Tumor angiogenesis is a critical target for cancer treatment and its inhibition has become a common anticancer approach following chemotherapy. However, due to the simultaneous activation of different compensatory molecular mechanisms that enhance tumor angiogenesis, clinically authorized anti-angiogenic medicines are ineffective. Additionally, medications used to treat cancer have an effect on normal body cells; nonetheless, more research is needed to create new cancer therapeutic techniques. With advances in molecular biology, it is now possible to use gene-editing technology to alter the genome and study the functional changes resulting from genetic manipulation. With the development of CRISPR/Cas9 technology, it has become a very powerful tool for altering the genomes of many organisms. It was determined that CRISPR/Cas9, which first appeared in bacteria as a part of an adaptive immune system, could be used, in modified forms, to alter genomes and function. In conclusion, CRISPR/Cas9 could be a major step forward to cancer management by providing patients with an effective method for dealing with cancers by dissecting the carcinogenesis pathways, identifying new biologic targets, and perhaps arming cancer cells with drugs. Hence, this review will discuss the current applications of CRISPR/Cas9 technology in tumor angiogenesis research for the purpose of cancer treatment.

It has recently been suggested that angiotensin-I converting enzyme (ACE) inhibitors decrease the risk of cancer. However, studies to date have not investigated esophageal carcinoma. Therefore, we investigated the inhibitory effect of ACE inhibitors on growth of esophageal carcinoma xenografts. We used the EC9706 cell line, which expresses the highest vascular endothelial growth factor (VEGF) mRNA level, to establish xenografts in 21 BALB/c nude mice. The mice were then randomly allocated to receive normal saline, perindopril (4 mg/kg), or benazepril (6 mg/kg). Five weeks later, the nude mice were sacrificed and all tumors were dissected and weighed. The number of microvessels was counted by immunostaining endothelial cells for CD31 and the microvessel density was assessed. The EC9706 cell line showed the highest expression of VEGF mRNA of four esophageal squamous cell carcinoma lines. After treatment, the average tumor inhibitory rate in the benazepril group was 45.4%, which was significantly higher than that of the control group (P < 0.05). Similar findings were observed when we used tumor weight as an index for tumor growth inhibition (P < 0.05). By contrast, there was no significant difference between the perindopril group and the control group (P > 0.05). The benazepril group appeared to show less vascularization than the control group (P < 0.05), but we did not find a significant difference between the perindopril group and the control group (P > 0.05). The EC9706 cell line showed the highest expression of VEGF mRNA level of the four esophageal squamous cell carcinoma cell lines examined. Benazepril inhibited the growth of esophageal carcinoma in vivo. The potential mechanism of benazepril seems to involve suppression of new vessel formation. Therefore, benazepril could be used as an effective agent for the treatment of esophageal carcinoma.

In the present study, four novel dienone cyclopropoxy curcumin analogs 1a-4a were synthesized by nucleophillic substitution reaction with cyclopropyl bromide. The tumor inhibitory and anti-angiogenic effects of the synthetic compounds were studied on mouse Ehrlich ascites tumor (EAT) in vivo. The compounds 1a-4a increased the life span (% ILS) of EAT bearing mice with corresponding significant reduction in ascites volume and cell number and induced apoptotic bodies in EAT cells. Anti-angiogenic studies of the compounds demonstrated significant reduction of microvessel density (MVD) in the peritoneum wall sections of mice and induced avascular zone in CAM model. Our findings demonstrate that the tumor growth inhibitory effects of synthetic dienone cyclopropoxy curcumin analogs 1a-4a could be mediated by promoting apoptosis and inhibiting tumor angiogenesis. However, the compounds need to be explored further to assess its clinical relevance.

Normal hepatocytes express pigment epithelium-derived factor (PEDF), an endogenous antiangiogenic factor. We hypothesized that decreased PEDF expression may be one mechanism driving hepatoblastoma growth, and in vivo gene transfer of PEDF could suppress neovascularization and limit tumor growth. PEDF functional activity was determined in vitro using endothelial cell migration assays and in vivo using a subcutaneous tumor model. HUH-6 human hepatoblastoma tumors were treated with hybrid adenoviral/adeno-associated viral expression vectors for PEDF (Hyb-PEDF, n = 4) or beta-galactosidase (Hyb-betagal, n = 4) daily for 4 d. Mitotic figures, microvascular density (MVD), PEDF, and VEGF expression were assessed. Hyb-PEDF treatment inhibited in vivo tumor growth (p < 0.008) and decreased MVD (p < 0.001), the number of mitotic figures (p < 0.001), and VEGF expression when compared with Hyb-betagal-treated tumors. HUH-6 expression of PEDF was dramatically reduced when cultured under hypoxic conditions and also when grown in vivo, and the addition of neutralizing anti-PEDF antibody increased the already high baseline angiogenic activity of the HUH-6 cell secretions in vitro (p < 0.04). PEDF is an important endogenous regulator of the liver vasculature. Augmenting intra-tumoral PEDF levels inhibits tumor growth by reducing angiogenesis and VEGF expression. Potent inhibitors of angiogenesis, such as PEDF, may be an effective alternative treatment for children with hepatoblastoma.

Angiogenesis refers to the formation of new blood vessels from an existing vasculature and is recognised as a necessary requirement for most tumours to grow beyond 1-2 mm in diameter. Factors established as playing a role in angiogenesis may be divided into two principal groups: (a) those that stimulate endothelial cell proliferation and/or elongation, migration and vascular morphogenesis including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived endothelial cell growth factor (PD-ECGF) and the tie and tek receptors, and (b) proteases and their receptors involved in the breakdown of basement membranes and the extracellular matrix (ECM) including the matrix metalloproteinases (MMPs), cathepsins and those involved in the plasmin cascade. Angiogenesis has been identified as a potential target for development of anticancer agents. The discovery of a range of naturally-occurring factors which negatively regulate angiogenesis, including the thrombospondins, angiostatin and endostatin, and the tissue inhibitors of MMPs (TIMPs), has given added impetus to this approach. Synthetic anti-angiogenic compounds have been developed, including TNP-470, carboxyamidotriazole, VEGF-tyrosine kinase inhibitors and MMP inhibitors (MMPI) which, like the naturally-occurring anti-angiogenic factors, inhibit angiogenesis in vitro and in vivo, and tumour development, growth and metastasis in vivo. Anti-angiogenic agents also enhance the antitumour activity of many conventional cytotoxic chemotherapeutic agents. Such combinations may have a particular role as adjuvant therapies following surgical resection of primary tumours. Unlike tumour cells, tumour associated endothelial cells do not develop resistance to anti-angiogenic agents. Furthermore, anti-angiogenic agents are generally cytostatic rather than cytotoxic. As such, these agents are, in general, likely to be administered over long periods of time. Therefore, as well as having proven antitumour efficacy, an anti-angiogenic compound will need to be well-tolerated if it is to become established in the clinical management of patients with malignant disease.

To inhibit tumor-induced angiogenesis, the VEGF signaling pathway was targeted using AAV vectors encoding a VEGF decoy receptor, a truncated, soluble form of the murine VEGF receptor-2 (tsFlk-1). This approach initially had significant anti-neuroblastoma efficacy in murine xenograft models of local and metastatic disease, but when higher circulating levels of tsFlk-1 were established, tumor growth was more aggressive than even in control mice. Part of the mechanism for this apparent tumor resistance was increased human VEGF expression by the tumor cells. However, further investigation revealed that although a greater amount of VEGF could be bound by higher levels of tsFlk-1, more VEGF also existed in an unbound state and was, therefore, available to support angiogenesis. This novel, tumor-independent mechanism for resistance to antiangiogenic strategies suggests that careful titering of angiogenesis inhibitors may be required to achieve maximal antitumor efficacy and avoid therapy resistance mediated, in part, by ligand bioavailability. This has important implications for therapeutic strategies that use decoy receptors and other agents, such as antibodies, to bind angiogenic factors, in an attempt to inhibit tumor neovascularization.

Thyroid carcinomas are suitable targets for gene therapy because they can be highly lethal on one hand, while being susceptible to specific tumour targeting on the other hand. Several gene therapy modalities have been evaluated so far in experimental models of thyroid cancer, including tumour suppressor gene replacement, oncogene inhibition, suicide gene therapy, immunotherapy, antiangiogenesis, and viral oncolysis. All of these strategies have shown promising results, but clinical studies are lacking. Based on the clinical experience achieved in a pilot study in patients with advanced thyroid cancer and on clinical results in other types of solid cancer, it is suggested that combined gene therapy approaches, as well as multimodality therapeutic regimens, including gene therapy and conventional treatments, should be pursued to achieve clinically significant results.

The purpose of this study was to evaluate the ability of adeno-associated virus (AAV) vector-mediated delivery of pigment epithelium-derived factor (PEDF) to inhibit neuroblastoma (NB) xenograft growth. Pigment epithelium-derived factor was chosen for this study because, in addition to being a potent inhibitor of angiogenesis, it is capable of inducing neuronal differentiation.

Cohorts of mice received either recombinant AAV encoding human PEDF (rAAV-hPEDF) at a range of doses or control vector via tail vein. Subsequent hPEDF expression was measured by enzyme-linked immunoassay. After 6 weeks, the mice were given human NB cells by retroperitoneal injection and then killed 5 weeks later. Tumor weight, microvessel density, tumor differentiation, apoptosis, and levels of intratumoral vascular endothelial growth factor (VEGF) expression were determined at that time. In subsequent cohorts of mice, AAV-mediated murine PEDF expression was tested against both human NB xenografts and murine tumors.

In a series of in vitro studies, PEDF was shown to inhibit endothelial cell activation and to stimulate differentiation of NB cell lines. After tail vein injection of rAAV-hPEDF, stable transgene expression was generated and correlated with levels of vector administration. Human NB xenograft growth was restricted by hPEDF in a dose-dependent fashion. Intratumoral VEGF expression and microvessel density were decreased, and tumor cell apoptosis was increased in PEDF-treated mice.

Treatment with PEDF had a significant impact on NB growth in mice when delivered continuously using a gene therapy-mediated approach. The activity of PEDF appears to be mediated in part by inhibition of tumor-induced angiogenesis through down-regulation of tumor-elaborated VEGF, with subsequent intratumoral apoptosis. Furthermore, hPEDF was able to induce NB differentiation in vitro and in vivo. In addition, antitumor efficacy was seen when mouse PEDF was used to treat syngeneic murine tumors. In our murine models, gene therapy-mediated delivery of PEDF appears promising for the treatment of neuroblastoma.

A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages including the potential for sustained expression. The purpose of this study was to attempt to restrict the growth of disseminated neuroblastoma through delivery of a truncated, soluble form of the vascular endothelial growth factor receptor-2 (VEGFR-2 fetal liver kinase [Flk]-1), a decoy receptor for VEGF.

Mice underwent portal vein injection of vector, either AAV-CAGG-tsFlk-1 or control vector. Subsequent truncated soluble fetal liver kinase-1 (tsFlk-1) expression was confirmed and quantified by ELISA. After 8 weeks, mice were given human neuroblastoma cells through the tail vein to establish disseminated disease and were sacrificed after 40 days. The weight of liver metastasis was measured. Intrahepatic tumor microvessel density and levels of apoptosis were determined. Survival was assessed in a second cohort of mice.

After intraportal injection of AAV-CAGG-tsFlk-1, high-level, stable transgene expression was generated. Sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug (BD Biosciences) neovascularization in vivo, suggesting that a systemic state of angiogenesis inhibition had been established. After neuroblastoma injection, mice expressing tsFlk-1 had a smaller tumor burden in the liver when sacrificed, as compared with control mice. The decrease in tumor weight in the liver correlated with lower intratumoral microvessel density and higher levels of tumor cell apoptosis in the tsFlk-1-treated tumors, supporting the hypothesis that decreased angiogenesis had occurred. In a second cohort of mice, survival of tsFlk-1-expressing mice after tumor cell challenge was longer than in control mice.

Longterm in vivo expression of a functional VEGF inhibitor was established using an AAV-mediated gene therapy approach, and it demonstrated antiangiogenic and anticancer efficacy in a murine model of disseminated neuroblastoma.

Since angiogenesis is essential for the growth of any solid tumor, emerging efforts are being made to develop antiangiogenic therapy. To date, however, no antiangiogenic agent has become widely available for the clinical setting. Angiotensin I-converting enzyme (ACE) inhibitors are commonly used as antihypertensive agents and it has recently been suggested that they decrease the risk of cancer. Studies have found that an ACE inhibitor, perindopril, is a potent inhibitor of experimental tumor development and angiogenesis at a clinically comparable dose. The potent angiogenic factor, vascular endothelial growth factor (VEGF), is significantly suppressed by perindopril and also inhibits VEGF-induced tumor growth. In vitro studies showed that perindopril is not cytotoxic to either tumor cells or endothelial cells. Since perindopril is already in widespread clinical use without serious side effects, it may represent a potential new strategy for anticancer therapy.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.