| Part oneTHE EXPRESSION OF 5-LIPOXYGENASE IN PANCREATIC CANCER AND ITS CORRELATION WITH COX-2 AND VEGFObjective.To investigate the expression of 5-LOXmRNA and 5-LOX protein in pancreatic cancer tissue and their correlation with pancreatic cancer clinicopathological characters.Meanwhile we study the relationship between 5-LOX expression and expression of COX-2 ,VEGF and supply rational data for the prevention and treatment of pancreatic cancer.Methods:From Dec.2001 to Dec.2002,we gathered 35 pancreatic cancer new tissue samples from general surgery ,Qilu Hospital of Shandong University and Shandong tumor hospital,and gathered 46 pancreatic cancer paraffinimbeded tissue samples between Jun.1998 and Dec. 1999 .The expression of 5-LOX protein in pancreatic cancer was detected by immunohistochemistry, the expression of 5-LOXmRNA,COX-2mRNA,VEGFmRNA by semi-quantitive reverse transcriptase-polymerase chain reaction method.We analyzed the relationship between their expression and clinicopathological features of pancreatic cancer.Results:1. Expression of 5-LOX mRNA , C0X-2mRNA, VEGF mRNA in pancreatic cancer tissue and their correlation with pancreatic cancer clinicopathological characters: 26 pancreatic cancer tissue samples could be detected a 416bp band,the positive rate of 5-LOX mRNA was 74.3% (26/35 ) ; 28 pancreatic cancer tissue samples could be detected a 305bpband,the positive rate of COX-2 mRNA was 80% ( 28/35) ; 21 pancreatic cancer tissue samples could be detected a 418bp band,the positive rate of VEGF mRNA was 60%( 21/35 ).Expression of 5-LOX mRNA, C0X-2mRNA had no correlation with location , histology, differentiation of pancreatic cancer and was associated with clinical stages; Expression of VEGFmRNA had no correlation with location, histology of pancreatic cancer and was associated with its differentiation and clinical stages.2.Relationship between 5-LOX mRNA, C0X-2mRNA and VEGF mRNA in pancreatic cancer tissue: 17 pancreatic cancer tissue samples showed VEGF mRNA in 26 showing 5-LOX mRNA, 5 pancreatic cancer tissue samples showed no VEGF mRNA in 9 showing no 5-LOX mRNA. Expression of 5-LOX mRNA , VEGF mRNA is coordinated in pancreatic cancer(p<0.05).Using the comparative content of them by correlation statistic analysis, 5-LOX mRNA and VEGF mRNA were correlated(r= 0.535,p<0.01). 19 pancreatic cancer tissue samples showed VEGF mRNA in 28 showing COX-2 mRNA, 5 pancreatic cancer tissue samples showed no VEGF mRNA in 7 showing no COX-2 mRNA. Expression of COX-2 mRNA > VEGF mRNA is coordinated in pancreatic cancer(p<0.05).Using the comparative content of them by correlation statistic analysis, COX-2 mRNA and VEGF mRNA were correlated(r=0.340,p<0.05).3.Relationship between 5-LOX mRNA and C0X-2mRNA in pancreatic cancer tissue: 19 pancreatic cancer tissue samples showed COX-2 mRNA in 26 showing 5-LOX mRNA, all pancreatic cancer tissue samples showed COX-2 mRNA in 9 showing no 5-LOX mRNA,and all pancreatic cancer tissue samples showed 5-LOX mRNA in 9 showing no COX-2 mRNA. COX-2 mRNA and 5-LOX mRNA were not correlated(p>0.05),no coordinated.4. Expression of 5-LOX protein in all kinds of pancreatic tissues: The positive rates of 5-LOX were 73.9%,20%,30%,0 respectively in pancreatic cancer,benign tumor,chronic inflammtion and normal tissues.Theexpression of 5-lipoxygenase in pancreatic cancer was significantely higher than that in other three kinds of tissues( x 2=12.95 > 5.24 > 6.76,/?<0.01,/?<0.05,/?<0.01). No correlation was found between normal tissues and benign tumor,chronic inflammtion. There was no correlation between the expression 5-LOX and the location,differentiation,histology types.III-IV stages of pancreatic cancer showed strong expressions of 5-LOX than that in I~II stages of pancreatic cancer ( x 2=4.57, /K0.05) . Expression of 5-LOX protein was all median or intense positive staining,no weak positive staining in pancreatic cancer metastasis.5. Relationship between expression of 5-LOX protein and survival rates in pancreatic cancer patients:24 cases were positive,9 cases negative in 33 pancreatic cancer patients with radical and palliative operation,and there was no statistical difference between expression of 5-LOX protein and survival rates in pancreatic cancer patients.Conclusions:1. 5-LOX mRNA and protein were overexpressed in pancreatic cancer. The expression of them reflected the progress and was not associated with the prognosis of pancreatic cancer.2. COX-2mRNA and VEGFmRNA were overexpressed in pancreatic cancer and associated with the clinical stages of pancreat ic cancer.3.5-LOX mRNA ? C0X-2mRNA and VEGF mRNA were correlated;the expression 5-LOX and COX-2 were both crossed and supplemental each other,not correlated(p>0.05),no coordinated. Part twoTHE EFFECTION OF 5-LOX/COX-2 INHIBITOR ON PANCREATIC CANCER CELLObjective:It has been verified that overexpression of 5-LOX/COX-2was correlated with the carcinogenesis and development of pancreatic cancer. We want to study the effect of 5-LOX/COX-2 inhibitor DHDMBF30 on proliferation and apoptosis of pancreatic cancer to further offer new drug for the prevention and treatment of pancreatic cancer.Methods:Investigate the effect of 5-LOX/COX-2 inhibitor DHDMBF30 on proliferatio and apoptosis of pancreatic cancer cell line Capan2 by MTT assay,FCM, microscpe and electron microscpe.Results:1.Effection of DHDMBF30 on proliferation of cell line Capan2: DHDMBF30 could inhibit the proliferation of cell line Capan2 ;after 24h of DHDMBF30 action,the inhibition rate of high concentration of DHDMBF30 significantly rose,but to a certain concentration the inhibition rate did not markedly enhance and a platform appeared(ICso=12 u mol/L).2Effection of DHDMBF30 on apoptosis of cell line Capan2:We used FCM to detect apoptosis peak of cell line Capan2 and found that contrast group had apoptosis peak before Go/Gi phase,the apoptosis peak of experimental group was markedly higher than that of contrast group.By Gimsa staining and scanning electron microscope we found that with the prolong of DHDMBF30 action time,the volume of Capan-2 cell gradually reduced,nuclear chromatin deeply stained> gathered and fractured,which sometime had apoptosis body.3.Expression of 5-LOX > COX-2 and VEGF gene and 5-LOX protein in cell line Capan-2 and effectoin of DHDMBF30 on them:Results of contrast group cell line Capan-2 RT-PCR were analyzed , a 416bp> a 304bp and 418bp band were detected; in experimental group we gained the same results,but the brightness of band decreased.which showed that there were expression of mRNA and protein 5-LOX, COX-2 and VEGF in cell line Capan-2,the inhibitor could inhibit them.By immunohistochemical we found that 5-LOX protein was positive in contrast group cell line Capan-2,which was brown granule and localized in cell cytoplasm or nuclear envelope; inexperimental group the staining was weak.Conclusions1. DHDMBF30 could inhibit the proliferation of cell line Capan2,induce its apoptosis.2.The mechanism of DHDMBF30 inhibiting the proliferation of cell line Capan2 and inducing its apoptosis could mainly be through inhibiting or down-regulating activity of 5-LOX, COX-2 and VEGF.3.DHDMBF30 may be one ideal drug for the prevention and therapy of pancreatic cancer.Part threeINHIBITION OF GROWTH OF HUMAN PANCREATIC CANCER IN NUDE MICE MODEL BY DHDMBF30Objective: 5-LOX/COX-2 inhibitor DHDMBF30 could inhibit the proliferation of cell line Capan2,induce its apoptosis in vitro. We want to study the effect of DHDMBF30 on human pancreatic cancer in nude mice model,investigate whether DHDMBF30 can become kind of drug for clinic.Methods: Cell line Capan2 were inoculated percutaneously on outer thigh to 12 nude mices,they were randomized into 2 groups.From the 48th h, DHDMBF30 and PBS were given. The nude mice models were observed,VEGFmRNA and protein were detected with RT-PCR and immunohistochemistry.Results:The tumor weight of DHDMBF30 group were (1.35 ±0.47) g ; PBS control group were (2.92 + 0.73) g(P<.01) .Expression of VEGF mRNA and protein in DHDMBF30 group was markedly decreased.Conclusions:1.DHDMBF30 can inhibit the growth of pancreatic cancer in nude mice.
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