Blockage of angiotensin II type I receptor decreases the synthesis of growth factors and induces apoptosis in C6 cultured cells and C6 rat glioma

Angiotensin II (Ang II) is a main effector peptide in the renin–angiotensin system and participates in the regulation of vascular tone. It also has a role in the expression of growth factors that induce neovascularisation which is closely associated to the growth of malignant gliomas. We have shown that the selective blockage of the AT1 receptor of angiotensin inhibites tumour growth, cell proliferation and angiogenesis of C6 rat glioma. The aim of this study was to study the effects of the blockage of AT1 receptor on the synthesis of growth factors, and in the genesis of apoptosis in cultured C6 glioma cells and in rats with C6 glioma. Administration of losartan at doses of 40 or 80 mg kg−1 to rats with C6 glioma significantly decreased tumoral volume and production of platelet-derived growth factor, vascular endothelial growth factor and basic fibroblast growth factor. It also induced apoptosis in a dose-dependent manner. Administration of Ang II increased cell proliferation of cultured C6 cells which decreased by the administration of losartan. Our results suggest that the selective blockage of AT1 diminishes tumoral growth through inhibition of growth factors and promotion of apoptosis.

The renin angiotensin aldosterone system (RAAS) has an important role in the regulation of blood pressure and fluid electrolyte balance. Angiotensin II (Ang II), a major participant in the RAAS, was initially described as a vasoconstrictor, but recent studies have revealed that it also participates in cell growth, cell differentiation and apoptosis (Stoll et al, 1995;Escobar et al, 2004), and has a role in cell migration and conformation of the extracellular matrix (Coker et al, 2001). Some reports indicate that Ang II induce neovascularisation (Fernandez et al, 1985;Le Noble et al, 1991;Andrade et al, 1996) due to stimulation of growth factors, such as platelet-derived growth factor (PDGF) (Khachigian et al, 2000;Cook et al, 2002), transforming growth factor beta b (TGFb) (Kagami et al, 1994;Ohta et al, 1994;Hamaguchi et al, 1999;Weigert et al, 2002), insulin-like growth factor 1 (IGF-1) (Brink et al, 1999;Haddad et al, 2003), basic fibroblast growth factor (bFGF) (Peng et al, 2001), vascular endothelial growth factor (VEGF) (Otani et al, 1998;Tamarat et al, 2002) and angiopoietin 2 (Otani et al, 2001). Angiotensin II also induces the expression of proto-oncogenes in smooth vascular muscle cells, including c-fos, c-jun, c-myc, erg-1, VL-30, and the activator of the protein 1 complex (Cook et al, 2002), Interestingly, many of these effects are inhibited by the blockage of the AT 1 receptor (Fujiyama et al, 2001). Angiotensin II has a dual and paradoxical intervention in apoptosis through its receptors AT 1 functioning as antiapoptotic and AT 2 functioning as proapoptotic (Stoll et al, 1995).
Glioma is the most frequent primary tumour of the brain. Malignant gliomas are still associated with poor prognosis, the mean survival time of patients with glioblastoma multiforme (GBM) is 1 year, and it has not changed significantly for the last three decades (Lopez-Gonzalez and Sotelo, 2000). Glioblastoma multiforme is accompanied by extensive angiogenesis which is essential for tumoral growth and invasiveness; it also produces a vast amount of growth factors such as PDGF, VEGF, HGF and FGF (Strugar et al, 1995;Arrieta et al, 1998Arrieta et al, , 2002Moriyama et al, 1999;Schmidt et al, 1999). In brain parenchyma there is a local RAAS, some neurons, glial cells and glioma cells express renin, angiotensinogen and receptors for Ang II (AT 1 and AT 2 ) (Ganong, 1984, Ariza et al, 1988Fogarty et al, 2002;Juillerat-Jeanneret et al, 2004). We have previously shown that the blockage of AT 1 receptors in rats with C6 glioma inhibited tumour growth, cell proliferation and angiogenesis . The aim of this study was to determine in vitro (in cultured C6 glioma cells) and in vivo (in rats with C6 glioma) the effects of the blockage of the AT 1 receptor on the synthesis of growth factors and its relation with cell proliferation and apoptosis.

Glioma induction
C6 glioma cells (American Tissue Culture Collection Rockville, MD, USA) were cultured under sterile conditions at 371C in a humid environment with 5% CO 2 in Dulbecco's modified Eagle's medium (DME) (Sigma chemical company, Saint Louis Missouri, USA) supplemented with 10% foetal bovine serum (GIBCO, New York, USA). After the cultures became confluent, the cells were washed with saline solution and harvested; 10 7 C6 cells from this source were inoculated intraperitoneally in a male Wistar rat; 2 weeks later, a large tumour was obtained (Guevara and Sotelo, 1999), it was mechanically dispersed in saline solution (1 : 1) at 41C, 10 7 cells from this source were subcutaneously injected in the left thigh of 40-day-old Wistar rats . All animals developed a noticeable tumour within 2 weeks. All animals used in this study were handled in accordance to the guidelines of the coordinating committee on cancer research for the welfare of animals with experimental neoplasms.

Blockage of AT 1 in vivo
When the tumour had reached a diameter of 1.5 cm, the rats were randomly assigned either to the control group (n ¼ 40), to the losartan 40 mg Kg À1 group (L40) (n ¼ 40), or to the losartan 80 mg Kg À1 group (L80) (n ¼ 40). Losartan (Merck-Sharp & Dohme, Mexico) was given orally, once a day, for 30 days. At the end of the experiment, 20 rats from each group were anaesthetised and perfused by intracardiac route with 10% formalin in saline solution for histological study. Before perfusion, the animals were bled by intracardiac puncture to analyse haematological and chemical parameters in blood; the body weight of animals from all groups remained similar throughout the study. The tumour was dissected and its volume was determined by water displacement. The tumours from nonperfused animals were kept at À701C until analysis.

Histological analysis and apoptosis in vivo
For microscopic study, the tumour was embedded in paraffin: 5 mm sections were stained with haematoxylin and eosin. Apoptosis was detected by DNA fragmentation using TdT incorporation of nucleotides on 3 0 ends of DNA (TUNEL technique). Briefly, 5 mm of glioma C6 tumour sections was dewaxed and immersed in 3% H 2 O 2 to block endogenous peroxidase; after washing with distilled water, proteinase K (20 mg ml À1 ) was applied to the specimens for 15 min at room temperature. Detection of apoptosis in situ was made by ApopTag Peroxidase (Oncor, Gaithersburg, MD, USA) that detects fragmented DNA and was performed according to the manufactureŕs conditions. Sections were counterstained with methyl-green. Positive control sections were prepared by nicking DNA with DNase and negative control sections were prepared by substituting with distilled water for working strength TdT enzyme. The proportion of apoptotic cells was expressed as apoptotic rate, which represented the number of apoptotic cells among 1000 glioma C6 nucleated cells, excluding segmented neutrophils due to their short lifespan.

Determination of growth factors in vivo
In all, 10 samples from each experimental group were unfrozen, weighted, and homogenised at 41C (1 : 1) in saline solution; 70 mg ml À1 of phenylmethylsulphonyl fluoride (PMSF) was added for inhibition of proteases, the lysate was centrifuged at 6500 g for 20 min and kept at À701C until analysis. Tissue contents of bFGF, VEGF, PDGF and HGF were measured by enzyme-linked immunosorbent assay (ELISA) (R&D system, Minneapolis, MN, USA). Each sample was assayed by duplicate and reported as means7s.d.

Treatment of C6 cells in culture
We separated C6 glioma cells in four groups: control cells (without treatment); cultures treated with Ang II (10 À7 M); cultures treated with Losartan (10 À5 M) (Stroth et al, 2000) and cultures treated with both Ang II and Losartan (L -A) at the same doses. All cultures were kept in a serum-free medium at 371C, in humidified environment with 5% CO 2 . The culture medium used for cells incubated for long periods was changed every 24 h containing either Ang II, losartan or Ang ll plus losartan at the mentioned doses.

Cell viability of C6 glioma in culture
For quantification of cell viability, 6.5 Â 10 5 glioma cells were cultured in microtitre plates (96 wells) with 100 ml culture medium and incubated for 24 h in a humidified atmosphere; 10 ml of tetrazolium salts (MTT) was added to each well and left for 4 h; these salts were cleaved into a coloured formazan product by metabolically active cells; afterwards 100 ml of solubilisation solution was added, the plates were allowed to stand overnight inside the incubator. After checking for complete solubilisation of the purple formazan crystals, the absorbance was measured at 570 nm, the reference wavelength was 650 nm. We obtained the percentage of viable cells from three assays, the control was used as reference (100%). Two replicates were perfomed using the same dosages.

Apoptotic rate in vitro by flow cytometry
For quantification of apoptosis by flow cytometry, 10 6 C 6 glioma cells were trypsinised and transferred to 1.5 ml Eppendorf tubes and cultured with DME culture medium either alone (controls) or with Losartan 10 À5 M, or with Ang II 10 À7 M, or with the mixture of losartan/Ang II (LA). Cultured cells were maintained at 371C with 5% CO 2 during 6, 12, 24, 48 or 96 h. The culture medium of those experimental groups which needed a long time of incubation was changed every day. At the end of the incubation period, the cells were centrifuged at 2000 g, the supernatant was discarded. Induced apoptosis was detected by flow-cytometric analysis of the permeabilised, propidium-iodide-stained cells (Telford et al, 1992). Samples (10 6 cells ml À1 ) were washed once in PBS and the pellets were re-suspended in 80% ethanol at 41C for 60 min. To detect apoptosis by flow cytometry, fixed cells were centrifuged, re-suspended in 1 ml PBS, and kept at 371C for 20 min before staining with a solution of 0.1% Triton X-100, 0.1 mM EDTA(Na) 2 , 5 U ml À1 RNase and 20 mg ml À1 propidium iodide in PBS. Samples were stored in the dark at room temperature and analysed with a Facscalibur Registered Trademark (Becton Dickinson & Co., San Jose, CA, USA). Using the cell quest software (San Jose CA, USA), cell number (10 4 ) was detected. Cell percentages in the different phases of the cell cycle were estimated according to the Fox's method (Lacombe et al, 1988). The assays were made by triplicate.

Apoptotic rate in vitro by ELISA
For quantification of apoptosis by ELISA, 10 5 C 6 glioma cells were trypsinised and transferred to 1.5 ml Eppendorf tubes and cultured with DME culture medium either alone (controls) or with Losartan 10 À5 M, or with Ang II 10 À7 M, or with the mixture of LA. Cultured cells were maintained at 371C with 5% CO 2 during 6, 12, 24, 48 or 96 h. The culture medium of those experimental groups which needed a long time of incubation was changed every day. At the end of the incubation period, the cells were centrifuged at 2000g, the supernatant was discarded and the cells were re-suspended in 1 ml of culture medium with 10% DMSO and kept in liquid nitrogen until processed. At that time, the vials were warmed at 371C and the cells were washed with culture medium by centrifugation at 2000g. The cells were then resuspended with incubation buffer (Cell Death detection ELISA Kit, Boehringer Mannheim catalogue: 1544675) at 41C during 30 min. The cellular lysate was centrifuged at 20 000g for 10 min and 400 ml of the supernatant were removed (cytoplasmic fraction) and diluted 1 : 10 with incubation buffer, absorbance was measured at 405 nm, the substrate solution was used as blank. The assays were made by triplicate; 10 6 C6 cells in hypertonic buffer and 10 6 C6 cells with camptotensin were used as positive controls.

Statistical analysis
Values were expressed as means7s.d.; for the quantification of apoptotic rate, the kappa value was used as the interobserver variability value. Statistical analysis was made using SPSS v10 software. Comparisons between groups were made by ANOVA and Tukey tests. Statistical significance was set at a P-value of 0.05.

Effect of the blockage of AT 1 receptor on tumoral growth and apoptotic rate in vivo
All animals survived until the end of the experiment. In all controls, the tumour grew to a very large size (over 6 cm diameter); there was no case of spontaneous involution. When compared to controls (mean volume: 55710 cm 3 ) a significant decrease in tumour volume was seen in animals treated with L40 (3077 cm 3 ) and with L80 (1979 cm 3 ) (Po0.05 and 0.01, respectively). Results were similar to those from our previous report . The apoptotic rate was higher in the L80 group (84.274.1) than in controls (2378) or in the L40 group (13.674.3) (Po0.01); no differences were seen when L40 group was compared with controls ( Figure 1). Comparisons of haematological and chemical blood parameters measured at the end of the study showed no differences between groups.

Effects of Ang II, losartan and LA on cell cultures
The percentage of viable cells was significantly smaller in cultures treated with L -A at 6 and 12 h as compared with controls (P ¼ 0.01 and 0.05, respectively); however, no differences were seen later, at 24, 48 and 96 h. The percentage of viable cells was higher in cells cultured with Ang II at 48 and 96 h as compared to controls (P ¼ 0.04 for both determinations). Treatment with Losartan had no effect on cell viability (Figure 3).

Apoptosis rate in vitro by flow cytometry
A significative increase of apoptosis was observed in cells treated with L -A particularly at 6 h as compared to controls (P ¼ 0.05) and with the losartan group (P ¼ 0.012), and at 12 h as compared with the groups Ang II (P ¼ 0.007) and losartan (P ¼ 0.001), which was maintained at 24 h with losartan (P ¼ 0.007). No differences were found at 48, 72 and 96 h ( Figure 4A).

Apoptosis rate in vitro by ELISA
Cells cultured with Ang II showed increased apoptotic rate at 6 h when compared with losartan (P ¼ 0.012) and at 12 h when compared to controls (P ¼ 0.012). When compared with controls, apoptosis was increased in cells treated with L -A (P ¼ 0.007), with losartan (P ¼ 0.003) and with Ang II (P ¼ 0.011) at 6 h, as well as the cells treated with L -A at 12 h (P ¼ 0.05). No differences were found at 24, 48, 72 and 96 h ( Figure 4B).
Viable cell ( (Stefanik et al, 1991;Westermark et al, 1995;Arrieta et al, 2002;Steiner et al, 2003); their concentrations are related to vascular density, radioresistance, cell proliferation, degree of malignancy and patients survival (Westermark et al, 1995;Gorski et al, 1999). Angiotensin II stimulates the production of these growth factors (Peng et al, 2001;Tamarat et al, 2002); this effect can be blocked by AT 1 antagonists but not by AT 2 antagonists (Tamarat et al, 2002). Normal and neoplasic astrocytes including C6 glioma cells express AT 1 and AT 2 receptors for Ang II Fogarty et al, 2002). We have previously shown in experimentally induced C6 glioma in rats that the selective blockage of AT 1 receptor reduces cell proliferation, angiogenesis and tumour growth . However, the participative mechanisms were unclear. In this study, we found that the blockage of AT 1 decreases the synthesis of the growth factors VEGF, PDGF and bFGF, coincident with the reduction of tumour size, cell proliferation and vascular density. The effect of losartan in the synthesis of growth factors is more intense in PDGF, which is inhibited with the blockage of AT1 in several experimental models; nonetheless, VEGF and FGF syntheses are also statistically reduced. However, the reduction of angiogenesis previously reported might be a consequence of VEGF and FGF inhibition due to a blockage of PDGF's stimuli, this would explain why the effect is not dose-dependent on losartan. Although HGF is also overexpressed in malignant gliomas and related to the degree of malignancy (Arrieta et al, 2002) and Ang II also participates in its synthesis (Matsumoto et al, 2003), in this study no effect on HGF contents was observed after blockage of the AT 1 receptor. Additionally, our results in vitro show that the reduction on cell viability within the first h of Ang II plus losartan administration was also associated with the induction of apoptosis but only during the first 12 h, whereas the administration of Ang II alone stimulates cell proliferation during 24 h after exposure. Increase of apoptosis in glioma C6 in vivo was seen only at high doses of losartan (80 mg Kg À1 ); this effect was also obtained in cultured C6 cells with the simultaneous administration of losartan and Ang II; it seems that the selective blockage of AT 1 may lead to disequilibrium of AT 1 /AT 2 relation that promotes AT 2 receptor stimulation which, in turn, could increase its proapoptotic effects. It is also likely that, in this condition, the presence of Ang II promotes apoptosis, as this effect is prevented by deletion of the AT 2 receptor gene (Yamada et al, 1996). Our findings are in agreement with studies in PC12W cells and neurons from newborn rats, showing that apoptosis is increased either by the selective stimulation of AT 2 receptor or by the blockage of AT 1 receptor simultaneous to the administration of Ang ll (Stoll et al, 1995;Yamada et al, 1996;Goldenberg et al, 2001;Suzuki et al, 2002).
Subucutaneous C6 glioma, in contrast to brain glioma, allowed us to measure tumour growth for a longer time and to determine a potential therapeutic effect when the drug is administered for long periods. However, similar results are to be expected in brain glioma as the blood-brain barrier is interrupted in brain tumours, facilitating the entrance of drugs into the tumour.
Angiogenesis and apoptosis are primordial features of malignant tumours, constituting attractive therapeutic targets; drugs that inhibit angiogenesis or promote apoptosis could be associated to cytotoxic agents to improve antitumoral therapy. The fact that multifunctional hormonal systems, such as the reninangiotensin -aldosterone system, influence tumour growth and angiogenesis provides interesting pathways to the study of cancer. The antineoplastic activity obtained by the selective blockage of AT 1 in malignant glioma seems to be mediated by two different mechanisms, inhibition of the synthesis of growth factors and promotion of apoptosis, providing a potential therapeutic adjuvant for malignant gliomas.