Cooperative effect of roscovitine and irradiation targets angiogenesis and induces vascular destabilization in human breast carcinoma.

Angiogenesis is considered as an essential process for tumour development and invasion. Previously, we demonstrated that cyclin-dependent kinase inhibition by roscovitine induces a radiosensitization and a synergistic antitumoral effect in human carcinoma but its effect on the microenvironment and tumour angiogenesis remains unknown. Here, we investigated the effect of the combination roscovitine and ionizing radiation (IR) on normal cells in vitro and on tumour angiogenesis in MDA-MB 231 tumour xenografts. We observed that the combination roscovitine and IR induced a marked reduction of angiogenic hot spot and microvascular density in comparison with IR or roscovitine treatments alone. The Ang-2/Tie-2 ratio was increased in presence of reduced vascular endothelial growth factor level suggesting vessel destabilization. In vitro, no radiosensitization effect of roscovitine was found in endothelial, fibroblast, and keratinocyte cells. IR potentiated the antiproliferative effect of roscovitine without inducing apoptosis in endothelial cells. Roscovitine decreased IR-stimulated vascular endothelial growth factor secretion of MDA-MB 231 and endothelial cells. A reduction in the endothelial cells invasion and the capillary-like tube formation in Matrigel were observed following the combination roscovitine and IR. This combined treatment targets angiogenesis resulting in microvessel destabilization without inducing normal cell toxicity.

Canstatin gene electrotransfer combined with radiotherapy: preclinical trials for cancer treatment.

Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alpha v beta3 and alpha v beta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.

A study of the immunological response to tumor ablation with irreversible electroporation.

Immune cell recruitment during the treatment of sarcoma tumors in mice with irreversible electroporation was studied by immunohistochemistry. Irreversible electroporation is a non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the membrane. Employing irreversible electroporation parameters known to completely ablate the tumors without thermal effects we did not find infiltration of immune cells probably because of the destruction of infiltration routes. We confirm here that immune response is not instrumental in irreversible electroporation efficacy, and we propose that irreversible electroporation may be, therefore, a treatment modality of interest to immunodepressed cancer patients.

A gene transfer comparative study of HSA-conjugated antiangiogenic factors in a transgenic mouse model of metastatic ocular cancer.

Different antiangiogenic and antimetastatic recombinant adenoviruses were tested in a transgenic mouse model of metastatic ocular cancer (TRP1/SV40 Tag transgenic mice), which is a highly aggressive tumor, developed from the pigmented epithelium of the retina. These vectors, encoding amino-terminal fragments of urokinase plasminogen activator (ATF), angiostatin Kringles (K1-3), endostatin (ES) and canstatin (Can) coupled to human serum albumin (HSA) were injected to assess their metastatic and antiangiogenic activities in our model. Compared to AdCO1 control group, AdATF-HSA did not significantly reduce metastatic growth. In contrast, mice treated with AdK1-3-HSA, AdES-HSA and AdCan-HSA displayed significantly smaller metastases (1.19+/-1.19, 0.87+/-1.5, 0.43+/-0.56 vs controls 4.04+/-5.12 mm3). Moreover, a stronger inhibition of metastatic growth was obtained with AdCan-HSA than with AdK1-3-HSA (P=0.04). Median survival was improved by 4 weeks. A close correlation was observed between the effects of these viruses on metastatic growth and their capacity to inhibit tumor angiogenesis. Our study indicates that systemic antiangiogenic factors production by recombinant adenoviruses, particularly Can, might represent an effective way of delaying metastatic growth via inhibition of angiogenesis.

Respective roles of TNF-alpha and IL-6 in the immune response-elicited by adenovirus-mediated gene transfer in mice.

The immunogenicity of recombinant adenoviruses (Ad) constitutes a major concern for their use in gene therapy. Antibody- and cell-mediated immune responses triggered by adenoviral vectors hamper long-term transgene expression and efficient viral readministration. We previously reported that interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha play an essential role in both the acute phase and antibody response against Ad, respectively. As TNF-alpha controls the immune response and the development of the immune system, we examined here the consequence of blockade of TNF-alpha activity through Ad-mediated gene delivery of a dimeric mouse TNFR1-IgG fusion protein on transgene expression from a second Ad. Ad encoding TNFR1-IgG (AdTNFR1-Ig) was injected intravenously along with Ad encoding beta-galactosidase or alpha1-antitrypsin transgene in wild-type (IL-6(+/+)) but also in IL-6-deficient mice (IL-6(-/-)) to analyze how TNF-alpha and IL-6 diminish liver gene transfer efficacy. Blockade of TNF-alpha leads to increased transgene expression in both wild-type and IL-6(-/-) mice due to a reduced inflammatory response and to diminished recruitment of macrophages and NK cells towards the liver. Antibody responses against adenoviral particles and expressed transgenes were only delayed in AdTNFR1-Ig-treated wild-type mice, but were markedly reduced in AdTNFR1-Ig-treated IL-6(-/-) mice. Finally, treatment of mice with etanercept, a clinically approved anti-TNF-alpha drug, confirmed the importance of controlling proinflammatory cytokines during gene therapy by adenoviral vectors.

Dynamic assessment of antiangiogenic therapy by monitoring both tumoral vascularization and tissue degeneration.

Tumor growth is dependent both on endothelial and tumor cells. The aim of this study was to investigate dynamically whether changes in tumor vasculature implicate tumor tissue degeneration during antiangiogenic therapies. In order to quantify intra-tumor vascularization and necrosis, we have used ultrasound technology. This study has identified essential parameters needed to quantify specifically and sensitively the number of microvessels and the extent of necrosis in xenografted human carcinomas during natural tumor evolution, using contrast-enhanced high-frequency ultrasonography with (HFCDUS) or without (HFUS) color Doppler. We showed that quantification of intra-tumor microvessels between HFCDUS and immunohistochemistry is correlated using an anti-CD31 antibody. Furthermore, quantification of tumor necrosis with HFUS was confirmed by histological examination of hematoxylin-eosin-saffranin-stained sections over the observation period. Subsequently, for the assessment of novel angiogenic inhibitors, HFCDUS and HFUS were used to elucidate the underlying dynamics linking vessel inhibition and tumor eradication. We describe a novel application for HFCDUS/HFUS that constitutes an effective, convenient, and non-invasive method for clinical assessment of angiogenic inhibitors. In conclusion, we showed that tumor cells abruptly became necrotic following an antivascular therapy, whereas untreated tumors were protected from degeneration by a significant blood supply.

The tumor suppressor activity induced by adenovirus-mediated BRCA1 overexpression is not restricted to breast cancers.

The BRCA1 (breast cancer 1) breast cancer susceptibility gene is recognized as responsible for most familial breast and ovarian cancers and is suggested to be a tissue-specific tumor suppressor gene. In this report, we investigated the tissue specificity of tumor inhibitory activities induced by a recombinant adenovirus coding for wild-type BRCA1 (wtAdBRCA1). We demonstrated a pronounced in vitro antiproliferative effect on H1299 lung and HT29 colon cells upon infection with AdBRCA1. We describe a prolonged G1 cell cycle arrest associated with a decrease in the hyperphosphorylated form of Rb, suggesting that the Rb/E2F pathway is implicated in BRCA1-induced cell growth arrest. We also observed a significant antitumor effect in these pre-established subcutaneous tumors after in situ delivery of AdBRCA1, although these two tumors do not express wt p53, and also estrogen alpha and beta, progesterone and androgen receptors. Moreover, BRCA1 can induce a strong prolonged cell cycle arrest and apoptotic cell death but no significant antiangiogenic effect in H1299 tumors. Finally, our data indicate that intratumor administration of wtAdBRCA1 significantly inhibits growth of lung and colon steroid hormone-independent tumors.

n-3 polyunsaturated fatty acids decrease mucosal/epidermal reactions and enhance antitumour effect of ionising radiation with inhibition of tumour angiogenesis.

The purpose of this study was to evaluate the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs) on normal tissue (lip mucosa) and tumour growth when combined with ionising radiation. The oral region (snout) of C57 black mice was irradiated with 16.5 Gy and n-3 PUFAs (100 microl) were injected intravenously for 2 weeks. After exposure to irradiation, the degree and duration of the acute reactions decreased significantly when mice were treated with n-3 PUFAs as compared to the control group. Interestingly, the range of the reactions in the n-3 PUFAs-treated group compared favourably to the group receiving amifostine (27.5 mg/kg i.v.). the effect of n-3 PUFAs was further evaluated in HEP-2 human carcinoma xenograft transplanted in nude mice. An inhibition of tumour growth was observed when mice were treated with n-3 PUFAs alone and this effect was maximal when combined with irradiation. Similar results were obtained using eicosapentaenoic acid. The effect of n-3 PUFAs was associated with inhibition of angiogenesis and tumour proliferation, and significantly decreased expression of cyclooxygenase-2. In conclusion, n-3 PUFAs administration decrease mucosal response, while moderately enhancing the antitumour effect of irradiation. The magnitude of the differential effect suggests that n-3 PUFAs need to be further investigated in the clinic.

Systemic administration of a recombinant adenovirus encoding a HSA-Angiostatin kringle 1-3 conjugate inhibits MDA-MB-231 tumor growth and metastasis in a transgenic model of spontaneous eye cancer.

We previously reported that intratumoral injection of AdK3, a recombinant adenovirus encoding human angiostatin kringle (K) 1 to 3, inhibits tumor vascularization and tumor growth. To reduce the serum clearance of this factor, we constructed an adenovirus (AdK3-HSA) that carries a chimeric gene encoding a fusion protein between angiostatin K1-3 and human serum albumin (HSA). This conjugate inhibited endothelial cell proliferation as efficiently as K1-3. K3-HSA serum concentrations in immunodeficient mice systemically injected with AdK3-HSA were dramatically higher than in AdK3-injected mice. Furthermore, the growth of MDA-MB-231 tumors grafted into nude mice that had been injected intravenously with AdK3-HSA was inhibited by 79% (versus 17% with AdK3). In TRP-1/SV40 Tag transgenic mice, which spontaneously develop eye tumors with brain metastases, intravenous injections of AdK3-HSA in newborn mice blocked metastatic dissemination efficiently and significantly, and prolonged survival by 3 weeks. After 2 months, only 46% of AdK3-HSA-treated animals developed micrometastases, whereas 94% of the AdCO1-injected group displayed numerous macrometastases. Nevertheless, ocular tumor growth was not modified because of impaired diffusion of the conjugate in the eye compartment. Our results show that HSA genetic coupling is an efficient way to increase the pharmacokinetics of circulating angiogenic inhibitors and thus their antitumoral activity.

BRCA1 carries tumor suppressor activity distinct from that of p53 and p21.

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route.

Adenovirus-mediated gene transfer in canine eyes: a preclinical study for gene therapy of human uveal melanoma.

BACKGROUND: Melanomas of the uveal tract are the most common intraocular malignancies in adults, with an incidence of six cases per million adults per year. Enucleation, which may enhance the dissemination of tumour cells into the systemic circulation, is still required for eyes with large tumours. Gene therapy is proposed as a new therapeutic approach for uveal melanoma management. METHODS: The potential of adenovirus-mediated gene transfer to normal eyes of two laboratory Beagles and in an iris tumour of a Great Dane were evaluated. Replication-defective adenoviral vectors (Adbetagal) were used to assess the feasibility, efficiency and safety of direct adenoviral delivery to the anterior chamber of normal eyes and to an iris tumour. The expression of angiostatin into the aqueous humour following an adenoviral-mediated delivery of human angiostatin (AdK3) was also investigated. RESULTS: The ciliary body was the area preferentially transduced after adenoviral injection into the anterior chamber. It was also demonstrated that a direct intratumoral injection of a recombinant adenovirus efficiently transduces a canine uveal melanoma. Western blot analysis performed on the aqueous humour revealed that the expression of the angiostatin recombinant protein in the aqueous humour correlated with the dose of AdK3 administered. Lymphocyte infiltrates at the site of AdK3 injection indicated induction of a strong cellular immune response, and humoral immune responses developed in all three dogs. CONCLUSIONS: The present study involving adenovirus-mediated gene transfer to dog eyes provides an essential basis for gene therapy treatment of uveal melanoma-bearing patients.

Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice.

Systemic administration of Ad5-based recombinant adenovirus leads to preferential transduction of the liver. Using this property, we have assessed the potential of venous viral injection to deliver a recombinant antiangiogenic adenovirus to treat cancer dissemination and improve survival. The results demonstrate that venous injection of adenovirus AdmATF, which encodes a secretable mouse ATF (amino-terminal fragment of urokinase) known to inhibit angiogenesis, suppressed angiogenesis induced by colon cancer metastasis growth in mice liver and improved survival. Nude mice were injected intravenously with 5 X 10(9) PFU of AdmATF and subsequently challenged after a 3-day interval by intrasplenically injected human colon carcinoma cells (LS174T, 3 x 10(6)) that home to liver. Microscopic inspection revealed that, within the AdmATF-pretreated mice (n = 8), the size and number of liver-metastasized nodules on day 30 were remarkably reduced (80% in number, p < 0.05) compared with control mice (n = 7) pretreated in parallel with a control adenovirus. Metastatic growth-related liver weight gain was also inhibited up to 90%. AdmATF-specific capability that offers liver resistance to the apparition and growth of liver metastasis was shown to correlate with the inhibition of peritumoral and intratumoral angiogenesis (reduced by 79%, p < 0.01 as shown by anti-vWF immunostaining of liver sections) and a twofold increase in tumor necrotic area and an eightfold increase in apoptotic tumor cell number. This protective effect was still observed when the mice were challenged 10 days after venous AdmATF injection (visible metastasis nodules: 6.3+/-3.1, n = 7 for control mice versus 2.7+/-2.9, n = 10 for treated mice, p < 0.05). More importantly, the mean survival has been prolonged from 45.1 days (n = 9) to 83.3 days (n = 10, p < 0.05). Altogether, the high efficacy, although transient, in this experimental mice model strongly advocates the plausibility of transforming the liver into a dissemination resistant organ by antiangiogenic gene therapy through systemic delivery approach.

Efficient, repeated adenovirus-mediated gene transfer in mice lacking both tumor necrosis factor alpha and lymphotoxin alpha.

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (LTalpha), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-alpha/LTalpha-/-), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-alpha/LTalpha-/- and TNF-alpha/LTalpha+/- mice versus TNF-alpha/LTalpha+/+ mice links antibody levels to TNF-alpha/LTalpha gene dosage. Due to the absence of neutralizing antibodies, the TNF-alpha/LTalpha knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-alpha in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.