The Biologic Response Of Human Breast Cancer Line To ～(131)I－labeled Anti-Her-2 Monoclonal Antibody

Objectives1. To establish a suitable human Breast Cancer cell line expressing high level of human Epidermal Growth Factor Receptor-2 (her-2) and the control cell lines in the experiment for evaluation of the characteristics of each cell to predict the possible influences to the result.2. To explore the cytotoxicity toward the her-2-positive human Breast Cancer cells which treated with ¹³¹I-labeled Herceptin, as well as some possible effects of inducing apoptosis and their mechanisms.3. To compare the effects of cytotoxicity toward the her -2-positive human Breast Cancer cells which treated with ¹³¹I-labeled Herceptin or the ¹³¹I alone or Herceptin in order to preliminarily assess the potent therapeutic value of ¹³¹I-labeled Herceptin and to based on further animal trials and clinical trials for the use of ¹³¹I-labeled Herceptin to the treatment of human Breast Cancer in the future.Materials and methods1. Three cell lines including the human Breast Cancer cell lines SK-BR-3, MCF-7 and human Lung Cancer cell line A549 were cultured. The growth curves should be established by the MTT assay during 8 days, and the her-2 expression rates were evaluated by a flow cytometer in the exponential growth phase of these three cell lines. The conjugation rate of ¹³¹I-labeled Herceptin was also measured by binding assay and indirect ELISA assay.2. The ¹³¹I was labeled with Herceptin by IODO-GEN assay, and then theimmunological activity was evaluated with indirect ELISA at the time of before and after 131I labeled to Herceptin.3. In order to assess cytotoxicity in the her-2 positive cell line treated with 131Mabeled Herceptin, SK-BR-3 cell line was incubated and then added to the various concentrations of 131I-labeled Herceptin or 131I alone or Herceptin respectively. Then, (Dthe dose-effect curves were drawn out according to the data of MTT assay; (2)the suitable dose was carried out according the dose-effect curves as above to assess the different killing effect on SK-BR-3 among three treated groups. Breast Cancer cell line MCF-7 or human Lung Cancer cell line A549 was identifiably treated as the control; And (3)the changes of the morphology and survival rates on the killed SK-BR-3 in each group would be observed. After SK-BR-3 was treated with 131I-Herceptin, the cell growth curve which was treated with were drawn out and Mitosis Index (MI) were also surveyed with Gimesa stain.4. To compare of apoptosis on over-express her-2 cell line in all treated groups, the suitable dose which was drawn out from the dose-effect curves as above, that was, the specific activity was 125,MCi/ml of radioactivity and the concentration of Herceptin was 145jug/ml of monoclonal antibody. 125yaCi/ml of radioactivity of 131I-Herceptin or 125,MCi/ml of radioactivity 131I alone or \45[igjm\ Herceptin was added to over-express her-2 cell line SK-BR-3 at a concentration of 5xl05cells/ml in each hole with lOQul fresh medium, the control group added to partes aequales of complete culture medium. After 8 hours incubation, all groups of cells were stained with annexinV-FITC and propidium iodide (PI) to survey the effect of inducing apoptosis, and then PI stained analyzed with FACS in a flow cytometer to evaluate cell DNA histograms.Results1. The expression rate of her-2 on the SK-BR-3, MCF-7 and A549 was 99.04%, 7.55% and 0.44% respectively, and the survival rates in all cell lines were more than 95%. All cell lines were adherent, and the conjugation rate in 131I-labeled Herceptin was 30.9%, 4.3%, 1.8% respectively in the fixed cells. The volume of three kinds of cell bodies was approximation, and the condition of cell culture medium andsubculturing were identical. However, the express rate of her-2 was significant difference. Moreover, the binding rate of radioactivity in the group of SK-BR-3 was the highest by Binding Assay. The SK-BR-3 could therefore be employed as the group of over-express her-2 in the study and the MCF-7, A549 were fit for the groups of the control.2. Indirect ELISA assay showed that immunological activity of Herceptin was no significantly difference between the groups of before and after 131I was labeled (p=0.92). 131I-Herceptin possessed both immunological activity and radiative activity for this reason. It can therefore pay a role of intervention factor in the study.3. The killing effect could be found on SK-BR-3 that treated with 131I-labeled Herceptin and related with a dose-dependent. In the interval of the specific activity from 15.6^Ci/ml to lOOQaCi/ml in 131I-labeled Herceptin or 131I, the effective concentration of Herceptin from 15.6jug/m\ to lOOQwg/ml, 1311-labeled Herceptin caused significantly stronger killing effect on SK-BR-3 than the 131I or Herceptin alone (p<0.05). According to the dose-effect curves, the suitable specific activity of 125fiCi/ml was adopted to the experiment. 125,?Ci/ml of 131I-Herceptin or 125/iCi/ml 131I or 145^g/ml Herceptin was added to the medium of all groups of experiment as well as the groups of control. As the result, there could be found as following: (Dthe most powerful killing effect could be found in the group of SK-BR-3 treated with 131I-Herceptin. The most killing rate was occurred at the time of 96hs after incubation, and survival rate was 78.9% only. On the contrast, killing effects were almost absent in the groups treated with Herceptin or 131I alone. The cells in these groups were appearance alive, however, the increased granulation would be observed in the mesoplast under a light microscope and the survival condition treated cells was inferior to normal; ?the cells were occurrence proliferation in the groups of treated with 1 'i alone; ?cells vacuolar degeneration, nuclear fragmentation and membrane disruption could be observed in most cells in the group of treated with 131I-Herceptin which were necrosis. Only a few cells were observed decreased size, cytoplasm concentration, shrinkage of nuclear and membrane, just like the change of apoptosis. The growth process of inhibition was detected by MI assay manifested that the maineffects in the group of treated cells with 131I-labeled Herceptin were inhibited karyomitosis in SK-BR-3 cell line and the condition of karyomitosis were significantly decreased in the group of treated cells with 131I-Herceptin. The MI value was the lowest in the group, 4.3% only. The MI value in the group of treated cells with 131I or Herceptin was 31.6% or 29.2%. It was 25.9% in the control' MI.4. After the 131I-Herceptin or 131I or Herceptin was presented in the medium of incubating SK-BR-3 for 8 hours, the apoptosis rate was 40.37%, 47.81% and 29.42% in the group of them respectively, and the control group of untreated was only 26.17%. The apoptosis rate in the group of untreated or Herceptin was significantly lower than that in 131I-Herceptin or 131I alone(p<0.05).5. To compare to the apoptotic peak (hypodiploid peak) in all groups with DNA histograms demonstrated that: I31I-Herceptin group was 4.71%, 13II group was 1.71%, Herceptin group was 2.06% and the control group was 0.81% o The apoptosis rate in the 131I-Herceptin group was significantly higher than that in the other groups (p<0.05). 77.72% cells in 131I-Herceptin group cut off G1/G2 stage. The rate of cells in 131I group and Herceptin group cut off G1/G2 stage, only account of 51.76% and 48.19%respectively. 33.36% cells in the 131I group and 39.59% cells in the Herceptin group were discerned to cut off GO/M stage. These data demonstrated that proliferation of SK-BR-3 was still occurrence after control the factors were dealing with. On the contrary, the 131I-Herceptin groups was only 6.7% at GO/M stage, it showed that proliferation of SK-BR-3 was inhibited by 131I-Herceptin. The statistics analysis showed that all data were significance difference among all groups (p<0.05).Conclusions131I-labeled Herceptin pay an efficient role of killing effect in SK-BR-3 cell line which is over-expressing her-2 in vitro. The capability of 131I- Herceptin of cytotoxicity towards SK-BR-3 is significant stronger than the 131I alone or Herceptin with long killing effect. The major representations in SK-BR-3 cell line treated with 131I- Herceptin include appearance of cells enlargement, vacuolar degeneration, nuclear fragmentation and membrane disruption that were necrosis. Our data therefore demonstrate that 131I-Herceptin is a promising agent for targetingradioimmunotherapy treatment with her-2 over-expressing Breast cancer.