MGMT和Survivin在乳腺癌中的表达及其临床意义.doc

MGMT和Survivin在乳腺癌中的表达及其临床意义 作者：王发亮，薄爱华，芦广萍，侯金超，吴荣薇【摘要】 目的：研究MGMT和Survivin在乳腺癌中的表达及其临床意义。方法：福尔马林固定，石蜡包埋乳腺癌和腺瘤标本，采用SABC免疫组织化学方法检测MGMT和Survivin在这些组织中的表达。结果：MGMT和Survivin在乳腺癌和乳腺腺瘤中的表达有显著性差异。MGMT在乳腺癌中的表达与患者的年龄、淋巴结转移有关，而Survivin仅与淋巴结转移有关。另外，MGMT和Survivin之间具有相关性（r=0.48,P<0.01）。结论：MGMT和Survivin的异常表达与乳腺癌的淋巴结转移有关；MGMT和Survivin可以作为判断乳腺癌发生和预后的重要指标；检测它们的表达可以指导临床上化疗方案的制定。 【关键词】 O6甲基鸟嘌呤DNA甲基转移酶；生存素；乳腺肿癌 Breast cancer is a common female malignant tumor.In recent years,the incidence of breast cancer is increasing in China while operation is the main therapeutic method nowadays.Because the genesis mechanism of breast cancer has not been clarified,and the effective pathway for predication was deficient,thus most patients with breast cancer were diagnosed at late stage,and they have missed the best opportunity for an operation.The curative rate will be increased if the pathogenesis is illuminated.It is usually considered that abnormal accumulation of gene mutation and the strong effects of Antiapoptosis proteins play important roles in the progression of tumor genesis.Recently an investigation has shown that MGMT and Antiapoptosis protein Survivin play important roles in the progression of breast cancer.In this study,to investigate the effects of MGMT and Survivin in the formation and progression of breast cancer,the expression of them in breast adenoma and breast cancer was detected. 1 MATERIALS AND METHODS 1.1 Clinical Data All cases were selected from the First Affiliated Hospital of Hebei North University,including 121 cases with breast cancer and 22 cases with breast adenoma.Among the 121 cases with breast cancer,57 cases were left,others were right,the that of patients with carcinoma was 47.8(2977 years),and the that of patients with adenoma was 35.7(2251 years).All the patients were not treated before specimens were taken.Among them,67 cases had lymph node metastasis,and 54 cases hadnt.All tissues were fixed with 10%formalin and embedded with paraffin,and two serial sections of 5m were cut from the paraffin blocks.Then the sections were placed on PolyLLysineCoated glass slides. 1.2 Immunohistochemical staining Each specimen was dewaxed,dehydrated,and then cooked for antigen retrieval in citric acid buffer.Slides were then incubated for 10 min in 0.3% hydrogen peroxide to quench endogenous peroxidase and then they were incubated overnight at 4 with respective antibodies such as rat monoclonal antihuman MGMT and rabbit polyclonal antihuman Survivin(both from Wuhan Boster Biological Technology Ltd).Slides were incubated for 30 min in the second antibody and then for 30 min in streptavidinperoxidase.The reaction products were visualized by 3,3diaminobenzidine tetrahydrochroloride(DAB).All positive MGMT and Survivin protein,stained brown yellow,were localized in the cytoplasm of the cells. 1.3 Data analysis Data analysis was carried out using SPSS statistical software package and was verified with 2test and spearmans rank.The value with P<0.05 was considered statistically significant. 2 RESULTS The positive rate of MGMT in breast cancer(57.85%)was higher than that in breast adenoma(13.64%)(2=14.56,P<0.01).While the positive rate of MGMT in 2944 yearold group(48.78%)was lower than that in the 6177 yearold group(86.96%),there was significant difference between the two groups(2=9.16,P<0.01),and there was significant difference between lymph node metastasis group and lymph node without metastasis group(2=20.55,P<0.01)(Table 1). As Table 1 shows,there was significant difference in the expression of Survivin between breast adenoma and breast cancer(2=8.77,P<0.01);its expression was significantly higher in lymph node metastasis group than that without lymph metastasis group(2 =15.90,P<0.01).Table 1 The expression of MGMT and Survivin in breast cancer and breast adenoma GroupsnMGMT(+)n%Survivin(+)n%Lymph node metastasiswith675176.124973.13without541935.192037.04Clinical pathological mechanism Infiltrating ductal carcioma804758.754353.75Infiltrating lobular carcioma412356.102663.41Positionleft573052.633357.89right644062.503656.25Age 2944412048.782253.664560573052.633256.146177232086.961565.22Breast cancer 1217057.856957.02Breast adenoma22313.64522.73 注:Compared with the group without lymph node metastasis,P<0.01;compared with the 6177yearold group,P<0.01;compared with the breast adenoma group,P<0.01 After spearmans rank correlation analysis,it could be found that in breast cancer the expression of MGMT was correlated with Survivin(r=0.48,P<0.01)(Table 2). Table 2 The relationship between MGMT and Survivin in breast cancer SurvivinN1MGMT+-+695415-521636N21217051 注:The expression of MGMT was correlated with that of Survivin(r=0.48,P<0.01) 3 DISCUSSION Humans MGMT gene,having a span of 170Kb1,locates in 10q26.Its expressing product is a kind of direct DNA repairase,having the ability to enable the methylation damage gene to obtain very good repair.It acts as a strong character in the process of gene repair in normal cells.This research discovered that it is also playing a similarly vital role in the occurrence and developing process of breast cancer. The positive rate of MGMT in breast adenoma was lower(13.64%),while,it was higher(57.85%)in breast cancer,the comparison between the two groups was remarkable(P< 0.01),it states clearly that MGMT is playing a vital role in the forming process of breast cancer.Its reason may be that once protooncogene in a cell has a sudden mutation,its product possibly can activate the DNA methylation enzyme through some way,thus causing the guanine in DNA to have the methylation in the O6 position,so something would be wrong in the process of pyrimidine and purine bases pairing(GCGT),or the O6 methyl guanine has the crossing link response to the cytosine on side chain,they both will cause difficulty in the process of DNA duplication.These exceptional changes may activate endonuclease through in examination system in the cell division cycle,then it will cause DNA to be fragment,the chromatin will shrink firmly,the cell will go to opoptosis.But once the MGMT gene promoter goes to demethylation,the MGMT gene will transform into the opening condition,its product MGMT protein can reposit the methyl from the methylation guanine to its own cysteine sulfhydryl,and thus it causes the cancer cell smoothly to get through the checking point in cell division cycle,and the cancer cell will obtain the infinite multiplication ability. This gives a suggestion us to assume that he if the expression of she MGMT in breast cancer patients is positive,it means that he himself or she herself has the ability to repair the methylation damage,therefore in the chemotherapy process it is not proper to use chloroNethylNnitrosourea(CENU),1,3bis(2chloroethyl)1nitrosoure(BCNU),MNNG and so on,which belong to the kind of chemotherapy medicine causing the DNA methylation,but other types of chemotherapy medicine can be used instead,such as Streptozotocin(STZ)2 or O6Benzylguanine(O6BG)3 to suppress the activeness of MGMT,more over gene silence technology can be used to reduce the activeness of MGMT in tumor cells46,and then the MGMT gene transfered to the normal tissue to strengthen the ability of normal cells to repair the DNA alkylating agent damage,and the tolerance of alkylating agent was enhanced710,then patients were given with the alkylating agent treatment. The positive rate of MGMT in 2944 yearold group was 48.78%(20/41),in 6177 yearold group was 86.96%(20/23),the comparison between the groups was remarkable(P< 0.01),this suggests that in the old group the proportion of demethylation of MGMT promoter is higher,the opening MGMT gene provides the safeguard for cancer cells smooth multiplication.This is possibly an important reason why the originating rate of breast cancer in old women is high,and way the breast cancer is difficult to treat and easy to recur in the old age crowd. Survivin gene,having a span of 14700bp 11,locates in 17q25.It doesnt express in the normal tissues(thymus exception)12,but it has a high expression in many kinds of tumors13,in many kinds of precancerous lesions and(or)benign pathological change(including breast adenoma,colon polyp,and so on13),others have also examined the Survivin expression,it may means that the secondary expression of Survivin possibly happens before the malignant changes or after the balance of the cell proliferation and death detunes. In this research,the positive rate of Survivin in breast adenoma was 22.73%(5/22),while it was 57.02%(33/65)in the breast cancer,the comparison between the two groups has a significance difference(P< 0.01).This means that Survivin plays a vital role in the occurrence process of breast cancer,the result also has further confirmed Survivins function of antiapoptosis.Survivin relies heavily on the cell cycle when it adjusts apoptosis,especially in G2M period.In the period Survivin and the cell cycle protein(p34cdc2/cyclin)B1 kinase bind together,and the former is phosphorylated by the later,this process carries on a key function in the protection of mitotic installment.The Survivins special function is causing resistivity of the tumor cells to paclitaxel and the radiotherapy treatment1416.The suggestion that the antagonist of Survivin should be used first if the expression of Survivin is high in breast cancer patients,then continue to treat with chemotherapy and radiotherapy. The expressions of MGMT and Survivin in the breast cancer were both higher than those in the breast adenoma,the comparison between the two groups has a significance difference(P< 0.01).The positive rates of MGMT and Survivin in lymph node metastasis group respectively were 76.12%(51/67)and 73.13%(49/67),those in the nonlymph node metastasis group were 35.19%(19/54)and 37.04%(20/54),the two proteins both have significance differences in the groups comparison(P< 0.01).So it can be inferred that the two proteins possibly have a common function in promoting the partial capillarity lymph vessel to form,and in the end,resulting in the cancer cells to transfer,therefore the two proteins in the breast cancer are symbol of bad prognosis. On clinical formulation of therapeutic schedule can be made by examining the expressions of MGMT and Survivin.If one or both of them have positive expression,in the course of surgery treatment,even if the tumescent lymph node nearby cancer stove cant be attention discovered,a lymph nodes sweeping should be made,a suitable auxiliary radiotherapy and chemotherapy should be given,and the development of the patients should be paid. Moreover,the statistical results also showed that the expressions of MGMT and Survivin in the breast cancer are moderately interrelated(r=0.48,2=27.43,P< 0.01).There are still no information about the relevant mechanism in domestic and foreign.It is possible that the process of phosphorylation of Survivin causes the CpG Island to go to methylation 17,and then the MGMT gene go to open.【参考文献】 1 Nakatsu Y,Hattori K,Hayakawa H,et al.Organization and expression of the human gene for O6methylguanineDNAmethyltransferaseJ.Mutat Res DNA Repair,1993,293(2):1191322 Panella TJ,Smith DC,Schold SC,et al.Modulation of O6alkylguanineDNA alkyltransferasemediated carmustine resistance using streptozotocin:a phase I trialJ.Cancer Res,52(9):245624593 Friedman HS,Kokkinakis DM,Pluda J,et al.Phase I trial of O6benzylguanine for patients undergoing surgery for malignant gliomaJ.J Clin Oncol,1998,16(11):357035754 Manome Y,Yoshinaga H,Watanabe M,et al.Adenoviral transfer of antisenses or ribozyme to O6methylguanineDNA methytransferase mRNA in brain tumor model resistant to chloroethylnitrosoureaJ.Anticancer Res,2002,22(4):202920365 Ji SP,Zhang YP.Modulation of O6methylguanineDNA methytransferase mediated(4amino2methyl5Pyrimidinyl)methyl3(2Chloroethyl)3nitrosourea resistance by antisense RNAJ.Cancer Detect Prev,1999,23(5):4224276 Zhang QW,Ohannesian DE,Erickson LC.et al.Hammerhead ribozymemediated sensitization of human tumor cells after treatment with 1,3Bis(2chloroethyl)1nitrosourea chemotherapyJ.Antibiot Gene Ther,2004,309(2):5065147 Wu M,Kelley MR,Hansen WK,et al.Reduction of BCNU toxicity to lung cells by highlevel expression of O6methylguanineDNA methytransferaseJ.Am J Physiol Lung Cell Mol Physiol,2001,280(4):7557618 Zieske SP,Gerson SL.Lentiviral transduction of P140k MGMT into human CD34+ hematopoietic progenitors at low multiplicity of infection confeis significant resistance to BG/BCNU and allows selection in vitroJ.Mol Ther,2002,5(1):3813879 Ragg S,Welliver MX,Bailey J,et al.Direct rev