Immunohistochemistry of somatostatin receptors (SSTRs) in hepatocellular carcinoma. (A) Negative control. (B–F) Double staining immunohistochemistry with a smooth muscle actin ( a -SMA) and SSTR1–5 in the same patient. We observed intense immunoreaction with SSTR1–4, and to a lesser degree with SSTR5. Within the tumour, double positive cells were seen (arrow, B insert). These cells are clearly activated hepatic stellate cells. Original magnification 100 6 , inserts 400 6 . 

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... by automatic sequencing using the ABI prism 310 (Perkin Elmer). To elucidate effects of somatostatin on tumour progression, SSTR subtype specific agonists were used: L-797,591 (SSTR1 agonist), L-779,976 (SSTR2 agonist), L-796,778 (SSTR3 agonist), L-803,087 (SSTR4 agonist), and L-817,818 (SSTR5 agonist) (Merck Research Laboratories, Rahway, New Jersey, USA). These substances are non-peptide somatostatin agonists with a very high subtype specificity, except for L-817,818 which has affinity not only for SSTR5 but also some for SSTR1. Although these substances have been used in animal cells, they were designed and developed as agonists for human SSTRs. The affinity of the agonists was tested in transfected CHO-K1 cells expressing each of the five human SSTRs. 18 In addition to their specificity, these agonists have a prolonged half life. Biological effects of the agonists has been tested in several cell lines and these effects were SSTR subtype specific. 19 Cell proliferation was measured by using the colorimetric 5-bromo-2 9 deoxyuridine (BrdU) cell proliferation ELISA (Boehringer, Mannheim, Germany), as described previously. 20 After trypsinisation, HepG2, HuH7, and HSCs were plated in 96 well dishes (Falcon, Becton Dickinson Labware, Meylan, France) at a density of 15 6 10 3 , 15 6 10 3 , and 6 6 10 3 cells per well, respectively. All cells were suspended in DMEM supplemented with 2% fetal calf serum. After 24 hours, media were renewed and cells exposed for another 24 hours to different concentrations of SSTR agonists (10 2 12 to 10 2 8 mol/l) in the presence of 10 m mol BrdU. Platelet derived growth factor (PDGF-BB 10 ng/ml), a strong mitogen for HSCs, 21 was added to the medium of HSCs (R & D Systems Europe, Abingdon, UK). In each experiment, all conditions were tested 10 times, and every experiment was performed in triplicate. To allow comparison of proliferation between different groups, results were expressed as percentages and compared with controls normalised to 100%. Mean (SD) values were calculated for all groups. Somatostatin induced apoptosis was studied by the TUNEL reaction according to the manufacturer’s instructions (In Situ Cell Death Detection Kit; Roche diagnostics, Belgium). After trypsinisation, HepG2, HuH7, and HSCs were plated in 96 well dishes at a density of 2.5 6 10 3 , 2.5 6 10 3 , and 1 6 10 3 cells per well, respectively. All cells were suspended in DMEM supplemented with 2% fetal calf serum. As apoptosis has been attributed to stimulation of SSTR2 and 3, different concentrations (10 2 12 to 10 2 6 mol/l) of L-779,976 and L-796,778, agonists of SSTR 2 and 3, respectively, were studied. After 24 and 72 hours of treatment, apoptotic cells were counted and expressed as a percentage of total cells. Experiments were performed in triplicate. The influence of the five SSTR agonists on migration of the hepatoma cell lines and HSCs was investigated, as described previously, 22 using Boyden invasion chambers with 8 m m pore size filters coated with Matrigel basement membrane matrix (Falcon, Becton Dickinson Labware, Bedford, Massachusetts, USA). PDGF-BB is a very strong chemoattractant for human HSCs 21 whereas hepatocyte growth factor (HGF) increases the invasiveness of HepG2 and HuH7 tumour cells. 22 To investigate the possibility that somatostatin inhibits PDGF- BB induced migration of HSCs or HGF induced migration of HepG2 and HuH7, cells were seeded in the upper well of Boyden chambers in the presence of PDGF-BB or HGF in the lower chamber. In these experiments, 2.5 6 10 4 hepatoma cells or HSCs were seeded onto the filter. HGF 20 ng/ml (R & D Systems Europe, Abingdon, UK) was added to the lower chamber when hepatoma cells were seeded in the upper chamber, or PDGF-BB 10 ng/l was added when HSCs were present in the upper chamber. Subsequently, 10 2 9 mol/l of SSTR agonists were added to the cells in the upper chamber. After 12 and 48 hours for experiments with HSCs and hepatoma cells, respectively, cells in the upper chamber were wiped with a cotton swab, and the filters were fixed in methanol and stained with haematoxylin. Cells invading the lower surface of the filter were counted in 10 high power fields using a Zeiss Axioplan microscope. Results were expressed as a percentage, relative to controls normalised to 100%. Experiments were performed in triplicate, except for controls and experiments with L-797,591 (agonist of SSTR1) which were performed six times. One way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons and the Mann-Whitney test were used when appropriate. Results are presented as means (SD). A p value of , 0.05 was considered statistically significant. Analysis was performed using Instat software (GraphPad software inc., San Diego, California, USA). Using well validated specific antibodies against the five recognised SSTRs, we were able to localise the different SSTRs in sections of normal liver (fig 1), cirrhosis (fig 2), and HCC (fig 3). Expression of the various receptors varied between different specimens and cells (table 3). In fig 1, normal liver is presented. Hepatocytes and sinusoidal cells did not stain for any of the five receptors. Cholangiocytes, on the other hand, consistently expressed SSTR subtypes 1, 3, and 4. Immunoreaction with SSTR subtypes 1 and 4 was more intense than with SSTR subtypes 2, 3, and 5 which were detected in, respectively, two, three, and one of the three analysed specimen. Furthermore, a weak immunoreaction of SSTR1 with endothelial cells of a small number of small arteries was found (not shown). In cirrhotic liver, expression of SSTRs significantly changed compared with normal liver. Hepatocytes within cirrhotic nodules expressed SSTR1 (fig 2B, C), and in 4/6 of the examined specimens, specimens also expressed SSTR2 (fig 2F, G), SSTR3 (fig 2J, K), SSTR4 (fig 2N, O), and SSTR5 (fig 2R, S). In most of the studied cases, we also found cells which double stained for all five SSTRs and a -SMA. These cells, clearly activated HSCs, were located at the interface of septa and cirrhotic nodules (fig 2C, G, K, O). In some specimens, we observed HSCs within hepatic nodules that stained for PDGFR- b and SSTR1 (fig 2D) or SSTR3 (fig 2L). In fig 3, immunohistochemistry for HCC is presented. Most of the tumours stained with SSTR subtypes 1 and 2 whereas 50% reacted with SSTR subtypes 3, 4, and 5 (table 3). In some tumours, non-tumoral cells double stained for a -SMA and SSTR subtypes 1, 2, and 3 (fig 3B, table 3). To verify the results of double staining immunohistochemistry, isolated cell lines were exposed to the different SSTR antibodies. Human HSCs were positive for all five SSTRs although immunoreaction with SSTR3 was less intense. Similarly, HepG2 expressed the five SSTR subtypes, again less clearly SSTR3. Finally, HuH7 reacted with all SSTRs, except for SSTR3 (data not shown). Messenger RNA of the five SSTRs was detected both in normal and pathological liver tissue (fig 4). We identified a single band for all receptors, except SSTR4 for which three bands were found in some patients. Sequencing demonstrated that the middle band (635 nucleotides), which was most intense, corresponded to SSTR4. This was in agreement with the findings of Talme et al who used the same primers for SSTR4. 23 All investigated samples were definitely positive for SSTR subtypes 1 and 2. For these receptors, no important variations between different patients or between cirrhosis and tumour samples from the same patient were observed. Expression of mRNA of SSTR subtypes 3, 4, and 5 was almost undetectable in some patients whereas a significant band was observed in others. In two of six patients, surrounding cirrhotic tissue expressed SSTR4 and 5 mRNA more clearly than the tumour of these patients. When examining expression of mRNA of the five SSTRs in different cell lines, we were able to demonstrate the presence of all receptors in HSCs, HepG2, and HuH7 cells (fig 5). In agreement with the results of liver tissue, a single band was demonstrated for SSTR subtypes 1, 2, 3, and 5 whereas multiple bands were found for SSTR4. Again, the middle band (635 nucleotides) was shown to correspond to SSTR4. Proliferation of hepatoma cells and HSCs was stimulated with 2% fetal calf serum or PDGF-BB, respectively. As expected, PDGF-BB induced a marked increase in the proliferation of HSCs. 21 Proliferation of hepatocellular carcinoma cell lines HepG2 and HuH7, and HSCs did not change in the presence of different concentrations of various SSTR agonists compared with cells treated with PDGF-BB alone (data not shown). In preliminary experiments, no apoptotic effects of somatostatin agonists were observed. As somatostatin induced apoptosis has been attributed to activation of SSTR2 and 3, definitive experiments were performed with these agonists. No effects were observed in any of the three cell lines after 24 hours. Even when cells were treated for 72 hours with L-779,976 or L-796,778, no difference in apoptosis between treated and untreated cells was detected (fig 6). In the presence of chemotactic stimuli PDGF-BB or HGF in the lower part of the Boyden migration chamber, all three cell types migrated through the pores to the lower surface of the membrane (fig 7A). Among the five SSTR agonists tested, only addition of L-797,591 (SSTR1 agonist) significantly reduced migration (fig 7B). Compared with untreated control cells, migration of HepG2, HuH7, and HSCs significantly decreased to 88 (7)% (p , 0.05), 83 (11)% (p , 0.05), and 67 (13)% (p , 0.01), respectively. In this study, we have described expression of SSTR subtypes in normal human liver, in cirrhosis, and in HCC. Furthermore, we showed that migration of hepatoma cells and HSCs was reduced via activation of SSTR subtype 1. However, the subtype specific somatostatin agonists used in this study had no effect on proliferation or apoptosis. Expression of SSTRs in human liver has not been ...

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... Meylan, France) at a density of 15 6 10 3 , 15 6 10 3 , and 6 6 10 3 cells per well, respectively. All cells were suspended in DMEM supplemented with 2% fetal calf serum. After 24 hours, media were renewed and cells exposed for another 24 hours to different concentrations of SSTR agonists (10 2 12 to 10 2 8 mol/l) in the presence of 10 m mol BrdU. Platelet derived growth factor (PDGF-BB 10 ng/ml), a strong mitogen for HSCs, 21 was added to the medium of HSCs (R & D Systems Europe, Abingdon, UK). In each experiment, all conditions were tested 10 times, and every experiment was performed in triplicate. To allow comparison of proliferation between different groups, results were expressed as percentages and compared with controls normalised to 100%. Mean (SD) values were calculated for all groups. Somatostatin induced apoptosis was studied by the TUNEL reaction according to the manufacturer’s instructions (In Situ Cell Death Detection Kit; Roche diagnostics, Belgium). After trypsinisation, HepG2, HuH7, and HSCs were plated in 96 well dishes at a density of 2.5 6 10 3 , 2.5 6 10 3 , and 1 6 10 3 cells per well, respectively. All cells were suspended in DMEM supplemented with 2% fetal calf serum. As apoptosis has been attributed to stimulation of SSTR2 and 3, different concentrations (10 2 12 to 10 2 6 mol/l) of L-779,976 and L-796,778, agonists of SSTR 2 and 3, respectively, were studied. After 24 and 72 hours of treatment, apoptotic cells were counted and expressed as a percentage of total cells. Experiments were performed in triplicate. The influence of the five SSTR agonists on migration of the hepatoma cell lines and HSCs was investigated, as described previously, 22 using Boyden invasion chambers with 8 m m pore size filters coated with Matrigel basement membrane matrix (Falcon, Becton Dickinson Labware, Bedford, Massachusetts, USA). PDGF-BB is a very strong chemoattractant for human HSCs 21 whereas hepatocyte growth factor (HGF) increases the invasiveness of HepG2 and HuH7 tumour cells. 22 To investigate the possibility that somatostatin inhibits PDGF- BB induced migration of HSCs or HGF induced migration of HepG2 and HuH7, cells were seeded in the upper well of Boyden chambers in the presence of PDGF-BB or HGF in the lower chamber. In these experiments, 2.5 6 10 4 hepatoma cells or HSCs were seeded onto the filter. HGF 20 ng/ml (R & D Systems Europe, Abingdon, UK) was added to the lower chamber when hepatoma cells were seeded in the upper chamber, or PDGF-BB 10 ng/l was added when HSCs were present in the upper chamber. Subsequently, 10 2 9 mol/l of SSTR agonists were added to the cells in the upper chamber. After 12 and 48 hours for experiments with HSCs and hepatoma cells, respectively, cells in the upper chamber were wiped with a cotton swab, and the filters were fixed in methanol and stained with haematoxylin. Cells invading the lower surface of the filter were counted in 10 high power fields using a Zeiss Axioplan microscope. Results were expressed as a percentage, relative to controls normalised to 100%. Experiments were performed in triplicate, except for controls and experiments with L-797,591 (agonist of SSTR1) which were performed six times. One way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons and the Mann-Whitney test were used when appropriate. Results are presented as means (SD). A p value of , 0.05 was considered statistically significant. Analysis was performed using Instat software (GraphPad software inc., San Diego, California, USA). Using well validated specific antibodies against the five recognised SSTRs, we were able to localise the different SSTRs in sections of normal liver (fig 1), cirrhosis (fig 2), and HCC (fig 3). Expression of the various receptors varied between different specimens and cells (table 3). In fig 1, normal liver is presented. Hepatocytes and sinusoidal cells did not stain for any of the five receptors. Cholangiocytes, on the other hand, consistently expressed SSTR subtypes 1, 3, and 4. Immunoreaction with SSTR subtypes 1 and 4 was more intense than with SSTR subtypes 2, 3, and 5 which were detected in, respectively, two, three, and one of the three analysed specimen. Furthermore, a weak immunoreaction of SSTR1 with endothelial cells of a small number of small arteries was found (not shown). In cirrhotic liver, expression of SSTRs significantly changed compared with normal liver. Hepatocytes within cirrhotic nodules expressed SSTR1 (fig 2B, C), and in 4/6 of the examined specimens, specimens also expressed SSTR2 (fig 2F, G), SSTR3 (fig 2J, K), SSTR4 (fig 2N, O), and SSTR5 (fig 2R, S). In most of the studied cases, we also found cells which double stained for all five SSTRs and a -SMA. These cells, clearly activated HSCs, were located at the interface of septa and cirrhotic nodules (fig 2C, G, K, O). In some specimens, we observed HSCs within hepatic nodules that stained for PDGFR- b and SSTR1 (fig 2D) or SSTR3 (fig 2L). In fig 3, immunohistochemistry for HCC is presented. Most of the tumours stained with SSTR subtypes 1 and 2 whereas 50% reacted with SSTR subtypes 3, 4, and 5 (table 3). In some tumours, non-tumoral cells double stained for a -SMA and SSTR subtypes 1, 2, and 3 (fig 3B, table 3). To verify the results of double staining immunohistochemistry, isolated cell lines were exposed to the different SSTR antibodies. Human HSCs were positive for all five SSTRs although immunoreaction with SSTR3 was less intense. Similarly, HepG2 expressed the five SSTR subtypes, again less clearly SSTR3. Finally, HuH7 reacted with all SSTRs, except for SSTR3 (data not shown). Messenger RNA of the five SSTRs was detected both in normal and pathological liver tissue (fig 4). We identified a single band for all receptors, except SSTR4 for which three bands were found in some patients. Sequencing demonstrated that the middle band (635 nucleotides), which was most intense, corresponded to SSTR4. This was in agreement with the findings of Talme et al who used the same primers for SSTR4. 23 All investigated samples were definitely positive for SSTR subtypes 1 and 2. For these receptors, no important variations between different patients or between cirrhosis and tumour samples from the same patient were observed. Expression of mRNA of SSTR subtypes 3, 4, and 5 was almost undetectable in some patients whereas a significant band was observed in others. In two of six patients, surrounding cirrhotic tissue expressed SSTR4 and 5 mRNA more clearly than the tumour of these patients. When examining expression of mRNA of the five SSTRs in different cell lines, we were able to demonstrate the presence of all receptors in HSCs, HepG2, and HuH7 cells (fig 5). In agreement with the results of liver tissue, a single band was demonstrated for SSTR subtypes 1, 2, 3, and 5 whereas multiple bands were found for SSTR4. Again, the middle band (635 nucleotides) was shown to correspond to SSTR4. Proliferation of hepatoma cells and HSCs was stimulated with 2% fetal calf serum or PDGF-BB, respectively. As expected, PDGF-BB induced a marked increase in the proliferation of HSCs. 21 Proliferation of hepatocellular carcinoma cell lines HepG2 and HuH7, and HSCs did not change in the presence of different concentrations of various SSTR agonists compared with cells treated with PDGF-BB alone (data not shown). In preliminary experiments, no apoptotic effects of somatostatin agonists were observed. As somatostatin induced apoptosis has been attributed to activation of SSTR2 and 3, definitive experiments were performed with these agonists. No effects were observed in any of the three cell lines after 24 hours. Even when cells were treated for 72 hours with L-779,976 or L-796,778, no difference in apoptosis between treated and untreated cells was detected (fig 6). In the presence of chemotactic stimuli PDGF-BB or HGF in the lower part of the Boyden migration chamber, all three cell types migrated through the pores to the lower surface of the membrane (fig 7A). Among the five SSTR agonists tested, only addition of L-797,591 (SSTR1 agonist) significantly reduced migration (fig 7B). Compared with untreated control cells, migration of HepG2, HuH7, and HSCs significantly decreased to 88 (7)% (p , 0.05), 83 (11)% (p , 0.05), and 67 (13)% (p , 0.01), respectively. In this study, we have described expression of SSTR subtypes in normal human liver, in cirrhosis, and in HCC. Furthermore, we showed that migration of hepatoma cells and HSCs was reduced via activation of SSTR subtype 1. However, the subtype specific somatostatin agonists used in this study had no effect on proliferation or apoptosis. Expression of SSTRs in human liver has not been studied in detail. In one study, SSTR2 was present in low amounts 24 although in another study using northern blot analysis, no relevant amounts of mRNA for any of the five SSTRs could be detected. 11 With the very sensitive RT-PCR technique, we were able to demonstrate the presence of all five SSTRs in normal human liver. By means of immunohistochemistry, we demonstrated that bile ducts, but not hepatocytes or sinusoids, immunoreacted with specific SSTR antibodies. Furthermore, SSTR1 was present in the endothelial cells of some small blood vessels, which is in agreement with findings in normal extrahepatic human blood vessels. 25 In a somatostatin 14/28 receptor binding study, levels of SSTRs were either absent, low, or very high in hepatitis and cirrhosis. 5 We also found considerable variation in SSTR expression in cirrhosis. Receptor subtypes 1 and 2 were prominent in most of the studied samples but SSTR 3, 4, and 5 were clearly present in some and absent in other specimens. As we did not use a quantitative PCR method, we cannot draw firm conclusions on the variation in mRNA expression. However, care was taken to start with equal amounts of mRNA, as proven by amplification of b -actin mRNA. Additionally, immunohistochemistry results ...