Original Article

Cancer Gene Therapy (2008) 15, 535–542; doi:10.1038/cgt.2008.20; published online 18 April 2008

Antiangiogenic systemic gene therapy combined with doxorubicin administration induced caspase 8 and 9-mediated apoptosis in endothelial cells and an anti-metastasis effect

M Peled1,2,3, A Shaish1,2, S Greenberger1,2, A Katav2, I Hodish1,2, D Ben-Shushan2, I Barshack1,4, I Mendel1, L Frishman1,2, R Tal1,2, L Bangio1, E Breitbart1 and D Harats1,2

  1. 1Vascular Biogenics Ltd., Or Yehuda, Israel
  2. 2The Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel Hashomer, Israel
  3. 3Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
  4. 4The Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel

Correspondence: Professor D Harats, Vascular Biogenics Ltd., 6 Jonathan Netanyahu Street, Or Yehuda, 60376, Israel. E-mail: dror@vbl.co.il

Received 28 July 2007; Revised 11 January 2008; Accepted 21 February 2008; Published online 18 April 2008.



Ad-PPE-Fas-c is an adenovector that expresses Fas-c under the control of the modified pre-proendothelin-1 (PPE-1) promoter. Fas-c is a chimeric death receptor containing the extracellular portion of tumour necrosis factor 1 receptor (TNFR1) and the transmembrane and intracellular portion of Fas. We recently demonstrated that Ad-PPE-Fas-c induced Fas-receptor-mediated endothelial cell apoptosis. Previously, doxorubicin was shown to enhance Fas-receptor clustering and the induction of its cascade. Therefore, the goal of this work was to test whether doxorubicin augments the capacity of Ad-PPE-Fas-c to induce endothelial cell apoptosis and to elucidate whether either the death-receptor-mediated apoptotic cascade or the mitochondria-associated apoptotic cascade is involved in the combined treatment effect. We found that a combined treatment of Ad-PPE-Fas-c and doxorubicin synergistically induced a reduction in endothelial cell viability and apoptosis. z-IETD-FMK, a caspase-8 inhibitor, and z-LEHD-FMK, a caspase-9 inhibitor, significantly decreased apoptosis induced by the combined treatment. Systemically administered combined therapy significantly reduced the lung metastases burden (70%) in mice as compared to each treatment alone. Thus, a combined treatment of Ad-PPE-Fas-c gene therapy and chemotherapy may be effective in the treatment of metastatic diseases and both the Fas cascade and the mitochondria-associated cascade are essential for this effect.


Fas, doxorubicin, apoptosis, metastasis, adenovirus, angiogenesis



Growing tumours rely on the oxygen and nutrients supplied through the newly formed capillary network.1 During oncogenic transformation, the cells undergo an angiogenic switch and develop the ability to promote angiogenesis. Disrupting tumour-associated angiogenesis by the induction of endothelial cell apoptosis is becoming a promising new strategy to control tumour growth. The antiangiogenic approach to antitumour treatment has several advantages over anticancer treatment that is directed at the tumour cells, as these new vessels are readily accessible by i.v. administration and the endothelial cells (ECs) lining these vessels possess relatively few mutations.

Apoptosis is a genetically regulated mechanism of cell death. Death receptor- or mitochondrion-dependent apoptosis is initiated by the recruitment and activation of initiator caspases in the apoptosis signaling pathways. In death-receptor-mediated apoptosis, the engagement of death receptors leads to the formation of the death-inducing signaling complex that contains the death receptors, adaptor proteins and caspase-8.2, 3, 4, 5 In mitochondrion-dependent apoptosis, release of cytochrome c into the cytosol results in the formation of apoptosome containing cytochrome c, Apaf-1 and caspase-9.6, 7, 8 Both pathways lead to the activation of downstream caspases, including caspase-3, caspase-6 and caspase-7,7, 9 which, in turn, lead to the degradation of cellular and nuclear proteins.

The induction of direct and specific EC apoptosis is assumed to cutoff the tumour's blood supply. In a previous work,10 we found that the introduction of a chimerical death receptor made up of the extracellular portion of tumour-necrosis factor 1 receptor (TNFR1) and the transmembrane and intracellular region of Fas2 induced apoptosis in endothelial cells. ECs were specifically susceptible to apoptosis induction with an adenovector expressing the chimerical receptor (Fas-c) gene under the regulation of a modified pre-proendothelin-1 (PPE-1) promoter (Ad-PPE-Fas-c). The specificity of the PPE-1 promoter to angiogenic ECs over normal nonactivated ECs was established by us previously, using green fluorescent protein (GFP) and luciferase-expressing vectors controlled by this promoter.11 A systemic injection of Ad-PPE-Fas-c to the tail vein resulted in growth retardation of the tumour, reduction of tumour mass with central tumour necrosis in the model of B16 melanoma and a reduction of Lewis lung carcinoma (LLC) lung metastasis, with minimal systemic toxicity.10

Doxorubicin (Dox) is a common chemotherapy agent widely used in the treatment of a variety of cancers, including leukaemias, sarcomas and breast cancer. Dox was shown to enhance FAS receptor clustering and the induction of its cascade.12 Moreover, it was demonstrated to be effective as an antitumour and anti-metastasis agent in combination with other FAS-associated agents in several models, including chemoresistant breast cancers,13 bladder cancer and prostate cancer.14 FAS receptor is part of the FAS-chimera transgene in the Ad-PPE-Fas-c vector.10 Thus, we sought to improve its apoptotic effect, by the death-receptor-dependent apoptosis, with Dox. The combined treatment of Ad-PPE-Fas-c and Dox was tested in the current study in vitro in ECs, and in vivo in a LLC mouse model. The question of the exact apoptotic cascade involved in the apoptotic induction of the combined treatment of Ad-PPE-Fas-c and Dox was also addressed in this study, by using specific caspase-8 and -9 apoptosis inhibitors, for the inhibition of the death receptor- or mitochondrion-dependent apoptosis, respectively.


Materials and methods


Bovine aortic ECs (BAECs) were a kind gift from N Savion (Goldschleger Eye Institute, Sheba Medical Center, Israel). Human embryonic kidney cells (293 cells) were purchased from American Type Culture Collection (Rockville, MD, USA). Passages 20–26 of 293 cells were used. D122-96 (LLC) cells were kindly provided by L Eizenbach (Weizmann Institute of Science, Rehovot, Israel). Cells were cultured in DMEM supplemented with 10% fetal calf serum and 100Uml−1 penicillin/streptomycin. All the cells were maintained at 37°C in a 5% CO2 humidified incubator.


Adenovectors Ad-PPE-Fas-c and Ad-PPE-GFP have been described previously.10, 11The expansion, purification and titration analyses of all vectors used were performed as described previously.10, 11 The titre used in this study was determined by a plaque assay and the absorbency of the dissociated virus at A260nm (1 A260nm unit=1.1 × 1012 viral particles (vp)ml−1) was used as additional information. Particle/plaque-forming unit (PFU) ratios were usually between 10:1 and 100:1. Thus, the multiplicity of infection (MOI) of 1000 vp is equivalent to an MOI of 10–100 PFU. The MOI was calculated according to PFU. Unless otherwise specified, Ad-PPE-GFP was used as a vector control and medium as a mock control.

Cytotoxicity assays and Chou–Talalay analysis

Bovine aortic ECs were seeded in a 96-well microtitre plate at 104 cells200μl−1 per well. After an overnight incubation, cell monolayers were infected with the recombinant adenoviruses at the indicated MOI. Dox (Sigma-Aldrich, St Louis, MO, USA), at the indicated concentrations, was added to the culture media 48h after infection. Next, 5ngml−1 of human TNF-α (Sigma-Aldrich) was added to the culture media 96h after infection, and cell viability was assessed 3h after the addition of TNF-α. TNF-α is the ligand of the chimeric receptor TNF-Fas-chimera10 and was used as the inducer of apoptosis in all of the in vitro experiments.

Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Briefly, MTS solution was added to each well. The optical density was then measured by a spectrophotometer at 490nm. Cell viability was calculated as a percentage of the non-transduced cells’ viability.

Dose–response curves were fit to Chou–Talalay lines,15 which are derived from the law of mass action and are described by the equation log(fa/fu)=m log Dm log Dm, in which fa is the fraction affected (percent cell death), fu is the fraction unaffected (percent cell survival), D is the dose, Dm is the median effect dose (the dose causing 50% of cells to be affected, that is, 50% survival) and m is the coefficient signifying the shape of the dose–response curve. Chemotherapy and virus were then added in combinations in a ratio equaling the ratio of their median effect doses, with each dose in each experiment plated in triplicate and each experiment performed three times. After fitting the combined dose–response curve from a single representative experiment to a Chou–Talalay line, Chou–Talalay combination indices (CI) were calculated for each fa using the equation CI=(D1/Dx1)+(D2/Dx2)+(D1)(D2)/((Dx1)(Dx2)), which defines CI for mutually nonexclusive treatment regimens (the CI reported in this article was derived assuming mutually nonexclusive treatments, although this interpretation did not change under the mutually exclusive assumption, in which the third term in the CI equation is eliminated), in which Dx1 and Dx2 are the chemotherapy and virus doses required to achieve a particular fa, respectively, and D1 and D2 are the doses of the two combined required to achieve the same fa. Levels of interaction are defined as follows: CI greater than 1.1 indicates antagonism, CI between 0.9 and 1.1 indicates additivity, CI between 0.8 and 0.9 indicates slight synergy, CI between 0.6 and 0.8 indicates moderate synergy, CI between 0.4 and 0.6 indicates synergy, and CI less than 0.4 indicates strong synergy.16 According to the protocol described by Chou and Talalay,16 CI values lack 95% confidence intervals, but the use of the above ranges to interpret CIs takes variability into account.

Annexin V/7-aminoactinomycine-D staining

Bovine aortic ECs were seeded in a 6-well plate at 5 × 105 cells3ml−1 per well. After an overnight incubation, cell monolayers were infected with the recombinant adenoviruses at 50 MOI. Dox (Sigma, St Louis, MO, USA), 50nM, were added to the culture media 48h after infection and 5ngml−1 of human TNF-α (Sigma-Aldrich) were added to the culture media 96h after infection. Annexin-7-aminoactinomycine-D (V/7-AAD) staining was performed 3h after the addition of TNF-α. BAECs were trypsinized and washed twice with phosphate-buffered saline and then stained with phycoerythrin-labelled annexin V/7-AAD according to the manufacturer's instructions (Annexin V/7-AAD kit, BD Biosciences, Palo Alto, CA, USA). Briefly, a washed cell pellet was resuspended in 0.2ml binding buffer at a concentration of 5 × 106 cellsml−1; 5μl of annexin V together with 5μl 7-AAD were added to 100μl cell suspension. BAECs were incubated for 15min at 25°C in the dark and 400μl binding buffer was added. The samples were analysed by flow cytometry.

Flow cytometry

Flow cytometry was performed using FACS-Calibur (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The fluorescence of 10000 events was measured. In general, this resulted in a situation in which viable, apoptotic and dead subpopulations could be adequately assessed.

Caspase activity assay

Caspase-8 and caspase-9 activity was measured using colorimetric assay kits (caspase-8 assay kit, Sigma-Aldrich; caspase-9 assay kit, Calbiochem, San Diego, CA, USA) according to the manufacturer's instructions. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labelled substrate. Briefly, BAECs were seeded in a 6-well plate at 5 × 105 cells3ml−1 per well. After an overnight incubation, cell monolayers were infected with the recombinant adenoviruses at 50 MOI. This was followed by the addition of 50nM Dox (Sigma) to the culture media 48h after infection, and of 5ngml−1 human TNF-α (Sigma-Aldrich) 96h after infection; caspase activity was assessed 3h after the addition of TNF-α. BAECs were washed with phosphate-buffered saline and lysed with cell lysis buffer (caspase-8 assay kit, Sigma-Aldrich; caspase-9 assay kit, Calbiochem). Samples were incubated on ice for 20min and centrifuged in a microcentrifuge at 20000g for 11min at 4°C to precipitate the cellular debris. Lysed cells were incubated with 10μl of substrate specific for caspase-8 or caspase-9 in a 96-well plate for 24h at 37°C. The specific substrate used was Ad-IETD-pNA for caspase-8 and LEHD-pNA for caspase-9. The caspase activity was measured in an enzyme-linked immunosorbent assay reader, according to the manufacturer's instructions provided with the assay kits.

Caspase inhibitors

The caspase-8 inhibitor, z-IETD-fmk, or the caspase-9 inhibitor, z-LEHD-fmk (Calbiochem, Bad Soden, Germany) diluted in dimethylsulphoxide was used at the respective concentrations 30min before the addition of Dox. The inhibitors were replenished after 48h, 30min before the addition of TNF-α. Dimethylsulphoxide was added to the control wells at the maximal concentration of dimethylsulphoxide used in the experiment.


C57BL/6J mice, 12–14 weeks old (Harlan Laboratories Ltd., Jerusalem, Israel), were used. All animal procedures were approved by the Animal Care and Use Committee of Sheba Medical Center.

Lewis lung carcinoma model

Fifty microliters containing 5 × 105 LLC cells in 0.9% NaCl solution were injected into the footpad of C57BL/6 mice. When the tumour thickness was more than 7mm (14 days following injection of LLC), it was removed to induce lung metastasis growth.11 Five days after primary tumour resection, 1011 vp per 100μl of Ad-PPE-Fas-c, Ad-PPE-GFP or 100μl of saline (control) was systemically injected into the tail veins. Four days after the injection of adenovectors, Dox or saline was injected i.p. for 5 consecutive days. TNF-α was not used in the in vivo experiments because it is abundant in the tumour's microenvironment7 and it was not effective in augmenting the anti-metastatic effect in previous in vivo experiments that were performed with Ad-PPE-Fas-c.10

Once five of the saline-injected control mice had died of metastasis, every mouse that reached the same day after tumour resection on which the fifth control mouse died was sacrificed. Primary tumour diameter (to assess tumour growth) and lung weight were measured. Lungs were fixed in formalin and embedded in paraffin.


Von Willebrand factor (VWF) was assessed by immunohistochemistry. Formalin-fixed, paraffin-embedded tissues were heated and then deparaffinized using xylene and declining grades of ethanol before being rehydrated in 0.1% Triton X-100 for 10min. Immunohistochemistry was performed using the Blood Vessel Staining Kit (Chemicon, Temecula, CA, USA) according to the manufacturer's instructions.

Microvessel density (MVD) was quantified by counting the number of microvessels per 400 × HPF (high power field) over six randomly selected fields. A microvessel was defined as a discrete VWF+cluster or single cell adjacent to a lumen.

Statistical analysis

All values are reported as mean±s.e. Statistical analyses were done by Student's t-test.



Doxorubicin enhanced endothelial cell death induced by Ad-PPE-Fas-c in vitro

To test whether Dox enhances the reduction in EC viability induced by adenovirus-mediated Fas-c gene transfer, cell viability was analysed. MTS assay showed that the median effect doses (ED50=dose causing 50% death) of Ad-PPE-Fas-c alone (that is, MOI of 1000) or of a combination of Dox+Ad-PPE-GFP (that is, 500nM Dox+500 MOI Ad-PPE-GFP) were reduced 10- to 20-fold by the combined treatment of Dox+Ad-PPE-FAS-c (that is, 50nM Dox+50 MOI Ad-PPE-Fas-c). Chou–Talalay combination indices were less than 0.4 (Figure 1b), which indicates strong synergy.16 Ad-PPE-GFP serves as a control for vector-induced toxicity, which is not related to the transgene encoded by the vector.

Figure 1.
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Interaction between Ad-PPE-Fas-c and doxorubicin (Dox) in bovine aortic endothelial cells (BAECs). (a) The ED50 values represent the dose of the adenovector required to induce 50% cell death in combination with or without Dox compared with untreated BAECs. (b) Chou–Talalay analysis of BAEC cells treated with Ad-PPE-Fas-c and Dox. Combination index (CI) is plotted as a function of fraction affected (Fa). CI less than 0.4 indicates strong synergy.16 The experiment was repeated three times, each in triplicate. The data shown are from one representative experiment.

Full figure and legend (73K)

Ad-PPE-Fas-c and doxorubicin induce apoptosis in vitro in endothelial cells

Apoptosis of BAEC cells was assessed by measurement of annexin V by fluorescence-activated cell sorting after the cells were treated with the Ad-PPE-Fas-c or Ad-PPE-GFP (50 MOI), with or without Dox (50nM). The percentage of apoptotic cells (annexin V positive/7-AAD negative) present was 4% for mock- or Ad-PPE-GFP-treated cells, 7% for Ad-PPE-Fas-c-treated cells, 14% for Dox- or Ad-PPE-GFP+Dox-treated cells and 36% for Ad-PPE-Fas-c+Dox-treated cells (Figure 2).

Figure 2.
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The combined treatment of Ad-PPE-Fas-c and doxorubicin (Dox) induces apoptosis in bovine aortic endothelial cells (BAECs). Annexin V (FITC) and 7-aminoactinomycine-D (AAD) staining of BAECs after treatment with Ad-PPE-Fas-c or Ad-PPE-GFP (50 multiplicity of infection), with or without Dox (50nM). Fluorescence-activated cell sorting analyses of BAECs were performed 3h following treatment with tumour-necrosis factor-α (5ngml−1). Percentages represent the annexinV-positive/7-AAD-negative cells (early apoptotic) cells. Results depict one representative of three independent experiments.

Full figure and legend (224K)

Caspase-8 and -9 activities in ECs are induced by Ad-PPE-Fas-c and doxorubicin

Apoptosis is induced either by the death receptor pathway, which includes caspase-8 activation, or by the mitochondrion-dependent pathway, which includes caspase-9 activation.

To investigate the involvement of the different apoptotic pathways in Ad-PPE-Fas-c- and Dox-induced apoptosis, caspase-8 and 9 activities were tested. Ad-PPE-Fas-c at 50 MOI or Dox (50nM), with or without the control vector Ad-PPE-GFP, induced an increase in caspase-8 and 9 activity, by twofold to fourfold, compared to mock-treated cells (Figure 3). The combined treatment of Ad-PPE-Fas-c and Dox resulted in a 3.5-fold or 9-fold increase in caspase-8 or 9 activity, respectively, compared to mock-treated cells (P<0.05) (Figure 3). The results suggest that both apoptotic pathways are involved in the combined treatment-induced apoptosis.

Figure 3.
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Combined chemotherapy- and gene-therapy-induced caspase-8 and -9 activation in bovine aortic endothelial cells (BAECs). Cells were incubated with 50nM doxorubicin, Ad-PPE-GFP (GFP) and Ad-PPE-FAS-c (Fas) (50 multiplicity of infection) as described in the Methods and harvested. (a) Caspase-8 and (b) caspase-9 activity was measured in BAECs. Caspase activity is presented as the fold increase when compared with an uninduced, negative control. Values are mean±s.e. of three separate experiments performed in triplicates. *P<0.05, when comparing the corresponding treatment with mock.

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Treatment with the caspase inhibitors z-LEHD-FMK or z-IETD-FMK prevented Ad-PPE-Fas-c and doxorubicin-induced cytotoxicity

To further investigate the involvement of the different apoptotic pathways in Ad-PPE-Fas-c and Dox-induced cytotoxicity, BAECs were treated with 100μM z-LEHD-FMK, a selective caspase-9 inhibitor, and z-IETD-FMK, a selective caspase-8 inhibitor (Figure 4). No reduction in cell viability was induced by Ad-PPE-GFP, Ad-PPE-Fas-c alone, Dox alone or Ad-PPE-GFP+Dox under the conditions used in this experiment (data not shown).

Figure 4.
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Inhibition of Ad-PPE-FAS-c- and doxorubicin-induced cytotoxicity by caspase-8 and 9 inhibitors. Bovine aortic endothelial cells were incubated with 100μM z-LEHD-FMK (caspase-9 inhibitor) or 100μM z-IETD-FMK (caspase-8 inhibitor) and treated as described with 50nM doxorubicin, 5ngml−1 tumour-necrosis factor-α, Ad-PPE-GFP and Ad-PPE-FAS-c (50 multiplicity of infection). Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay 96h following vector transduction. Values are mean±s.e. of triplicates. *P<0.05 when comparing the corresponding treatment with mock, **P<0.05 when comparing the corresponding treatment with no inhibitor and ***P<0.05 when comparing the corresponding treatment with one-inhibitor treatment.

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The caspase inhibitors prevented the combined treatment of Ad-PPE-Fas-c and Dox-induced cytotoxicity in BAECs, as measured by viability assay (MTS). Caspase-8 and -9 inhibitors increased cell viability from 27 to 74 and 61% (P<0.05), respectively (Figure 4). When the cells were treated with both inhibitors, viability was increased to 92% (P<0.05, compared to a single inhibitor) (Figure 4).

These results suggest that both caspase-8 and caspase-9 are required for apoptosis induction by the combined treatment.

Antitumoral activity of Ad-PPE-Fas-c and doxorubicin

The ability of Dox to enhance antitumoral activity of Ad-PPE-Fas-c was tested in a mouse model of LLC metastases. Mice bearing lung metastases of LLC were intravenously injected with 1011 vp Ad-PPE-Fas-c. Control animals were injected with saline or 1011 vp Ad-PPE-GFP. Ad-PPEx-GFP served as a negative control that can reduce tumour burden by an immunological mechanism.17 Four days following virus injection, mice were administered i.p. with 2.5mgkg−1 (body weight) Dox or saline for 5 consecutive days. Mice were killed when five saline-treated mice died of metastases. Average lung metastasis weights of mice injected with Ad-PPE-Fas-c, Dox, or Ad-PPE-GFP and Dox were reduced by 50–60%, compared to those in the saline-treated group (Figure 5a). The combined treatment of Ad-PPE-Fas-c and Dox reduced lung metastasis weights by 90%, compared to saline. Moreover, a significant (P<0.05) reduction of 60–70% was also observed when Ad-PPE-Fas-c+Dox treatment compared with each treatment alone. Lung surfaces of most Ad-PPE-Fas-c+Dox-treated mice were almost free of tumours, but the lungs of other treatment groups were partly covered by metastases and almost completely replaced by tumour tissue in the saline-treated group (Figure 5b).

Figure 5.
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Effect of Ad-PPE-FAS-c and doxorubicin (Dox) on Lewis lung carcinoma lung metastases burden. Mice were randomized into different groups (n=10). Ad-PPE-FAS-c, saline or Ad-PPE-GFP was administered i.v. at 1 × 1011 particles per dose. Dox was administered i.p. at 2.5mgkg−1 per dose, 4 days following adenovector injection. (a) Metastases weight was measured by subtraction of the average normal wet organ weight (200mg) from each tumour-bearing organ. The values represent the mean±s.e. *P<0.05 compared to saline; **P<0.05 compared to either therapy alone. (b) Representative lung surfaces of treated versus untreated mice. Arrows: Lewis lung carcinoma metastases. (c) Von Willebrand factor staining of blood vessels in the lung metastases. All images were obtained with a × 400 objective on an Olympus B × 51 light microscope. (d) Microvascular density. Values are mean±s.e. *P<0.05 compared to green fluorescent protein (GFP) and GFP+Dox.

Full figure and legend (158K)

The effect on MVD in the lung metastases was examined using VWF immunostaining (Figures 5c and d). Ad-PPE-GFP+Dox and Ad-PPE-Fas-c each did not reduce MVD significantly compared to Ad-PPE-GFP, however, the combination of Ad-PPE-Fas-c and Dox significantly reduced MVD when compared to Ad-PPE-GFP and Ad-PPE-GFP+Dox (P<0.05).

Mild hepatitis was observed in all the Ad-PPE-GFP- or Ad-PPE-FAS-c-treated groups, demonstrated both by liver enzymes (SGOT, SGPT) and by histopathological analysis (data not shown). This could be attributed to the known liver toxicity induced by adenovirus.17 No mortality or morbidity was observed by other parameters, including: weight, creatinine, urea, uric acid and lactate dehydrogenase.



In the current study, we used a proapoptotic mechanism, utilizing adenoviral gene transfer plus chemotherapy, to kill the ECs of the emerging blood vessels feeding the tumour. We demonstrated that both caspase-8 and caspase-9 are essential for the combined treatment effect and that this treatment has a synergistic effect on lung metastasis inhibition.

In a previous study, Ad-PPE-Fas-c was used to induce angiogenic-specific apoptosis in ECs and to reduce LLC lung metastases.10 Here, by using Dox, we maximized the efficacy of Ad-PPE-Fas-c, based on the mechanism of Fas-induced apoptosis.

Doxorubicin is a common chemotherapy agent widely used in the treatment of a variety of cancers. Although it has been shown to induce programmed cell death,6, 18, 19, 20, 21, 22 the mechanisms by which Dox operates appear to vary, depending on the cell type analysed. Thus, FAS/FASL interactions have been reported to mediate the drug-induced apoptosis in several tumour cell lines,12 but not in others.23 Previously, it was shown that Dox induced both apoptosis and CD95 (FAS receptor) upregulation in human primary endothelial cells through a p53-dependent mechanism.24 According to this work, however, the upregulation of CD95 was not involved in the Dox-induced activation of the caspase cascade that led to the apoptosis of human primary endothelial cells. This conclusion relied on the fact that anti-CD95- or anti-CD95L-blocking antibodies did not inhibit Dox-mediated apoptosis in human primary endothelial cells. On the other hand, Micheau et al.12 showed that Dox and other chemotherapeutic drugs induce FAS ligand-independent FAS receptor trimerization in leukaemic and colon carcinoma cell lines and that the FAS cascade is partially essential for the apoptosis induced by the chemotherapeutic drugs tested. However, a third research project, performed by Kataoka et al.,23 demonstrated that a FAS cascade-specific inhibitor (FLIP) did not prevent apoptosis induced by Dox in FLIP stably transfected jurkat cells, a leukaemic cell-line. To conclude, two works claimed that Fas cascade is not essential for apoptosis induction by Dox,23, 24 although Fas expression is elevated,24 and Micheau et al.12 claimed it is partially essential through a ligand-independent mechanism.

Our hypothesis, relying mainly on the study by Micheau et al.,12 is that Dox treatment for ECs with enhanced expression of FAS-c will induce trimerization of FAS-c and apoptosis.

Here, we showed that Dox concentrations as low as 50nM increased EC apoptosis in cells transduced with Ad-PPE-Fas-c. In vitro experiments were performed in the presence of TNF-α to mimic the TNF-α-enriched tumor microenvironment.7 However, similar in vitro results were obtained in the absence of TNF-α, using an increased titre of Ad-PPE-Fas-c to exert cell death (data not shown).

The cytotoxic effect was mediated by caspase-8 and -9 activation and was blocked by the caspase-8 inhibitor, z-IETD-FMK, and the capase-9 inhibitor, z-LEHD-FMK. Although our initial hypothesis regarding the synergism between Ad-PPE-FAS-c and Dox was based exclusively on the Fas cascade, we observed that caspase-9 was also essential for the synergistic effect. Caspase-9 is not part of the Fas cascade, but rather a part of the mitochondria-associated apoptotic cascade.7, 8 When ECs were treated with Ad-PPE-Fas-c (1 × 103 MOI) alone and TNF-α, the caspase-8 inhibitor (z-IETD-FMK) blocked the cytotoxic effect, whereas the caspase-9 inhibitor (z-LEHD-FMK) did not block it (data not shown). That is, caspase-9 is not essential for apoptotic induction by Fas-c, but it is essential for apoptotic induction by the combined treatment. These results are in accordance with Lorenzo et al.,24 whose research demonstrated the induction of caspase-9 in human ECs treated with Dox.

The results suggest that both apoptotic pathways are essential for the complete combined treatment effect. Ad-PPE-Fas-c induced the FAS cascade, as expected, while it appears that the addition of Dox resulted in the induction of the mitochondria-associated cascade as well. Indeed, links between the death receptor cascade and the mitochondrial pathway were shown to exist at different levels. Upon death receptor triggering, activation of caspase-8 may result in cleavage of Bid, a Bcl-2 family protein with a BH3 domain, which in turn translocates to mitochondria to release cytochrome c—thereby initiating a mitochondrial amplification loop.25 In addition, cleavage of caspase-6 downstream of mitochondria may feed back to the receptor pathway by cleaving caspase-8.26 Thus, one might postulate that by triggering both pathways, there would be an enhancement in the apoptotic effect of each pathway alone; that is, if Dox induces the mitochondria-associated cascade and Ad-PPE-Fas-c the FAS cascade, each cascade could enhance the other, culminating in cell death.

We also tested whether FAS-c gene therapy combined with chemotherapy can be used for inhibiting lung metastasis. The LLC model was selected to test the combined treatment because of the rich vascularity in the lung metastases and the response of this model to antiangiogenic therapy.27, 28, 29 There are several advantages to antiangiogenic therapy: it targets no particular tumour lineage and, thus, can be applied to a variety of solid tumours; it enables an easy reach of target tissue upon intravenous administration, and it was demonstrated to have an amplification effect on tumour killing since one EC is known to support the nutritional needs of approximately 100 tumour cells.30 Using the LLC model, we demonstrated that the combined treatment of Ad-PPE-Fas-c and Dox elicits a greater antitumoral effect than either therapy alone. Moreover, a significant reduction in lung metastases-MVD was observed in the Ad-PPE-Fas-c+Dox-treated mice, implying that ECs targeting is at least one of the mechanisms by which the combined treatment inhibited metastases.

In summary, we found that administration of Ad-PPE-Fas-c and Dox induced EC apoptosis by the FAS and mitochondria-associated cascade and significantly suppressed LLC lung metastases in mice. The metastasis suppression was enhanced by the combined treatment of FAS-c gene therapy and chemotherapy, compared with either therapy alone. This finding suggests that a combination of FAS-c gene therapy and chemotherapy may have clinical applications for a treatment aimed to minimize tumour burden in cancer patients either due to residual tumours after primary tumour resection or to the dormant micrometastases.



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