of the most promising ones is based on the knowledge that angiogenesis, the mechanism of formation of new blood vessels coupled with the degradation of the extracellular matrix for metalloproteinases, could be crucial in the development of this tumor. The principal molecule implicated in angiogenesis process of ovarian cancer is the vascular endothelial growth factor (VEGF). Several studies are now in progress to clarify its role as a diagnostic tool or its therapeutic implication. Presently, there is no indication for the use of VEGF in a preliminary diagnosis seeing that an increase in levels can be seen in both benign and malignant ovarian conditions. VEGF is also responsible for an increase in vascular permeability and is directly related to symptoms such as ascites and pleural effusion, both of which are frequent in ovarian cancer. Several papers have analised the role of VEGF as a prognostic factor and some of them do confirm VEGF as an independent prognostic factor in ovarian cancer. VEGF and the metalloproteinase system coupled with angiogenesis are currently being evaluated as therapeutic targets but no positive results have yet to be seen in this field.