| Hepatocellular carcinoma (HCC) is one of the most common tumors in China. Only 20% of HCC patients have opportunity to perform resection, and the 5-year survival rate is limited to about 35% after surgery. Usually no potentially curative therapy is possible in the patients with unresectable tumor or recurrence after surgery. In the past few years, non-cytotoxitic drugs were appeared to be potentially benefit for treatment of HCC.It has been reported that octreotide, an analogue of somatostatin, and cyclooxygenase-2 (COX-2) inhibitors are able to inhibit the growth of HCC. However the underlying mechanisms of these two drugs are much less known. Our group has reported that aspirin, non-selective COX inhibitor, combined with octreotide could arrest the growth of HCC synergically. The enhanced effects may be related with inhibition of COX-2 and cellular signal transduction system.Aims of the study1. Do the COX-2 inhibitors with different selectivity to COX-2 havedifferent effects on the growth of HCC?2. Which selective COX-2 inhibitor would show the best inhibitive effect on HCC?3. Whether rofecoxib, combined with octreotide would enhance the inhibition effects of proliferation of HCC in vitro and in vivo?4. What would be the mechanisms behind the inhibition?Methods1. Cell proliferation is evaluated by 3H-thymidine incorporation into DNA.2. The TdT-mediated dUTP nick end labeling assay (TUNEL) and Annexin V labeling assay were used to detect the cellular apoptosis.3. Human hepatocellular carcinoma cells SMMC7721 were implanted orthotopically in the liver of nude mice, and then nude mice were treated with either octreotide (100 g-kg-1 d-1) combined with rofecoxib (30mg -kg-1 -d-1) or rofecoxib only for eight weeks.4. COX-2 gene in HCC cells was detected by reverse transcription polymerase chain reaction (RT-PCR) technology.5. The expression of PCNA and VEGF was detected by immuno-cytochemistry or immunohistochemistry.6. The HCC microvascular density was examined by immunohisto-chemical staining with factor VIII antibody.7. The expressions of COX-2, c-fos, ERK, P655 I B and IKK weredetected by western blot.8. Gelatin Zymography and RT-PCR technology were used to detect the expression of matrix metalloproteinase-2 (MMP-2) progelatinase and MMP-2 gene.9. The DNA binding activity of AP-1 and NF- B was mesured by electrophoretic mobility sift assay (EMSA).Results1. Three selective COX-2 inhibitors (meloxicam, celecoxib, rofecoxib) can inhibit 3H-thymidine incorporation into SMMC7721 cells. The inhibition effects show a concentration dependence, the higher selective on COX-2, the stronger inhibition on HCC cells.2. After treated with meloxicam, celecoxib, rofecoxib, at concentration of1 10-5mol/L for 24 hours, the apoptosis rate of HCC was 14.6% 2.79% , 21.6% 3.59% , 27.1% 3.56% respectively, and which was significantly higher than that in the controls(only few apoptosis cells was observed).3. COX-2 mRNA was detected by RT-PCR, and the expression of which was decreased by rofecoxib.4. The expression of ERK1/2 in HCC cells were significantly decreased, after treated with octreotide(1 10-7mol/L) or rofecoxib (1 10-5mol/L) for 24 hours, when compared with the controls. The inhibition effect of octreotide combined with rofecoxib on the expression of ERK1/2in HCC, was much stronger than that of either of them.5. The expression of PCNA, VEGF, c-Fos, P65, I B, IKK / in HCC cells, were significantly decreased, after treated with rofecoxib (l 10-5mol/L) for 24 hours, and which was no significant difference between octreotide(1 10-7mol/L) group and the controls, octreotide(1 10-7mol/L) can further down regulate the decreased effection which was induced by rofecoxib.6. The expression of MMP-2 protein and mRNA in HCC cells, were decreased by rofecoxib significantly, and which was no significant difference between octreotide ( 1 10-7mol/L) group and the controls, octr... |
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