| Hepatocellular carcinoma (HCC) is a common malignancy in China with poor prognosis. About 80%~90% of HCC in China are associated with hepatitis B virus (HBV). Thus an important task for caring HCC patients is to look for an effective therapy to improve outcome.Using cell lines and animal model, we have previously identified that two kinds of non-cytotoxic antineoplastics—somatostatin analogue (SSTA) and selective cyclooxygenase-2 (COX-2) inhibitor are able to inhibit HCC proliferation and to induce apoptosis in a synchronizing way. Additionally, in the HCC cell transfected with hepatitis B virus X protein (HBX), the expression of somatostatin receptor (SSTR) is decreased. And the inhibitory effect of SSTA is depressed.Recent clinical trials on HCC patients treated with SSTA revealed discrepant results. So it is necessary to explore other associated factors which may potentially influence on effect of SSTA in vivo. The anti-tumor effect of SSTA and COX-2 inhibitor depends on whether there are SSTR and COX-2 expressed in tumor tissues. Yet the expression level of SSTR and COX-2 in HCC tissues is not clear now. It is also unknown whether HBX could affect SSTR expression in vivo, and subsequently reduce the anti-tumor effect of SSTA. This study is to explore the expression development of SSTR subtypes and COX-2 during carcinogenesis and progression of HCC, to investigate the correlation between SSTR, HBX and alpha-fetoprotein (AFP) expression. It would be helpful with further studies to explain the corresponding pharmacological and clinical topics, such as causes of discrepant clinical effect, the possible effective rate, the optimal time of medication, and selection of patients.AIMSTo answer the following questions:1. During the pathological course of HCC progression, how dose the expression of SSTRs in human liver tissues change?2. What is the expression level of each SSTR subtype in tumor tissues of HBV-associated HCC patients?3. Whether the expression of HBX can affect the expressions of SSTR subtypes in HCC?4. How does COX-2 expression correlate with HCC development from cirrhosis? What's the relationship between COX-2 and SSTR expression? What's the best time for single use or combined use of SSTR and COX-2 inhibitor?5. Is there any correlation between SSTRs expression and AFP expression in HCC? Is serum AFP concentration related to level of SSTRs expression in HCC? Can it be used to select patients for SSTA therapy?METHODS 1. The expression of SSTR1-5 subtypes in different human liver tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR).2. The specificities of human SSTR1-5 RT-PCR products were confirmed by DNA sequencing.3. The level of SSTR subtype proteins in human liver tissues were detected by immunohistochemistry (IHC).4. The SST concentration in human liver tissues was examined by radioimmunoassay (RIA).5. The expression of HBX protein in human liver tissues was detected by immunohistochemistry (IHC).6. The expression of COX-2 protein in human liver tissues was detected by western blot.7. The AFP concentration in HCC tissues and peripheral blood of HCC patients was measured by enzyme-linked immunosorbent assay (ELISA).RESULTS1. The mRNA of human SSTR1-5 subtypes could be detected in all human liver tissues: normal liver (5 cases) 20.0-80.0%; chronic hepatitis (14 cases) 57.1-85.7%; cirrhotic liver (40 cases) 60.0-92.5%; adjacent-HCC liver (40 cases) 90.0-97.5%; HCC tissues (40 cases) 62.5-87.5%, respectively. This indicates a tendency of expression level: normal liver HCC, P<0.05.2. Sequencing and gene arrangement comparisons indicate that the specificities of SSTR1-5 RT-PCR products were 96%-100%. 3. The protein level of SSTR subtypes is as: normal liver (5 cases) 20.0-60.0%, 0.13-0.81; chronic hepatitis (14 cases) 42.9-71.4%, 1.02-4.41; cirrhotic liver (40 cases) 47.5-67.5%, 1.93-5.15; adjacent-HCC liver (40 cases) 85.0-95.0%, 23.57-32.56; HCC tissues (40 cases) 47.5-70.0%, 5.93-10.13, respectively. A similar tendency as mRNA level: normal liver < chronic hepatitis < hepatic cirrhosis << adjacent-HCC>HCC, P<0.05.4. The positive staining of SSTR1-5 were seen in cytomembrane and cytoplasm of hepatic cells with irregularly scattered distribution in normal liver tissues, chronic hepatitis and hepatic cirrhosis; yet the expression of SSTRs rapidly increased in adjacent-HCC tissue, with a pattern of "surrounding portal vein" and "facing portal vein" on single cell. This feature is the more close to tumor region the more obvious.5. Blood vessel with SSTRs expression was rarely seen in liver tissues of normal, chronic hepatitis and cirrhosis. Yet positive staining on blood vessel is markedly increased in adjacent-HCC liver tissues and the more close to tumor region the more obvious. The positive staining was seen on the endothelium cytomembrane of portal vein, but never visible on central veins and hepatic artery.6. Bile duct with SSTR 1,2,4 expressed were occasionally seen in chronic cholangitis cases. Yet SSTR3,5 were never observed.7. The level of SSTRs expression in HCC was far lower than that in adjacent-HCC liver tissue (P<0.05), yet sill higher than that of hepatic cirrhosis (P<0.05). Blood vessel was less seen in HCC tissue, where SSTRs positive blood vessel were even less. The ratios of SSTR1-5 protien / mRNA positive rate in HCC were: 61%, 80%, 80%, 79%, 93%, respectively. The efficiency of SSTR5 translation is the highest.8. The SST levels in normal liver tissues (5 cases), chronic hepatitis (14 cases), cirrhotic liver (40 cases), adjacent-HCC liver (40 cases), and HCC (40 cases) were 16.2±2.4, 78.3±14.1, 140.6±30.4, 618.1±129.6 and 352.8±74.4, respectively. There is a significant difference between each group (P<0.05).9. There is a positive linear correlation between SSTR1-5 proteins expression (IOD data) and SST level among different types of human liver tissues (r = 0.899, 0.907, 0.908, 0.928, 0.919, P<0.05).10. The expression (IOD data) of HBX in cirrhotic liver tissues (40 cases), adjacent-HCC liver tissues (40 cases) and HBV-associated HCC tissues (40 cases) were: 55.6%, 5.75±2.13; 91.7%, 7.21±3.21; 23.1%, 7.17±2.79, respectively, indicating levels in hepatic cirrhosis> HCC, P<0.05.11. The analysis of linear correlation and curve fitting shows that there is neither a straight line correlation (r = 0.382, 0.581, 0.512, 0.174, 0.378, respectively, P>0.05) nor a curvilinear correlation (r = 0.403, 0.614, 0.569, 0.386, 0.507, respectively, P > 0.05) between SSTR1-5 expression and HBX expression in the liver tissues previously described.12. COX-2 expression (OD data of COX-2 strip / OD data ofβ-actin strip) in cirrhotic liver tissues (40 cases), adjacent-HCC liver tissues (40 cases) and HCC tissues (40 cases) is as: 95.0%, 0.256±0.038; 82.5%, 0.199±0.032; 77.5%, 0.138±0.014, respectively, indicating level in hepatic cirrhosis>adjacent-HCC>HCC, P<0.05. 13. There is a negative straight line correlation between SSTR1-5 and COX-2 expression during the development of cirrhosis→adjacent-HCC (r = -0.802, -0.897, -0.822, -0.870, -0.885, reaspectively, P<0.05); yet the correlation of the upper two changes into positive straight line correlation during the development of adjacent-HCC→HCC [r = 0.5319(P>0.050), 0.789, 0.617, 0.713, 0.735, P<0.05.].14. In 40 cases of HCC patients, 29 cases were serum AFP positive, while 11 cases negative. There is a significant positive straight line correlation between serum AFP concentration and AFP expression in HCC tissue (r = 0.938, P<0.01).15. The analysis of curve fitting revealed a positive two shape parameters curve correlation between SSTR1-5 and AFP expression in HCC (r = 0.786, 0.901, 0.841, 0.845, 0.904, respectively, P<0.05). AFP expression in HCC either too high or too low would inhibit the expression of SSTR1-5.16. The analysis of curve fitting shows that there is a positive two shape parameters curve correlation between SSTR1-5 expressions of HCC and serum AFP concentration (r = 0.882, 0.901, 0.877, 0.854, 0.903, respectively, P<0.05). Low expression of SSTR1-5 in HCC is correlated to either too high or too low serum AFP level.17. Serum AFP levels at 200-600 ng/ml, 300-800 ng/ml, 200-600 ng/ml, 200-800 ng/ml, 200-800ng/ml are associated with higher expressions of SSTR1-5 (50%CI) in HCC.CONCLUSIONS 1. In normal human liver tissue, SSTRs expression is low suggesting that this anti-proliferation regulator rarely intervenes in proliferation of normal liver cell.2. In the development of hepatic cirrhosis from HBV, the liver cell undergoes a course of ordinal regeneration→out-of-order regeneration. Accordingly, SSTRs expression is slowly increased at a relatively low level. This indicates that the compensated regeneration of liver cell at the meantime initiated the inhibitory mechanism of proliferation --SST-SSTRs.3. In precancerous lesion, the peaked SSTRs expression, accumulation surrounding portal vein, prominent internalization and recirculation of SST-SSTRs aggregation all suggest that about 90% liver cells mobilize the anti-proliferation regulator~SST-SSTRs to adjust abnormal proliferation. This indicates SST-SSTRs is also actively involved in the anti-tumor mechanism.4. The markedly up-regulation of SSTR2,5 can be used to distinguish precancerosis and hepatic cirrhosis, but also facilitate SSTA to prevent progression of precancerosis. It is the optimal stage to use SSTA. For those patients with precancerous pathological changes, about 90% of them would benefit from SSTA therapy.5. In HCC cell, the altered genome has SSTRs level reduced to 10-30%. This probably accelerates the malignant proliferation of tumor.6. During progression of HCC, the level of SST correlates to SSTRs expression. It not only suggests that there are up-regulated SSTRs in these pathological stages, but also indicates that extra-hepatic factors are related to SST's inhibitory effects on tumor-genesis.7. About 60% HCC patients are candidates for SSTA use. Yet about 40% HCC are SSTRs negative, further more, SSTRs expression is markedly decreased in the progressive stage of HCC. So SSTA may have minimal effect on those end-stage HCC patients. This could explain discrepant effects of SSTA on different HCC patients. It is estimated that the effective rate of SSTA treating HCC is about 60%.8. The high expression of HBX in HCC and adjacent-HCC regions indicates that HBX has a close relationship to carcinogenesis and development of HCC.9. In HBV-associated HCC patients, HBX does not influence SSTRs expression. About 60% of HBV-associated HCC patients are SSTRs positive, which offer a beneficial clue for SSTA therapy.10. During the sequential development of HCC from cirrhosis, the gradually decreased COX-2 indicates an important role of chronic inflammation involved in carcinogenesis. So the inhibitory proliferation effect of COX-2 inhibitor is the earlier the better.11. Precancerous and early stages of HCC are the optimal time for combined use of SSTA and COX-2 inhibitor.12. The serum AFP concentration of HCC patients rises with the expression level of AFP in HCC tissues.13. Either too high or too low AFP expression in HCC would inhibit the expressions of SSTR1-5 in HCC.14. Either too high or too low of serum AFP level indicates low expression of SSTR1-5 in HCC. Serum AFP levels ranging between 200-800ng/ml are associated with higher expressions of SSTR1-5 in HCC. Those patients would be suitable candidates for SSTA.
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