Study On Preparation Of PAMAM-NK4 Nanocomposite And Their Inhibitory Effect On The Growth Of Breast Cancer In Vitro And In Vivo

Background and objective:Breast cancer is one of the most common malignant tumors in global women. The incidence of breast cancer in global area has risen, and it has been a severe threaten to the health and life of women. Since 1990s, the growth rate of the incidence of breast cancer in China has been more than twice of the global area, and has been the highest among all the malignant cancer of Chinese women. Therefore, the comprehensive therapy of breast cancer has been one of the most heating topics in Chinese women health. At present, the main treatment of breast cancer has been turned from traditional operation to the comprehensive treatment combined by many methods including operation, chemo-therapy, radiation therapy, and endocrine therapy etc. Among them, operation and radiation therapy belong to partial treatment, which have limited coverage area; chemo-therapy belongs to general treatment, which damages normal tissue in various degree while killing tumor cells, and which leads to toxic side effect and drug resistance that influence the final result of chemo-therapy.With the development of the studies in cytobiology and molecular biology, we have better understanding that the incidence and development of tumor diseases are the result of multiple genes working together. Therefore, the gene therapy of breast cancer becomes a heating topic of studies nowadays. Gene therapy of breast cancer is a method to translocate functional or normal genes into certain tissues or cells to correct defective genes or to endue them with specific function so as to treat with cancer. The key to the success of gene therapy is the efficient gene translocation and expression, so it is essential to select target genes and carries.Hepatocyte growth factor (HGF) is a cytokine produced by interstitial cells. It combines to receptor c-Met and has autophosphorylation, which promotes the development, invasion, metastasis, and angiogenesis of cancer. Reports have shown that the high expression of HGF played an important role in the incidence, development, invasion, and metastasis of breast cancer. NK4 was first found out as specific HGF antagonist, which insisted of N-terminal amino acid and four Kringle zones of HGF. As part of HGF, NK4 was a competitive antagonist to the combination of HGF and c-Met and the angiogenesis so as to compress the proliferation, invasion, and metastasis of tumor cells. Studies have shown that NK4 has anti-tumor effect in many kinds of malignant tumors such as pancreatic cancer, cervical carcinoma, and hepatic cancer, and the conclusion has been confirmed on nude mice models. Therefore, NK4 gene has been chosen as target gene in this experiment to study the anti-tumor effect of NK4 gene in breast cancer.To transfect target gene into tumor issue precisely, the selection of carrier has been essential. At present, gene carriers mainly include virus vector and non-virus vector including lipidosome and cationic polymer. Though the transfection effect of virus vector has been high, the autoimmunogenicity and cytotoxity as well as the difficulty of preparation, the small capacity of target genes, and the low specificity of targeting have limited its clinical application. Lipidosome carried long fragment of target genes while not having high specification, which meant that it was not the best carrier. Polyamidoamine dendrimers (PAMAM) is one of the most commonly used cationic polymer. The surface of PAMAM with positive charge amino group could have electrostatic interaction with negative charge phosphate group on DNA dominant chain, which formed stable combination. This has not only promoted the DNA transfection effect but the stability of PAMAM-DAM combination. Also it had some advantages as follows:1.It had no autoimmunogenicity and would not lead to immunoreactions.2. It had no inherent toxicity and cytotoxity.3. It would protect target genes from the damage of organism blood, tissue and cells complements, and different kinds of enzyme, so as to enlarge the enduring and expression of target genes in cells.Therefore, fifth genereation of PAMAM was selected as gene carrier in this experiment to prepare PAMAM-NK4 nanocomposite. Target gene NK4 was transfected into breast cancer cells to detect the transfection effect of nanocomposite and to analyze its function on breast cancer in vitro. Human breast cancer cell nude rat model was constructed to detect the anti-cancer effect of nanocomposite on breast cancer in vitro and in vivo, and to provide a new thread for gene therapy of breast cancer.Methods:1. Preparation and identification of the PAMAM-NK4 nanocomposite1.1 Cell culture of MCF-7 and MDA-MB-231 human breast cancer cellsThe culture medium of the MCF-7 and MDA-MB-231 breast cancer cell lines were changed when the cells were first bought from the institute and were put into the 37℃ incubator with 0.05 CO2. The growth states of cell were observed by scanning electron microscope. The nutrient medium was replaced timely according to the speed of cell growth, about two to three days each time.1.2 Preparation of the PAMAM-NK4 nanocompositePut the purified G5 PAMAM and the recombinant plasmid pcDNA3.1-NK4 into PBS solution at the charge ratio of 5. The mixture was stirred for 15 seconds and placed at room temperature for 30 minutes.1.3 Transfer the cell lines of the MCF-7 and MDA-MB-231 human breast cancerThe nanocomposite PAMAM-NK4 was diluted into 100 nmol/L with serum-free DMEM. The two cell lines were transferred by 2ml PAMAM-NK4 suspension liquid and incubated at 37℃ in 5% CO2,4 hours added serum medium to culture.1.4 Test the transfection efficiency in witroTransfection of the cells was observed under the inverted fluorescence microscope after 24 hours. The transfection efficiency was detected by green fluorescent expression.2. Study the role of the PAMAM-NK4 nanocomposite in the breast cancer cells2.1 Cell proliferation was determined by MTT assayThe human breast cancer cell lines MDA-MB-231, MCF-7 in logarithmic growth phase were divided into three groups,5 in each group:the control group, the blank plasmid group and the PAMAM-NK4 group.40μl MTT solution was added to each pore, then supernatant was abandoned after 4 hours. Cells were then added with 150μl DMSO solution and stirred for 10 minutes. Optical density (OD) of each hole was tested at the wavelength of 570 nm by Enzyme-Linked Immunosorbent Assay. The proliferation inhibition rate was calculated as (1-OD value of experimental group/OD value of control group)×100%.2.2 The protein expression of NK4 was detected by Western blotting assayThe cell proteins were extracted from the control group, the blank vector group and the experiment group in logarithmic growth phase by cell lysate. BCA method was used to measure the total protein concentration of each sample. SDS-PAGE electrophoresis, transferred to film, and then closed with 5% skim milk TBST solution, and then added with HGF monoclonal antibody, and the secondary antibody labeled horseradish peroxidase in which β-actin was used as the internal standard. Use the ECL chemiluminescence kit to development after washing 3 times. Scan film and analyze the gray value of the strip using IPP6.0 software.2.3 Flow cytometry was used to analyze the cell apoptosis rateCell suspension of each group was digested by pancreatin after cultured for 24 hours. The pre-staining cells were washed with cold PBS and re-suspended in staining buffer. Adding an appropriate amount of flourescent labeled Annexin V reagents and PI, incubating for 15 minutes at room temperature. After added with staining buffer, the cells were immediately analyzed on a flow cytometer. The morphology of apoptosis was observed under fluorescent microscope and the apoptosis, necrosis percentage was caculated.3. Investigate the effect of PAMAM-NK4 on the tumor growth in nude mice3.1 Establish the xenografts of human breast cancer cell in nude miceThe MDA-MB-231 cell in logarithmic growth phase were digested by pancreatin and re-suspended in cultural medium. Forty nude mice were vaccined 0.2ml(about 1×107 MDA-MB-231) cells under the second nipple on the left chest fat pad. After the vaccination, the growth of tumor nodules were observed and measured every two days, and everyday after seventh days. Use vernier caliper to measure and record the long diameter(a) and the short diameter (b), calculate the tumor volume (V). V=ab2/2.3.2 The effect of PAMAM-NK4on the tumor growth in nude miceThe mice were divided into four groups randomly,10 in each group:the blank group, in which 0.2ml 0.9% Nacl was injected into the tumor and tumor-surrounding, the blank vector group, in which 0.2ml plasmid solution (including PAMAM 100μg) was injected into the tumor and tumor-surrounding, the PAMAM-NK4 group, in which 0.2ml plasmid solution (including PAMAM-NK4 100μg) was injected into the tumor and tumor-surrounding, and the positive control group, in which 0.2ml (including Adriamycin 100μg) solution was injected intraperitoneally. All nude mice were continuously injected for 7 days and were sacrificed on day 30 after treatment. The tumor was removed completely. Weight and tumor size were measured.3.3 The NK4 protein expression levels in transfected tumors were detected by Western blotting0.2g organization of each group was taken out, shredded with scissors and milled in lysis buffer. After centrifuged for 10 minutes, the total supernatant cellular protein was aspirated, and the protein concentration was measured by BCA method. SDS-PAGE electrophoresis, transferred to film, and the closed with TBST solution, and then added HGF monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled horseradish peroxidase. The NK4 protein expression in different samples was detected with ECL chemiluminescence.Statistical analysisAll results in this study was performed using SPSS 19.0, the measured data showed as mean±standard deviation (x±s). If there is variance equality existed, analysis of variance should be adopted, and then the two-two comparisons with LSD method should be choosed. If there is no variance equalitu existed. Welch method should be adopted in multiple groups, then the two-two comparisons with Dunnet’s T3 method should be used, P<0.05 for the statistically significant difference.Results1. Breast Cancer cells were effectively transfected with PAMAM-NK4 nanoparticlesThe green fluorescent expressions in breast cancer MDA-MB-231, MCF-7 cells were detected under inverted fluorescence microscope after the transfected of PAMAM-NK4 nanocomposite. After 24 hours of the transfection, green fluorescence began to appear, and then gradually increased, reach it peak at 72 hours. The fluorescence intensity gradually weakened after 72 hours.2. PAMAM-NK4 nanocomposite can effectively inhibit cell proliferation of the human breast cancerThe results of MTT showed that proliferation ability had no significant difference between the blank vector group and the control group. Compared with the control group, the OD value of the experiment group was remarkable decreased, MDA-MB-231 [(0.27±0.06) vs (0.54±0.06)], MCF-7 [(0.30±0.05) vs (0.43±0.05)], the difference was statistically significant (both of the P value<0.05). The inhibition rate of MDA-MB-231 cell was 50.4%, significantly higher than MCF-7 cell which was 30.6%, the difference was statistically significant (p=0.01).3. The effect of PAMAM-NK4 nanocomposite on the NK4 protein expression in human breast cancer cellThe results of Western blot showed that the average expressive level of endogenous HGF in MDA-MB-231, MCF-7cells were (0.88±0.03) and (0.35±0.01) respectively. There was no significant difference between the blank plasmid group and the control group (P>0.05). The average expressive level of NK4 protein in the experiment group cells were (2.26±0.02) and (1.26±0.01) respectively. It demonstrates that after the transfection the protein expression in treatment group was significantly increased compared with the control group.4. PAMAM-NK4 nanocomposite increased the apoptosis rate of the human breast cancer cellThere was no significant difference between the blank plasmid group and the control group (P>0.05). Apoptosis rate of the MDA-MB-231, MCF-7 cell line without treatment by transfection were (2.26±0.06)% and (2.40±0.04)%. After the transfection of PAMAM-NK4 nanocomposite the apoptosis rate were (2.90±0.04)% and (2.90±0.06)% respectively, showing that the experiment group had relative higher percentage of apoptosis, and that the difference was statistically significant (P<0.05).5. Successfully established the xenografts of human breast cancer cell in nude mice.1 week after inoculated MDA-MB-231 cells, small bulge appeared at the inoculation site on four group of nude mice. The tumors grow slowly and the skin of each group nude mice was generally good. Tumorigenicity rate was 100%.6. PAMAM-NK4 nanocompisities inhibits the growth of the tumor model in nude mice.The tumor final volume of each group were respectively (2.42±0.62) cm3, (2.38±0.64) cm3, (1.50±0.43) cm3 and (1.64±0.31) cm3, and the tumor weight of those were (3.86±1.12) g, (3.48±1.07) g, (1.41±0.47) g and (1.54±0.38) g. The tumor volume and weight had no significant difference between the blank group and the blank plasmid group (P<0.05). The results also showed that the treatment group and the positive control group of nude mice were remarkable inhibited while there was no significantly difference between the treatment group and the positive control group (P>0.05).7. The effect of PAMAM-NK4 nanocomposite on the NK4 protein expression in xenograft tumorThe blank group and the blank vector group had a small amount of HGF expression. The expressive level of blank group was (0.82±0.05) and there was no significantly difference among the blank vector group, the positive control group and the blank group (P>0.05). The protein expression in treatment group was significantly increased compared with blank group, the expressive level was up (3.44±0.07). It showed that the treatment group could express the exogenous NK4 protein.Conclusions1. PAMAM-NK4 nanocomposites can successful transfer target gene-NK4 into breast cancer cell lines. They can significantly express the exogenous NK4 protein, inhibit the proliferation of breast cancer cells and increase the apoptosis rate. The tumor growth of MDA-MB-231 cells was inhibited more effectively than MCF-7 cells.3. The xenografts of human breast cancer MDA-MB-231 cell were successfully established in nude mice. The tumor in nude mice was remarkably inhibited by PAMAM-NK4 nanocomposite and could express exogenous NK4 protein. The tumor growths were significantly inhibited by PAMAM-NK4 nanocomposite and Adriamycin while there was no significantly difference between them.