Experimental And Clinical Study On The Influence Of Activating CD40-CD40L Path On Proliferation Of Breast Cancer Cells

BackgroundNowadays, breast cancer is one of the most common malignant tumors. Likeother malignant tumors, the occurrence and development of breast cancer are due tothe weakness and insufficiency of the immune function of body. And as a result thebody lacks the strong and powerful immunologic surveillance or killing on tumorcells. Tumors cells breed rapidly and partially invade the body. Moreover, they makethe metastasis in the distance. As the immune function is one of the most importantfactors which decides breast cancer patients' prognosis, the immune treatment hasalready guided an important direction for both breast cancer treatment and breastcancer studies.According to the characteristics of immune reaction, the immunity could bedivided into two types—specific and none-specific immunity. Specific immunity isdivided into three stages—antigen presentation, activation of immune cells andimmune reaction, among which the activation of immune cells is the most important.Lymphcytes are the main immune cells, and to activate them acquires two relatedstimulated signals. One is called anti-specific signal, or the first signal induced byTCR/CD3. T cell antigen receptor acquires the first signal or anti-specific signal assoon as it recognizes MHC-antigen-complex expressed by APC. The other is called anti-none-specific signal or the second signal induced by mutual simulatedmolecules such as B7-CD28. Neither the two signals could be missing during thebody anti-tumor immune process. When T cells owns the first signal but lacks thesecond signal, it is in a state of none-response, or anergy. Corresponding with this,according to the characteristics of tumor treatment, the immune treatments of tumorcould be divided into two types: Specific and none-specific immunity. The specificimmunity treatment is of great importance for the immune research and applicationas well. At present the strengthening of the specific immunity treatment has beenimplemented mainly through the enhancement of immungenicity on tumor antigenpeptide, release the immune tolerance and inhibition of body on tumors, strengthenthe specific active immune cells' immune-antigen-reaction. But regrettably, so far,although having target tropism and lacking of severe poisonous or side-effects, pureimmune treatments have not certainly obtained any breakthrough in curative effect.Now operation, radiotherapy and chemotherapy have brought revolutional progressto patients' treatment effect. A large number tumor cells were killed. The distantmetastasis rates had been reduced, and the survival rates have been unceasinglyenhanced. More and more many scholars, both at home and abroad, have noted thatoperation, radiotherapy and chemotherapy injury, because of lacking of better targettropism, the immune function as soon as a large number tumor cells been killed.Chemotherapy is typical case. On one hand, a large number tumor cells are killedand the burden of tumor is alleviated. Thus tumor cells induced-immune inhibitionon the body will be released. On the other hand, as the chemotherapy medicine killsa large number of tumor cells, it injuries hemopoietic system such as immune systemand bone marrow, also it inhibits the production of immune cells and influence thebody immune function. For instance, chemotherapy medicine ADM and Docetaxalare widely used for breast cancer and well recognized that could achieve the best therapeutic effect. Both have severe bone marrow inhibition and are poisonous to theheart. And they are threats to patients. The problem clinically wanted to be solved isthat reducing ADM and docetaxal's dosage without lowering the therapeutic effect.In recent years, more and more discoveries have been made that activatingcostimulatory signals can take anti-tumor effect in many aspects. Also they enhancethe sensitivity of tumor cells to chemotherapy medicine because of clear treatmenttarget tropism. Judging from the strategy of tumor therapy, the best therapeutic effectcould be gained by taking anti-tumor effect on various levels. And it could diminishthe opportunities for toralence. According to this finding, in this study, we plan tocombine activating costimulatory signal with chemotherapy for breast treatment. Wehope make better treatment effect, and reduce chemotherapic drugs' dosage withoutlowering the therapeutic effect.ObjectivesThe aim of this research are1. to study the states of the costimulatory pathmammary CD40-CD40L(CD154), B7- CD28 for breast cancer, and the influence of surgery, chemotherapyto the costimulatory path CD40-CD40L (CD 154), B7-CD28;2. to explore the influence and possible mechanism of CD40-CD40L, B7- CD28for breast cancer cells' proliferation;3. to explore the Synergism and possible mechanism of combing CD40-CD40Lwith ADM or Docetaxal for breast cancer cells' proliferation.Research SignificanceCombing CD40-CD40L with chemotheary may clear tumor cells effectively andimprove the therapeutic effect. Also it may enhance the sensitivity of tumor cells to chemotherapy medicine thus reducing the poisonous or side-effects caused by anumber of chemotherapy medicine. So the breast cancer immmune therapy whichtreats CD40- CD40L as the target will probably explore a new way for bettering ofcommon used clicinal therapeutic methods of breast cancer. We hoped this researchcan provide the preliminary experimental basis for this mentality.Methods1. Among 70 patients with breast diseases, 48 patients out of 70 suffer from localadvanced breast cancer is divided into two groups: Group A (underwent modifiedradical mastectomy, n=22), Group B (underwent modified radical mastectomy aftertwo courses of properative chemotherapy. The regimen is CAF. n=26). And the rest22 patients with none-maliganant breast diseases make up the Group C (controlgroup, received goitre excision of breast gland, n=22). Label-gathering:Twice. Firstly,to prick 2ml venous blood from each patient without eating and drinking anything inthe three groups at the very beginning of treatment in the morning. Secondly, whenGroup A and Group C are on the third day after operation and Group B is on thethird day after the second chemotherapy, prick 2ml venous blood from each withouteating and drinking anything in the morning. With flow cytometry to analyse theexpression of above mentioned labels CD3⁺T, CD4⁺T, CD8⁺T, CD 16⁺CD56⁺NK andcostimulatory molecule and their ligands CD40,CD40L,CD80,CD86,CD28.2. With flow cytometry to analyse the expression of CD40,CD80 and CD86 ofMCF-7,SK-BR-3,MDA-MB-435,T47D,ZR-75-30 and original generation breastcancer cells(come from Group A and Group B).Compare the expression of CD40,CD80 and CD86 of original generation breast cancer cells before and after thechemotherapy.3. With method MLC to cause proliferation of Balb/c,KM and C57BL/6J inbred strain male-mouse's spleen cells. With method MTT to test the proliferation level oflymphocytes after interrupteding with anti-CD154 and anti-CD80. With methodMTT to test the lymphocytes'immune states,immune regulation and antigenspecification of immune regulation. With method MTT to test the influence of KMcells+rmIL-2 on the lymphocytes'immune states.4. With method MTT to test the proliferation level ofMDA-MB-435,MDA-MB-231after interrupting with rhCD40L,rhCD40L+IFN-Y,rhCD40L+ADM,rhCD40L+Docetaxal.5. With method PI to test the periodic distributions of breast cancer cellMDA-MB-435 and MDA-MB-231 after interrupting with rhCD40L.6. With method MTT to test the cell membrane molecules CD80,CD86,CD54(ICAM-1),CD95 (Fas),CD95L (FasL),CD95L,HLA-DR of MDA-MB-435and MDA-MB-231' cell membrane molecules.7. With method PCR to test apoptosis related gene bcl-xl and bax's change ofMDA-MB-435 and MDA-MB-231.8. Statistics analysis: The data expressed with (?)±S. With Independent-Samples TTest to compare means between two groups (Levene's Test for non-equality ofvariaces). With One-Way ANOVA Test to compare means between many groups(AsP＜0.05, for equality of variances, method LSD Test compare means among groups,then method Dunnett compare means between every group and control group, fornon-equality of variances, with method Tamhane), a two ways compares.α=0.05,With SPSS13.0 to accomplish the data processing.Results1. The number of CD4⁺T and CD16⁺CD56⁺NK cells in peripheral blood of breastcancer patients was smaller while the number of CD8⁺T, NK cells was larger ascompared with the normal control group. There were significant differences respectively (P＜0.05). The number of CD4⁺T was smaller compared with thenormal control group (P＜0.05) after operation or chemotherapy. CD3⁺T,CD4⁺T,CD16⁺CD56⁺NK decreased after operation+chemotherapy (P＜0.05) and CD80,CD86,CD28 decreased after operation+chemotherapy (P＜0.05). CD80 and CD86decreased after operation (P＜0.05). CD86 decreased and CD40 increased afterchemotherapy (P＜0.05). CD80,CD86,CD40L decreased and CD40 increased afteroperation+chemotherapy (P＜0.05).2. The percentages of HLA-DR molecules' expression in 5 breast cancer cells weredifferent as compared with HBL-100. The expression levels of CD40 moleculeswere all higher as compared with HBL-100. Among all CD40 molecules, there weresignificant differences at the expression levels as MCF-7,T47D,ZR-75-30 grouprespectively (P＜0.05). The expression levels of CD80 molecules were all lower ascompared with HBL-100. Among all CD80 molecules, there were significantdifferences at the expression levels as SK-BR-3,MDA-MB-435,MCF-7,T47Dgroup respectively (P＜0.05). The expression levels of CD86 molecules were alllower as compared with HBL-100. Among all CD86 molecules, there weresignificant differences at the expression levels as SK-BR-3,MDA-MB-435,MCF-7group (P＜0.05).3. The proliferation level of lymphocytes decreased after interrupting withanti-CD154 mono-clonal antibodies (P＜0.05). The proliferation level oflymphocytes decreased after interrupting with anti-CD154+anti-CD80 (P＜0.05). The lymphocytes proliferate rarely after interrupting with rmlL-2 or KM cellsalone. The lymphocytes proliferate rarely after interrupting with rmIL-2 or KM cellsalone (P＞0.05).The lymphocytes proliferate obviously after interrupting withrmIL-2+KM cells together (P＜0.05 ). The lymphocytes proliferate rarely afterinterrupting with anergy in MLC(BALB/c+KM) (P＞0.05). The lymphocytes proliferate obviously after interrupting with anergy in MLC(BALB/c+C57BL/6J) (P＜0.05).4. MDA-MB-435,MDA-MB-231 proliferate rarely after interrupting with rhIL-2(P＞0.05). The CD40-CD40L costimulatory path activated by rhCD40L couldinhibit the proliferation of breast cancer cells. It held up the proliferation of breastcancer cells at stage G₁. After 48 hours, MDA-MB-435' number of stage G₁increased obviously (P＜0.05), the number of stage S,G₂ decreased obviously (P＜0.05). After 72 hours, MDA-MB-231' number of stage G₁ increased obviously (P＜0.05), the number of stage S decreased obviously (P＜0.05). MDA-MB-231'CD80,CD86,CD54 (ICAM-1),CD95 (Fas) increased obviously (P＜0.05),CD95L( FasL)decreased obviously(P＜0.05). After 4,24 hours,bax/bcl-xl increasedobviously (P＜0.05).5. MDA-MB-435,MDA-MB-231 proliferate rarely after interrupting withrhIL-2+IFN-γ(P＞0.05). MDA-MB-435,MDA-MB-231 proliferate rarely afterinterrupting with rhCD40L+ADM,rhCD40L+Docetaxel (P＞0.05).Conclusions1. There is injury of immune function in local advanced breast cancer patients.Operation,chemotherapy may aggravates this injury. The costimulatory signals wereweakened. Operation, chemotherapy may aggravates this injury;2. Because of the changes of breast cancer's HLA-DR molecules and costimulatorymolecules, weaken costimulatory signals may cause the immune escape of breastcancer cells.3. The lymphocytes proliferate obviously after interrupting costimulatory pathCD40-CD40L,B7-CD28 with anti-CD154 or anti-CDS0 lonely. The lymphocytesproliferate obviously after interrupting costimulatory path CD40-CD40L,B7-CD28 with anti-CD154 and anti-CD80 together. The anergy has immunosuppression andantigen specification. The anergy may be reversal.4. The CD40-CD40L costimulatory path activated by rhCD40L could inhibit theproliferation of breast cancer cells. It held up the proliferation of breast cancer cellsat stage G₁. The costimulatory pathway of rhCD40L activated CD40-CD40L(CD 154) and IFN-γ,cyclical specific chemotherapeutics such as ADM,docetaxelplay the cooperative effect on the inhibition of proliferation of breast cancer cells.