The presence of angiotensin type 2 (AT2) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and their role in the induction of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point to a new, unique approach enabling the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT2 receptor.

CLINICAL PERSPECTIVES

  • The clinical benefit of chemotherapy and radiotherapy of patients suffering from uterine leiomyosarcoma is limited. Conventional chemotherapy does not target non-cycling tumour cells or cells, which exited from the mitosis.

  • Our study demonstrates that the AT2 receptor is substantially up-regulated in non-cycling human leiomyosarcoma cells. Exposure of quiescent, but not proliferating leiomyosarcoma cells to the high-affinity, non-peptide AT2 receptor agonist, C21, induces rapid cell death.

  • Patients receiving chemotherapy in the advanced-stage of disease may benefit from the treatment with C21 in terms of a better therapeutic response and longer progression-free survival.

INTRODUCTION

Recent studies have provided evidence of an intracrine, intracellular renin–angiotensin system (RAS), defined by the presence of all components of the RAS within cells [1]. Angiotensin II (Ang II), the main effector peptide of the RAS, exerts its effects via high-affinity binding to at least two seven-transmembrane domain receptors, the angiotensin type 1 (AT1) and type 2 (AT2) receptor. In the adult organisms, the AT1 receptor is almost ubiquitously expressed in various tissues and its stimulation induces cell proliferation and tissue hypertrophy. The Ang II–AT1 receptor system, often up-regulated in a number of human cancers, promotes angiogenesis, tumour growth and invasion of malignant cells [2]. Tissue RASs are also present in the female reproductive organs and stimulation of the AT1 receptor contributes to the malignant transformation and proliferation of tumour cells in gynaecological cancers [3]. The AT2 receptor does not couple to classical heterotrimeric G-proteins, rather the activation of signalling cascades participating in the regulation of various intracellular processes is mediated by the AT2 receptor interacting proteins (ATIPs) which bind to the intracellular C-terminal region of the receptor. The majority of ATIPs identified to date are related to cancer suppression [46]. Unlike the AT1 receptor, the expression of the AT2 receptor in the adult organism remains restricted to a few organs and tissues such as the adrenal medulla, brain and ovarian follicles [2]. Generally, the AT2 receptor counteracts the proliferation-promoting actions of growth factors and the AT1 receptor, but its exact role in cancer cell biology has not yet been established [2,7]. Previous advancements in the RAS have discovered the mitochondrial localization of AT2 receptors and their involvement in NO generation, protection and reduction in the age-related mitochondrial dysfunction in various human and mouse non-tumour cells [8]. The intracellular distribution of AT2 receptors has not yet been investigated in tumour cells. The human myometrium harbours the AT2 receptor and its expression varies during the menstrual cycle and pregnancy, allowing for the possibility that the receptor is involved in the regulation of cell proliferation, differentiation and apoptosis [9,10]. We, therefore, studied the expression of AT2 receptors in human uterine leiomyosarcoma cells and control human uterine smooth muscle cells (HutSMC), including their presence in mitochondria, and assessed their roles in the induction of apoptosis and cell death. We employed Compound 21 (C21), a new, orally active, non-peptide, high-affinity AT2 receptor agonist (Ki value of 0.4 nM for the AT2 receptor and Ki value of >10 μM for the AT1 receptor) [11]. A few peptide AT2 receptor agonists have been designed based on the modification of the Ang II molecule. The Ang II-like peptides are not orally active, have short plasma half-lives, and, therefore, limited clinical applicability. The synthetic peptide CGP42112A, a high-affinity ligand for AT2 receptors (Ki < 1 nM), has widely been used in experimental work aimed to study the role of AT2 receptors in various physiological functions, but has never been developed into a drug for clinical use. Very recently, pharmacological effects of novokinin, a designed peptide AT2 receptor agonist (Ki < 7 μM) have been reported [12]. A new Ang II-like peptide AT2 receptor agonist LP2 is a cyclic Ang II derivative with one exchanged amino acid developed by Lanthio Pharma, Groningen, The Netherlands. LP2 is currently in preclinical testing and is indicated for the treatment of idiopathic pulmonary fibrosis. At the present time, C21 is the only non-peptide, orally active AT2 receptor agonist which is currently in the final stage of preclinical testing [13]. C21 has been tested in a broad variety of cardiovascular and neurological models, such as myocardial infarction, hypertension-induced vascular remodelling, stroke, spinal cord injury and Alzheimer's disease, however, besides one study [14], the AT2 receptor agonist has not yet been used in cancer research. Using LC–MS (liquid chromatography–mass spectrometry) analysis we demonstrate that C21 is able to pass through the cell membrane of SK-UT-1 cells and to induce rapid cell death through its interaction with the intracellular AT2 receptors.

MATERIALS AND METHODS

Antibodies

Rabbit anti-cleaved caspase 3 antibody (Cell Signaling Technology), mouse anti-Bcl-2 monoclonal antibody, mouse anti-Bax monoclonal antibody (Santa Cruz Biotechnology), monoclonal anti β-actin antibody (Sigma–Aldrich), anti-rabbit, anti-goat and anti-mouse IgGs (Amersham), Fluor®488 donkey anti-rabbit IgG (Molecular Probes), goat anti-human AT2 receptor polyclonal antibody (for Western blot) (Santa Cruz Biotechnology) and rabbit anti-human AT2 receptor antibody (for immunofluorescence) (Abcam) were used in the present study. The specificity of the AT2 receptor antibodies was tested by Western blot analysis using protein extracts from PC12W cells expressing solely the AT2 receptor.

Chemicals

The angiotensin receptor ligand Ang II was purchased from Sigma–Aldrich and losartan was generously supplied by Dr R. Smith (DuPont Merck Pharmaceutical Company, Wilmington, DE, USA). PD 123177 was a kind gift from Joan Keiser (Park Davis, Ann Arbor, Michigan, USA), C21 was provided by Dr U.M. Steckelings (Institute of Molecular Medicine, Odense, Denmark). All other chemicals and kits are mentioned in the text and if not otherwise stated, they were purchased from Sigma–Aldrich or Merck.

Cell cultures

The human leiomyosarcoma cell line, SK-UT-1 cells (HTB-114, American Type Culture Collection), was obtained from the European Collection of Animal Cell Cultures. The cells kindly provided by Dr Hendrik Ungefroren (University Hospitals of Schleswig-Holstein, Campus Kiel, Germany) [15], were cultured in a medium consisting of DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 1:1 (Invitrogen) supplemented with 10% heat inactivated FCS (FCS Superior; Biochrom AG), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM of L-glutamine. To obtain quiescent cells, SK-UT-1 cells were maintained in a medium containing 0.1% FCS for 48 h before the treatment. Primary HutSMC purchased from PromoCell (PromoCell Heidelberg), were cultured in the ready-use smooth muscle cell growth medium complex (PromoCell Heidelberg) containing 100 U/ml penicillin, 100 μg/ml streptomycin. To obtain quiescent cells, HutSMC were cultured in DMEM/Ham's F-12 1:1 (Invitrogen) medium supplemented with 1% heat inactivated FCS. Both cell types grew as an adherent monolayer at 37°C in a humidified atmosphere of air/CO2 (19:1). For cell passages, 1× trypsin/EDTA was used. Ang II was used at a concentration of 10−7 M. Unless otherwise stated, the concentration of C21 in the incubation medium was 10−6 M. Losartan, a selective, non-peptide AT1 receptor antagonist, and PD 123177, a selective, non-peptide AT2 receptor antagonist, were used at concentrations of 10−5 M and 10−6 M, respectively. The concentrations of the AT1- and AT2-receptor antagonists were established in preliminary experiments using the assessment of apoptosis and the quantification of [18F]2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake, respectively. The apoptosis induced by AT2 receptor stimulation in SK-UT-1 cells was evaluated by staining for dUTP nick-end labelling (TUNEL) and by DAPI (Invitrogen Molecular Probes) immunofluorescence analysis after concomitant treatment of quiescent SK-UT-1 cells with Ang II and various doses of losartan (10−7, 10−6, 10−5 and 10−4 M). Losartan concentration increased the Ang II-induced apoptosis up to a concentration of 10−5 M, higher concentrations of the AT1 receptor antagonist were not more effective. Losartan alone did not exert any apoptotic effects. 18F-FDG uptake reliably reflects the rate of cell proliferation [16]. The concomitant treatment of cells with Ang II and 10−6 M PD 123177 (selective activation of AT1 receptors) resulted in the highest 18F-FDG incorporation; a higher concentration of the AT2 receptor antagonist (10−5 M) was not more effective. PD 123177 alone (10−6 M) did not exert any effects. However, it is worth noting that incubation of quiescent SK-UT-1 cells with PD 123177 alone at a concentration of 10−5 M and higher may exert cytotoxic effects as evidenced by increased release of lactate dehydrogenase (LDH) into the incubation medium (data not shown). Identical concentrations of angiotensin receptor ligands were also employed in a number of studies carried out in various tumour cell lines. Losartan and PD 123177 were added to the incubation medium 30 min prior to Ang II or C21. Various doses of C21 (10−7, 10−6, 10−5 M) were tested to investigate the effects of C21 on rapid cell death in quiescent SK-UT-1 cells. The effects could already be observed at a concentration of 10−7 M, however, more profound effects were detected at higher concentrations (10−6 and 10−5 M) (Figure 3a).

Isolation of RNA and RT-PCR

Total RNA was isolated from proliferating or quiescent SK-UT-1 cells using Trizol-reagent and dissolved in RNase-free water. The RNA quantity and quality were determined by a spectrophotometer. First-strand synthesis (5 μg total RNA) was carried out with SuperScript II Reverse Transcriptase using Oligo-dT12–18 oligonucleotides (Invitrogen Life Technologies). The equality of the reverse-transcribed cDNA was verified by RT (reverse transcription)-PCR using intron spanning primers for β-actin (Clontech). The PCR temperature profile used was 94°C (5 min) hot start, followed by 30 cycles at 94°C (45 s), 56–60°C (45 s), 72°C (1 min), and 7 min at 72°C to ensure double-stranded cDNA. Optimal PCR conditions were established by determining the ratio between the signal strength and the number of PCR cycles. The saturation of PCR reactions for the AT1 receptor was reached at 30 and for the AT2 receptor at 55 cycles (data not shown). The PCR products were separated by electrophoresis on 1.0–1.5% agarose gels, stained with 0.5 μg/ml ethidium bromide and visualized by UV illumination. Amplification for the β-actin control was verified for correct size (1128 bp) to ensure the quality of cDNA and lack of contamination with the genomic DNA.

Oligonucleotides

The following oligonucleotides were used for RT-PCR cDNA: AT1 receptor: sense (5′-GCA TTG ATC GAT ACC TGG CT-3′) and antisense (5′-TTA CAT TAT CTG AGG GGC GG-3′) primers were used for PCR amplification to yield a 669-bp product; AT2 receptor: sense (5′-GCT TGT GAA CAT CTC TGG CA-3′) and antisense (5′-TTC ATT AAG GCA ATC CCA GC-3′) primers. The PCR amplification yielded a 582-bp product. β-Actin: sense (5′-ATG GAT GAT GAT ATC GCC GCG-3′) and antisense (5′-CAT GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3′) primers.

Preparation of subcellular fractions

Mitochondrial and cytosol proteins were isolated as described elsewhere with minor modifications [17,18]. Briefly, fresh collected cell pellets were washed twice with ice-cold PBS and gently homogenized in a pre-cooled Dounce Homogenizer containing 2 ml of ice-cold isolation medium (250 mM mannitol, 20 mM Tris, 1 mM EGTA, 1 mM EDTA, 0.3% w/v BSA and 1% phosphatase and protease inhibitors). The homogenates were stored in an ice bath for 30 min and then centrifuged at 450g for 10 min at 4°C. Subsequent centrifugation of the supernatants (18,000g for 10 min) generated the cytosolic fraction. The mitochondrial pellets were washed twice with isolation medium, re-suspended firstly in PBS, then in CelLytic M Cell Lysis Reagent (Sigma–Aldrich), containing 1% of the Halt Protease & Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and incubated in an ice bath for 30 min. The mitochondrial suspension was briefly sonicated and centrifuged to remove insoluble material (15,000g at 4°C for 15 min). Supernatants were normalized for total mitochondrial protein content (BCA Protein Assay Kit, Thermo Scientific) and stored at −80°C. The mitochondrial pool contained high amounts of the matrix protein GRP75 while nuclear proteins (histone H1) were completely absent.

Preparation of the whole cell extraction for Western blot analyses

To obtain the whole cell fraction, cells were lysed in CelLytic M Cell Lysis Reagent containing 1% of the Halt Protease & Phosphatase Inhibitor Single-Use Cocktail. After a short incubation (5 min at 95°C), the lysates were sonicated and centrifuged (15,000g at 4°C for 15 min) to remove insoluble materials. The protein concentration in the supernatant was measured by the BCA Protein Assay Kit (Thermo Scientific).

Western blot analyses

Equivalent to 20 μg of total proteins per lane were loaded and separated on 12% SDS-polyacrylamide gels and transferred to Immobilon-P Transfer Membrane (PVDF) (Millipore). The membranes were blocked and incubated overnight with the primary antibody against the AT2 receptor (1:1000), cleaved (activated) caspase-3 (1:1000), Bax-protein (1:1000) or Bcl-2 protein (1:2000). On the following day, the membranes were washed and incubated with the horseradish peroxidase-conjugated secondary antibody. Western blots were developed with Amersham ECL Plus Western Blotting Detection Reagents on high performance chemiluminescence film (Amersham Hyperfilm ECL, GE Healthcare Life Sciences). For the re-staining, the blots were stripped in Restore Western Blot Stripping Buffer (Thermo Scientific), washed and blotted again. To normalize the protein content of each lane, all membranes were stained with protein staining kit or blotted with anti-β-actin antibody (1:10000). For the densitometry analysis, the films were scanned and quantified using the quantification software (Quantity One, Bio-Rad Laboratories).

Immunofluorescence analysis and confocal laser microscopy

Quiescent cells grown on fibronectin coated cover slips were exposed to vehicle for 24 h. MitoTracker® Red CMXRos (0.125 μM) (Sigma–Aldrich) was added to the culture medium. Thirty minutes thereafter, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT), washed three times with PBST and permeabilized with 0.2% saponin in 0.1% BSA for 30 min at RT. After incubation in the block solution containing 2% BSA, the cells were incubated with rabbit anti-human AT2 receptor antibody (1:1000) (Abcam) at 4°C overnight. Following a wash step with PBST, the cells were incubated for 1 h at RT in the dark with the secondary antibody, Alexa Fluor®488-conjugated donkey anti-rabbit antibody (Molecular Probes) in 1% BSA. The cells were washed, dried and mounted in Prolong Gold antifade reagent. Immunofluorescence analyses were carried by a confocal laser scanning microscope (LSM 510, equipped with an Axiovert 100M; Carl Zeiss). The picture represents one single plane from a z-stack. All micrographs were made with a 63× objective. Co-localization was analysed with ImageJ. The correlation between the AT2 receptor and the mitochondrial components was quantitatively assessed by the split co-localization coefficients M1 and M2 (Mander's co-localization coefficients) [19].

Apoptotic DNA ladder

Approximately 2×106 cells/well were exposed to Ang II alone or Ang II combined with losartan or PD 123177 for 24, 36 or 48 h and lysed thereafter in 200 μl of lysis buffer according to the manufacturer's recommendation (Apoptotic DNA Ladder Kit, Roche Diagnostics). 100% propan-2-ol was used to precipitate the DNA. After several washing steps, 3 μg of total DNA from each sample was loaded on 1% agarose gel. The DNA fragments were stained with 5 μg/ml ethidium bromide solution and the DNA images were documented.

Staining for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)

SK-UT-1 cells (4×104/well), cultured in 24-well plates were exposed to Ang II ± losartan or PD 123177 for 48 h. Internucleosomal DNA fragmentation was detected by the TUNEL method (In Situ Cell Death Detection Kit POD, Roche Diagnostics) as recommended by the manufacturer with slight modifications. The treated cells were fixed using 4% paraformaldehyde, incubated in 0.3% H2O2 in methanol and kept in permeabilization solution. After a wash step, all cells were incubated in a 50% TUNEL mixture. Following incubation with Converter-POD (horse radish peroxidase), the cells were treated with 3–3′-diaminobenzidine tetrahydrochloride (DAB) diluted with POD buffer. The labelled cells were examined and photographed under an inverted light microscope. Cells serving as the negative control were incubated with 50% TUNEL mixture without labelling enzyme. The cells pre-treated with DNase (100 μg/ml) served as the positive control.

Cell counting

SK-UT-1 cells and HutSMC were exposed to the AT1- and AT2-receptor ligands, washed with PBS and detached from the culture dish with trypsin/EDTA solution. The cell suspension was centrifuged at 110g for 5 min. The cell pellets were re-suspended in PBS and the cell suspension (20 μl) was mixed with 20 μl of Trypan Blue solution. The cell numbers were ascertained in a haemocytometer (Omnilab, Germany).

Lactate dehydrogenase (LDH) assay

Assessment of the cell membrane integrity and cytotoxicity based on the measurement of LDH activity released from damaged cells into the culture medium were determined by the Cytotoxicity Detection Kit (Roche Diagnostics) according to the manufacturer's recommendation.

Liquid chromatography–mass spectrometry (LC–MS) analysis of C21

The LC–MS analysis of C21 in the incubation medium and cell lysates was carried out on an ESI Quattro Premier XE Mass Spectrometer (Waters) with an Agilent 1100 HPLC pump, auto sampler and diode array detector using a Nucleosil-100-5 HD C18 analytical column (4.1 mm × 100 mm; Macherey Nagel). For the gradient elution of the sample, mobile phase A (water containing 0.1% formic acid) and mobile phase B (methanol containing 0.1% formic acid) were used. The linear LC gradient of the mobile phase B increased from 0% to 100% in 30 min. The total run time was 35 min. The stock solution of C21 (1 mM) was prepared in ultra-pure water and appropriate dilutions were made shortly before the sample injection. The electrospray mass spectrometer was operated in positive ion mode, the cone temperature was set to 100°C and the cone voltage to 3000 V. Nitrogen was used as the ion gas at a flow rate of 8 l/min. The mass spectrometer was optimized using constant infusion with the integrated infusion pump of the Quattro Premier XE Mass Spectrometer. The LC–MS data were acquired and processed using MassLynx Software (Waters).

Statistical analyses

All values are expressed as the mean ± SEM. The numbers of samples and separate experiments are given in brackets. The distribution of the sampled data was analysed by Kolmogorov–Smirnov test and the homogeneity of variances was tested by Bartlett's test. One-way analysis of variance (ANOVA) followed by post-hoc Bonferroni test for pairwise comparison was used to analyse the effects of the AT1- and AT2-receptor stimulation with Ang II (± losartan or PD 123177) on TUNEL-positive cell numbers and the AT2 receptor activation with C21 (±PD 123177) on SK-UT-1 cell numbers, LDH release and Bax/Bcl-2 ratio. Statistical analysis of differences in protein levels of Bcl-2, Bax and activated caspase-3 was carried out by paired t-test and two-tailed P values were adjusted for multiple comparisons using Bonferroni correction. Student's t-test for unpaired samples was used to compare the effects of C21 on the density of AT2 receptors in mitochondria, the numbers of HutSMC and LDH release. Statistical significance was accepted for P<0.05.

RESULTS

Expression of Ang II receptor subtypes in SK-UT-1 cells

Both angiotensin receptor subtypes are expressed in SK-UT-1 cells (Figure 1a). The AT1 receptor mRNA was higher in proliferating than in quiescent SK-UT-1 cells. The AT2 receptor mRNA was unambiguously detected only in quiescent SK-UT-1 cells indicating that the receptor abundance is low during cell proliferation but the receptor is increasingly expressed when the cell cycle is arrested. Although no significant differences in the AT2 receptor protein were detected in the whole cell fraction (Figure 1a, lower panel), the AT2 receptor was clearly up-regulated in mitochondria and in the cytosol of quiescent SK-UT-1 cells (Figure 1b). Two bands of the blot indicate a high degree of receptor glycosylation (Figure 1a). The localization of the AT2 receptor in mitochondria of quiescent SK-UT-1 cells grown on fibronectin coated cover slips was also confirmed by confocal laser microscopy. The AT2 receptor fluorescence (green) matched the distribution of the mitochondrial fluorescence marker MitoTracker Red (red); the spatial overlap of the AT2 and mitochondrial signals is shown in Figure 1c iii and iv. The correlation between the green (AT2 receptor) and the red (MitoTracker Red) components, assessed by the split co-localization coefficients M1 and M2, was high. The co-localization coefficients M1=0.85 and M2=0.99 indicate that the images are almost identical. Our data demonstrates, i) an augmented expression of the AT2 receptor in SK-UT-1 cells which entered quiescence and, ii) cell compartment-specific distribution of the receptor, with abundant AT2 receptor protein levels in mitochondria.

Expression and intracellular distribution of the AT2 receptor in SK-UT-1 cells

Figure 1
Expression and intracellular distribution of the AT2 receptor in SK-UT-1 cells

(a) Expression of AT1 and AT2 receptors in human uterine leiomyosarcoma (SK-UT-1) cells. Upper panel: AT1 receptor mRNA in proliferating and quiescent SK-UT-1 cells. Lower panel: AT2 receptor mRNA and the AT2 receptor protein in the whole cell fraction. (b) Densities of the AT2 receptor protein in the cytosol and mitochondria in proliferating and quiescent SK-UT-1 cells (the data is representative of three separate experiments). (c) The co-localization of the AT2 receptor and mitochondria studied by confocal laser microscopy. i) AT2 receptors stained with fluorescent antibody (green); ii) mitochondrial staining with MitoTracker Red; iii) the co-localization of the AT2 receptor and mitochondria (the coincident signal appears yellow), quantitatively assessed by the split co-localization coefficients M1 and M2 (Mander's co-localization coefficients); iv) higher magnification of a cell showing the overlapping of the AT2 receptor immunoreactivity in mitochondria (white arrow). The presented immunofluorescence data is representative of six separate experiments (63× oil immersion).

Figure 1
Expression and intracellular distribution of the AT2 receptor in SK-UT-1 cells

(a) Expression of AT1 and AT2 receptors in human uterine leiomyosarcoma (SK-UT-1) cells. Upper panel: AT1 receptor mRNA in proliferating and quiescent SK-UT-1 cells. Lower panel: AT2 receptor mRNA and the AT2 receptor protein in the whole cell fraction. (b) Densities of the AT2 receptor protein in the cytosol and mitochondria in proliferating and quiescent SK-UT-1 cells (the data is representative of three separate experiments). (c) The co-localization of the AT2 receptor and mitochondria studied by confocal laser microscopy. i) AT2 receptors stained with fluorescent antibody (green); ii) mitochondrial staining with MitoTracker Red; iii) the co-localization of the AT2 receptor and mitochondria (the coincident signal appears yellow), quantitatively assessed by the split co-localization coefficients M1 and M2 (Mander's co-localization coefficients); iv) higher magnification of a cell showing the overlapping of the AT2 receptor immunoreactivity in mitochondria (white arrow). The presented immunofluorescence data is representative of six separate experiments (63× oil immersion).

Activation of the cell membrane AT2 receptors induces apoptosis in quiescent SK-UT-1 cells

Figure 2a shows the time course of the AT2 receptor-induced DNA fragmentation in quiescent SK-UT-1 cells. Activation of the membrane AT2 receptors in quiescent SK-UT-1 cells was achieved by a concomitant treatment with Ang II and the selective AT1 receptor antagonist, losartan for 24, 36 and 48 h. Treatment for 36 h resulted in a strong fragmentation of DNA; the effect was less pronounced in cells treated 24 or 48 h (Figure 2a). No fragmentation of DNA was observed in the presence of the selective AT2 receptor antagonist, PD 123177, indicating that the DNA fragmentation was triggered by AT2 receptor activation (Figure 2b). Indeed, stimulation of AT2 receptors in quiescent SK-UT-1 cells increased the numbers of cell positively stained for TUNEL (n=10) (F3,36=15.652, P<0.001). The highest apoptotic rates were detected in cells stimulated with Ang II and losartan (Figures 2c and 2d). The DNA fragmentation (Figure 2b), TUNEL staining (Figures 2c and 2d) and the tendency of the activated caspase-3 to increase (n=6) (Figure 2e) indicate that activation of the membrane AT2 receptors in quiescent SK-UT-1 cells does activate the apoptotic pathway but does not increase the rate of cell death as evidenced by unaltered cell numbers [cell numbers × 10−4 detected 24 h after the onset of the treatment (n=6): vehicle: 11.3±0.9; Ang II: 8.9±0.8; Ang II+losartan: 8.8+1.1; Ang II+PD 123177: 9.4±1.3] and unaltered release of LDH [LDH release into the incubation medium quantified 24 h after the onset of the treatment (n=9): vehicle: 0.065±0.005; Ang II: 0.063±0.005; Ang II+losartan: 0.060+0.007; Ang II+PD 123177: 0.074±0.009]. Moreover, the apoptotic effects induced by stimulation of the membrane AT2 receptors with Ang II were temporary and no longer detectable 72 h after the onset of the treatment. Activation of the membrane AT1 receptors or exposure of SK-UT-1 cells to losartan alone did not exert any effects (data not shown).

Angiotensin II (Ang II) induces apoptosis in quiescent SK-UT-1 cells

Figure 2
Angiotensin II (Ang II) induces apoptosis in quiescent SK-UT-1 cells

(a) Time course of the AT2 receptor-induced DNA fragmentation in quiescent SK-UT-1 cells. (b) Effects of AT1- and AT2-receptor activation in quiescent SK-UT-1 cells with Ang II upon the DNA fragmentation assessed 36 h after the onset of the treatment with vehicle, Ang II, Ang II+losartan (Los; stimulation of AT2 receptors) and Ang II+PD 123177 (PD; stimulation of AT1 receptors). The autoradiograph shows that AT2 receptor stimulation induced a strong DNA fragmentation. (c) Apoptosis analysed by TUNEL staining in quiescent SK-UT-1 cells treated with vehicle, Ang II, Ang II+losartan and Ang II+PD 123177 for 48 h. (d) The numbers of cells positively stained for TUNEL (n=10). Statistical comparison with vehicle treated cells: ** P<0.01, *** P<0.001, and with cells exposed to Ang II: ## P<0.001, calculated by one-way ANOVA followed by a post hoc Bonferroni test from pairwise comparisons. (e) Western blot analysis of the activated caspase-3 in quiescent SK-UT-1 exposed to vehicle, Ang II, Ang II+losartan and Ang II+PD 123177 for 24 h (n=6). No significant differences were detected (paired t-test; two-tailed P values were adjusted for multiple comparisons using the Bonferroni correction).

Figure 2
Angiotensin II (Ang II) induces apoptosis in quiescent SK-UT-1 cells

(a) Time course of the AT2 receptor-induced DNA fragmentation in quiescent SK-UT-1 cells. (b) Effects of AT1- and AT2-receptor activation in quiescent SK-UT-1 cells with Ang II upon the DNA fragmentation assessed 36 h after the onset of the treatment with vehicle, Ang II, Ang II+losartan (Los; stimulation of AT2 receptors) and Ang II+PD 123177 (PD; stimulation of AT1 receptors). The autoradiograph shows that AT2 receptor stimulation induced a strong DNA fragmentation. (c) Apoptosis analysed by TUNEL staining in quiescent SK-UT-1 cells treated with vehicle, Ang II, Ang II+losartan and Ang II+PD 123177 for 48 h. (d) The numbers of cells positively stained for TUNEL (n=10). Statistical comparison with vehicle treated cells: ** P<0.01, *** P<0.001, and with cells exposed to Ang II: ## P<0.001, calculated by one-way ANOVA followed by a post hoc Bonferroni test from pairwise comparisons. (e) Western blot analysis of the activated caspase-3 in quiescent SK-UT-1 exposed to vehicle, Ang II, Ang II+losartan and Ang II+PD 123177 for 24 h (n=6). No significant differences were detected (paired t-test; two-tailed P values were adjusted for multiple comparisons using the Bonferroni correction).

C21 induces rapid cell death in quiescent SK-UT-1 cells and activates mitochondria-mediated apoptosis

We next studied the effects of AT2 receptor activation by C21. Surprisingly, exposure of quiescent SK-UT-1 cells to C21 induced rapid cell death in a time- and concentration-dependent fashion (Figure 3a). About 70% of quiescent SK-UT-1 cells died within 24 h of treatment with a single dose of C21. The cytotoxic effects of C21 were reversed by PD 123177, clearly implying an AT2 receptor-dependent mechanism (n=6) (F2,15=40.193, P<0.001) (Figure 3b, left panel). A dramatic increase in the LDH release into the media (n=9) (F2,24=16.132, P<0.001) (Figure 3b, right panel) indicates that a large portion of cells exposed to C21 underwent necrosis or, alternatively, aponecrosis. The cells lost their typical spindle form and showed morphological changes occurring with necrosis such as cell swelling and/or fractionation and formation of vacuoles. Bcl-2 (n=6) and Bax (n=6) proteins and cleaved caspase-3 (n=6) were quantified in SK-UT-1 cells, which escaped from the cell death induced by a 24 h incubation with C21. The anti-apoptotic Bcl-2 protein was reduced and the pro-apoptotic protein, Bax was up-regulated (Figure 3c). Increased Bax/Bcl-2 ratio (Figure 3c, right panel) and activation of the executioner caspase-3 (Figure 3d) indicate the entry of SK-UT-1 cells into mitochondria-mediated apoptotic cell death. Interestingly, quiescent SK-UT-1 cells which escaped the rapid cell death after a 24 exposure to C21 (10−6 M) displayed significantly lower densities of AT2 receptors in mitochondria (n=7) (Figure 3e). These findings substantiate the relevance of the augmented intracellular AT2 receptor levels for the cytotoxic activity of C21 in quiescent SK-UT-1 cells. The long-term effects of C21 were studied in quiescent and proliferating SK-UT-1 cells exposed to vehicle or C21 for 5 days. All quiescent SK-UT-1 cells were killed off upon a 5-day exposure to a single dose of C21 (Figure 3f). In contrast, C21 was ineffective against proliferating SK-UT-1 cells cultured in the growth medium (Figure 3g).

C21 induces rapid cell death and activates mitochondrial apoptosis in quiescent SK-UT-1 cell

Figure 3
C21 induces rapid cell death and activates mitochondrial apoptosis in quiescent SK-UT-1 cell

(a) SK-UT-1 cells treated with vehicle remained attached to the culture dish. SK-UT-1 cells exposed to C21 detached from the culture dish and lost their typical spindle form. (b) Effects of treatment of quiescent SK-UT-1 cells with vehicle, C21 and C21+PD 123177 (PD) upon cell numbers (left panel, n=6) and LDH release into the incubation medium (right panel, n=9). Statistical comparison with vehicle treated cells: *** P<0.001, and with cells treated simultaneously with C21 and PD 123177: †††P<0.001, calculated by one-way ANOVA followed by a post hoc Bonferroni test for pairwise comparisons. (c) Western blot analysis of the Bcl-2 (left panel, n=6) and Bax (middle panel, n=6) proteins and, (d) activated caspase-3 (n=6) in quiescent SK-UT-1 cells exposed to vehicle, C21 and C21+PD 123177 for 24 h. Statistical comparison with vehicle treated cells: * P<0.05, ** P<0.01, calculated by paired t-test; two-tailed P values were adjusted for multiple comparisons using the Bonferroni correction. (c) right panel, Bax/Bcl-2 ratio (n=6). Statistical comparison with vehicle treated cells: ** P<0.01, calculated by one-way ANOVA followed by a post hoc Bonferroni test for pairwise comparisons. (e) Density of the AT2 receptor in mitochondrial fractions of SK-UT-1 cells exposed to vehicle or C21 for 24 h (n=7); * P<0.05, compared with vehicle treated cells (Student's t-test). (f) Effects of a 5-day exposure of quiescent SK-UT-1 cells to vehicle or C21 (Cresyl Violet staining). C21 was added to the media only once at the beginning of the experiment. (g) C21 was ineffective against proliferating SK-UT-1 cells cultured in the growth medium.

Figure 3
C21 induces rapid cell death and activates mitochondrial apoptosis in quiescent SK-UT-1 cell

(a) SK-UT-1 cells treated with vehicle remained attached to the culture dish. SK-UT-1 cells exposed to C21 detached from the culture dish and lost their typical spindle form. (b) Effects of treatment of quiescent SK-UT-1 cells with vehicle, C21 and C21+PD 123177 (PD) upon cell numbers (left panel, n=6) and LDH release into the incubation medium (right panel, n=9). Statistical comparison with vehicle treated cells: *** P<0.001, and with cells treated simultaneously with C21 and PD 123177: †††P<0.001, calculated by one-way ANOVA followed by a post hoc Bonferroni test for pairwise comparisons. (c) Western blot analysis of the Bcl-2 (left panel, n=6) and Bax (middle panel, n=6) proteins and, (d) activated caspase-3 (n=6) in quiescent SK-UT-1 cells exposed to vehicle, C21 and C21+PD 123177 for 24 h. Statistical comparison with vehicle treated cells: * P<0.05, ** P<0.01, calculated by paired t-test; two-tailed P values were adjusted for multiple comparisons using the Bonferroni correction. (c) right panel, Bax/Bcl-2 ratio (n=6). Statistical comparison with vehicle treated cells: ** P<0.01, calculated by one-way ANOVA followed by a post hoc Bonferroni test for pairwise comparisons. (e) Density of the AT2 receptor in mitochondrial fractions of SK-UT-1 cells exposed to vehicle or C21 for 24 h (n=7); * P<0.05, compared with vehicle treated cells (Student's t-test). (f) Effects of a 5-day exposure of quiescent SK-UT-1 cells to vehicle or C21 (Cresyl Violet staining). C21 was added to the media only once at the beginning of the experiment. (g) C21 was ineffective against proliferating SK-UT-1 cells cultured in the growth medium.

Liquid chromatography–mass spectrometry (LC–MS) analysis of C21 penetration into SK-UT-1 cells

SK-UT-1 cells (approximately 107 cells per well) were exposed to C21 (10−6 M) for 1 h. The concentration of C21 was determined in 50 μl of the culture medium. Fifty microliters of the incubation medium contained approximately 24 ng of C21 (Figure 4I). To remove C21 from the culture medium, the cells were washed three times with ice-cold PBS and 50 μl of the third washing solution was used for the determination of C21 concentrations (Figure 4II). Then, the cells were lysed, centrifuged and the concentration of C21 was measured in 50 μl of the supernate. The molecular weight of C21 detected in our experiment and that reported previously are identical (476.6) [10]. The concentration of C21 in the cytosol was 13% of that in the culture medium (approximately 60 fg/100 cells) (Figure 4III). We demonstrate for the first time that C21 penetrates through the cell membrane of SK-UT-1 cells and reaches high intracellular concentrations.

Liquid chromatography-mass spectrometry (LC-MS) analysis of intracellular concentrations of Compound 21 (C21) in SK-UT-1 cells

Figure 4
Liquid chromatography-mass spectrometry (LC-MS) analysis of intracellular concentrations of Compound 21 (C21) in SK-UT-1 cells

MS chromatograms of C21 in the culture medium (50 μl) 1 h after the onset of the incubation (I), in the third washing medium (PBS) (II) and in the cell lysates (50 μl of supernatant) (III). The concentration of C21 in the culture medium was 10−6 M. C21 penetrates the cell membrane and the detected concentration in the cytosol was 13% of that in the culture medium (approximately 60 fg/100 cells).

Figure 4
Liquid chromatography-mass spectrometry (LC-MS) analysis of intracellular concentrations of Compound 21 (C21) in SK-UT-1 cells

MS chromatograms of C21 in the culture medium (50 μl) 1 h after the onset of the incubation (I), in the third washing medium (PBS) (II) and in the cell lysates (50 μl of supernatant) (III). The concentration of C21 in the culture medium was 10−6 M. C21 penetrates the cell membrane and the detected concentration in the cytosol was 13% of that in the culture medium (approximately 60 fg/100 cells).

C21 does not exert cytotoxic effects in quiescent HutSMC

Slightly higher levels of the AT2 receptor protein were detected in quiescent HutSMC than in SK-UT-1 cells and approximately the same amounts of the receptor protein were found in the mitochondrial compartments of both cell types (Figure 5a). However, the dose of C21 (10−6 M), which induced rapid cell death in quiescent SK-UT-1 cells, was ineffective in HutSMC. Treatment of quiescent HutSMC with C21 for 24 h (Figure 5b) or 5 days (Figure 5d) did not alter the cell morphology (Figures 4b and 4d) nor did it modify cell numbers (Figure 4c, left panel) (n=5) and the LDH release into the incubation medium (Figure 5c, right panel) (n=5).

C21 does not exert cytotoxic effects in quiescent HutSMC

Figure 5
C21 does not exert cytotoxic effects in quiescent HutSMC

(a) The levels of the AT2 receptor in the whole cell and mitochondrial fractions of quiescent SK-UT-1 cells and human uterine smooth muscle cells (HutSMC). (b) HutSMC exposed to C21 for 24 h remained attached to the culture dish and display normal morphology. (c) Exposure of HutSMC to C21 for 24 h did not alter the cell numbers (n=5) nor did it modify the release of LDH into the culture medium (n=5); Veh, vehicle. (d) C21 does not exert cytoxic effects in quiescent HutSMC exposed to the AT2 agonist for 5 days.

Figure 5
C21 does not exert cytotoxic effects in quiescent HutSMC

(a) The levels of the AT2 receptor in the whole cell and mitochondrial fractions of quiescent SK-UT-1 cells and human uterine smooth muscle cells (HutSMC). (b) HutSMC exposed to C21 for 24 h remained attached to the culture dish and display normal morphology. (c) Exposure of HutSMC to C21 for 24 h did not alter the cell numbers (n=5) nor did it modify the release of LDH into the culture medium (n=5); Veh, vehicle. (d) C21 does not exert cytoxic effects in quiescent HutSMC exposed to the AT2 agonist for 5 days.

DISCUSSION

Recent research has provided evidence of an intracellular RAS system in a variety of non-malignant cells including cardiac, renal, hepatic, vascular and myometrial cells [1,20]. The present study reports for the first time on the distribution of AT2 receptors in intracellular compartments of the human leiomyosarcoma cells and provides evidence for the existence of a functional RAS system in tumour cells. Our results demonstrate that the abundance of AT2 receptors is low during cell proliferation but the receptor is substantially up-regulated when the cell cycle is arrested. The most significant novel findings of the study are the AT2 receptor-mediated activation of the intrinsic, mitochondrial apoptotic pathway in quiescent SK-UT-1 cells and the evidence that C21 passes through the cell membrane of SK-UT-1 cells and induces rapid cell death via interaction with the intracellular AT2 receptors.

We could reliably detect the AT1 and the AT2 receptor and demonstrate that SK-UT-1 cells in fact express both types of Ang II receptor. Similarly to other tumour cell lines, such as PC12W or R3T3 cells, the AT2 receptor was up-regulated in the quiescent state [21]. Considerable amounts of the AT2 receptor protein were detected in mitochondria of quiescent SK-UT-1 cells. The higher molecular mass of the receptor detected in mitochondria results from a different degree of glycosylation [22]. AT2 receptors localized in the cell membrane do not contribute to the mitochondrial AT2 receptor pool, because unlike the AT1 receptor, which rapidly internalizes upon binding of an agonist, activation of the membrane AT2 receptors is not followed by its interaction with β-arrestins and internalization [23,24]. The high density of the AT2 receptor in mitochondria of non-cycling SK-UT-1 cells rather results from its increased transcription initiated by the withdrawal of growth factors [25,26].

As with other cell types, activation of the AT2 receptors in quiescent SK-UT-1 cells with Ang II promoted apoptosis as evidenced by DNA fragmentation and TUNEL-staining. The apoptotic mechanisms triggered by stimulation of the membrane AT2 receptors imply the down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL or the dephosphorylation and inactivation of the Bcl-2 protein by mitogen-activated protein kinase-phosphatase-1 [27,28]. However, there were substantial differences between the rates of cell death induced by Ang II and C21. A 24 or 48 h stimulation of AT2 receptors with Ang II did induce apoptosis in about one third of quiescent SK-UT-1 cells but did not lead to immediate cell death as evidenced by unaltered cell numbers. Alternatively, an ongoing cell proliferation could compensate for the apoptotic cell loss, because starvation media are not completely devoid of growth factors and numerous signalling pathways are constitutively activated even under serum starvation conditions. Therefore, even weak growth factor signalling can maintain survival and drive proliferation of malignant tumour cells [25]. In contrast, exposure of quiescent SK-UT-1 cells to C21 induced rapid cell death. The most probable explanation for the substantial distinction between the effects induced by Ang II and C21 are the different sites of their actions. The membrane AT2 receptor does not internalize upon binding of Ang II (see above) and the AT1 receptor antagonist, losartan, completely prevented the cellular uptake of the Ang II/AT1 receptor complex, the subsequent cytoplasmic release of Ang II and, consequently, its interaction with intracellular AT2 receptors [23,24]. In contrast, C21 penetrates through the cell membrane and reaches high intracellular concentrations. While both, AT2 receptor ligands activate the membrane receptors, only C21 can interact with intracellular AT2 receptors. The present results provide evidence that the rapid cell death induced by C21 in quiescent SK-UT-1 cells results from its interaction with intracellular, most probably mitochondrial, AT2 receptors. A non-specific, overall cell cytotoxicity of C21 can be excluded as the quiescent HutSMC did not die after exposure to the compound, indicating that AT2 receptor-mediated cell death is cell type-specific.

Quiescent SK-UT-1 cells which survived a 24 h exposure to C21, displayed features of apoptosis and, even more importantly, lower densities of the AT2 receptor in mitochondria than vehicle-treated cells. These findings raise an interesting question of whether these cells escaped from the rapid cell death because the densities of AT2 receptors in mitochondria were already lower at the beginning of the treatment with C21, or because these cells succeeded in rapidly transporting AT2 receptors outside mitochondria after the exposure to C21, thus preventing an effective interaction of the agonist with the mitochondrial AT2 receptor. We have observed that multiple passages of SK-UT-1 cells dramatically reduced the AT2 receptor density in mitochondria and increased the AT2 receptor immunoreactivity in the cytosol. In these cells, C21 did not trigger an immediate cell death and the cells began to die at later time points (72 h) (data not shown).

The cytotoxic effects of C21 were confined to quiescent uterine leiomyosarcoma cells; the normal uterine smooth muscle cells were not affected. The AT2 receptor in the human myometrium is abundantly expressed and the rate of its expression is controlled by a variety of physiological regulatory mechanisms [9,10]. On the other hand, the rate of AT2 receptor expression and its subcellular trafficking in leiomyosarcoma cells strongly depend on the cellular state. In proliferating cells, the expression and levels of the AT2 receptor are low. Consequently, the effects of its interaction with C21 are negligible. However, both, the augmented expression of intracellular AT2 receptors and their interaction with C21 are decisive for the induction of rapid cell death in quiescent SK-UT-1 cells. Cancer cells that fail to receive sufficient growth factor signalling or nutrients reduce energy metabolism and undergo cycle arrest [25,26]. The augmented synthesis and increased transfer of the AT2 receptor to mitochondria may result from the modified gene expression patterns which help tumour cells to overcome the withdrawal of growth factors and to re-enter proliferation. Based on the data reported by Abadir et al. [8], it is tempting to speculate that upon growth factor withdrawal, the mitochondrial AT2 receptors may exert protective effects, such as suppression of respiration and O2 consumption and reduction in reactive-oxygen species production by ligand-independent receptor signals [29]. However, binding of C21 to the receptor eventually leads to excessive NO production resulting in strong inhibition of respiration, subsequent mitochondrial dysfunction and cell death.

In summary, our study shows that AT2 receptors are over-expressed in non-cycling human leiomyosarcoma cells and display high densities in mitochondria. We demonstrate that the high-affinity, non-peptide AT2 receptor agonist, C21, crosses the cell membrane and exerts profound cytotoxic effects via activation of intracellular AT2 receptors. The majority of cells died within 24 h after exposure to C21, the remaining cells underwent apoptosis and died at later time points. The cytotoxic actions of C21 were exclusive for quiescent tumour cells as C21 was devoid of any cytotoxic or apoptotic effects in HutSMC. The present data provide evidence for the existence of a RAS system in tumour cells and may constitute a solid basis for follow-up studies focused on the role of the intracellular RAS and especially the AT2 receptor in tumour cell biology. Our findings can also help to better understand the processes occurring in non-cycling tumour cells or tumour cells forced to enter quiescence and contribute thus to the development of new molecularly targeted therapies for malignant tumours.

Abbreviations

     
  • Ang

    II, angiotensin II

  •  
  • AT1

    angiotensin type 1

  •  
  • AT2

    angiotensin type 2

  •  
  • ATIP

    AT2 receptor interacting proteins

  •  
  • C21

    Compound 21

  •  
  • DMEM

    Dulbecco’s modified Eagle’s medium

  •  
  • 18F-FDG

    [18F]2-fluoro-2-deoxy-D-glucose

  •  
  • HutSMC

    human uterine smooth muscle cells

  •  
  • LC–MS

    liquid chromatography–mass spectrometry

  •  
  • LDH

    lactate dehydrogenase

  •  
  • RAS

    renin–angiotensin system

  •  
  • RT

    reverse transcription

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling

AUTHOR CONTRIBUTION

Yi Zhao conceived the study, performed the experimental work and collected most of the data. Ulf Lützen contributed to the design of experiments and to acquisition of the funding and approved the manuscript. Jürgen Fritsch and Stefan Schütze carried out confocal laser microscopy experiments and performed the analysis of the data, Maaz Zuhayra performed the LC–MS detection of C21 and analysed the data, Ulrike Steckelings provided C21 and approved the manuscript. Chiara Recarti contributed to the design of the study and made critical revision of the manuscript. Pawel Namsolleck participated in the conception and design of the study, critically reviewed the manuscript and contributed to creating its final version. Thomas Unger contributed to the interpretation of the data, revised the article for intellectual content and made the final approval of the article. Juraj Culman conceived and designed the study, interpreted the data, performed statistical analyses, supervised the study and drafted and approved the article.

FUNDING

This research was not supported by specific grant from any funding agency in the public, commercial or not-for-profit sectors.

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Author notes

1

Both authors contributed equally to this work.