Expression Of TRAIL And Survivin In Gastric Carcinoma And Inducement Of Gastric Carcinomous Apoptosis In Vitro

Gastric carcinoma is one of the most common malignant tumors in our country , which greatly affects human health and life. Actually the multidisciplinary therapies of gastric carcinoma include surgery, chemotherapy and immunity therapy. The effect , however , has not shown a significant improvement. The occurrence and development of gastric carcinoma is not only related to the abnormal hyperplasia of tumor cell, but also to the abnormal apoptosis of the tumor cells. The unusual mechanism of cell apoptosis is very important in the occurrence of tumor and the anti-tumor treatment. It may be become a basic strategy to treat the gastric carcinoma by using the intervene apoptosis technology that the target is to induce the tumor cell.Apo2 ligand or tumour necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL) is one of the several members of the tumour necrosis factor (TNF) gene superfamily that induce apoptosis through engagement of death receptors (DRs). Apo2L/TRAIL interacts with an unusually complex receptor system of two DRs and three decoys. Various studies show that TRAIL potently induces apoptosis in a broad range of man transformed cell lines and cancer cell lines, but not in many normal cells. It seems to be an ideal factor for creating a selective and powerful antitumor effect.TRAIL can improve the apoptosis through its peculiar receptor; DR4 and DR5 are death receptors they can transform the death message sign, then induce the rapidity, enormous reaction of apoptosis. DcRl and DcR2 is decoy receptors, they can not transformthe message because they absent or partly absent the death domain, so only to combine the receptor competently to play a role in the protection.Indeed, human recombinant soluble TRAIL induces apoptosis in a variety of cancer cell lines, includes cell lines derived from cancers of the colon, lung,breast, prostate, pancreas, kidney, central nervous system and thyroid, as well as from leukaemia and multiple myeloma. But the resistance of some cancer cells to TRAIL present the main obstacles for clinical experimentation. Chemotherapeutic drugs are used in the treatment of most cancers; however, these drugs are only partially effective in the treatment of tumors. Therefore, new ways of treating cancer must be developed, combined treatment of TRAIL with chemotherapeutic drugs will provide a synergistic apoptotic response . TRAIL might enhanced the effect of ADM and 5-Fu in our experiment.Survivin is an inhibitor of apoptosis protein (IAP), the expression of Survivin gene has a tightly relation to the occurrence of gastric carcinoma, play a key role in the carcinogenesis of tumor. The study indicated Survivin can degrade the target gene, inhibit the duplicate, transcription and processing of after transcription, induce the automatic apoptosis of cell and obviously inhibit the growth of tumor, then to provide a new target for the gene therapy of tumor.TRAIL induces the apoptosis of cell by two ways, any factor that can change the level or activity of the apoptosis in this two ways can probably affect the apoptosis that induced by TRAIL. Survivin can directly inhibit the activity of tail common way of two apoptosis way, which is Capase-3. To use the Survivin ASODN to inhibit the expression of Survivin gene will revising the resistance of tumor cell to TRAIL in some way.Now the study about TRAIL and its receptors is only single immuchemical or the observe of RT-PCR. There was no report about the intervene of drug to improve the sensitive and TRAIL and apoptosis. We want to explore from the next factors (l)Using the technology of immunhistochemistry SP , in situ hybridization and RT-PCR to test the relation between the expression of TRAIL and its receptors protein and mRNA in gastriccarcinoma and the adjacent carcinoma tissue; (2)Using the RT-PCR and the immunhistochemistry to detect the expression of protein and mRNA of Survivin in gastric carcinoma and the adjacent tissue to explore the mechanism of Survivin on apoptosis and angiogenisis of gastric carcinoma , analysis the relation between Survivin and TRAIL; (3) To observe the function of hrsTRAIL(human recombined soluble TRAIL) combination with chemotherapeutic agents and Survivin ASODN in human gastric cancer cell line BGC-823 . Part one:The study about the relation between apoptosis and expression of protein and mRNA of TRAIL and its receptors in gastric carcinoma Materials and Methods:1. Using the technology of immunohistochemistry SP ,in situ hybridization, RT-PCR to detect the expression of protein and mRNA of TRAIL and DR4,DR5,DcRl,DcR2 in 76 cases of gastric adenocarcinoma, 32 cases of moderately to severely atypical hyperplasia tissues , 22 cases of intestinal metaplasia tissues, 21 cases of atrophic gastritis tissues and 76 cases of normal mucosa of gastric adenocarcinoma2. Using the RT-PCR to test the relative level expression of the mRNA of TRAIL and its receptors DR4, DR5, DcRl, DcR2 in the cases of gastric carcinoma, adjacent carcinoma tissues and the normal gastric mucosa.3. To indicate the apoptosis index (AI) in these tissues by using TUNEL technology, analysis the relationship between AI and the expression of protein and mRNA.of TRAIL and its receptors.4. Statistical analysis: All the dates were analyzed by SPSS 10.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [x ±s] The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable are analyzed by the correlation analysis.The level of significant difference isa =0.05. Results :1. The result of immunochemical and in situ hybridization indicates: The expression of TRAIL protein and mRNA decreased gradually in normal mucosa, atrophic gastritis, intestinal metaplasia, moderately to severely atypical hyperplasia tissues and gastric adenocarcinoma. Protein and mRNA levels in normal mucosa, atrophic gastritis, intestinal metaplasia were significant higher than it in gastric adenocarcinoma; and the levels in normal mucosa was significant higher than it in moderately to severely atypical hyperplasia tissues {P <0.05). DR4 and DR5 protein and mRNA expression level had not significant difference among the every group but the level of protein and mRNA of DcRl,DcR2 have significant difference(P <0.05).2 The expression of TRAIL mRNA level in normal mucosa and adjacent carcinoma tissue was significant higher than it in gastric adenocarcinoma by RT-PCR(P <0.05);3. The relative level of expression of TRAIL protein and mRNA gradually decrease among the different differentiation of gastric carcinoma (P <0.05). the protein and mRNA of every receptor is not significance difference with the gastric carcinoma differentiation.4. It was statistically significant of AI between the TRAIL protein and mRNA positive group and negtive group (P<0.05);whereas no obvious correlation was determined among DR4, DR5, DcRl protein and mRNA and AI (P>0.05) .5. The level of DR5 protein and mRNA was higher than that of DR4, but it had no statistically significance (P>0.05) .6.There was positive relation between the protein and mRNA of TRAIL and its receptors.Part twoThe mRNA and protein expression of Survivin and its correlation with theclinicopathological factors in gastric carcinomaMaterials and Methods:1.Using the technology of immunohistochemistry SP and RT-PCR to detect the expressionof protein and mRNA of Survivin in 76 cases of gastric carcinoma and normal gastric mucosa accordingly.2. Labelling MVD by CD34 in these tissue.3. Statistical analysis: All the dates were analyzed by SPSS 10.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [ x + s] The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable are analyzed by the correlation analysis.The level of significant difference is a =0.05.Result:1. The expression of protein and mRNA of Survivin was not examined in the normal gastric muscoa but expressed highly in the gastric carcinoma, the results showed a correlation with invasion,lymph node metastasis and TNM stage (P<0.05) .2. hi gastric carcinoma negative correlation was showed between AI and the expression of Survivin protein and mRNA and the correlation was showed between MVD and the expression of Survivin protein and mRNA3. In gastric carcinoma the level of TRAIL protein and mRNA was negatively associated with Survivin (P<0.05)Part threeEffects of hrsTRAIL combination with chemotherapeutic agents andSurvivinASODN in human gastric cancer cell line BGC-823Chapter oneEffects of hrsTRAIL combination with chemotherapeutic agents in human gastriccancer cell line BGC-823Materials and Methods:1. The proliferation inhibitory and apoptosis rates of BGC-823 cells were measured byMTT assay and flow cytometry and TUNEL method respectively after treatment byhrsTRAIL combination with 5-Fu, ADM .2. The levels of Survivin> Caspase-3 protein in BGC-823 cells was examined by Western blot.3. The expression of DR4, DR5, DcRl and DcR2 was detected by immunohistochemstry and RT-PCR respectively. The morphologic changes were observed by microscope.4. Statistical analysis: All the dates were analyzed by SPSS 10.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [x ±s] The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable are analyzed by the correlation analysis.The level of significant difference isa =0.05. Results:1. There was significant differences between the proliferation inhibitory rate of BGC-823 cells in 10 ng/ml,100 ng/ml,300ng/ml hrsTRAIL groups and control groups(P<0.05) . A cooperative effect of 10 ng/ml,100 ng/ml,300ng/ml hrsTRAIL and lOOmg/L 5-Fu on proliferation inhibitory rate of BGC-823 cells was found (P<0.05 ) ; and the same results were found in 100 ng/ml,300ng/mlhrsTRAIL combined with 0.1 mg/1^ 1.0mg/L> lO.Omg/LADM (P<0.05) .2. TUNEL and FCM revealed that ADM and 5-Fu could enhance TRAIL induced apoptosis of gastric cancer cell line BGC-823, the trend was the same as the result of MTT.3. There was no significant change of expression of TRAILR protein and mRNA before and behind therapy (P>0.05) .4. The level of survivin protein enhanced in group of 50mg/L5-Fu,1.0 mg/L ADM and hrsTRAIL (10 ng/ml) ; moreover, the increased trend became low accompany with the increase of concentration ofhrsTRAILand combined with 5-Fu and ADM (P>0.05) .5. The level of Caspase-3 protein increased in group of 50mg/L5-Fu,1.0 mg/L ADM and hrsTRAIL (10 ng/ml) sigularly; moreover, the increased trend enhanced accompanywith the increase of concentration of hrsTRAILand combined with 5 -Fu and ADM,thereinto there was significant difference of the level of Caspase-3 protein in group ofhrsTRAIL (300 ng/ml) combined with 5-Fu group and hrsTRAIL (300 ng/ml) combinedwith ADM group (PO.05) .6. The change in different degrees was observed in different groups by microscope..Chapter twoStudies on treatment of Survivin ASODN combination with hrsTRAIL of humangastric cancer cell line BGC-823Materials and Methods:1. The morphologic changes of human gastric cancer cell line BGC-823 .were observed by microscope.2. The proliferation inhibitory and apoptosis rates of BGC-823 cells were measured by MTT assay and flow cytometry respectively after the treatment of Survivin ASODN combination with hrsTRAIL.3. The levels of Survivin, Caspase-3 protein and mRNA in BGC-823 cells were examined by Western blot and RT-PCR.4.Statistical analysis: All the dates were analyzed by SPSS 10.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [ x ± s] The comparison of positive rates uses the Chi-square, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable are analyzed by the correlation analysis.The level of significant difference is a =0.05. Results:1. Apoptosis cells were detected in different stages by microscope2. There was significant difference between the proliferation inhibitory rate of BGC-823 cells in Survivin ASODN combination with hrsTRAIL groups and Survivin ASODN group sigularly (PO.05); there was significant difference of all therapy groupcompared with control group and N-ODN group (P<0.05) .3. The apoptosis rates of gastric cancer cell line BGC-823 increased gradually accompany with the enhancement of concentration of Survivin ASODN,the trend was as the change of inhibitory rate.4. The level of survivin protein and mRNA in every therapy group was lower than control group and N-ODN group; O.lumol/L ASODN could not affect the the level of survivin mRNA obviously(P>0.05),but the same concentration ASODN could change the ASODN protein (PO.05) ; The level of survivin protein decreased according to the concentration ASODN increased, but no correlation was found between three groups and control group and N-ODN group(P>0.05).5. The level of Caspase-3 protein and mRNA in every therapy group was higher than control group and N-ODN group (PO.05) ; The level of Caspase-3 protein and mRNA increased according to the concentration ASODN increased, it has significant difference between Survivin ASODN combination with hrsTRAIL groups and Survivin ASODN groupsigularly (PO.05) .Conclusions:1. For the first time, we detected the expression of TRAIL and receptors protein and mRNA in gastric carcinoma, adjacent gastric carcinoma tissues by combining with immunohistochemistry,in situ hybridization and RT-PCR methods.2. The expression of TRAIL and receptors protein and mRNA was decreased gradually in normal mucosa, atrophic gastritis, intestinal metaplasia, moderately to severely atypical hyperplasia and gastric carcinoma tissues, it showed there was lost of TRAIL in the process of development of gastric carcinoma and indicated that TRAIL was correlative with carcinogenesis of gastric carcinoma.3. The expression of TRAIL protein and mRNA had significant relationship with tumors differentiation (PO.05). It had no statistically significance between TRAIL and receptors protein and mRNA and the clinical pathology of gastric carcinoma.4. The protein and mRNA of DR4, DR5 was expressed widely in the gastric carcinoma and adjacent gastric carcinoma, but there was difference expression of protein and mRNA of DcRl, DcR2 between the gastric carcinoma and adjacent gastric carcinoma, it indicates the reason that TRAIL can induces the apoptosis of tumor cell is the result of the combining between the TRAIL and TRAILR.5. Survivin can inhibit the apoptosis of gastric carcinoma; improve the angiogenesis of gastric carcinoma and the occurrence, development of gastric carcinoma.6. The expression of Survivin protein and mRNA was negative relationship to the positive expression of TRAIL protein and mRNA in gastric carcinoma (P<0.05). Survivin and TRAIL are two important targets in the apoptosis treatment of gastric carcinoma cells.7. The treatment of hrsTRAIL can inhibit the gastric carcinoma cell BGC-823 lines in some way. The coordination action of hrsTRAIL combining 5-Fu and ADM to kill the gastric carcinoma cell BGC-823 is achieved by inducing the apoptosis of cells.8. It was no relationship between the cell membrane distribution of Death receptor and Decoy receptors and the effect of cell apoptosis inducing by ADM , 5-Fu combination with hrsTRAIL.9. Survivin and Caspase-3 might play an important role in resistant characteristic of hrsTRAIL in gastric carcinoma cell BGC-823.lO.Using Survivin ASODN to restrain the expression of Survivin can inhibit the growth ofgastric carcinoma effectively. The inhibitory effect on the cell growth could increased bydown -regulation of Survivin gene.11 .Transfection of Survivin ASODN could increase the effect of apoptosis by hrsTRAIL;Using the Survivin ASODN to restrain the Survivin can reverse partly the insistence ofgastric carcinoma cell to hrsTRAIL.