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Hyperoxia retards growth and induces apoptosis and loss of glands and blood vessels in DMBA-induced rat mammary tumors

Hyperoxia retards growth and induces apoptosis and loss of glands and blood vessels in... Background: This study investigated the effects of hyperoxic treatment on growth, angiogenesis, apoptosis, general morphology and gene expression in DMBA-induced rat mammary tumors. Methods: One group of animals was exposed to normobaric hyperoxia (1 bar, pO = 1.0 bar) and another group was exposed to hyperbaric hyperoxia (1.5 bar, pO = 1.5 bar). A third group was treated with the commonly used chemotherapeutic drug 5- Fluorouracil (5-FU), whereas animals housed under normal atmosphere (1 bar, pO = 0.2 bar) served as controls. All treatments were performed on day 1, 4, 7 and 10 for 90 min. Tumor growth was calculated from caliper measurements. Biological effects of the treatment, was determined by assessment of vascular morphology (immunostaining for von Willebrandt factor) and apoptosis (TUNEL staining). Detailed gene expression profiles were obtained and verified by quantitative rtPCR. Results: Tumor growth was significantly reduced (~57–66 %) after hyperoxic treatment compared to control and even more than 5-FU (~36 %). Light microscopic observations of the tumor tissue showed large empty spaces within the tissue after hyperoxic treatment, probably due to loss of glands as indicated by a strong down-regulation of glandular secretory proteins. A significant reduction in mean vascular density (30–50%) was found after hyperoxic treatment. Furthermore, increased apoptosis (18–21%) was found after hyperoxic treatment. Conclusion: Thus, by increasing the pO in mammary tumor tissue using normobaric and moderate hyperbaric oxygen therapy, a significant retardation in tumor growth is achieved, by loss of glands, reduction in vascular density and enhanced cell death. Hyperbaric oxygen should therefore be further evaluated as a tumor treatment. Page 1 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 Background Due to the apparent link between blood supply, oxygena- Growth of solid tumors depends on adequate supply of tion and tissue growth, it has for a long time been a mis- oxygen and nutrients. There are, however, marked differ- conception that HBO per se could have a tumor- ences in the vascular network in the different regions of promoting effect. There are now several lines of evidence the tumor. In the centre, there is typically a hypoxic milieu showing that this is not the case [13]. In a rat model of due to structural and functional vessel disturbances (per- dimethyl-α-benz-anthracene (DMBA)-induced mammary fusion- and diffusion-limited O delivery), while in the adenocarcinomas, we have recently demonstrated a sig- periphery there is generally a denser vascular network nificant decrease in mammary tumor size after repeated with subsequent improved blood flow. While normal tis- HBO treatment at 2 bar for 90 minutes [14]. These para- sue can compensate for such an O deficiency by raising doxical data indicate that an increase in the delivery of the blood flow, large tumor areas cannot adequately physically dissolved O in the tumor tissue by hyperbaric counteract the restriction in O supply and therefore hyperoxic treatment may suppress its growth. develop hypoxia. Thus, the HbO saturation is signifi- cantly lower in tumors than in normal surrounding tissue, The present study was initiated to see if 1.5 bar (pO2 = with a gradual reciprocal decrease as the tumor increases 1.5) as well as pure oxygen at normal atmospheric pres- in size [1,2]. sure (1 bar, pO = 1.0) would have a similar effect. The aim of the present study was therefore to find the least It is now widely accepted that hypoxia promotes tumor pressure gradient that gave a therapeutic effect on tumor growth, angiogenesis and reduce the effect of chemo- and growth. Therapeutic efficacy was determined by assessing radiation- therapy [3-6]. We might therefore expect that tumor growth, angiogenesis, apoptosis, general morphol- an increase in the oxygen-content in tumor tissue might ogy and gene expression profiling. have the opposite effect. Methods Hyperbaric oxygen treatment (HBO) offers one possibility Animal model of mammary tumors to increase the oxygen content in various tissues [7-10]. Female Sprague-Dawley (Møllegård, Denmark) rats (n = The use of HBO in cancer therapy has been aimed at 35) were used. Mammary tumors (adenocarcinomas) improving the radiation response in solid tumors [10] as were induced by dimethyl-α-benz-anthracene (DMBA) well as to improve healing of normal tissue after radiation dissolved in olive oil and given to the 7 week rat by gavage injury [11]. The increase in tissue pO during and after at a dose of 16 mg. The experiments were performed when HBO treatment is due to enhanced transport of soluble the rats were 13–15 weeks old, having reached a body oxygen. The physically solved oxygen at normobaric air weight of 250–300 g and developed tumors along the pressure is approximately 0.3 ml O /l00 ml blood, with a mammary crest. Tumor size was measured externally by corresponding HbO of approximately 21 ml/100 ml calipers on day 1, 4, 7, and 11 and estimated according to blood. By breathing 100% oxygen at normobaric pres- the following formula: π/6 · (a) (b), where a is the short- sure, the amount of physically soluble oxygen increases 6 est transversal diameter and b is the longest transversal times (1.8 ml O /100 ml blood). If the atmospheric pres- diameter. The examinations were performed by the same sure is elevated to 3 bar in the presence of 100% O , the person, with no knowledge about the various exposures of amount of oxygen delivered to the tissue would increase the rats. All measurements were performed during isoflu- to 6.0 ml O /100 ml blood, which is even sufficient to ran (Shering-Plough AS, Narum, Denmark) and N2O support resting tissue independent of the O contribution anesthesia (Ohmeda: BOC Health Care, Weast-Yorkshire, from hemoglobin [12]. When oxygen is in solution, it can England). The study was approved by the Norwegian reach physiologically obstructed areas that are inaccessi- Committee for Animal Research (Oslo, Norway). ble to the HbO -containing red blood cells. In line with this, several investigators have measured a significant Experimental groups and treatment design delay in washout (15–60 min) of the pO in different Four separate groups of rats were studied (for details see tumors after HBO treatment [3,7-9]. Table 1). Table 1: The experimental groups. Gas Total pressure (bar) pO2 (bar) Drug No. of rats Group 1 Air 1.0 0.2 NaCl 10 Group 2 Air 1.0 0.2 5-FU 10 Group 3 Oxygen 1.0 1.0 NaCl 8 Group 4 Oxygen 1.5 1.5 NaCl 8 The hyperbaric oxygen treatement and 5-FU (0.2 mg/kg i.p.) were given on day 1, 4, 7 and 10. Page 2 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 Hyperbaric chamber and oxygen exposure procedure pH6.0) and then microwaved for 5 min. The slides were 130 l hyperbaric chamber with an internal diameter of 50 washed in PBS before terminal deoxynucleotidyl trans- cm was used. For inspection and video supervision there ferase was applied to each slide and incubated for 1 hr at are two windows in the chamber wall. Penetrators for gas 37°C in a humidified chamber. A stop/washing buffer inlet and outlet run through the chamber's end wall. The was used before applying the converter POD (anti-fluores- chamber and rat cage were cleaned and degreased (96– cein antibody conjugated with peroxidase as reporter 100% ethanol) before starting exposure with pure oxygen. enzyme) for 30 min. Diaminobenzidine (DAB) was used All electrical installations were disconnected. The rats as a chromogen and the slides were counterstained with were placed in litter-free cages (590 × 385 × 200 mm) dur- hematoxylin. The density of apoptotic cells per mm of ing HBO exposure. All rats were showered slightly with the tumor viable zone was determined using a counter water prior to entering the pressure chamber, due to the grid (10 random vision fields × 40). The percentage of danger of fire in a pure oxygen atmosphere. Four petrid- apoptotic cells is expressed as a percentage of total cells. ishes with water were placed on the chamber floor to additionally humidify the atmosphere. To initiate the Von Willebrand factor For detection of vascular endothelial cells we stained for treatment, 100% oxygen was introduced into the chamber as air was simultaneously flushed out. The oxygen concen- Von Willebrand factor. The rabbit anti-human polyclonal tration in the chamber was monitored continuously by an antibody against Von Willebrand factor (DAKO) was oxygen cell (C3, Middelsborough, England). When oxy- diluted in TrisBSA to a 1:250 and than to a 1:500 dilution. gen was above 98%, the rats of group 3 were kept at this The tissue sections were incubated for 45 min and 75 min level for 90 min, while in group 4 the chamber was pres- respectively with the primary antibody, washed three surized with oxygen over approximately 2 min to 1.5 bar, times for 5 min in TBS and further incubated with anti- and this pressure was maintained throughout 90 min. rabbit IgG (DAKO Cytomation Envision Kit, Labelled Pol- During the 90 min period, the chamber was flushed twice ymer – HRP Anti Rabbit) for 35 min. All incubations were for 5 min (at 30 and 60 min) with pure oxygen in both performed in a humidity chamber. The tissue was then series. The temperature was held at approximately 22°C washed three times with PBS. Detection was carried out and the humidity at approximately 100%. The CO con- using a DAB chromogen, which resulted in a positive tent was kept low by use of a scrubber material (Sodasorb, brown staining. Harris Haematoxylin (Merck, Damstadt, Molecular Products United Drug Co Ltd, Essex, UK) Germany) was used for nuclear counterstaining. Negative placed inside the chamber floor. control slides were obtained by omitting the primary anti- body. Quantification of the microvessels was performed Morphological analyses in 10 consecutive fields in both the center and in the The animals were sacrificed with pentobarbital during iso- periphery and averaged as vessels/mm . Approximately fluran and N2O anesthesia, and the tumors were dissected 50 blood vessels in both the periphery and central part of out (one tumor from each rat). One part of the tumor was the tumor were randomly selected for diameter measure- fixed in 4% buffered formalin, processed and embedded ments. in paraffin. Tissue sections of each specimen were stained using Harris Haematoxylin and Eosin (H & E, Merck, All the specimens were examined in a Nikon light micro- Damstadt, Germany). The sections were analyzed with scopy (THP Eclipse E600, Nikon Corporation, Tokyo, regards to apoptosis (TUNEL assay) and blood vessel den- Japan) and the images captured with a Nikon Digital sity (von Willebrand factor). Indirect immunohistochem- Camera (DXM 1200F, Nikon Corporation, Tokyo, Japan). istry was performed by the EnVisison™ + System, horse- The image processing and analysis system LUCIA, version radish peroxidase and 3'3'-diaminobenzidine (DAB) 4.8 (Laboratory Imaging Ltd, Prague, The Check Republic) (DAKO, Glostrup, Denmark) method, as described in the was used. manufacturer's protocol. RNA extraction and microarray-based expression analysis TUNEL staining Specimens of tumor tissue (approximately 2 × 1 × 0.3 cm) Apoptosis was examined by the terminal transferase- were immersed in ice cold RNA-later solution (Ambion, mediated dUTP nick end-labeling (TUNEL) method (Boe- Foster City, CA), and kept at 4°C over night. On the fol- hringer Mannheim, Mannheim, Germany), performed lowing day, the tissue tubes were frozen at -20°C until according to the manufacturers recommendations. The RNA isolation. The tumor tissue samples were homoge- DNA strand breaks in apoptotic cells are labelled by nised by a Kinematica Polytron homogenizer. Total RNA attaching biotin-or digoxygenin conjugated dUTP in a was extracted using the QIAGEN RNAeasy Lipid kit, and reaction catalyzed by exogenous terminal deoxynuclepti- the amount and quality of the extracted RNA was meas- dyl transferase (TdT-assay) or DNA polymerase. For anti- ured by the NanoDrop ND-1000 Spectrophotometer and gen retrieval the slides were put into citrate buffer (0.1 M, Page 3 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 the Agilent 2100 Bioanalyzer. Purified total RNA was Identification of genes with significant differential expres- stored at -80°C until use. sion in hyperoxic exposed tumors was carried out using 'significance analysis of microarrays' (SAM) (16). The nor- Twenty microgram total RNA was reversely transcribed malized dataset from J-Express was imported into the and fluorescently labelled using the Agilent Fluorescent TM4 Microarray Software Suite Multi Experiment Viewer Direct Label Kit (G2557A), according to the manufac- 3.1 (MeV) (TIGR, US). Signal intensities in all HBO turer's instructions. To screen for gene expression changes treated 'test' samples were compared to those of the in mammary tumors as a result of hyperoxic exposure, untreated 'control' samples. The analysis threshold was set competitive hybridisations of samples from the treated to a highly conservative false discovery rate of zero. tumors (Group 3, 100% O , 1 bar) against the untreated Real-Time PCR Analysis tumors (normal air, 1 bar) were performed, where four hyperoxia-treated tumor samples were each compared to Total RNA was extracted, quality controlled and stored as a pool of ten control tumors. Test samples were labelled described above. From each of the samples, 50 ng RNA with Cy5 and control samples labelled with Cy3. After the was reverse transcribed to cDNA using TaqMan RT rea- labelling reactions, the dye-incorporation ratio was deter- gents (Applied Biosystems, Foster City, CA). The final con- mined using a spectrophotometer, showing ratios within centrations of the reagents were as follows: 1 × TaqMan RT 10 to 20 pmol per µg cDNA. The amount of the Cy- buffer, 5.5 mM MgCl , 2 mM dNTP mixture, 2.5 mM ran- labelled cDNA was checked by measuring its optical den- dom hexamers, 0.4 U/µl RNase inhibitor and 1.25 U/µl sity in the NanoDrop ND-1000 Spectrophotometer, at Multiscribe reverse transcriptase in RNase-free water to a OD 260/280 nm for DNA purity and at OD 550 nm and total volume of 50 µl. The reaction mix was incubated at 650 nm for dye incorporation. The amount of cDNA out- 25°C for 10 min (primer annealing), 48°C for 30 min put was 1–2 µg and the dye incorporation was about 5% (synthesis) and 95°C for 5 min (enzyme inactivation). for Cy3 and 3% for Cy5. The resulting cDNA samples were stored at -20°C. All sub- sequent real-time PCR experiments were performed on an All hybridisations were carried out on Agilent 22 k Rat ABI Prism 7900 HT sequence detector system using 384- Oligo Microarrays (G4130A), with 22,575 60-mer probes well plates. The PCR reaction solution for parotid secretory representing over 20,000 rat genes, ESTs and EST clusters. protein (Psp), prolactin induced protein (Pip) and common For hybridisation, the Agilent 60-mer oligo microarray salivary protein 1 (Csp1) contained 1 µl 20 × TaqMan Gene processing protocol (V 4.1, SureHyb 22 k chamber/SSC Expression Assays (Applied Biosystems) and 10 µl 2 × wash) was strictly followed. Briefly, the entire labelling TaqMan Universal PCR Master Mix. The acidic ribosomal reactions for Cy3-labelled control samples and Cy5- protein P0 (P0) reaction solution contained 10 µl 2 × labelled test samples were combined and applied to the SYBR green (Medprobe), together with forward and microarray together with control targets and hybridisation reverse primers (Sigma-Aldrich) in a final concentration buffers provided by the Agilent in situ hybridization plus of 300 nM. All PCR reactions contained 4 µl of cDNA reac- kit (5184–3568). The microarrays were then incubated in tion mix and RNasefree water to a total volume of 20 µl. an Agilent hybridisation oven (G2545A) with rotation for The real-time PCR was run as follows: 50°C for 2 min 17 hours at 60°C. On the following day, microarrays were (UNG incubation) and 95°C for 10 min (AmpliTaq Gold washed and immediately dried using an ultra pure filtered activation), followed by 40 cycles of 95°C for 10 s and stream. All operations were in accordance with the N 60°C for 1 min. For each sample, the gene expression was manufacturer's instructions. quantified by the standard curve method and normalized against the expression of the ribosomal protein P0 gene. After drying, the slides were scanned for fluorescence sig- The standard curve consisted of five points that were nals by the Agilent G2565AA Microarray Scanner with a obtained by a two-fold serial dilution of control RNA, pixel size of 10 nm. The TIFF images generated by the starting out at 250 ng, together with a 'non-template' con- scanning process were imported into GenePixPro 5.0 soft- trol; all performed in triplicate. P0 was preferred as the ware (Axon Instruments Inc) for primary data analysis. endogenous control over GAPDH and b-actin, which Control spots and spot artifacts were flagged in GenePix both gave qualitatively similar results in pilot experiments for subsequent filtering and background-corrected signal (data not shown). intensity values were imported into the J-Express Pro V2.6 software (MolMine, Bergen, Norway)[15]. Flagged spots Statistics were filtered and the red versus green signal intensities of Results were expressed as means ± SD unless indicated each spot were adjusted by global normalization to reduce otherwise. Differences between groups were assessed by the impact of dye bias in the data sets. unpaired, two-tailed Student's t-test or the Mann-Whitney U test. P < 0.05 was considered significant. Page 4 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 spot" areas (areas of high vascular density) in both the Results Tumor growth tumor centre and periphery were examined. The mean A total of 36 tumors were studied (one from each rat). The vascular density was markedly reduced in the tumors tumor size ranged from 1.2 to 2.3 cm prior to the start of treated with hyperoxia compared to controls (Table 3). the experiments (Day1). The marked increase in tumor The ratio of central/peripheral vessel density is given in size found in controls (group 1, n = 10) over a period of Table 3. The diameter of the remaining tumor vessels was, 11 days (p < 0.001), was reduced by administering 5-FU however, increased after hyperoxic treatment compared to to the animals (group 2, n = 10, p < 0.02). A further reduc- control, as can be seen in Table 3. The results also show tion in tumor size was observed at day 11 after both 1.0 that the diameter of the peripheral vessels is larger than in bar (group 3, n = 8) and 1.5 bar (group 4, n = 8) hyperoxia the central parts in both control and at 1 bar hyperoxic treatment as compared to controls (p < 0.01) and 5-FU treatment. The ratio of central/peripheral vessel diameter treated rats (p < 0.05). Our previous study at 2 bar, 100% is given in Table 3. The relationship between tumor vol- O significantly attenuated tumor growth in the same ume and mean vessel density is visualized in Fig 3. tumor model (included in Fig. 1) [14]. Taken together Tumor cells these results indicate a reduction in growth that is dose- dependent on pO . The percentage of apoptotic cells in TUNEL-stained tissue section was significantly increased (18–21%) after hyper- Down-regulation of glandular secretory proteins oxic treatment compared to controls as shown in Table 3. Since these results confirmed our previous findings that hyperoxia might attenuate the growth of rat mammary Discussion tumors, we decided to perform a global gene expression The present study shows that exposing rats to normobaric profiling to search for differentially expressed genes and (1 bar) or hyperbaric (1.5 bar) hyperoxia (100% O ), four their biological function. By comparison of global gene times for 90 minutes, over a period of 10 days signifi- expression level in 5 hyperoxia treated tumors with a pool cantly retards tumor growth. In the present model this of 10 control tumors, we notably observed a small cluster treatment showed more efficacy than the commonly used (n = 5) of genes involved in glandular secretion that were chemotherapeutic drug 5-FU. Hyperoxic treatment did all strongly down-regulated by the exposure of the tumors affect the mammary tumors by significantly down-regu- to 100% O (Table 2). These proteins included parotid lating glandular genes that may have implications in secretory protein (Psp), common salivary protein 1 (Csp1) and tumor growth (loss of glands), reducing vascular density prolactin induced gene (Pip), cystein-rich secretory protein and enhancing apoptosis. (crisp-1) and proline rich proteoglycan 2 (Prpg2). All these genes displayed moderate to high expression levels in the HBO is not a tumor promoter control tumors, but after a total of 6 hours of hyperoxic HBO has been used in combination with both chemo- treatment their expression was dramatically diminished, therapy and radiotherapy to enhance the pO in the oth- with a down-regulation ranging from 5–33 times in the erwise hypoxic tumor tissue and thereby potentiate the individual tumors. To verify the microarray results, we effect of these treatments [10]. HBO has also been used for used quantitative real- time PCR, which confirmed that wound healing, treatment of necrotic tissues and recovery the expression of Psp, Pip and Csp1 was markedly dimin- of radiation-injured tissues because of its ability to pro- ished in the hyperoxia-exposed tumors (Table 2). mote angiogenesis and thereby enhance blood supply to the injured area [10,11]. However, it has for a long time General morphology been a misconception that HBO per se could have a tumor The gene expression data indicated that HBO treatment promoting effect. There are now several lines of evidence was associated with attenuation of glandular function in showing that this is not the case [13]. The present work the mammary tumor. Subsequently the E & H stained support previous conclusions from our group demon- tumor tissue sections, clearly demonstrates that normo- strating that HBO has a significant inhibitory effect on baric and as well as hyperbaric hyperoxia treatment mammary tumor growth in rats [14]. Also two studies on induced large areas of empty space ("vacuoles") both in mice injected with various tumor cell lines exposed to the central parts (Fig 2A) and in the periphery (Fig 2B) of 70% O for 3 weeks showed a reduced number of lung the tumor tissue compared to control (Table 3), thereby colonies derived from mammary carcinoma MT-7 cells indicating a loss of glandular tissue. The morphology of 1 and of lung-tumor cell lines [17,18]. Some cell lines have, and 1.5 bar sections were identical. however been shown to be oxygen resistant [17], which indicates differences in the oxygen sensitivity in different Tumor vessels tumor-types. In order to analyse the impact of hyperoxia on the induc- tion of vessel formation, the vascular densities in "hot Page 5 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 ** *** *** *** 24 68 10 12 Days Control (21% O ) 5-FU 1 bar (100% O ) 1.5 bar (100% O ) 2 bar (100% O ) + 5-FU (Stuhr et al 2004) 2 bar (100% O ) (Stuhr et al 2004) * p < 0.05, ** p< 0.02 , *** p< 0.01 vs control # p < 0.05 vs 5-FU Th Figure 1 e effect of normoxic and hyperoxic treatment on tumor growth compared to controls and 5-FU treated The effect of normoxic and hyperoxic treatment on tumor growth compared to controls and 5-FU treated. Treatments were given on day 1, 4, 7 and 10. Values represent means ± SE. Page 6 of 10 (page number not for citation purposes) Tumor growth (% of initial volume) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 Table 2: Hyperoxia-induced changes in expression of secretory proteins in DMBA-induced mammary tumors in rats Gene Name Gene symbol Change in expression level RefSeq Probe ID Array Q-PCR Parotid secretory protein Psp 3.0E-02 1.0E-04 NM_052808 A_43_P12746 Prolactin induced protein Pip 5.1E-02 6.0E-04 NM_022708 A_43_P12308 Cysteine-rich secretory protein 1 Crisp1 4.6E-02 ND NM_022859 A_42_P457387 Common salivary protein 1 Csp1 5.8E-02 7.1E-02 NM_133622 A_42_P562492 Proline-rich proteoglycan 2 Prpg2 2.2E-01 ND NM_172065 A_42_P765648 a) The fold change in expression level was determined as the ratio between HBO-treated- and control-tumors. Values correspond to fold decrease, i.e. down-regulation of expression. The data are means of ratios obtained from four HBO-treated tumors compared to a pool of control tumors. ND: Not determined. Hyperoxia changes the morphology of the tumor by iogenesis. Normally, hyperoxic treatment is known to inducing loss of glands enhance angiogenesis in wound healing, in necrotic ulcers In addition to the tumor growth retardation, the mor- and after radiation injury [10,11]. Surprisingly, the phology of heamatoxylin-eosin stained tumor tissue present study demonstrates that hyperoxia reduce the showed significant changes after hyperoxic treatment. mean vessel density in mammary tumors. This indicates Most pronounced were the empty spaces in the tumor tis- that hyperoxia induce an anti-angiogenic effect in tumor sue, which suggests a loss of glands in the mammary tissue tissue which is opposite to what is expected in "normal" as indicated by a strong turn-off of glandular secretory tissues. Another, surprising finding was the increase in proteins (parotid secretory protein, prolactin induced vascular diameter both in the periphery and central parts protein and common salivary protein-1) (Table 2). One of the tumor, since HBO is generally know to induce vaso- of the glandular secretory proteins, prolactin, stimulates constriction. This vasodilatory effect might indicate that tumor growth and motility of human breast cancer cells the tumor is struggling to elevate its flow to compensate and a recent review suggested that antagonizing prolactin for the loss of blood vessels and thereby prevent starva- should be evaluated as a tumor treatment [19]. It is there- tion. fore very surprising that hyperoxic treatment alone could down-regulate the prolactin induced protein and therby Many tumor models have demonstrated a close correla- most likely also prolactin. tion between local oxygen deficiency and the production of HIF-1α and VEGF [21,22]. HIF-1α activity as well as Hyperoxia has an anti-angiogenic effect VEGF secretion is tightly regulated by the oxygen-level, Hypoxia induce the formation of new tumor blood ves- and in the well oxygenated state they are rapidly degraded sels necessary for further tumor growth [20]. One might [24]. Thus by increasing the pO in tumor tissue with nor- therefore expect that hyperoxia should prevent tumor ang- mobaric and hyperbaric oxygen it is reasonable to assume Table 3: Area fraction of vacuoles, number of apoptotic cells, mean vascular density, vascular diameter and ratio central/peripheral vessel density and vessel diameter in controls and in tumors treated with hyperoxia. Values are given as means ± SD. Control 1 bar, 21% O Test 1 bar, 100% O Test 1.5 bar, 100% O 2 2 2 pO = 0.21 pO = 1.0 pO = 1.5 2 2 2 Area fraction vacuoles "empty spaces" 0.01 ± 0.04 0.11 ± 0.05 *** 0.21 ± 0.02 *** Apoptotic cells (% of total cells) 61% ± 6.4 85% ± 7.3 *** 88% ± 5.0 *** Mean vascular density (number of vessels/mm ) Centrally 98 ± 18.6 50.1 ± 17.4*** 69.8 ± 18.5* Periphery 79.5 ± 19.2 38.5 ± 19.3*** 51.8 ± 26.8* Vascular diameter (µm) Centrally 19.1 ± 4.9 23.1 ± 9.1 ** 23.2 ± 10.1** Periphery 10.9 ± 3.9†† 12.5 ± 4.7 *†† 23.9 ± 10.0 *** Ratio central/peripheral vessel density 1.2 ± 0.7 1.6 ± 1.3 1.5 ± 1.1* Ratio central/peripheral vessel diameter 1.9 ± 0.8 2.0 ± 1.2 1.1 ± 0.6*** *P < 0.05, **P < 0.01, ***P < 0.001 vs control (1 bar, air) ††P < 0.01 vs centrally Page 7 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 tr E Figure 2 xol (left) and during hypero amples of eosin-hematoxylin xic treatment (rig stained tumor-tissu ht, 1 bar, pO e of the cen = 1.0) tral (A) and peripheral (B) part of the mammary tumor in con- Examples of eosin-hematoxylin stained tumor-tissue of the central (A) and peripheral (B) part of the mammary tumor in con- trol (left) and during hyperoxic treatment (right, 1 bar, pO = 1.0). The images under A are scaled to the same magnification (× 4) and the images under B to the same magnification (× 10). Scale bar indicate 500 µm (A) and 100 µm (B). Page 8 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 When level of free oxygen radical production exceeds Control endogenous cellular antioxidant capacity it can create an "oxidative stress" which can lead to cell death [30]. Free oxygen radicals have previously been shown to induce apoptosis and also inhibit angiogenesis [31]. In the litera- 160 ture it seems as if ROS has "two faces". It appears as 1.5 bar though the action of free radicals on normal and tumor cells are opposite. When free radicals attack normal cells, DNA damage can occur, whereas ROS in tumor cells has 1 bar an unexpected, but highly beneficial action, namely inhi- bition of tumor cells [review:31]. Since the supoxide dis- mutase (SOD) activity in most tumors has been shown to be lower than in normal tissue, this might make the tumors even more susceptible to increased ROS activity during hyperoxic exposure. So, a moderate increase in Tumor volume (mm ) ROS activity by HBO might be beneficial in tumors. Th m Figure 3 m e relationship between tumo 2 before and after hyperoxic treatment r size and blood vessels per Conclusion The relationship between tumor size and blood vessels per The present study shows that both normobaric (1 bar, mm2 before and after hyperoxic treatment. Values repre- pO = 1.0) and hyperbaric hyperoxia (1.5 bar, pO = 1.5) sents mean ± SE. 2 2 significantly retards mammary tumor growth in rats. Hyperoxic treatment did affect the mammary tumors by inducing loss of glands, reducing vascular density and enhancing apoptosis. The mechanisms behind these changes are still not known. Hyperbaric oxygen treatment that VEGF and HIF-1α would have been down-regulated. should therefore be further evaluated as adjuvant tumor The anti-angiogenic effect found in the present study treatment. would correspond to this hypothesis. Competing interests Hyperoxia induces apoptosis The author(s) declare that they have no competing inter- We found the amount of normal cells (cells with intact ests. nucleus) in the tumor to be decreased after hyperoxic treatment. It is therefore important to know that hyper- Authors' contributions oxic treatment in the present range (1 -1.5 bar, pO = 1– AR carried out the morphology, immunoassay (von wille- 1.5) and time exposed (90 min) have never been shown brandt and Tunnel staining), statistical analysis and to induce cell death of other tissues of the body, including hyperbaric exposures. lungs which are known to be most susceptible to HBO (UHMS) [25]. A review by O'Reilly stated that cell prolif- CS and VMS carried out the gene expression profiling and eration (of normal tissue) in adult rats, mice and monkeys PCR. exposed to lethal levels of oxygen (>90%) was unaffected for the first 48, and alveolar epithelium relative unaffected RB and RKR participated in the design and helped draft until after 72 hours [26]. However, a recent report [27] on the manuscript. in vitro benign and malignant mammary epithelial cells showed that HBO (97.9% oxygen) at a high pressure (2.4 LEBS conceived the study, supervised the hyperbaric expo- bar) inhibited epithelial cell proliferation. Furthermore, sures and wrote the manuscript. an organised, genetically programmed cell death was found in the tumor tissue after hyperoxic treatment as All authors read and approved the final manuscript. indicated by elevated amount of tunnel-positive apop- totic cells. Acknowledgements The present study has been supported by the Norwegian Microarray Con- sortium (NMC), a national FUGE technology platform (Functional Genom- Potential mechanism? ics in Norway), the Norwegian Cancer Society and the Norwegian We might speculate that all or at least some of the changes Research Council. we have mentioned after both normobaric and hyperbaric hyperoxia are due to free oxygen radicals (ROS). ROS activity is known to be elevated during hyperoxia [28,29]. Page 9 of 10 (page number not for citation purposes) MVD (vessels/mm ) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 25. Clark JM: Oxygen toxicity. In The Physiology and Medicine of Diving References 4th edition. Edited by: Bennet P, Elliot D. WB Saunders Company Ltd; 1. Vaupel P, Kallinowski F, Okunieff P: Blood flow, oxygen and nutri- 1993:121-169. ent supply, and metabolic microenvironment of human 26. O'Reilly MA: DNA damage and cell cycle checkpoints in tumors: a review. Cancer Res 1989, 49:6449-65. Review hyperoxic lung injury: braking to facilitate repair. Am J Physiol 2. Höckel M, Vaupel P: Tumor hypoxia: definitions and current Lung Cell Mol Physiol 2001, 281:291-305. clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001, 27. Granowitz EV, Tonomura N, Benson RM, Katz DM, Band V, Makari- 3:266-76. Review Judson GP, Osborne BA: Hyperbaric oxygen inhibits benign and 3. Brizel DM, Lin S, Johnson JL, Brooks J, Dewhirst MW, Piantadosi CA: malignant human mammary epithelial cell proliferation. The mechanisms by which hyperbaric oxygen and carbogen Anticancer Res 2005, 25:3833-42. improve tumor oxygenation. Br J Cancer 1995, 72:1120-1124. 28. Yamaguchi KT, Stewart RJ, Wang HM, Hudson SE, Vierra M, Akhtar 4. Teicher BA: Hypoxia and drug resistance. Cancer Metastasis Rev A, Hoffman C, George D: Measurements of Free Radicals from 1994, 13:139-68. Review smoke inhalation and oxygen exposure by spin trapping and 5. Semenza GL: HIF-1 and human disease: one highly involved ESR spectroscopy. Free Radic 1992, 16:167-174. factor. Genes Dev 2000, 14:1983-91. Review 29. Narkowicz CH, Vial JH, McCartney P: Hyperbaric oxygen therapy 6. Helczynska K, Kronblad A, Jogi A, Nilsson E, Beckman S, Landberg G, increases free radical levels in the blood of humans. Free Rad- Pahlman S: Hypoxia promotes a dedifferentiated phenotype in ical Res Comm 1993, 19:71-80. ductal breast carcinoma in situ. Cancer Res 2003, 1:1441-4. 30. Halliwell B, Gutteridge JM: Role of free radicals and catalytic 7. Beppu T, Kamada K, Yoshida Y, Arai H, Ogasawara K, Ogawa A: metal ions in human disease: an overview. Methods Enzymol Change of oxygen pressure in glioblastoma tissue under var- 1990, 186:1-85. ious conditions. J Neurooncol 2002, 58:47-52. 31. Das UN: A radiacl approach to cancer. Med Sci Monit 2002, 8. Becker A, Kuhnt T, Liedtke H, Krivokuca A, Bloching M, Dunst J: 8:79-92. Oxygenation measurements in head and neck cancers dur- ing hyperbaric oxygenation. Strahlenther Onkol 2002, 178:105-8. 9. Kinoshita Y, Kohshi K, Kunugita N, Tosakki T, Yokota A: Preserva- Pre-publication history tion of tumor oxygen after hyperbaric oxygenation moni- The pre-publication history for this paper can be accessed tored by magnetic resonance imagin. Br J Cancer 2000, here: 82:88-92. 10. Al-Waili NS, Butler GJ, Beale J, Hamilton RW, Lee BY, Lucas P: Hyperbaric oxygen and malignancies: a potential role in radi- http://www.biomedcentral.com/1471-2407/7/23/prepub otherapy, chemotherapy, tumor surgery and phototherapy. Med Sci Monit 2005, 11:279-89. Epub 2005 Aug 26 11. Feldmeier JJ, Hampson NB: A systemic review of the literature reporting the application of hyperbaric oxygen prevention and treatment of delayed radiation injury:an evidence-based approach. Undersa Hyperbar Med 2002, 29:4-30. 12. Gill AL, Bell CAN: Hyperbaric oxygen: its uses, mechanisms of action and outcomes. Q J Med 2004, 97:385-395. 13. Feldmeier J, Carl U, Hartmann K, Sminia P: Hyperbaric oxygen: does it promote growth or recurrence of malignancy? Under- sea Hyperb Med 2003, 30:1-18. 14. Stuhr LE, Iversen VV, Straume O, Maehle BO, Reed RK: Hyperbaric oxygen alone or combined with 5-FU attenuates growth of DMBA-induced rat mammary tumors. Cancer Lett 2004, 210:35-40. 15. Dysvik B, Jonassen I: J-Express: exploring gene expression data usimg Java. Bioinformatics 2001, 17:369-70. 16. Tusher VG, Tibshirani R, Chu G: Significance analysis of micro- arrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001, 98:5116-21. 17. Margaretten NC, Witschi H: Effects of hyperoxia on growth characteristics of metastatic murine tumors in the lung. Can- cer Res 1988, 48:2779-83. 18. Lindenschmidt RC, Margaretten N, Griesemer RA, Witschi HP: Mod- ification of lung tumor growth by hyperoxia. Carcinogenesis 1986, 7:1581-6. 19. Clevenger CV, Furth PA, Hankinson SA, Schuler LA: The role of prolactin in mammary carcinoma. Endocrine Reviews 2003, 24:1-27. 20. Folkman J: Clinical application of research on angiogenesis. N Engl J Med 1995, 333:1757-1763. Publish with Bio Med Central and every 21. Thews O, Wolloscheck T, Dillenburg W, Kraus S, Kelleher DK, Kon- erding MA, Vaupel P: Microenvironmental adaptation of exper- scientist can read your work free of charge imental tumors to chronic vs acute hypoxia. Br J cancer 2004, "BioMed Central will be the most significant development for 13:1181-9. 22. Vaupel P: The role of hypoxia-induced factors in tumor pro- disseminating the results of biomedical researc h in our lifetime." gression. Oncologist 2004, 9:10-17. Sir Paul Nurse, Cancer Research UK 23. Maxwell PH, Dachs GU, Gleadle JM, Nicholls LG, Harris AL, Stratford IJ, Hankinson O, Pugh CW, Ratcliffe PJ: Hypoxia-inducible factor- Your research papers will be: 1 modulates gene expression in solid tumors and influences available free of charge to the entire biomedical community both angiogenesis and tumor growth. Proc Natl Acad Sci 1997, peer reviewed and published immediately upon acceptance 94:8104-9. 24. Marxsen JH, Schmitt O, Metzen E, Jelkmann W, Hellwig-Burgel T: cited in PubMed and archived on PubMed Central Vascular endothelial growth factor gene expression in the yours — you keep the copyright human breast cancer cell line MX-1 is controlled by the O2 availability in vitro and in vivo. Ann Anat 2001, 183:243-249. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 10 of 10 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Hyperoxia retards growth and induces apoptosis and loss of glands and blood vessels in DMBA-induced rat mammary tumors

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Publisher
Springer Journals
Copyright
Copyright © 2007 by Raa et al; licensee BioMed Central Ltd.
Subject
Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
ISSN
1471-2407
eISSN
1471-2407
DOI
10.1186/1471-2407-7-23
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17263869
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Abstract

Background: This study investigated the effects of hyperoxic treatment on growth, angiogenesis, apoptosis, general morphology and gene expression in DMBA-induced rat mammary tumors. Methods: One group of animals was exposed to normobaric hyperoxia (1 bar, pO = 1.0 bar) and another group was exposed to hyperbaric hyperoxia (1.5 bar, pO = 1.5 bar). A third group was treated with the commonly used chemotherapeutic drug 5- Fluorouracil (5-FU), whereas animals housed under normal atmosphere (1 bar, pO = 0.2 bar) served as controls. All treatments were performed on day 1, 4, 7 and 10 for 90 min. Tumor growth was calculated from caliper measurements. Biological effects of the treatment, was determined by assessment of vascular morphology (immunostaining for von Willebrandt factor) and apoptosis (TUNEL staining). Detailed gene expression profiles were obtained and verified by quantitative rtPCR. Results: Tumor growth was significantly reduced (~57–66 %) after hyperoxic treatment compared to control and even more than 5-FU (~36 %). Light microscopic observations of the tumor tissue showed large empty spaces within the tissue after hyperoxic treatment, probably due to loss of glands as indicated by a strong down-regulation of glandular secretory proteins. A significant reduction in mean vascular density (30–50%) was found after hyperoxic treatment. Furthermore, increased apoptosis (18–21%) was found after hyperoxic treatment. Conclusion: Thus, by increasing the pO in mammary tumor tissue using normobaric and moderate hyperbaric oxygen therapy, a significant retardation in tumor growth is achieved, by loss of glands, reduction in vascular density and enhanced cell death. Hyperbaric oxygen should therefore be further evaluated as a tumor treatment. Page 1 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 Background Due to the apparent link between blood supply, oxygena- Growth of solid tumors depends on adequate supply of tion and tissue growth, it has for a long time been a mis- oxygen and nutrients. There are, however, marked differ- conception that HBO per se could have a tumor- ences in the vascular network in the different regions of promoting effect. There are now several lines of evidence the tumor. In the centre, there is typically a hypoxic milieu showing that this is not the case [13]. In a rat model of due to structural and functional vessel disturbances (per- dimethyl-α-benz-anthracene (DMBA)-induced mammary fusion- and diffusion-limited O delivery), while in the adenocarcinomas, we have recently demonstrated a sig- periphery there is generally a denser vascular network nificant decrease in mammary tumor size after repeated with subsequent improved blood flow. While normal tis- HBO treatment at 2 bar for 90 minutes [14]. These para- sue can compensate for such an O deficiency by raising doxical data indicate that an increase in the delivery of the blood flow, large tumor areas cannot adequately physically dissolved O in the tumor tissue by hyperbaric counteract the restriction in O supply and therefore hyperoxic treatment may suppress its growth. develop hypoxia. Thus, the HbO saturation is signifi- cantly lower in tumors than in normal surrounding tissue, The present study was initiated to see if 1.5 bar (pO2 = with a gradual reciprocal decrease as the tumor increases 1.5) as well as pure oxygen at normal atmospheric pres- in size [1,2]. sure (1 bar, pO = 1.0) would have a similar effect. The aim of the present study was therefore to find the least It is now widely accepted that hypoxia promotes tumor pressure gradient that gave a therapeutic effect on tumor growth, angiogenesis and reduce the effect of chemo- and growth. Therapeutic efficacy was determined by assessing radiation- therapy [3-6]. We might therefore expect that tumor growth, angiogenesis, apoptosis, general morphol- an increase in the oxygen-content in tumor tissue might ogy and gene expression profiling. have the opposite effect. Methods Hyperbaric oxygen treatment (HBO) offers one possibility Animal model of mammary tumors to increase the oxygen content in various tissues [7-10]. Female Sprague-Dawley (Møllegård, Denmark) rats (n = The use of HBO in cancer therapy has been aimed at 35) were used. Mammary tumors (adenocarcinomas) improving the radiation response in solid tumors [10] as were induced by dimethyl-α-benz-anthracene (DMBA) well as to improve healing of normal tissue after radiation dissolved in olive oil and given to the 7 week rat by gavage injury [11]. The increase in tissue pO during and after at a dose of 16 mg. The experiments were performed when HBO treatment is due to enhanced transport of soluble the rats were 13–15 weeks old, having reached a body oxygen. The physically solved oxygen at normobaric air weight of 250–300 g and developed tumors along the pressure is approximately 0.3 ml O /l00 ml blood, with a mammary crest. Tumor size was measured externally by corresponding HbO of approximately 21 ml/100 ml calipers on day 1, 4, 7, and 11 and estimated according to blood. By breathing 100% oxygen at normobaric pres- the following formula: π/6 · (a) (b), where a is the short- sure, the amount of physically soluble oxygen increases 6 est transversal diameter and b is the longest transversal times (1.8 ml O /100 ml blood). If the atmospheric pres- diameter. The examinations were performed by the same sure is elevated to 3 bar in the presence of 100% O , the person, with no knowledge about the various exposures of amount of oxygen delivered to the tissue would increase the rats. All measurements were performed during isoflu- to 6.0 ml O /100 ml blood, which is even sufficient to ran (Shering-Plough AS, Narum, Denmark) and N2O support resting tissue independent of the O contribution anesthesia (Ohmeda: BOC Health Care, Weast-Yorkshire, from hemoglobin [12]. When oxygen is in solution, it can England). The study was approved by the Norwegian reach physiologically obstructed areas that are inaccessi- Committee for Animal Research (Oslo, Norway). ble to the HbO -containing red blood cells. In line with this, several investigators have measured a significant Experimental groups and treatment design delay in washout (15–60 min) of the pO in different Four separate groups of rats were studied (for details see tumors after HBO treatment [3,7-9]. Table 1). Table 1: The experimental groups. Gas Total pressure (bar) pO2 (bar) Drug No. of rats Group 1 Air 1.0 0.2 NaCl 10 Group 2 Air 1.0 0.2 5-FU 10 Group 3 Oxygen 1.0 1.0 NaCl 8 Group 4 Oxygen 1.5 1.5 NaCl 8 The hyperbaric oxygen treatement and 5-FU (0.2 mg/kg i.p.) were given on day 1, 4, 7 and 10. Page 2 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 Hyperbaric chamber and oxygen exposure procedure pH6.0) and then microwaved for 5 min. The slides were 130 l hyperbaric chamber with an internal diameter of 50 washed in PBS before terminal deoxynucleotidyl trans- cm was used. For inspection and video supervision there ferase was applied to each slide and incubated for 1 hr at are two windows in the chamber wall. Penetrators for gas 37°C in a humidified chamber. A stop/washing buffer inlet and outlet run through the chamber's end wall. The was used before applying the converter POD (anti-fluores- chamber and rat cage were cleaned and degreased (96– cein antibody conjugated with peroxidase as reporter 100% ethanol) before starting exposure with pure oxygen. enzyme) for 30 min. Diaminobenzidine (DAB) was used All electrical installations were disconnected. The rats as a chromogen and the slides were counterstained with were placed in litter-free cages (590 × 385 × 200 mm) dur- hematoxylin. The density of apoptotic cells per mm of ing HBO exposure. All rats were showered slightly with the tumor viable zone was determined using a counter water prior to entering the pressure chamber, due to the grid (10 random vision fields × 40). The percentage of danger of fire in a pure oxygen atmosphere. Four petrid- apoptotic cells is expressed as a percentage of total cells. ishes with water were placed on the chamber floor to additionally humidify the atmosphere. To initiate the Von Willebrand factor For detection of vascular endothelial cells we stained for treatment, 100% oxygen was introduced into the chamber as air was simultaneously flushed out. The oxygen concen- Von Willebrand factor. The rabbit anti-human polyclonal tration in the chamber was monitored continuously by an antibody against Von Willebrand factor (DAKO) was oxygen cell (C3, Middelsborough, England). When oxy- diluted in TrisBSA to a 1:250 and than to a 1:500 dilution. gen was above 98%, the rats of group 3 were kept at this The tissue sections were incubated for 45 min and 75 min level for 90 min, while in group 4 the chamber was pres- respectively with the primary antibody, washed three surized with oxygen over approximately 2 min to 1.5 bar, times for 5 min in TBS and further incubated with anti- and this pressure was maintained throughout 90 min. rabbit IgG (DAKO Cytomation Envision Kit, Labelled Pol- During the 90 min period, the chamber was flushed twice ymer – HRP Anti Rabbit) for 35 min. All incubations were for 5 min (at 30 and 60 min) with pure oxygen in both performed in a humidity chamber. The tissue was then series. The temperature was held at approximately 22°C washed three times with PBS. Detection was carried out and the humidity at approximately 100%. The CO con- using a DAB chromogen, which resulted in a positive tent was kept low by use of a scrubber material (Sodasorb, brown staining. Harris Haematoxylin (Merck, Damstadt, Molecular Products United Drug Co Ltd, Essex, UK) Germany) was used for nuclear counterstaining. Negative placed inside the chamber floor. control slides were obtained by omitting the primary anti- body. Quantification of the microvessels was performed Morphological analyses in 10 consecutive fields in both the center and in the The animals were sacrificed with pentobarbital during iso- periphery and averaged as vessels/mm . Approximately fluran and N2O anesthesia, and the tumors were dissected 50 blood vessels in both the periphery and central part of out (one tumor from each rat). One part of the tumor was the tumor were randomly selected for diameter measure- fixed in 4% buffered formalin, processed and embedded ments. in paraffin. Tissue sections of each specimen were stained using Harris Haematoxylin and Eosin (H & E, Merck, All the specimens were examined in a Nikon light micro- Damstadt, Germany). The sections were analyzed with scopy (THP Eclipse E600, Nikon Corporation, Tokyo, regards to apoptosis (TUNEL assay) and blood vessel den- Japan) and the images captured with a Nikon Digital sity (von Willebrand factor). Indirect immunohistochem- Camera (DXM 1200F, Nikon Corporation, Tokyo, Japan). istry was performed by the EnVisison™ + System, horse- The image processing and analysis system LUCIA, version radish peroxidase and 3'3'-diaminobenzidine (DAB) 4.8 (Laboratory Imaging Ltd, Prague, The Check Republic) (DAKO, Glostrup, Denmark) method, as described in the was used. manufacturer's protocol. RNA extraction and microarray-based expression analysis TUNEL staining Specimens of tumor tissue (approximately 2 × 1 × 0.3 cm) Apoptosis was examined by the terminal transferase- were immersed in ice cold RNA-later solution (Ambion, mediated dUTP nick end-labeling (TUNEL) method (Boe- Foster City, CA), and kept at 4°C over night. On the fol- hringer Mannheim, Mannheim, Germany), performed lowing day, the tissue tubes were frozen at -20°C until according to the manufacturers recommendations. The RNA isolation. The tumor tissue samples were homoge- DNA strand breaks in apoptotic cells are labelled by nised by a Kinematica Polytron homogenizer. Total RNA attaching biotin-or digoxygenin conjugated dUTP in a was extracted using the QIAGEN RNAeasy Lipid kit, and reaction catalyzed by exogenous terminal deoxynuclepti- the amount and quality of the extracted RNA was meas- dyl transferase (TdT-assay) or DNA polymerase. For anti- ured by the NanoDrop ND-1000 Spectrophotometer and gen retrieval the slides were put into citrate buffer (0.1 M, Page 3 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 the Agilent 2100 Bioanalyzer. Purified total RNA was Identification of genes with significant differential expres- stored at -80°C until use. sion in hyperoxic exposed tumors was carried out using 'significance analysis of microarrays' (SAM) (16). The nor- Twenty microgram total RNA was reversely transcribed malized dataset from J-Express was imported into the and fluorescently labelled using the Agilent Fluorescent TM4 Microarray Software Suite Multi Experiment Viewer Direct Label Kit (G2557A), according to the manufac- 3.1 (MeV) (TIGR, US). Signal intensities in all HBO turer's instructions. To screen for gene expression changes treated 'test' samples were compared to those of the in mammary tumors as a result of hyperoxic exposure, untreated 'control' samples. The analysis threshold was set competitive hybridisations of samples from the treated to a highly conservative false discovery rate of zero. tumors (Group 3, 100% O , 1 bar) against the untreated Real-Time PCR Analysis tumors (normal air, 1 bar) were performed, where four hyperoxia-treated tumor samples were each compared to Total RNA was extracted, quality controlled and stored as a pool of ten control tumors. Test samples were labelled described above. From each of the samples, 50 ng RNA with Cy5 and control samples labelled with Cy3. After the was reverse transcribed to cDNA using TaqMan RT rea- labelling reactions, the dye-incorporation ratio was deter- gents (Applied Biosystems, Foster City, CA). The final con- mined using a spectrophotometer, showing ratios within centrations of the reagents were as follows: 1 × TaqMan RT 10 to 20 pmol per µg cDNA. The amount of the Cy- buffer, 5.5 mM MgCl , 2 mM dNTP mixture, 2.5 mM ran- labelled cDNA was checked by measuring its optical den- dom hexamers, 0.4 U/µl RNase inhibitor and 1.25 U/µl sity in the NanoDrop ND-1000 Spectrophotometer, at Multiscribe reverse transcriptase in RNase-free water to a OD 260/280 nm for DNA purity and at OD 550 nm and total volume of 50 µl. The reaction mix was incubated at 650 nm for dye incorporation. The amount of cDNA out- 25°C for 10 min (primer annealing), 48°C for 30 min put was 1–2 µg and the dye incorporation was about 5% (synthesis) and 95°C for 5 min (enzyme inactivation). for Cy3 and 3% for Cy5. The resulting cDNA samples were stored at -20°C. All sub- sequent real-time PCR experiments were performed on an All hybridisations were carried out on Agilent 22 k Rat ABI Prism 7900 HT sequence detector system using 384- Oligo Microarrays (G4130A), with 22,575 60-mer probes well plates. The PCR reaction solution for parotid secretory representing over 20,000 rat genes, ESTs and EST clusters. protein (Psp), prolactin induced protein (Pip) and common For hybridisation, the Agilent 60-mer oligo microarray salivary protein 1 (Csp1) contained 1 µl 20 × TaqMan Gene processing protocol (V 4.1, SureHyb 22 k chamber/SSC Expression Assays (Applied Biosystems) and 10 µl 2 × wash) was strictly followed. Briefly, the entire labelling TaqMan Universal PCR Master Mix. The acidic ribosomal reactions for Cy3-labelled control samples and Cy5- protein P0 (P0) reaction solution contained 10 µl 2 × labelled test samples were combined and applied to the SYBR green (Medprobe), together with forward and microarray together with control targets and hybridisation reverse primers (Sigma-Aldrich) in a final concentration buffers provided by the Agilent in situ hybridization plus of 300 nM. All PCR reactions contained 4 µl of cDNA reac- kit (5184–3568). The microarrays were then incubated in tion mix and RNasefree water to a total volume of 20 µl. an Agilent hybridisation oven (G2545A) with rotation for The real-time PCR was run as follows: 50°C for 2 min 17 hours at 60°C. On the following day, microarrays were (UNG incubation) and 95°C for 10 min (AmpliTaq Gold washed and immediately dried using an ultra pure filtered activation), followed by 40 cycles of 95°C for 10 s and stream. All operations were in accordance with the N 60°C for 1 min. For each sample, the gene expression was manufacturer's instructions. quantified by the standard curve method and normalized against the expression of the ribosomal protein P0 gene. After drying, the slides were scanned for fluorescence sig- The standard curve consisted of five points that were nals by the Agilent G2565AA Microarray Scanner with a obtained by a two-fold serial dilution of control RNA, pixel size of 10 nm. The TIFF images generated by the starting out at 250 ng, together with a 'non-template' con- scanning process were imported into GenePixPro 5.0 soft- trol; all performed in triplicate. P0 was preferred as the ware (Axon Instruments Inc) for primary data analysis. endogenous control over GAPDH and b-actin, which Control spots and spot artifacts were flagged in GenePix both gave qualitatively similar results in pilot experiments for subsequent filtering and background-corrected signal (data not shown). intensity values were imported into the J-Express Pro V2.6 software (MolMine, Bergen, Norway)[15]. Flagged spots Statistics were filtered and the red versus green signal intensities of Results were expressed as means ± SD unless indicated each spot were adjusted by global normalization to reduce otherwise. Differences between groups were assessed by the impact of dye bias in the data sets. unpaired, two-tailed Student's t-test or the Mann-Whitney U test. P < 0.05 was considered significant. Page 4 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 spot" areas (areas of high vascular density) in both the Results Tumor growth tumor centre and periphery were examined. The mean A total of 36 tumors were studied (one from each rat). The vascular density was markedly reduced in the tumors tumor size ranged from 1.2 to 2.3 cm prior to the start of treated with hyperoxia compared to controls (Table 3). the experiments (Day1). The marked increase in tumor The ratio of central/peripheral vessel density is given in size found in controls (group 1, n = 10) over a period of Table 3. The diameter of the remaining tumor vessels was, 11 days (p < 0.001), was reduced by administering 5-FU however, increased after hyperoxic treatment compared to to the animals (group 2, n = 10, p < 0.02). A further reduc- control, as can be seen in Table 3. The results also show tion in tumor size was observed at day 11 after both 1.0 that the diameter of the peripheral vessels is larger than in bar (group 3, n = 8) and 1.5 bar (group 4, n = 8) hyperoxia the central parts in both control and at 1 bar hyperoxic treatment as compared to controls (p < 0.01) and 5-FU treatment. The ratio of central/peripheral vessel diameter treated rats (p < 0.05). Our previous study at 2 bar, 100% is given in Table 3. The relationship between tumor vol- O significantly attenuated tumor growth in the same ume and mean vessel density is visualized in Fig 3. tumor model (included in Fig. 1) [14]. Taken together Tumor cells these results indicate a reduction in growth that is dose- dependent on pO . The percentage of apoptotic cells in TUNEL-stained tissue section was significantly increased (18–21%) after hyper- Down-regulation of glandular secretory proteins oxic treatment compared to controls as shown in Table 3. Since these results confirmed our previous findings that hyperoxia might attenuate the growth of rat mammary Discussion tumors, we decided to perform a global gene expression The present study shows that exposing rats to normobaric profiling to search for differentially expressed genes and (1 bar) or hyperbaric (1.5 bar) hyperoxia (100% O ), four their biological function. By comparison of global gene times for 90 minutes, over a period of 10 days signifi- expression level in 5 hyperoxia treated tumors with a pool cantly retards tumor growth. In the present model this of 10 control tumors, we notably observed a small cluster treatment showed more efficacy than the commonly used (n = 5) of genes involved in glandular secretion that were chemotherapeutic drug 5-FU. Hyperoxic treatment did all strongly down-regulated by the exposure of the tumors affect the mammary tumors by significantly down-regu- to 100% O (Table 2). These proteins included parotid lating glandular genes that may have implications in secretory protein (Psp), common salivary protein 1 (Csp1) and tumor growth (loss of glands), reducing vascular density prolactin induced gene (Pip), cystein-rich secretory protein and enhancing apoptosis. (crisp-1) and proline rich proteoglycan 2 (Prpg2). All these genes displayed moderate to high expression levels in the HBO is not a tumor promoter control tumors, but after a total of 6 hours of hyperoxic HBO has been used in combination with both chemo- treatment their expression was dramatically diminished, therapy and radiotherapy to enhance the pO in the oth- with a down-regulation ranging from 5–33 times in the erwise hypoxic tumor tissue and thereby potentiate the individual tumors. To verify the microarray results, we effect of these treatments [10]. HBO has also been used for used quantitative real- time PCR, which confirmed that wound healing, treatment of necrotic tissues and recovery the expression of Psp, Pip and Csp1 was markedly dimin- of radiation-injured tissues because of its ability to pro- ished in the hyperoxia-exposed tumors (Table 2). mote angiogenesis and thereby enhance blood supply to the injured area [10,11]. However, it has for a long time General morphology been a misconception that HBO per se could have a tumor The gene expression data indicated that HBO treatment promoting effect. There are now several lines of evidence was associated with attenuation of glandular function in showing that this is not the case [13]. The present work the mammary tumor. Subsequently the E & H stained support previous conclusions from our group demon- tumor tissue sections, clearly demonstrates that normo- strating that HBO has a significant inhibitory effect on baric and as well as hyperbaric hyperoxia treatment mammary tumor growth in rats [14]. Also two studies on induced large areas of empty space ("vacuoles") both in mice injected with various tumor cell lines exposed to the central parts (Fig 2A) and in the periphery (Fig 2B) of 70% O for 3 weeks showed a reduced number of lung the tumor tissue compared to control (Table 3), thereby colonies derived from mammary carcinoma MT-7 cells indicating a loss of glandular tissue. The morphology of 1 and of lung-tumor cell lines [17,18]. Some cell lines have, and 1.5 bar sections were identical. however been shown to be oxygen resistant [17], which indicates differences in the oxygen sensitivity in different Tumor vessels tumor-types. In order to analyse the impact of hyperoxia on the induc- tion of vessel formation, the vascular densities in "hot Page 5 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 ** *** *** *** 24 68 10 12 Days Control (21% O ) 5-FU 1 bar (100% O ) 1.5 bar (100% O ) 2 bar (100% O ) + 5-FU (Stuhr et al 2004) 2 bar (100% O ) (Stuhr et al 2004) * p < 0.05, ** p< 0.02 , *** p< 0.01 vs control # p < 0.05 vs 5-FU Th Figure 1 e effect of normoxic and hyperoxic treatment on tumor growth compared to controls and 5-FU treated The effect of normoxic and hyperoxic treatment on tumor growth compared to controls and 5-FU treated. Treatments were given on day 1, 4, 7 and 10. Values represent means ± SE. Page 6 of 10 (page number not for citation purposes) Tumor growth (% of initial volume) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 Table 2: Hyperoxia-induced changes in expression of secretory proteins in DMBA-induced mammary tumors in rats Gene Name Gene symbol Change in expression level RefSeq Probe ID Array Q-PCR Parotid secretory protein Psp 3.0E-02 1.0E-04 NM_052808 A_43_P12746 Prolactin induced protein Pip 5.1E-02 6.0E-04 NM_022708 A_43_P12308 Cysteine-rich secretory protein 1 Crisp1 4.6E-02 ND NM_022859 A_42_P457387 Common salivary protein 1 Csp1 5.8E-02 7.1E-02 NM_133622 A_42_P562492 Proline-rich proteoglycan 2 Prpg2 2.2E-01 ND NM_172065 A_42_P765648 a) The fold change in expression level was determined as the ratio between HBO-treated- and control-tumors. Values correspond to fold decrease, i.e. down-regulation of expression. The data are means of ratios obtained from four HBO-treated tumors compared to a pool of control tumors. ND: Not determined. Hyperoxia changes the morphology of the tumor by iogenesis. Normally, hyperoxic treatment is known to inducing loss of glands enhance angiogenesis in wound healing, in necrotic ulcers In addition to the tumor growth retardation, the mor- and after radiation injury [10,11]. Surprisingly, the phology of heamatoxylin-eosin stained tumor tissue present study demonstrates that hyperoxia reduce the showed significant changes after hyperoxic treatment. mean vessel density in mammary tumors. This indicates Most pronounced were the empty spaces in the tumor tis- that hyperoxia induce an anti-angiogenic effect in tumor sue, which suggests a loss of glands in the mammary tissue tissue which is opposite to what is expected in "normal" as indicated by a strong turn-off of glandular secretory tissues. Another, surprising finding was the increase in proteins (parotid secretory protein, prolactin induced vascular diameter both in the periphery and central parts protein and common salivary protein-1) (Table 2). One of the tumor, since HBO is generally know to induce vaso- of the glandular secretory proteins, prolactin, stimulates constriction. This vasodilatory effect might indicate that tumor growth and motility of human breast cancer cells the tumor is struggling to elevate its flow to compensate and a recent review suggested that antagonizing prolactin for the loss of blood vessels and thereby prevent starva- should be evaluated as a tumor treatment [19]. It is there- tion. fore very surprising that hyperoxic treatment alone could down-regulate the prolactin induced protein and therby Many tumor models have demonstrated a close correla- most likely also prolactin. tion between local oxygen deficiency and the production of HIF-1α and VEGF [21,22]. HIF-1α activity as well as Hyperoxia has an anti-angiogenic effect VEGF secretion is tightly regulated by the oxygen-level, Hypoxia induce the formation of new tumor blood ves- and in the well oxygenated state they are rapidly degraded sels necessary for further tumor growth [20]. One might [24]. Thus by increasing the pO in tumor tissue with nor- therefore expect that hyperoxia should prevent tumor ang- mobaric and hyperbaric oxygen it is reasonable to assume Table 3: Area fraction of vacuoles, number of apoptotic cells, mean vascular density, vascular diameter and ratio central/peripheral vessel density and vessel diameter in controls and in tumors treated with hyperoxia. Values are given as means ± SD. Control 1 bar, 21% O Test 1 bar, 100% O Test 1.5 bar, 100% O 2 2 2 pO = 0.21 pO = 1.0 pO = 1.5 2 2 2 Area fraction vacuoles "empty spaces" 0.01 ± 0.04 0.11 ± 0.05 *** 0.21 ± 0.02 *** Apoptotic cells (% of total cells) 61% ± 6.4 85% ± 7.3 *** 88% ± 5.0 *** Mean vascular density (number of vessels/mm ) Centrally 98 ± 18.6 50.1 ± 17.4*** 69.8 ± 18.5* Periphery 79.5 ± 19.2 38.5 ± 19.3*** 51.8 ± 26.8* Vascular diameter (µm) Centrally 19.1 ± 4.9 23.1 ± 9.1 ** 23.2 ± 10.1** Periphery 10.9 ± 3.9†† 12.5 ± 4.7 *†† 23.9 ± 10.0 *** Ratio central/peripheral vessel density 1.2 ± 0.7 1.6 ± 1.3 1.5 ± 1.1* Ratio central/peripheral vessel diameter 1.9 ± 0.8 2.0 ± 1.2 1.1 ± 0.6*** *P < 0.05, **P < 0.01, ***P < 0.001 vs control (1 bar, air) ††P < 0.01 vs centrally Page 7 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 tr E Figure 2 xol (left) and during hypero amples of eosin-hematoxylin xic treatment (rig stained tumor-tissu ht, 1 bar, pO e of the cen = 1.0) tral (A) and peripheral (B) part of the mammary tumor in con- Examples of eosin-hematoxylin stained tumor-tissue of the central (A) and peripheral (B) part of the mammary tumor in con- trol (left) and during hyperoxic treatment (right, 1 bar, pO = 1.0). The images under A are scaled to the same magnification (× 4) and the images under B to the same magnification (× 10). Scale bar indicate 500 µm (A) and 100 µm (B). Page 8 of 10 (page number not for citation purposes) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 When level of free oxygen radical production exceeds Control endogenous cellular antioxidant capacity it can create an "oxidative stress" which can lead to cell death [30]. Free oxygen radicals have previously been shown to induce apoptosis and also inhibit angiogenesis [31]. In the litera- 160 ture it seems as if ROS has "two faces". It appears as 1.5 bar though the action of free radicals on normal and tumor cells are opposite. When free radicals attack normal cells, DNA damage can occur, whereas ROS in tumor cells has 1 bar an unexpected, but highly beneficial action, namely inhi- bition of tumor cells [review:31]. Since the supoxide dis- mutase (SOD) activity in most tumors has been shown to be lower than in normal tissue, this might make the tumors even more susceptible to increased ROS activity during hyperoxic exposure. So, a moderate increase in Tumor volume (mm ) ROS activity by HBO might be beneficial in tumors. Th m Figure 3 m e relationship between tumo 2 before and after hyperoxic treatment r size and blood vessels per Conclusion The relationship between tumor size and blood vessels per The present study shows that both normobaric (1 bar, mm2 before and after hyperoxic treatment. Values repre- pO = 1.0) and hyperbaric hyperoxia (1.5 bar, pO = 1.5) sents mean ± SE. 2 2 significantly retards mammary tumor growth in rats. Hyperoxic treatment did affect the mammary tumors by inducing loss of glands, reducing vascular density and enhancing apoptosis. The mechanisms behind these changes are still not known. Hyperbaric oxygen treatment that VEGF and HIF-1α would have been down-regulated. should therefore be further evaluated as adjuvant tumor The anti-angiogenic effect found in the present study treatment. would correspond to this hypothesis. Competing interests Hyperoxia induces apoptosis The author(s) declare that they have no competing inter- We found the amount of normal cells (cells with intact ests. nucleus) in the tumor to be decreased after hyperoxic treatment. It is therefore important to know that hyper- Authors' contributions oxic treatment in the present range (1 -1.5 bar, pO = 1– AR carried out the morphology, immunoassay (von wille- 1.5) and time exposed (90 min) have never been shown brandt and Tunnel staining), statistical analysis and to induce cell death of other tissues of the body, including hyperbaric exposures. lungs which are known to be most susceptible to HBO (UHMS) [25]. A review by O'Reilly stated that cell prolif- CS and VMS carried out the gene expression profiling and eration (of normal tissue) in adult rats, mice and monkeys PCR. exposed to lethal levels of oxygen (>90%) was unaffected for the first 48, and alveolar epithelium relative unaffected RB and RKR participated in the design and helped draft until after 72 hours [26]. However, a recent report [27] on the manuscript. in vitro benign and malignant mammary epithelial cells showed that HBO (97.9% oxygen) at a high pressure (2.4 LEBS conceived the study, supervised the hyperbaric expo- bar) inhibited epithelial cell proliferation. Furthermore, sures and wrote the manuscript. an organised, genetically programmed cell death was found in the tumor tissue after hyperoxic treatment as All authors read and approved the final manuscript. indicated by elevated amount of tunnel-positive apop- totic cells. Acknowledgements The present study has been supported by the Norwegian Microarray Con- sortium (NMC), a national FUGE technology platform (Functional Genom- Potential mechanism? ics in Norway), the Norwegian Cancer Society and the Norwegian We might speculate that all or at least some of the changes Research Council. we have mentioned after both normobaric and hyperbaric hyperoxia are due to free oxygen radicals (ROS). ROS activity is known to be elevated during hyperoxia [28,29]. Page 9 of 10 (page number not for citation purposes) MVD (vessels/mm ) BMC Cancer 2007, 7:23 http://www.biomedcentral.com/1471-2407/7/23 25. Clark JM: Oxygen toxicity. In The Physiology and Medicine of Diving References 4th edition. Edited by: Bennet P, Elliot D. WB Saunders Company Ltd; 1. Vaupel P, Kallinowski F, Okunieff P: Blood flow, oxygen and nutri- 1993:121-169. ent supply, and metabolic microenvironment of human 26. O'Reilly MA: DNA damage and cell cycle checkpoints in tumors: a review. 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