Graduate School of Advanced Science and Engineering Waseda University
博 士 論 文 概 要
Doctoral Thesis Synopsis
論 文 題 目
Thesis Theme
The synergic effects of anti-HER2 antibody and polystyrene nanoparticles on response of human
carcinoma cells
申 請 者 (Applicant Name)
Thi minh phuc LE
リ フック ティ ミン
Department of Nanoscience and Nanoengineering, Research on Nanobiomaterials
May, 2018
No. 1
Human Epidermal growth factor Receptor (HER) family of transmembrane of tyrosine kinase receptors is composed of four members HER1, HER2, HER3 and HER4. These receptors share a common structure with extracellular ligand binding domain, transmembrane domain and intracellular tyrosine kinase domain. In monomeric state, all members in HER family are inactivated. Their activation requires binding to ligand and formation of homo- or heterodimers. After activated, HER family transmits signal to promote proliferation, invasiveness, angiogenesis and inhibition of apoptosis. In human, the abnormal activation or expression of receptors in HER family often leads to cancer. Unlike other receptors in HER family, HER2 is ligand-orphan receptor. HER2 does not activate by binding the ligand. The activation of HER2 requires the formation homodimers of HER2. This activation process occurs on HER2 overexpression cells without ligand. HER2 is also activated by formation of HER2-based heterodimers dependent upon ligand. This activation process relies initially on binding of ligand to HER2 partner, and then the receptor can dimerize with HER2. Following receptor dimerization, the activation of signal transduction pathways leads to proliferation, survival and angiogenesis. HER2 is expressed in many human cancer cells but not in normal cells. HER2 is good target for anti-cancer therapy using anti-HER2 antibody (Ab). Mechanisms for anti-cancer of anti-HER2 Ab include reduction number of HER2 on cell membrane, attenuation signal and prevention heterodimer formation.
Polystyrene nanoparticles (PS NPs) are widely used as a model for studying the interaction between NPs and cells due to their advantages including commercial availability, high quality, diverse sizes and shapes, biocompatibility, biological low-toxicity, and high functionality due to the presence of many chemical groups.
PS NPs are utilized in various research and clinical applications such as carrier for drug, bio-imaging, biosensors, photonics, self-assembling nanostructures, spray and exterior paints. The increasingly wide uses of PS NPs are leading to a corresponding rising in exposure levels for human, thereby high exposure levels induce the problem of potential risk to human health. Therefore, the investigations of potential interactions between PS NPs and cells could be essential.
Both PS NPs and anti-HER2 Ab are used in clinical applications for image diagnostic, drug carrier and treat cancer, however, we don’t know what is happening in our body when anti-HER2 and PS NPs are taken up together. Many studies focused on the effect of anti-HER2 Ab on cellular response, other studies investigated on the effect of PS NPs on cellular response, however, no study researched on the effect of anti-HER2 Ab and PS NPs on cellular response. I proposed the hypothesis: “Anti-HER2 Ab and PS NPs could be affected to cellular response of human cancer cells”. This Doctoral thesis studied 1) Expression anti-HER2 Ab with high yield used a new growth controlled medium, 2) Study on the effect of anti-HER2 Ab and EGF on cellular uptake of PS NPs and 3) Study on the effect of anti-HER2 Ab, EGF and PS NPs on cell death. In detail, the Doctoral thesis consists five chapters as follow.
Chapter I gives a general introduction on HER family and why HER2 is good target for cancer therapy using anti-HER2 antibodies. The anti-cancer mechanisms of anti-HER2 antibodies are also provided. In addition, this chapter describes characteristics and applications of PS NPs in bio-imaging, drug delivery systems.
Hypothesis and strategies are also mentioned in this chapter. Detailed strategies and results are described in
No. 2 chapter II-IV.
Chapter II presents an enhancement of anti-HER2 Ab expression level by growth controlled medium. In this chapter, transient expression system was used for anti-HER2 Ab expression. In transiently transfected cells, the recombinant antibody expression vector does not integrate into the host genome and does not replicate and is eventually lost through cycles of cell division over several days. Both cells with and without recombinant antibody expression vector are proliferation, but only cells with recombinant antibody expression vector able to produce antibodies, result in cell proliferation could not proportional to antibodies yield. Therefore, cell growth could be unnecessary for protein expression in transient expression system. To overcome the limitations, a new growth controlled medium named NPLTT medium (Non-Protein and Lipid medium for Transient Transfection) was developed with two purposes. One purpose is to enhance antibodies expression level by optimizing medium composition for antibody production. The other purpose is to control cell growth by development of medium capable of long-term culture at high density. In NPLTT medium, some amino acids were increased as a protein expression resource; sugars were increased as an energy source for cells. Ascorbic acid and glutathione were added to suppress cell damage due to antioxidant effect. In addition, 1% Pluronic F-68 was added to suppress shock absorption of cells during shaking culture and form cell clumps. To evaluate the effectiveness of new growth controlled medium in anti-HER2 Ab expression, all obtained results were compared between two expression systems. One of them is Free Style system, which is one of commercial high expression systems. The other of them is NPLAd system, which was developed by using adapted culture method in our laboratory. The results indicated that expression level in NPLAd system was higher than that in Free Style System. Molecular quality and biological activity of antibodies expressed by NPLAd system were almost the same with Ab of Free Style system. These results suggested that NPLAd system could be useful for recombinant antibody expression.
Chapter III shows anti-HER2 Ab inhibits EGF (epidermal growth factor)-induced cellular uptake of PS NPs. Cellular uptake of nanomaterials serves as key role for determining of nanotoxicity as well as for developing of target drug delivery strategies. Previous studies have showed that PS NPs could be internalized the cells depend on dose, size, time or cell cycle. In this study, I supposed that anti-HER2 Ab could be also affected to cellular uptake efficiency of PS NPs. A431 human epithelial carcinoma cell line, which is possess high level of HER1 and moderate level of HER2 on cell surface, was used for study. The results showed that EGF enhanced cellular uptake of PS NPs. This result suggested that HER1 and HER2 concerned with increasing in cellular uptake of PS NPs. To confirm role of HER1 and HER2 in this increase, three studies were done: 1) inhibition study using anti-HER2 Ab, 2) localization study of PS NPs and HER1 in the cells and 3) the effect of EGF on uptake of PS NPs by HeLa human cervical cancer cells, which were HER1 low expression cell line. The results indicated that anti-HER2 Ab decreased the uptake efficiency of PS NPs that enhanced by EGF.
Co-localization of PS NPs and HER1 were observed in cytoplasm. EGF did not affect to uptake ratio in HeLa cells. This result suggested that the effect of EGF on uptake of PS NPs was specific for HER1-overexpressed cell line. In conclusion, HER1 and HER2 played key role in EGF-enhanced cellular uptake of PS NPs. Anti-HER2 Ab inhibited EGF-enhanced cellular uptake of PS NPs.
No. 3
Chapter IV describes anti-HER2 Ab enhances PS NPs-induced cell death. Anti-HER2 Ab and PS NPs could be affected to cellular response of A431 cells. This chapter focuses on effect of anti-HER2 Ab on cell death. The results showed that EGF stimulated proliferation of A431 cells, however, cell numbers of A431 were dose-dependent decreased by PS NPs. To clarify why PS NPs inhibited cell growth, the effect of EGF on uptake was checked. The results showed that cellular uptake was increased by EGF. This result suggested that high cellular uptake ratio leads to an increased incidence of cell death. We also observed co-localization of NPs and HER1 in the cytoplasm. On the other hand, decrease in the number of A431 cells upon treatment with PS NPs could be due to decreased viability or an increase in cell death. The results indicated that PS NPs with or without EGF treatment reduced cell viability significantly. Altered the cell cycle towards an increasing death cell population in the sub G0/G1 phase. The cell population in the sub G0/G1 phase includes apoptotic cells and necrotic cells. Thus to find out actual reason, caspase 3 activity, which is critical enzyme in apoptosis, was checked. The results suggested that PS NPs and EGF induced cell death by apoptosis, but PS NPs induced cell death by necrosis. In addition, anti-HER2 Ab enhanced cell death induced by PS NPs. This finding could be useful for the safe and effective use of nanoparticles in clinical applications.
Chapter V deals about the general conclusions and discussion for future prospects. My findings are the first time showed that 1) anti-HER2 Ab affected to cellular response of A431 cells by inhibited uptake of PS NPs enhanced by EGF and 2) anti-HER2 Ab increased PS NPs–induced cell death. Both anti-HER2 Ab and PS NPs were used in clinical applications for image diagnostic and treatment of cancer, however, we don’t know what is happen in our body when used both of them together. Either anti-HER2 Ab would reduce PS NPs incorporation into cells or PS NPs would increase anti-HER2 Ab effecting. The information about the effects of anti-HER2 Ab and PS NPs on cellular response could be useful for their clinical applications.
No.1
早稲田大学 博士（工学） 学位申請 研究業績書
(List of research achievements for application of doctorate (Dr. of Engineering), Waseda University)
氏 名 LE Thi minh phuc
印
（
As of 07, 2018
） 種 類 別(By Type)
題名、 発表・発行掲載誌名、 発表・発行年月、 連名者（申請者含む）
(theme, journal name, date & year of publication, name of authors inc. yourself) Academic
papers ◯1. Le Thi Minh Phuc, Akiyoshi Taniguchi. Epidermal growth factor enhances cellular uptake of polystyrene nanoparticles by clathrin-mediated endocytosis. International Journal of Molecular Science. 2017, 18, 1301-1309.
◯2. Le Thi Minh Phuc, T. Sasaki, H. Shimizu, N.T.M. Huyen, N.T.T. Thuy, L.Q. Huan, Akiyoshi Taniguchi. Enhancement of recombinant antibody expression level by growth controlled medium. The Open Biotechnology Journal. 2018, 12, 78-85.
3. Le Thi Minh Phuc, Pham V.P., Le Q.H. Using electrochemical technique to detect HER2 antigen. Indian Journal of Applied Research. 2014, 4, 64-66.
4. Ngo T.T.H., Dam T.A., Le Thi Minh Phuc, Pham T.N., Le Q.H. Expression of gene encoding for polyepitope of early prostate cancer antigen (EPCA-2) in Escherichia coli.
Journal of Clinical Medicine. 2014, 80.
5. Le Thi Minh Phuc, Pham V.P., Dang T.M.L., La T.H., Le T.H., Le Q.H. Preparation of curcumin-loaded pluronic F127/chitosan nanoparticles for cancer therapy. Advances in Natural Sciences: Nanoscience and Nanotechnology. 2013, 4, 025001.
6. Le T.T.D., La T.H., Le Thi Minh Phuc, Pham V.P., Nguyen T.M.H., Le Q.H. Docetaxel and curcumin-containing poly(ethylene glycol)-block-poly(ε-caprolactone) polymer micelles. Advances in Natural Sciences: Nanoscience and Nanotechnology. 2013, 4, 025006.
7. Yoda T., Vestergaards M.C., Hamada T., Phuc Thi Minh Le, Takagi M. Thermo-induced vesicular dynamics of membranes containing cholesterol derivatives. Lipids. 2012, 47, 813-820.
No.2
早稲田大学 博士（工学） 学位申請 研究業績書
(List of research achievements for application of doctorate (Dr. of Engineering), Waseda University) 種 類 別
(By Type)
題名、 発表・発行掲載誌名、 発表・発行年月、 連名者（申請者含む）
(theme, journal name, date & year of publication, name of authors inc. yourself) Presentation Oral presentation
1. Polystyrene nanoparticles interfered to EGF-response of epithelial cell line. The 26th Intelligent Materials and Systems Symposium. Tokyo, Japan. 2017/1/11
2. Epidermal growth factor enhances cellular uptake of polystyrene nanoparticles by clathrin-mediated endocytosis. The 12th Nano-Biomedical Society Meeting. Tsukuba, Japan.
2017/11/9.
Poster presentation
1. Effecting of polystyrene nanoparticles on the growth of human squamous carcinoma cell line.
MANA international symposium. Tsukuba, Japan. 2016/3/9-11
2. Epidermal growth factor enhanced cellular uptake of polystyrene nanoparticles in human epithelial cell line. Tsukuba Medical Industry Cooperative Forum 2017. Tsukuba, Japan.
2017/01/20
3. Epidermal growth factor enhances cellular uptake of polystyrene nanoparticles by clathrin-mediated endocytosis. The 39th Conference of the Japan Biomaterials Society. Tokyo, Japan. 2017/11/21
