| Background： Gastric cancer (GC) is a highly aggressive and lethal malignantcarcinoma, most patients received treatment in advanced stage. Although the developmentand progress of modern medical technology and advanced open and laparoscopic minimallyinvasive techniques, the effect of surgery is getting better, however, because of its highlyaggressive characteristics, recurrence and metastasis is the leading cause of death inpatients with gastric cancer. Many signaling pathways involve in the mechanisms of tumorinvasion and metastasis, recent evidence suggests that aberrant activation of the SHHsignaling pathway is a common feature of many highly aggressive tumors. Therefore, thespecific inhibition of the activation of SHH signaling pathway may be a new therapeuticmeasures. The role and mechanisms of SHH signaling pathway in invasion and metastasisof gastric cancer and whether blocking SHH signaling pathway can inhibit gastric cancercell growth, invasion and metastasis is still unclear.Objective: To investigate the role and its possible mechanism of SHH signalingpathway in the development, invasion and metastasis of gastric cancer, to provide a furtherunderstanding of GC and theoretical basis of new diagnosis and treatment.Method:1. Collection of the gastric cancer tissues and normal gastric mucosa specimens.2. To observe the expression Shh, Ptch1, Gli1protein by immunohistochemicalstaining and analyzed the results of clinical parameters and pathological data.3. To observe the expression of Shh, Ptch1, Gli1protein in seven kinds of gastriccancer cell lines (MNK-45, SGC-7901, MNK-28, BGC-823, MGC-803, AGS, HGC-27) byWestern blot assay.4. Select the gastric poorly differentiated adenocarcinoma cell of MGC-803whichstrongest expression of SHH signaling pathway molecules Shh, Ptch1, Gli1protein assubsequent experimental cells. Treated cells using different concentrations of cyclopamine (2μmol/L,5μmol/L,10μmol/L), to observe changes of cell proliferation by MTT assay,to observe changes of DNA synthesis and cell cycle by flow cytometry.5. To observe the changes in cell morphology using inverted microscope microscope,to detect apoptosis.in situ TUNEL assay.6. To detect changes of cell invasion and exercise capacity by scratch healing assayand transwell chamber experiments.7. To detect the expression of Ptch1, Gli1, P21, Bcl2, E-cad proteins by Western blotassay.8. To observe the changes of cell invasion and metastasis in abdominal andsubcutaneous xenograft experiments.Results:1. Constructed113cases of gastric cancer tissues and60cases of normal gastrictissue biopsy successfully, sliced completely, uniform thicknessly, distinct nuclear stainingplasma, red and blue moderately.2. Expression of Shh, Ptch1Gli1which belong to SHH signaling pathway wassignificantly higher in gastric carcinoma than that in normal gastric mucosa. The positiverate of protein in gastric carcinoma and normal gastric mucosa were71.68%(81/113),84.96%(96/113),80.53%(91/113) and31.67%(19/60),28.33%(17/60),20.00%(12/60). Shh,Ptch1, Gli1protein expression levels showed a significant positive correlation in gastriccancer tissues with Spearman correlation analysis.3. The key components of SHH signaling pathway, Shh, Ptch1, Gli1proteinexpressed in varying degrees in seven gastric cancer cell lines. Shh is highly expressed inBGC-823, MGC-803, AGS, SGC-7901, positive expressed in MNK-28, low expressed inMNK-45and lowest in HGC-27. Ptch1is highly expressed in BGC-823, HGC-27,MGC-803, positive expressed in AGS and SGC-7901, lowly expressed in MNK-45andMNK-28. Gli1expressed in all of the seven kinds of gastric cancer cell lines, but theexpression was significantly weaker than Shh and Ptch1. Gli1expressed higher in MNK-45,MGC-803, AGS, MNK-28cells than in BGC-823, HGC-27, SGC-7901.4. Treated MGC-803cell using different concentrations of cyclopamine (2μmol/L,5μmol/L,10μmol/L) to inhibit the SHH signaling pathway, Ptch1and Gli1proteinexpression was significantly decreased. We found that a dose-dependent manner with increasing concentrations of cyclopamine by Western blot analysis.cyclopamine cansignificantly inhibit the growth and proliferation of MGC-803cell, which was positivelycorrelated with the time and dose. MTT results showed that the growth curve growthslowed with time with the same drug concentration intervention, While at the same timepoint, the greater the concentration of the drug, slower the cell growth and proliferation.Flow cytometry test results suggest that the DNA synthesis significantly reduced withcyclopamine interventions, cell cycle G0/G1was significantly increased while the numberof S-phase cells decreased significantly. Western blot analysis found that P21expressionwas significantly increased.5. After blocking SHH signaling pathway, cell morphology turned round, blunt, cellshrinkage, nuclear condensation, adherent cells fall off and suspended. TUNEL assay foundthat cytoplasm reduced, volume shrinkaged, nuclear condensation and fragmentationincreased. Reflective enhanced significantly and apoptosis rate increased with Reflectionfluorescence microscope. Western blot analysis found that the anti-apoptotic gene bcl-2downregulation6. Cell invasion and exercise capacity was significantly inhibited after theintervention of cyclopamine. Cell scratch healing assay showed that healing ability ofexperimental group were weakened, Transwell chamber found that cell migration andinvasion in vitro decreased, inhibition rates were77.1±16.2%å'Œ80.3±15.3%. E-cadherinprotein expression was significantly increased after cyclopamine intervention blockingSHH signaling pathway by Western blot analysis.7. Animal subcutaneous xenograft experiments showed that tumor growth is lowerand tumor volume is smaller in the experimental group than in the control group.Immunohistochemistry showed that Shh, Ptch1and Gli1protein expression were negative,positive, negative in the experimental group; while Shh, Ptch1and Gli1protein expressionwere positive, strongly positive, weakly positive in the control group. The number ofintra-abdominal tumor, tumor size and the total weight of the tumor in the experimentalgroup were less than control group of Nude mice Intraperitoneal xenograft tumor model.Conclusion: SHH signaling pathway expression abnormally elevated in gastriccarcinoma with tumor differentiation, depth of invasion, clinical stage, lymph nodemetastasis. Gastric cancer cell lines similar to this case, SHH signaling pathway activity is strongest in the strongly aggressive and highly malignant cell line MGC-803. BlockingSHH signaling pathway could significantly inhibit DNA synthesis, blocking cell cycleprogression, inhibit cell proliferation, and its mechanism may be associated with theupregulation of p21protein expression. Blocking SHH signaling pathway decreased gastriccancer cell viability, increased apoptosis, the mechanism may be associated with theinhibition of anti-apoptotic Bcl-2gene expression. Blocking SHH signaling pathway caneffectively inhibit gastric cancer cell invasion, migration and athletic ability, the mechanismmay be associated with upregulation of E-cad protein expression. Animal subcutaneousxenograft experiments also show that after blocking SHH signaling pathway in nude mice,the tumor growth is slow, the tumor volume is small. Nude mice Intraperitoneal xenograftexperiments showed that the number,the volume and the total weight of tumor is less withblocking SHH signaling pathway.Therefore, abnormal activation of SHH signaling pathway is one of the characteristicsof a high degree of gastric malignancy, it is involved in the development of gastric cancer,anti-apoptosis, invasion and metastasis regulation, blocking SHH signaling pathwayenables malignant behavior reduced in gastric cancer. To further study the specificfunctions and mechanisms of SHH signaling pathway, including the relationship with othersignal pathways, the safety and efficacy issues, is important for a better understanding ofthe mechanisms of the pathogenesis, invasion and metastasis in gastric cancer, can providea more efficient and Potential treatment. |
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