Apoptosis Mechanism Research Of Nanogold Rods Coated With MAb-EGFR Targeted Photothermal Effect On Human Hypopharyngeal Squamous Cell Carcinoma FaDu Cell Line

Objective:Hypopharyngeal carcinoma belongs to the head and neck squamous cell carcinoma(HNSCC),and the morbidity is about 95%among the primary Head and Neck tumors.Most hypopharyngeal carcinoma develops in the piriform fossa.It is one of the highest morbidity in the respiratory tract malignant tumor.Unfortunately,there is an upward trend in incidence in recent years.Due to its symptom is not obvious and easy to be neglect,so it is diagnosed until it already developed to middle-late mostly.How could we eradicate tumor,while preserving function of hypopharyngeal,reducing the recurrence and improving the survival rate,has been a clinical doctor exploration direction.Nano gold particles refers to the small particles of gold,the preparation of certain size and controllable aspect ratio of gold nanorodsss can be allowed in a very direct way cut their optical properties.When the aspect ratio of 3.9 of gold nanorodsss,near-infrared laser in800 nm wavelength exposure can cause vibration of surface conducting electrons coherent,and this is the phenomenon of surface plasmon resonance(surface plasmon resonance,SPR).Absorbing the light energy is converted into a lot of heat which is high enough relatively to destroy cancer cells without damage the surrounding normal tissues,so as to achieve the purpose of clearing tumor tissues at the cell level,which is plasma resonance light heat treatment(Plasmonic photothermal therapy,PPTT)theoretical basis.80%-90%of patients with head and neck squamous cell carcinoma(HNSCC)has been confirmed that EGFR overexpression on cell membrane.At present,EGFR monoclonal antibody(EGFR-mAb)is commonly used functional membranes.Many experiments show that the stable combination of EGFR-mAb/GNRs conjugate content can selectively kill head and neck squamous cancer cell with high expression of EGFR on membrane.This experiment used gold nanorods combined with EGFR monoclonal antibody to stimulate apoptosis of hypopharyngeal cancer cells targetedly under near infrared laser,and we tried to analyse the mechanisum in PI3K/Akt signal pathway.We study from the following aspects:(1)To observe the nanogold rods combined with mAb-EGFR gathering in hypopharyngeal carcinoma FaDu cells,and temperature variation of the cells.(2)To detect the cytotoxicity to tumor cells compared with 293T.as control cell line which has no EGFR expressed on membrane;(3)Detect apoptosis stimulated by nanogold rods combined with mAb-EGFR;(4)To analyse the apoptosis mechanism in PI3K/Akt/mTOR signaling pathway.Methods:The experiment used the aspect ratio of about 3.9 nm gold rods,its surface modification epidermal growth factor receptor monoclonal antibodies(epidermal growth factor receptor monoclonal antibody,mAb-EGFR).Then,FaDu cell line which had been interacted with nanogold rods was exposured by near infrared laser(808nm).The first part:the interaction of FaDu cell line and the nanogold rods coated with mAb-EGFR,and explore the concentration nanogold rods in vitro.We used confocal microscope to detect nanogold rods gathering in the cell,and the cytotoxicity analysis by lactate dehydrogenase method(LDH)method and the analysis of the temperature change.The second part:We tried to verify nanogold rods combined with mAb-EGFR could make FaDu cell line apoptosis under near infare laser.We used the methods those are Anexin V-PI staining and flow cytometry instrument,ELISA,and fluorescence microscopy.The third part:We tried to investigate in the PI3K/Akt signal pathway and DNA damage pathway to explainthe mechanisum of FaDu cell line's apoptosis of Photothermal caused by nanogold rods combined with mAb-EGFR,experiment using Western Blot immunoblot and the Q-PCR in vitro.We analysed PI3K/Akt signaling pathway Akt pathway,PTEN,P53 and other factors tendency and the possible mechanism of cell apoptosis.Our experiments were to explore new targeted therapeutic agents.Results:1.Nanogold rods coated with mAb-EGFR could enter FaDu cells significantly more than the nanogold rods which were unmodified by mAb-EGFR,and nanogold rods gathered in the cytoplasm,even can enter the nucleus.For 293T cells,the ones with mAb-EGFR which were into cells significantly less than the number of unmodified nanogold rods.Nanogold rods coated with mAb-EGFR for FaDu cells have the property of targeting.2.Add the same volume concentration(15%or 25%)of the mAb-EGFR/Au + NIR group,cytotoxicity of nanogold rods for FaDu cells is higher than 293 t cells(P<0.01).For 293 t cells cytotoxicity of the same concentration(15%to 25%)of Au + NIR group,cytotoxicity of mAb-EGFR/Au + of NIR group was obviously lower(P<0.01).3.The longer under the irritation of near infare laser,the higher temperature we got from nanogold rods gathering FaDu cells and 293T cells(P<0.01).And under the condition of the same dose of Nanogold rods coated mAb-EGFR for hypopharyngeal cancer FaDu cells was higher than that of 293T cells(P<0.01).4.We used Annexin V/PI flow cytometry method,Tunel method and Western Blot dBal-2/Bax,Caspase3 and 9 to verify nanogold rods with mAb-EGFR made FaDu hypopharyngeal cancer cell apoptosis.5.We used Q-PCR and Western Blot method to investigate sevral factors of the PI3K signaling pathway and DNA damage pathway,and EGFR-Au + NIR group's total AKT compared with other groups did not change significantly,but for P-AKT-S473 and P-AKT-T308,there is an obvious lower expression;PI3K,GSK3? in EGFR-Au + NIR,Au + NIR and EGFR-Au,Au processing factors under present obvious downward trend;P-PTEN in EGFR-Au + Au NIR,EGFR,EGFR processing factors have a rising trend;In DNA damage pathway,P53,the experimental group compared with control group obviously went up;ATR,P-CHK1 and P-CHK2 in EGFR-Au + Au NIR,EGFR,EGFR processing factors rising;P-PDK1 in EGFR and EGFR-Au has a rising trend in the processing factors,but expressed in EGFR-Au + NIR group reduced.Conclusions:1.Nanogold rods coated mAb-EGFR could enter FaDu Hypopharyngeal squamous carcinoma cells and 293T cells;2.When the OD value is about 1.3,the concentration of 15%-25%of the nanogold rods solution can effectively kill FaDu cancer cells.3.The in vitro experiment,we could determine the time of irritation under the near infrared laser is 6 minutes.4.Nanogold rods coated mAb-EGFR with irritation under the near infrared laser induced FaDu tumor cell apoptosis.5.Nanogold rods coated mAb-EGFR with irritation of near infrared laser,block the AKT phosphorylation,so that the PI3K/AKT/mTOR signaling pathwaysinactivation,promoted DNA damage the cancer cell,and induced Hypopharyngeal squamous carcinoma FaDu tumor cell apoptosis.