Attractive Target For Anti-angiogenic Therapy Research Articles

Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown.MethodsWe detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay.ResultsWe found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC.ConclusionsTaken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy.5QhhKt7hfqU82rJMQJ16V8Video

Synchronous inhibition of mTOR and VEGF/NRP1 axis impedes tumor growth and metastasis in renal cancer

Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of mTOR and the disruption of VEGF/NRP1 axis, respectively. We also exploited a liposomal formulation decorated with a proprietary tumor-targeting-peptide (TTP) to simultaneously deliver these two agents in a tumor-targeted manner. The TTP-liposomes encapsulating both Everolimus and EG00229 (EG-L) demonstrated higher in vitro and in vivo growth retardation than the single drug-loaded liposomes (E-L and G-L) in two different ccRCC models and led to a noticeable reduction in lung metastasis in vivo. In addition, EG-L displayed remarkable inhibition of tumor growth in a highly aggressive syngeneic immune-competent mouse model of ccRCC developed in Balb/c mice. Taken together, this study demonstrates an effective approach to achieve improved therapeutic outcome in ccRCC.

The role of SOX family members in solid tumours and metastasis.

Cancer is a heavy burden for humans across the world with high morbidity and mortality. Transcription factors including sex determining region Y (SRY)-related high-mobility group (HMG) box (SOX) proteins are thought to be involved in the regulation of specific biological processes. The deregulation of gene expression programs can lead to cancer development. Here, we review the role of the SOX family in breast cancer, prostate cancer, renal cell carcinoma, thyroid cancer, brain tumours, gastrointestinal and lung tumours as well as the entailing therapeutic implications. The SOX family consists of more than 20 members that mediate DNA binding by the HMG domain and have regulatory functions in development, cell-fate decision, and differentiation. SOX2, SOX4, SOX5, SOX8, SOX9, and SOX18 are up-regulated in different cancer types and have been found to be associated with poor prognosis, while the up-regulation of SOX11 and SOX30 appears to be favourable for the outcome in other cancer types. SOX2, SOX4, SOX5 and other SOX members are involved in tumorigenesis, e.g. SOX2 is markedly up-regulated in chemotherapy resistant cells. The SoxF family (SOX7, SOX17, SOX18) plays an important role in angio- and lymphangiogenesis, with SOX18 seemingly being an attractive target for anti-angiogenic therapy and the treatment of metastatic disease in cancer. In summary, SOX transcription factors play an important role in cancer progression, including tumorigenesis, changes in the tumour microenvironment, and metastasis. Certain SOX proteins are potential molecular markers for cancer prognosis and putative potential therapeutic targets, but further investigations are required to understand their physiological functions.

Angiopoietin-2 Induces Angiogenesis <i>via</i> Exosomes in Human Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. The expression of ANGPT2 in tumor-derived exosomes remains unknown. Methods: We detected the ANGPT2 expression in HCC-derived exosomes by the immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by the confocal laser scanning microscopy. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by the Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. Findings: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC. Interpretation: These results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy. Funding Statement: National Natural Science Foundation of China (81672420, 81702406, 81673433); National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province, 2017B090903004); Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Tissue and serum samples were collected from patients after obtaining informed consent in accordance with a protocol approved by the Ethics Committee of Sun Yat-sen Memorial Hospital (Guangzhou, China). All experimental procedures involving animals were performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85-23, revised 2011) and in accordance with the institutional ethical guidelines for animal experiments.

Predicting Glioblastoma Response to Bevacizumab Through MRI Biomarkers of the Tumor Microenvironment.

Glioblastoma (GB) is one of the most vascularized of all solid tumors and, therefore, represents an attractive target for antiangiogenic therapies. Many lesions, however, quickly develop escape mechanisms associated with changes in the tumor microenvironment (TME) resulting in rapid treatment failure. To prevent patients from adverse effects of ineffective therapy, there is a strong need to better predict and monitor antiangiogenic treatment response. We utilized a novel physiological magnetic resonance imaging (MRI) method combining the visualization of oxygen metabolism and neovascularization for classification of five different TME compartments: necrosis, hypoxia with/without neovascularization, oxidative phosphorylation, and aerobic glycolysis. This approach, termed TME mapping, was used to monitor changes in tumor biology and pathophysiology within the TME in response to bevacizumab treatment in 18 patients with recurrent GB. We detected dramatic changes in the TME by rearrangement of its compartments after the onset of bevacizumab treatment. All patients showed a decrease in active tumor volume and neovascularization as well as an increase in hypoxia and necrosis in the first follow-up after 3months. We found that recurrent GB with a high percentage of neovascularization and active tumor before bevacizumab onset showed a poor or no treatment response. TME mapping might be useful to develop strategies for patient stratification and response prediction before bevacizumab onset.

Abstract 5: The histone acetyltransferase inhibitor CPTH6 impairs tumor angiogenesis acting on both endothelial and cancer cells

Abstract Protein acetylation is typically catalyzed by enzymes with histone acetyltransferase (HATs) or histone deacetylase (HDACs) activity. To date, it has been identified the involvement of protein acetylation in different tumorigenic signaling events, including angiogenesis. The biological process of neo-vasculature formation from pre-existing blood vessels is widely considered to be an essential process to sustain tumor growth as well as to provide a route for tumor cell metastatization. In this context, the role exerted by HDACs in tumor angiogenesis is well known, whilst the role of HATs is largely unknown. Among molecules with HAT inhibitory activity, the thiazole derivative CPTH6 has been characterized by our group for its antitumor activity in different tumor models, including non-small cell lung cancer (NSCLC). In this study, we assessed the effect of CPTH6 on angiogenesis-related properties of both endothelial and NSCLC cells. The human umbilical vein endothelial cell (HUVEC) and H1299 NSCLC cell lines were used. HUVEC and H1299 morphogenesis was analyzed by plating cells on matrigel and evaluating their ability to organize capillary-like structures. The effect of CPTH6 on protein acetylation was assessed by WB analysis. Transwell supports were employed to evaluate HUVEC migration and invasion. Human Angiogenesis Antibody Array was used to test the conditioned media derived from CPTH6-treated H1299 cells. C57/BL6 and nude mice were used to perform matrigel plug assay and to evaluate tumor growth, respectively. IHC analysis of CD31 was employed to evaluate the number of intra-tumor vessels in tumor xenografts. The HAT inhibitor CPTH6 affected some endothelial cell functions in vitro. In particular, CPTH6 impaired HUVEC invasion, migration and differentiation abilities at doses that did not alter proliferation. Although CPTH6 did not affect histone H3 acetylation, it slightly reduced α-tubulin acetylation in HUVEC. In addition, CPTH6 decreased the neovascularization in vivo, as evidenced by the impairment of the VEGF-induced vascularization of matrigel plugs. Interestingly, CPTH6 affected also the angiogenesis-related properties of cancer cells. In particular, this compound reduced the ability of H1299 to organize capillary like structures and, conditioned media derived from CPTH6-treated H1299 cells, impaired HUVEC morphogenesis. Accordingly, CPTH6 reduced the secretion of some pro-angiogenic factors (VEGF, EGF, ANG, TIE-2, TNF-α) and, at the same time, increased the release of anti-angiogenic ones (Endostatin, PLG). Finally, in H1299-tumor xenografts, CPTH6 decreased significantly the number of intra-tumor vessels, even though it did not impair tumor growth. Overall, this study adds information to the role of HATs in tumor angiogenesis, and proposes HAT inhibition as an attractive target for antiangiogenic therapy of NSCLC. Citation Format: Marta Di Martile, Marianna Desideri, Simonetta Buglioni, Carla Azzurra Amoreo, Daniela Trisciuoglio, Donatella Del Bufalo. The histone acetyltransferase inhibitor CPTH6 impairs tumor angiogenesis acting on both endothelial and cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5.

Highlights of This Issue

Trop-2 is highly expressed in diverse epithelial cancers with limited presence on normal tissues. Here, Rossi and colleagues report redirected T-cell therapy targeting Trop-2 on solid tumors with a trivalent bispecific antibody (bsAb). Studies with human blood and animal models showed that interferon-α enhanced T-cell mediated antitumor activity significantly, without causing increased cytokine production that is often associated with adverse side effects. Additionally, this group discovered that following association of T cells with target cells, Trop-2, and possibly other tumor cell proteins, was translocated to the cell surface of T cells. This target/T-cell phenomenon mediated by bsAb warrants further investigation.MAPK reactivation through BRAF spliced variants, NRAS mutation, and RTK activation is the major resistant mechanism to BRAF inhibition in melanoma. In this study, Yadav and colleagues studied in vivo and in vitro melanoma models of acquired resistance to vemurafenib, and described that reactivation of MAPK and overexpression of cyclin D1 were common features across the resistant models. Treatment with LY2835219, a CDK4/6 selective inhibitor, was sufficient to induce growth regression in vemurafenib-resistant tumors and enhanced apoptosis in vemurafenib-resistant cells, suggesting that targeting cyclin-D1/CDK4 signaling by LY2835219 could be an effective strategy to overcome MAPK-mediated resistance to BRAF inhibitors.Endoglin has emerged as an attractive target for antiangiogenic therapy in various cancers. Only a few studies explored the effect of targeting tumor cell–specific endoglin. Pal and colleagues have analyzed the effects of targeting tumor cell–specific endoglin in pancreatic cancer. Their results indicate that endoglin-targeted antiangiogenic therapy in pancreatic cancer may offer an additional advantage by targeting the tumor cells as well. Moreover, inhibition of endoglin also contributed to drug sensitivity, making it ideal for combination therapies in case of chemotherapy-resistant tumors. Overall, their data suggests endoglin-targeted therapy could be a double-speared approach for pancreatic cancer treatment.Lymphangiogenesis in tumors is an integral step in the metastatic spread of cancer cells and there is a need to identify novel antilymphangiogenic agents. Astin and colleagues used a zebrafish-based chemical screen to identify compounds that have antilymphatic activity in both zebrafish and mouse models of lymphangiogenesis. One compound, kaempferol, a natural product found in plants, was shown to inhibit VEGFR signaling and was able to reduce the density of tumoral lymphatics and lymph node metastases in a breast cancer xenograft model. These findings reveal that zebrafish are a viable platform for the identification and development of mammalian antilymphatic compounds.

Tumor endothelial marker 7 (TEM-7): A novel target for antiangiogenic therapy

Antiangiogenesis has been validated as a therapeutic strategy to treat cancer, however, a need remains to identify new targets and therapies for specific diseases and to improve clinical benefit from antiangiogenic agents. Tumor endothelial marker 7 (TEM-7) was investigated as a possible target for therapeutic antiangiogenic intervention in cancer. TEM-7 expression was assessed by in situ hybridization or by immunohistochemistry (IHC) in 130 formalin-fixed paraffin-embedded (FFPE) and 410 frozen human clinical specimens of cancer plus 301 normal tissue samples. In vitro TEM-7 expression was evaluated in 4 human endothelial cell models and in 32 human cancer cell lines by RT-PCR and flow cytometry. An anti-TEM-7 antibody was tested in vitro on human SKOV3 ovarian and MDA-MB-231 breast carcinoma cells that expressed TEM-7 in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. In frozen tumor tissues, TEM-7 mRNA and protein was detected in all but one of the cancer types tested and was infrequently expressed in normal frozen tissues. In FFPE tumor tissues, TEM-7 protein was detected by IHC in colon, breast, lung, bladder, ovarian and endometrial cancers and in sarcomas. TEM-7 protein was not detected in head and neck, prostate or liver cancers. TEM-7 expression was restricted to the vasculature and was absent from tumor cells. In vitro, TEM-7 was not detected in human microvascular endothelial cells (HMVEC) or human umbilical vein endothelial cells (HUVEC) but was induced in endothelial precursor/progenitor cells (EPC) in the presence of the mitogen phorbol ester PMA. An anti-TEM-7 antibody mediated ADCC and phagocytosis in SKOV3 and MDA-MB-231 cell lines infected with an adenovirus expressing TEM-7. These data demonstrate that TEM-7 is a vascular protein associated with angiogenic states. TEM-7 is a novel and attractive target for antiangiogenic therapy.

The selective P-TEFb inhibitor CAN508 targets angiogenesis

Small molecule inhibitors of cyclin-dependent kinases (CDK) have been developed as anticancer drugs with cytostatic and cytotoxic properties, but some of them have also been shown to limit angiogenesis. Here, we report that the 3,5-diaminopyrazole CAN508 inhibits endothelial cell migration and tube formation. In addition, it reduces phosphorylation of the C-terminus of RNA polymerase II and inhibits mRNA synthesis in endothelial cells, in accordance with previous observations that it has high selectivity towards the positive transcriptional regulator P-TEFb. Moreover, CAN508 reduces expression of vascular endothelial growth factor by several human cancer cell lines. The findings suggest that P-TEFb may be an attractive target for anti-angiogenic therapy.

Targeting Vascular NADPH Oxidase 1 Blocks Tumor Angiogenesis through a PPARα Mediated Mechanism

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.

Analysis of expression of TGF-β1 receptor (ALK-1) in normal and tumor tissues by tissue microarrays

e22044 Background: TGF-β1 is an important angiogenic factor involved in different aspects of angiogenesis and vessel maintenance. TGF-β1 receptors consist of class I, class II and accessory receptors. Activin-like kinase I (ALK-1) is a class I TGF- β1 receptor which is almost exclusively expressed on endothelial cells. Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in the ALK-1 gene. Knock-out mice for ALK-1 die during gestation due to vascular malformation. ALK-1 may thus represent an attractive target for anti-angiogenic therapy. A therapeutic monoclonal antibody (PF-03446962) against ALK-1 has been developed which is currently in phase I testing. Here we describe target validation in normal and tumor tissue using tissue micro-arrays (TMAs). Methods: Normal tissue arrays comprised of 609 individual histological samples representing 76 human tissues. Multi-tumor arrays consisted of 3923 individual tumor samples from 87 tumor types. Consecutive sections of tissue arrays were stained for ALK-1 and CD31. CD31 immunohistology (IHC) was performed to ensure presence of vessels within tissue samples. ALK-1 staining was graded 0=absent, 1=weak, 2= moderate and 3=strong by an experienced pathologist blinded to tissue type. Results: ALK-1 staining of vessels in normal tissues was generally weak (mostly grade 1 and rarely grade 2) and was detectable in lymphatic tissues including tonsil, lymph nodes, thymus and spleen, lung, the entire GI tract including parotid, submandibular and sublingual glands as well as pancreas. ALK-1 positive vessels were also found within the female genital tract including placenta, uterus and ovary. In human tumor vessels ALK-1 expression showed high variability between tumor types. The highest ALK-1 expression rate was found in lung cancer (NSCLC 49%, SCLC 83%), neuroendocrine pancreas tumor (71%), colon cancer (50%), chondrosarcoma 50%, angiosarcoma 40% and NHL (44%). Conclusions: TMAs are an excellent tool to verify target expression in normal and tumor tissues. Results may be used to predict side effects of targeted therapy and to direct phase II testing of innovative agents in patients likely to respond. [Table: see text]

A New Kid on the Block

The formation of blood vessels through the process of angiogenesis is critical in normal vascular development and numerous vascular disorders. The most prominent stimulus for angiogenic processes in endothelial cells is vascular endothelial growth factor (VEGF), which regulates their migration, proliferation, and survival.1 The crucial role for this factor is documented by the lethal phenotype resulting from disruption of a single allele in mice.2,3 Binding of VEGF to its receptors activates several intracellular signaling molecules, among them phospholipase Cγ (PLCγ), protein kinase C (PKC), protein kinase D (PKD), and phosphatidyl-insitol-3 kinase (PI3K).1 The VEF-induced signaling events finally culminate in gene expression changes in the nucleus. See accompanying article on page 1782 A key regulator of gene expression is chromatin structure, which is largely determined by the acetylation status of histones. In general, acetylation of these nucleosomal proteins by histone acetyl transferases (HATs) stimulates transcription, whereas deacetylation by histone deacetylases (HDACs) leads to transcriptional repression. The HDAC family comprises 18 members in humans, which are classified based on their homologies to yeast proteins. …

Pericytes from human non-small cell lung carcinomas: An attractive target for anti-angiogenic therapy

Anti-angiogenic strategies have largely focused on endothelial cells and progenitors. However, pericytes are also an important component of vasculature. Perivascular cells from normal tissues have been widely reported, yet have not been extensively studied from human tumors. We have investigated pericytes from tumors of patients with lung cancer, the leader of cancer-related deaths in both men and women. Antibodies and magnetic beads were used to isolate cells from non-small cell lung carcinomas (NSCLC). The morphology of the pericytes was distinct with multiple elongated cytoplasmic extensions. Molecular expression of angiogenic genes was quantified by RT-PCR. Flow cytometric analysis shows that NSCLC pericytes express antigens such NG2 and VEGFR1 and present the ganglioside 3G5. The value of pericytes as models of tumor vasculature was demonstrated in cell-culture-based angiogenesis assays such as tube formation and proliferation. Results show that pericytes from some NSCLC but not all were able to maintain tubes networks on Matrigel. Pericyte function can be influenced by angiogenic growth factors or anti-angiogenic agents. Pericytes displayed invasive action against NSCLC clusters in the absence of other cell types. Perivascular cells contribute to the progression of disease and are an attractive target for anti-angiogenic therapy.

Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer.

Endoglin (CD105), a co-receptor in the TGF-beta receptor complex, is over-expressed on proliferating endothelial cells in the breast tumor neovasculature and thus offers an attractive target for anti-angiogenic therapy. Here we report the anti-angiogenic/anti-tumor effects achieved in a prophylactic setting with an oral DNA vaccine encoding murine endoglin, carried by double attenuated Salmonella typhimurium (dam-, AroA-) to a secondary lymphoid organ, i.e., Peyer's patches . We demonstrate that an endoglin vaccine elicited activation of antigen-presenting dendritic cells, coupled with immune responses mediated by CD8+ T cells against endoglin-positive target cells. Moreover, we observed suppression of angiogenesis only in mice administered with the endoglin vaccine as compared to controls. These data suggest that a CD8+ T cell-mediated immune response induced by this vaccine effectively suppressed dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature. It is anticipated that vaccine strategies such as this may contribute to future therapies for breast cancer.

Thalidomide in the treatment of patients with hepatocellular carcinoma

The treatment of patients with hepatocellular carcinoma (HCC) presents a major challenge, because associated cirrhosis limits the choice of chemotherapeutic agents. However, the abundant vascularity of HCC presents an attractive target for antiangiogenic therapy that potentially may be tolerated by cirrhotic patients. The current study was conducted to assess the antitumor activity, treatment tolerance, treatment-related toxicity, and patient survival after the administration of thalidomide in a Phase II trial. Thirty-seven HCC patients were accrued between March, 1999, and March, 2000. Initially, the dose of oral thalidomide was escalated from 400 mg per day during the first week to 1000 mg per day by the fifth week, delivering one-third of the dose in the morning and the remaining two-thirds of the dose in the evening prior to bedtime. Changes in the daily drug administration schedule were allowed based on tolerance. Response was assessed at 8-week intervals. Thirty-two of 37 registered patients were evaluable for response. One patient had a partial response (PR), 1 patient had a minor response (MR), 10 patients had stable disease (SD) (31%; 95% confidence interval [95%CI], 16-51%), and 20 patients) (61%; 95%CI, 42-78%) had disease progression. The most commonly encountered toxicity was somnolence, with Grade 3-4 somnolence (>or= 4 hours of sleep during normal waking hours) in 9 patients (35%) and Grade 2 somnolence (<or= 3 hours) in 30% of patients. In fact, only 48% of patients tolerated a daily dose>800 mg if it was delivered at bedtime. Grade 3-4 skin reactions were observed in 20% of patients, and exfoliative dermatitis was observed in 1 responding patient. The overall median survival was 6.8 months. With a 5% PR rate, a 5% MR rate, and a 31% SD rate, the results indicate that thalidomide mostly may offer HCC patients disease stabilization. It is possible that, at a different dosage, or combined with other chemotherapy agents, or with the use of a different thalidomide analogue, longer patient survival may be achieved. However, in view of the significant neurologic toxicity encountered among these commonly cirrhotic HCC patients, thalidomide monotherapy at the high doses studied cannot be recommended for the treatment of HCC.

Inhibition of Corneal Neovascularization by Recombinant Adenovirus Mediated Antisense VEGF RNA

The expression of vascular endothelial growth factor has been strongly implicated in the pathogenesis of conditions leading to inappropriate blood vessel growth in the eye. As such, vascular endothelial growth factor is an attractive target for anti-angiogenic therapies designed to treat neovascular eye diseases. Onesuch therapy, antisense gene therapy, is a technique based on the ability of single-stranded DNA or RNA sequences to alter the expression of targeted genes. Recombinant adenoviruses have demonstrated efficient ocular cell transduction with a high level of transgene production. Cauterization of the normally avascular rat cornea results in a strong neovascular response, making it an ideal animal model for the testing of anti-angiogenic therapies. In this study, a recombinant adenovirus system was assessed for the ability to express biologically relevant antisense RNA to reduce vascular endothelial growth factor expression in a rat model of corneal neovascularization. Recombinant adenovirus constructs expressing short and long antisense and sense vascular endothelial growth factor cDNA, under the control of cytomegalovirus major immediate early promoter or the RNA polymerase III promoter, VA1, were constructed. The expression of short and long antisense RNAs was demonstrated by Northern blot hybridization. All constructs were capable of producing RNA, and the highest level of antisense RNA production was detected in retinal pigment epithelial cells which had been transduced with the longer antisense cDNA construct under the control of the VA1 promoter. This construct was also the most efficient in reducing in vitro vascular endothelial growth factor production (P<0.05) and human endothelial cell proliferation. This construct was subsequently injected into rat eyes 24hr prior to cauterization of the cornea and antisense vascular endothelial growth factor expression was demonstrated by in situ hybridization. The resulting neovascular response was clearly inhibited at 4, 7 and 14 days post-cautery, compared to the control injections which demonstrated an intense neovascular response. Only one out of six eyes injected with the long antisense cDNA construct under the control of the VA1 promoter demonstrated any vascular response to cautery. The reduction in the neovascular response was correlated, with significantly lower amounts of vascular endothelial growth factor protein in the corneas (P=0.006). These observations suggest that the specific down-regulation of vascular endothelial growth factor production is sufficient to reduce the corneal neovascular response and that recombinant adenovirus might be a useful vehicle to produce antisense RNA in situ to down-regulate ocular gene expression.