| BackgroundGastric cancer is a malignant tumor of digestive system with high morbidity and mortality,which threatens the life and health of Chinese people.The early gastric cancer has metastasis tendency.So more than fifty percent of the patients are diagnosed in advanced disease.Although surgery is the main method in the treatment of gastric cancer,most patients had local invasion and distant metastasis at the time of diagnosis,loss of surgical opportunity,thus limiting the application of surgical methods and treatment effects.Chemotherapy is the preferred treatment for localrecurrence and advanced gastric cancer patients without surgical opportunity.However,the therapeutic effect is still unsatisfactory.The objective response rate to chemotherapy is only 20-40%for the patients,and the median overall survival time(OS)is 6-11 months.At present,there is a lack of standard chemotherapyregimen in the treatment of gastric cancer,so the survival situation of gastric cancer is not optimistic.Therefore,in recent years,many researchers are devoted to the discovery of new specific biomarkers and exploration for new treatment of gastric cancer,in order to improve the diagnosis rate of early gastric cancer,improve the prognosis of patients with gastric cancer.In the field of tumor biology,molecular targeted therapy has aroused widespread concern,in order to improve the specific antitumor effect,reduce the resistance and nonselective toxicity.Multiple clinical studies on the mechanisms of molecular targeted therapy for gastric cancer have been carried out,such as epidermal growth factor,insulin-like growth factor receptors,c-Met,cell cycle,apoptosis,key enzymes,mTOR signal pathway andangiogenesis regulation,immune checkpoint blockade.Trastuzumab is the first target drug to be approved for the standard treatment of gastric cancer.ToGA clinical study showed that trastuzumab combined with chemotherapy can improve the survival rate of HER-2 positive patients with advanced gastric cancer,combination therapy was significantly better than chemotherapy alone.However,the over expression rate and amplification numberof HER2 in gastric cancer is very low,it is reported to be only 6-23%,its application in clinical treatment is limited.In April 2014,on the based of the RAINBOW experimental results,human vascular endothelial growth factor receptor 2 antagonist ramucirumab was approved by FDA,used in combination with chemotherapy in the treatment of second-line or failure of first-line chemotherapy for advanced gastric and gastroesophageal junction adenocarcinoma.Mesylate apatinib,antiangiogenesistyrosinase inhibitor,independently developed in china,significantly improved OSin patients of second-line chemotherapy failure with advanced gastric cancer.In addition,the most of these targeted drugs for gastric cancer have not achieved satisfactory clinical results.Stathmin is an ubiquitous cell signaling molecule,which is a key protein that participates in microtubule polymerization,and may play an important role in mitosisor other various cellular processes.Through its phosphorylation and dephosphorylation,Stathmin regulates microtubule system dynamic balance,controls cell cycle,changescell biological behavior.The expression ofStathmin is regulated by many transcription factors and protein kinase/phosphatase,regulates microtubule dynamics,cell proliferation and activity,apoptosisof malignant tumor cellswhichis of great significance forthe occurrence and development of malignant tumor,molecular diagnosis and curative effect prediction.The recent research found that Stathmin was over-expressed in a variety of tumor tissues,correlated with tumor differentiation,lymph node metastasis,TNM stage of disease and the clinical prognosis.In previous study,we first analyzedthe expression of Stathmin gene in two gastric cancer cell lines(SGC7901 and MGC803),8 cases of resection of tumor tissue and paracancerous tissue,the results showed that high expression of Stathmin in twogastric cancer cell lines andtumor tissues and adjacent normal tissues showed no expression or only weak positive expression.Stathmin may play an important role in differentiation and proliferation.Inhibitionor blocking Stathmin could affect the mitosis,growth,metastasis,survival and tumor clone formation of tumor cells,the cell cycle was blocked in the G2/M phase and the apoptosis rate increased.Because its high expression wasclosely relatedwith microtubule dependent process may affect the chemotherapy efficacy of anti microtubule drugs,and lead to drug resistance of tumor cells.We hypothesize that anti Stathmin therapy with docetaxel may have a synergistic effect and because both of them take microtubule pathway as a drug target.Blocking theexpression and biological function of Stathmin may become a treatment for cancer.There is no system study between Stathminandoccurrence and development of gastric cancer.Theimportant role and exact mechanism of Stathminin gastric malignant tumor is not clear.On the basis of previous studies,we detected the expression of Stathmin in gastric cancertissues and adjacent normal tissues,and to study the relationship between the clinical diagnosis and prognosis characteristics of gastric cancer and Stathmin,provide ideas for screening molecular markers;The correlation between gastric cancer and sensitivity to chemotherapy and survival rate was analysed,and will be helpful toimprove prognosis and chemotherapy benefits in gastric cancer;Stathmin ASODN exertedeffects in growth and proliferation of gastric cancer cellsin vitro and in vivo experiments;Combination of Stathmin inhibitors with docetaxel could play synergistic effect on proliferation of gastric cancer cells.In brief,our study will provide clinical evidence for the new strategy ingastric cancertreatment and potential molecular target markers of gastric cancer.Part 1.The significance of Stathmin expression and and its correlation with chemotherapy efficacy and prognosisObjectives1.To detectthe expression of Stathmin in gastric cancer tissues and adjacent tissues by western blot and immunohistochemical method.2.To study the relationship betweenexpression of Stathmin anddifferentiation oftumor cells,invasion depthand lymph node metastasis ingastric cancer,to provide some ideas for the screening of molecular markers of gastric cancer.3.To investigate the correlation between theStathmin expression and the sensitivity to chemotherapy and survival rate,and to provide evidence for the clinical selection of chemotherapy and the prognosis of gastric cancer.MethodsThis experiment collected 56 cases withgastric cancer tissues and cancer adjacent tissueswith surgical resection(radical resection,palliative resection)andtissue biopsy from January 2009 to December 2009,in Shandong provincial Qianfoshan Hospital,and which were pathologically confirmed gastric cancer,were not receiving preoperative radiotherapy,chemotherapy or minimally invasive treatment,all sections were according to the national standard and the group of gastric cancer and pathology physicians review.The expression of Stathmin in gastric carcinoma in the study was detected using immunohistochemistry and western blot;The relationship between Stathmin expression and clinical pathological features of gastric cancer were assessed;Correlation between expression level of Stathmin and the DC(docetaxel and cisplatin)chemotherapy efficiency,clinical benefit rate,1 and 3 years survival ratewere explored,to provide the basis for the formulation of individualized treatment plan.Results1.Expression of Stathmin in gastric cancer tissues and adjacent tissuesThe results of immunohistochemistry showed that Stathmin positive color was pale brown or brown.Our results showed that majority of the cells in the cancer tissues were positive for Stathmin,while the staining was obviously less intense in the adjacent regions.In addition,we also detected the expression of Stathmin protein in gastric cancer and adjacent tissues by western blot method.The expression level ofStathminprotein was significantly increased in the tumor tissues.In all samples,the positive expression rate for Stathmin in the cancer tissues was 71.4%,while expression rate was only 37.5%in the adjacent regions(P<0.05);2.Stathmin expression and clinical and pathological analysis of gastric cancerBy analyzing the expression of Stathmin in gastric cancer and clinical pathological data,the expression of Stathmin was closely correlated with tumor differentiation(P<0.001),invasion depth(P<0.001),lymph node metastasis(P<0.001),negatively withgender(P>0.05)and age(P>0.05).3.Stathmin and chemotherapy efficacy and prognosisThe effectiverate and clinical benefit rate in the Stathmin-positive group were 10.5%and 52.6%,respectively,which were much lower than the Stathmin-negative group(23.1%and 69.2%,respectively)(the effective rate P=0.022,the clinical benefit rate P=0.020,P<0.05),with statistically significant difference.Two groups of patients were followed up for a period of 1 years and 3 years.There was no significant difference in the 1 yearsand 3 years survival rate of the two groups(P>0.05).ConclusionsThe positive expression of Stathmin protein in gastric carcinoma was higher than that of adjacent tissues.Expressionof Stathmin protein was positively correlated with tumor differentiationand clinical stage of TNM,whilenegtively correlated with gender and age group.The chemotherapy rate and the clinical benefit rate decreased inStathmin positive expression group,we have not found its expression has significant impact on survival.Therefore,the expression level of Stathminwill be a marker for the prognosis of gastric cancer,and can be used as a reference for assessing the efficacy of chemotherapy.Part2.Effects of Stathmin antisense oligodeoxynucleotides on the growth and tumorigenicity of gastric cancer cellsObjectives1.To study the effect of Stathmin ASODN on proliferation and apoptosis of gastric cancer cell line in vitro2.Toinvestigate the inhibitory effect of Stathmin ASODN on the tumor growth of tumor bearing micein vivo3.To evaluate the effect of docetaxel combined with Stathmin ASODN on the growth of gastric cancer cellsin vitroMethods1.Gastric cancer cell line SGC7901 was transfected with Stathmin ASODNorSODN.2.RT-PCR was used to detect the expression level of Stathmin in ASODN transfected cells,SODN transfected cells and control cells.The expression level of Stathmin protein was detected by western blot method.3.MTT method was used to detect the proliferation of cells.4.The cell doubling time of Stathmin ASODN,SODN treated SGC 7901 cells and non-transfected cells was observed.5.The effect of Stathmin ASODN on apoptosis of gastric cancer cells was analyzed by flow cytometry.6.The growth of gastric cancer in nude mice transfected with StathminASODN and SODN was observed,tumor diameter wasmeasured.Results1.Effects of StathminASODN on the proliferation of human gastric cancer cell line SGC 7901 in vitroStathmin ASODN or SODN was transfected into SGC 7901 cells.After transfecting cell at 72 h,the relative expression of Stathmin mRNA were 0.21 ±0.08,1.01 ± 0.43,and 1.44 ± 0.54 by RT-PCR in the ASODN-,SODN-transfected cells,and control cells,respectively.The level of Stathmin protein were decreased in the antisense oligonucleotides transfected cells than nottransfected cells by western blot assay.After treated with different concentrations of Stathmin ASODN in SGC 7901 cell,cell proliferation was suppressed obviously compared with the SODN group(P<0.05)by MTT assay.The inhibition effect reached the peak in 72 hours,the best treatment concentration is 20?mol/L.Cell doubling time prolonged.AnnexinV-FIT C(+)PI(-)early apoptotic cells were increased by flow cytometry.2.Effects of Stathmin ASODNon the tumor growth of tumor bearing mice in vivoThe growth of tumor transfected with Stathmin ASODN in nude mice was obviously slower than the control group.The mean diameter of gastric tumors transfected with Stathmin ASODN in nude mice was 4.04±10.17mm3,which wasmuch smaller than the SODN-treated group(8.01±0.21mm,),with significant difference between the two groups(P<0.05).3.Effects of StathminASODN combined with docetaxel on the proliferation of SGC 7901 cellsThe proliferation assessment showed that docetaxel inhibited the proliferation of SGC 7901 cells,in a dose-and time-dependent manner.When combined with the Stathmin ASODN,the inhibitory effects on the cell proliferation were enhanced,which were significantly stronger than either treatment alone(P<0.05).Conclusions1.Stathmin ASODN can significantly inhibit the proliferation of gastric cancer cells in vitro and in vivo.Stathmin may be a potential molecular marker and target for the treatment of gastric cancer.2.Stathmin ASODN will enhance the inhibitory effect of docetaxel on the proliferation of gastric cancer cells,indicating synergistic effect of the combination.Stathminwith chemotherapy may have synergistic anti-tumor effect,the clinical benefit of combination therapy will be greater. |
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