Killing Effects Of Mimetic Peptide Conjuncted To ¹³¹I With High Affinities For HER2 On Breast Cancer Cells In Vitro

Objective: To study the killing effects of mimetic peptide (B2-S22-AFA) conjuncted to 131I with high affinities for HER2 on breast cancer cells SKBR3 (with high HER2 level) and MDA-MB-231 (with low HER2 level) in vitro. Accordingly provide experimental data to targeting therapy for breast cancer and other tumors with HER2 overexpression.Methods: 1. Collect the breast cancer cells SKBR3 and MDA-MB-231at log phase cultured in vitro, quantitate the cells total protein in cell disruption lysate under ultraviolet light spectrophotometer by coomassie brilliant blue staining method, detect HER2 expression level on SKBR3 and MDA-MB-231 cells with Western Blot method.2. With different concentration of EGF promoting the breast cancer cells, detect the inhibiting effects of B2-S22-AFA on SKBR3 and MDA-MB-231 cells through methyl thiazolyl tetrazolium assay (MTT). And choose suitable concentration of EGF as following experiment conditions. 3. B2-S22-AFA is labeled with 131I by NBS (N-bromosuccinimide) method. After the termination of the reaction, the labeling rate is tested by trichloroacetic acid precipitating method. Then the product is purified by Sephadex G-25 column chromatography. Collect the eluate into tubes at a constant speed and detect the radioactivity of each tube. According to the radioactivity of the eluate and corresponding tube number, we can draw a curve of two waves. Collect the eluate of the protein wave and assay its radiochemical purity, specific activity, immnoreactivity. Detect the stability of the product. 4. Cell conjugation test, add B2-S22-AFA labeled with 131I to excess breast cancer cells SKBR3,MDA-MB-231, detect the intensity of radiation to evaluate the binding ratio of the products and the above two kinds of cells respectively. 5. With EGF promoting the cells'proliferation, we survey lethal effect of 131I-B2-S22-AFA on the cells SKBR3,MDA-MB-231 by MTT assay.Results: 1. Western Blot shows that HER2 protein expression is high on SKBR3 cell and low on MDA-MB-231 cell, SKBR3 cell appears positive strap and MDA-MB-231 cell has no strap. 2. Different concentration of EGF has different promotion to cell proliferation, the higher concentration, the stronger propagation. At the same concentration of EGF, the propagating effect is much stronger on SKBR3 cell than that on MDA-MB-231 cell, especially after 48h. 3. B2-S22-AFA almost has not depressant effect on MDA-MB-231 cell proliferation that lowly expresses HER2, but has obvious depressant effect on SKBR3 cell that overexpresses HER2. The inhibition ratio on SKBR3 reach to(30.3±3.5)%. 4. Throw NBS method, we label B2-S22-AFA with 131I. The method can yield labeling rate ranging from 64.8% to 69.6%. The labeled products appear bimodal curve after purification by gel filtration, the protein peak shows high, sharp and narrow, but salt volume low and smooth, there are not damage peak and polymerization peak. To 131I- B2-S22-AFA, its radiochemical purity exceeds 95%, and even reach to 86.3%, 77.4%, and 65.8% when placed at 37℃after 24h, 48h and 72h respectively. 5. The binding ratio of 131I- B2-S22-AFA to SKBR3 is nearly 10 times that to MDA-MB-231. 6. The peak inhibition effect of 131I -B2-S22-AFA on cells were found at 48h after incubation, with significantly the highest inhibition rate of (51.2±3.0 )% in SKBR3 ( with high expressing rate of HER2) group.The inhibition effect of 131I -B2-S22-AFA on SKBR3 is greatly stronger than that on MDA-MB-231(with low level of HER2) and other groups(P<0.05).Conclusions: 1. The results of Western Blot indicate that SKBR3 is HER2–positive cell (with high HER2 level) and MDA-MB-231 is HER2–negative cell (with low HER2 level). 2. EGF has promotion effect on the breast cancer cells both in high expression of HER2 cells such as SKBR3 and even in low expression of HER2 cells such as MDA-MB-231, its promoting proliferation effect on SKBR3 is obviously stronger than that on MDA-MB-231. 3. B2-S22-AFA can conjugate to the breast cancer cells expressing HER2, the higher expression level of HER2, the higher conjugation ratio. B2-S22-AFA has obvious depressant effect on breast cancer cells overexpressing HER2, but has less depressant effect on HER2 low expression cells. 4. To HER2 mimetic polypeptide B2-S22-AFA, marked with 131I throw NBS method shows harmless to its immunological activity and good to its stability. Futhermore, this method is very convenient. 5. 131I -B2-S22-AFA can conjugate to breast cancer cells overexpressing HER2 and inhibit their propagation obviously, which provides experimental data to nuclide targeted therapy for breast cancer and other tumors with HER2 overexpression.