Study On The Expression And Targeting Of Phospho -Akt And Survivin For Radiosensitization In Breast Cancer

Breast cancer requires continued use of different treatment modalities, including combinations of surgery,chemotherapy,radiotherapy,biotherapy and endocrinotherapy as a systemic disease.Radiotherapy of patients with breast cancer plays a crucial role in local control of the disease.Breast radiation reduces local relapses in the breast,chest wall and axilla. Meanwhile,for patients at high risk of local relapse,radiation therapy was also shown to improve total survival rate and reduce relapse rate and mortality rate.However,development of radioresistance in breast cancer cells presents a difficult problem in the course of treatment.Possible cellular mechanisms for resistance development common to various therapeutic regimens,including radiation,are the activation of phosphatidylinositol 3-kinase(PI3K)/Akt and/or the high level of survivin expression in tumor cells.In this study,the expression of p-Akt and survivin in breast cancer tissues, the impact on the expression of p-Akt and survivin in breast cancer treated by deguelin and radiation,the ability of deguelin to doubly block the expression of p-Akt and Survivin for radiosensitization and enhance antitumor effects in breast cancer cells and nude mice model were studied to explore the role of the expression of p-Akt and survivin in radioresistance of breast cancer and to provide the experimental foundation for clinical application by targeting the p-Akt and survivin strategy for radiosensitization of breast cancer.PART ONE The expression and clinical significance of p-Akt and survivin in breast cancer tissues[Objective]To investigate the expression of p-Akt and survivin in breast cancer tissues and their clinicopathological significance and guide the treatment of breast cancer.[Materials and methods]A total of 68 primary human breast carcinoma specimens were obtained from patients who underwent surgery at the Department of Breast and Endocrine Surgery,the First Affiliated Hospital of Nanjing Medical University,from June 2006 to December 2006.All the cases were confirmed by pathological examination and received no radiotherapy,chemotherapy and endocrinotherapy treatment prior to surgery. Normal breast tissues of 10 cases were used for control.The immunohistochemical method(streptavidin peroxidase,SP)was used to assess the expression of p-Akt,survivin,HER2,ER,PR and p53.And then to further analyze the correlations between the levels of p-Akt,survivin expression and other parameters.[Results]Ten normal human mammary tissues and sixty-eight breast cancer tissues were obtained.Of which,fifty-eight were invasive ductal carcinoma,eight ductal carcinoma in situ,two lobular carcinoma. Pathological stage(pTNM):sixteen patients belong to stageâ… ,thirty-five toâ…¡and seventeen toâ…¢-â…£.Positive immunostaining for p-Akt and survivin was detected in the cytoplasm and/or nuclei of tumor cells by SP immunohistochemical staining.The ratios of positive immunostaining expression separately were p-Akt 41.2%(28/68),survivin 51.5%(35/68), HER2 33.8%(23/68),ER 58.8%(40/68),PR 57.4%(39/68)and p53 55.9%(38/68).Positive immunostaining for p-Akt and survivin was not detected in the cytoplasm and/or nuclei of normal breast tissue cells.The biological parameters were further analyzed that the positive correlation was found betweeen expression of p-Akt with HER2 in tumor tissues(r value is 0.412,0.383;P value is 0.001,0.001,respectively).However,the expression of p-Akt is inverse correlation with the expression of PR(r value=-0.306, P value=0.011).In the clinical parameters,the expression of p-Akt has no correlation with the expression of patients'age(P value=0.861).The expression of p-Akt has correlation with the expression of pathological stages,the condition of lymph node metastasis and tumor sizes(r value is -0.326,-0.407,0.271;P value is 0.014,0.004,0.025,respectively).The expression of survivin has no correlation with the expression of patients' age,pathological stages and tumor sizes(P value is 0.785,0.424,0.434, respectively).The expression of survivin is positive correlation with the condition of lymph node metastasis(r value=0.299,P value=0.013). [Conclusions]The activation of Akt and survivin significantly contributes to the progression of breast cancer.Activated Akt and survivin may indicate poor prognosis of breast carcinoma,p-Akt and survivin may be as the new target of antitumor therapy.PART TWO Study on effects of radiotherapy on p-Akt and survivin expression in breast cancer cells and deguelin enhancing radiosensitization of breast cancer[Objective]To investigate the roles of p-Akt and survivin in radioresistance of breast cancer cells and deguelin targeting p-Akt and survivin for radiosensitization of breast cancer,and identify potential molecular markers to use in future clinical studies.[Materials and methods]MDA-MB-231 breast cancer cells were treated withγradiation,deguelin and deguelin combined withγradiation. MTT colorimetric method was used to assess cell growth inhibition.Western blot was used to detect levels of Akt,phospho-Ser⁴⁷³-Akt,survivin and cleaved caspase-3 expression.Tumor cell cycle and apoptosis in MDA-MB-231 were assayed by FCM.Cell survival fraction was assessed by clonogenic assay.[Results]The cell growth of MDA-MB-231 was inhibited in dose- and time-dependent manners.The inhibitory rates of the cells after the different doses of radiation were as follows:3Gy of radiation induced an inhibitory rate of(38±4)%and 4Gy of radiation induced an inhibitory rate of(56±7) %at 48h after treatments.MTT assay showed that proliferation inhibitory rates of cells with combined treatment were significantly higher than those with radiation alone(p＜0.01).3Gy of radiation combined with 10nmol/L deguelin induced a synergistic inhibitory effect in MDA-MB-231 cells. 10nmol/L deguelin could remarkably inhibit the expression of phospho-Ser⁴⁷³-Akt and survivin and enhance cleaved caspase-3 expression level in MDA-MB-231 cells.The levels of phospho-Ser⁴⁷³-Akt and cleaved caspase-3 increased and survivin expression did not alter after 3Gy of radiation.However,phospho-Ser⁴⁷³-Akt and survivin expression further significantly decreased and cleaved caspase-3 further significantly increased after deguelin combined with radiation treatment.Deguelin combined with radiation treatment induced a significant increase apoptosis as well as arrested cell cycle in the G₂-M phase in MDA-MB-231 cells(P＜0.01). Deguelin could decrease clonogenic cell survival and enhance radiosensitization of breast cancer.[Conclusions]Deguelin attenuates radiation-induced prosurvival Akt signaling and enhances the radiosensitivity of MDA-MB-231 cells,and the mechanisms for this action may include inhibiting phospho-Akt and survivin expression,increasing caspase-dependent apoptosis,and prolonging cell cycle arrest in the G₂-M phase. PART THREE Study on the ability of deguelin to doubly block the expression of p-Akt and survivin for radiosensitization of nude mice model in breast cancer[Objective]To investigate the role of deguelin to the nude mice model of breast cancer,as well as therapeutical effect on radiosensitization of nude mice model in breast cancer and provide the experimental foundation for future clinical study.[Materials and methods]Experimental animals were female(body weight,18±2g)and four- to five-week-old BALB/c nude mice(Shanghai Slake experimental animal limited Co.).Tumor xenografts were obtained by s.c injection of MDA-MB-231 single cell suspensions(1×10⁶)in left flank of BALB/c nude mice and allowed to grow in volume for approximately 20 days to a size of 100-140 mm³.Twenty-four mice were divided averagely into 4 groups as follows:Control group(group C,corn oil dissolvent,80μl/ mice),deguelin group(group D,deguelin 4mg/kg/mice),radiation treatment group(group RT,2Gy ⁶⁰Co radiation/mice),deguelin+radiation treatment group(group D+RT,deguelin 4mg/kg/mice+2Gy ⁶⁰Co radiation/mice). Corn oil dissolvent and deguelin were administered intragastrically 5 days a week for 6 weeks.Radiation treatment was administered by 2Gy ⁶⁰Co radiation 5 days a week for 6 weeks.Mice were weighed,and the tumor size was measured once a week with an electronic caliper.The tumor volume was estimated from two-dimensional tumor measurements by the formula: Tumor volume=length(mm)×width²(mm²)×π/6.The mice were sacrificed at two weeks after the treatment ended.Mice weight,tumor volume and weight were recorded.The tumor inhibitory rate was calculated. Partial tumor tissues were used by flow cytometry and western blot test.[Results]24 tumor xenografts mice were divided averagely into 4 groups in balanced mice weight and tumor size before treatment.Tumor growth curve displays tumor growth of control group is most rapid,the group RT takes second place,and the group D is in the third.However, tumor has shrinking tendency in the group D+RT.After treatment ended, average tumor volume(mm³)were group C:728.8±182.7,group RT:427.8±106.4,group D:260.8±68.4,and group D+RT:95.5±18.7.Average tumor weight(g)were group C:1.28±0.21,group RT:0.68±0.14,group D:0.42±0.06,and group D+RT:0.25±0.05.Average tumor inhibitory rate were group RT:46.88±10.95%,group D:67.19±5.01%,and group D+RT: 80.60±3.68%.Each treated group compared with control group has significance(P＜0.000).Flow cytometry results show that the effect of radiation treatment and deguelin treatment on cell cycle is similar.They can increase the G1 and G2/M stage cells and the apoptoic cells,decrease the S stage cells.The effect of radiation treatment is not as good as the effect of deguelin treatment. Deguelin combined with radiation treatment has the most effect on inducing apoptosis of tumor tissues and increasing G2/M stage cells.Each treated compared with control group has significance(P＜0.01).Western blot results show that radiation treatment can increase the expression of phospho-Ser⁴⁷³-Akt and survivin comparing to others group (P＜0.01).The expression of phospho-Ser⁴⁷³-Akt and survivin in group D and group D+RT decrease.Compared with group D,the effect of group D+RT is more obvious(P＜0.004).Cleaved caspase-3 express level increase in group RT,group D,and group D+RT.Cleaved caspase-3 express level is the most obvious in group D+RT(P＜0.001).[Conclusion]Radiation could induce an increasing the expression of phospho-Ser⁴⁷³-Akt and survivin in breast tumor tissue.Deguelin could inhibit this effect of radiation.Deguelin and radiation could increase cleaved caspase-3 express level and induce apoptosis in breast tumor tissue. Compared with deguelin alone treatment or radiation alone treatment, deguelin combined with radiation treatment significantly inhibited the growth of tumor xenograft.Deguelin enhanced the radiosensitivity of breast tumor xenograft.Meanwhile,combination treatment has also not increased the side effect of tumor xenograft mice.Deguelin enhancing the radiosensitivity of breast cancer is a new therapic strategy by targeting p-Akt and survivin.