The Effect Of ADCC Induced By Trastuzumab In Breast Cancer Cells After Plus Pertuzumab

Objective:Verify the expression levels of human epidermal growth factor receptor 2(HER-2),EPFR protein in different subtypes of human breast cancer cells,search the correlation of different expression levels of HER-2 and EGFR proteins in anti-dependent cell mediated cytotoxic(ADCC)mediated by trastuzumab;On the basis of this,search the effect of the increase of natural killer(NK)cell ratio on ADCC mediated by trastuzumab;search the reaction time of ADCC peak after adding trastuzumab;Explore the difference in ADCC of adding pertuzumab at different intervals after adding trastuzumab.The aim of this study was to explore more auxiliary treatment methods for breast cancer clinical treatment and to explore the possibility methods of enhancing the benefit of clinical double-target drugs.Methods:1.Different subtypes of human breast cancer cells were cultured,and the total protein was extracted.The contents of HER-2 and EGFR protein in different samples were determined by Western blot.According to the expression level of HER-2 protein,the overexpression and low expression cell lines were selected.2.Peripheral blood mononuclear cells(PBMCs)were isolated from whole blood of healthy adults by density gradient centrifugation.3.NK cells were isolated and purified from PBMCs by magnetic beads to select CD56+NK cells.Human myeloid leukemia cell line K562 was cultured and co cultured with purified NK cells.The cytotoxicity of NK cells was determined by flow cytometry.4.The purified NK cells were co incubated with breast cancer cell lines of different subtypes in different proportions,and the same amount of trastuzumab was added.Flow cytometry was used to determine the difference of ADCC under different proportions of NK cells and target cells.5.Within a certain range,the ADCC effect of trastuzumab on HER2 positive breast cancer cell line was determined by flow cytometry;After adding trastuzumab,the difference of ADCC effect in different reaction time was determined,and the reaction time to reach the peak of ADCC was explored.6.After adding trastuzumab to HER-2 positive breast cancer cell lines,pertuzumab was added at different intervals.Flow cytometry was used to detect the difference of its effect on trastuzumab mediated ADCC effect and the time to reach the peak of ADCC.Results:1.Western blot results showed that HER-2 and EGFR proteins were highly expressed in SK-BR-3 and BT-474 cell lines,but no or low expression in MDA-MB-231 and MCF-7 cell lines,and no expression in K562 cell line.2.Compared with PBMCs before separation,the purity of NK cells after separation and purification increased,and the proportion of CD56+NK cells increased significantly.3.The killing ability of NK cells in different healthy adults is different,and there are individual differences.4.When the ratio of NK cells to target cells increased,the ADCC effect increased in all subtypes of breast cancer cell lines,but after adding trastuzumab,the ADCC effect increased in HER-2 positive breast cancer cell lines,which was more significant than that in HER-2 negative breast cancer cell lines.In MDA-MB-231 cell line,ADCC effect may be inhibited.5.Within a certain range,changing the concentration of trastuzumab has little effect on ADCC.6.Adding trastuzumab for 4-24 hours,the peak value of ADCC was reached at 24 hours.7.When the concentration of trastuzumab and pertuzumab was 1:1,the killing ratio was more stable than 1:2.8.After the addition of trastuzumab and the addition of pertuzumab within 1 hour interval,the killing ratio increased significantly at 12 hours,and reached the peak at 20 hours.9.24 h after adding trastuzumab,the results showed that the peak of killing ratio was reached at 8h and 16h after adding pertuzumab at intervals of 0h,4h,8h,12h,16h and 20h,which was 8h earlier than that of the experimental group added trastuzumab alone.The peak of killing ratio was close to that of the group added pertuzumab at intervals of 1 h.Conclusion:In HER2 positive or HER2 negative human breast cancer cells,adding different proportions of CD56+NK cells stimulated by IL-2 increased,the killing ratio with the increase of the proportion of NK cells;When the ratio of effector cells to target cells was 9:1 and with the trastuzumab’s added,the killing ratio of HER-2 positive human breast cancer cells was significantly higher than that of HER-2 negative cells;The immunotherapy aimed at increasing the number of NK cells,stimulating the proliferation of NK cells and improving the function of NK cells may become as an adjuvant therapy for HER-2 positive breast cancer.With the increase of drug concentration,ADCC has saturation.Due to the limitation of objective reasons,many patients in the clinical treatment process can not be directly taking double target therapy,some patients were only received trastuzumab for single target therapy,some patients received single target therapy for a period of the time before double target therapy,we want to explore the difference of the clinical benefits of these patients.Through the experiments,we found that compared with the groups using trastuzumab alone,the groups using dual target drugs got more stable killing ratio and reached the peak of killing ratio earlier,and the peak of killing ratio between these groups was close.The effect of using double target drugs is better than that of using trastuzumab only,which is consistent with the clinical conclusion.Compared with adding pertuzumab after a period of time,there is no significant difference in the peak of the ADCC,or it can be proved that the patients who received double target therapy after single target therapy,the benefit of these patients in ADCC will not be inferior to that of patients receiving dual target therapy.