Study On The Molecular Mechanism Of Apoptosis In Laryngeal Squamous Cancer Cells Induced By Photothermal With AuNRs Functionally Modificated Using EGFRmAb

PART Ⅰ:Study on the expression of epidermal growth factor receptor in laryngeal squamous cell carcinoma tissue and Hep-2cell apoptosis induced by siRNA of silencing EGFR geneObjectives:Investigate expression of EGFR in tissue of laryngeal squamous cell carcinoma. Clarify the relationship between expression and pathological classification. Explore the influence of RNA interference on expression of EGFR mRNA gene and apoptosis in Hep-2laryngeal cancer cells.Methods:Collected36cases of laryngeal cancer specimens from January2008to March2011in the first and the third affiliated hospital of Kunming medical college. Normal controls groups came from13patients with total laryngectomy. Hep-2cell line served as the positive control. Western blot was used to detect the EGFR expression in cancer tissue, paracancer tissue and normal mucosa tissue. Quantity One software was used to quantitative analysis on image to obtain the relative protein expression of EGFR, and analyzed the relationship between them and the pathology classification. RNAi technology was used to interfere in expression of EGFRmRNA gene in Hep-2laryngeal cancer cells. RT-PCR was used to detect the inhibitive rate of EGFRmRNA gene. Western blot was used to test protein expression of EGFR. Flow cytometry(FCM) was used to further test the expressing inhibition of EGFR and the apoptotic rate.Results:(1) Order of expression of EGFR protein in the laryngeal cancer samples from high to low is laryngeal cancer tissue,paracancer tissue and normal mucosa tissue. The normal mucosa tissue shows no bands about EGFR protein in Western blot. Expression of EGFR have differences in laryngeal cancer tissue,paracancer tissue and normal tissue with different differentiation, P<0.05. All above show that the expression level of EGFR has correlation with different locations in laryngeal cancer samples and the tissue differentiation.(2) Obtain expression plasmid pSi-hEGFR of EGFRsiRNA successfully. Use RT-PCR to detect after transfecting Hep-2cells for48h. The results show that gene expression level of EGFR in Hep-2cells transfected with pSi-hEGFR plasmid is54%of the control cells. All above results demonstrate that transfection of pSi-hEGFR plasmid obviously inhibit the expression of EGFR gene. Western blot and Flow cytometry detection get consistent results. Further detection finds that the apoptosis of Hep-2laryngeal cancer cells reach at15.13%after transfecting with Si-hEGFR plasmid for48h, and7.03with transfecting with pSi-Negative plasmid. The difference is very significant, P<0.01. All above results demonstrate that RNAi technology may silence EGFR gene expression and induce Hep-2 cell apoptosis.Conclusions:EGFR is highly expressed in laryngeal cancer. In different locations the EGFR expression rate is not the same, cancerous tissue is higher than paracancer tissue, paracancer tissue is higher than normal mucosa tissue. RNAi technology silencing EGFRmRNA can inhibit the EGFR expression of Hep-2laryngeal cancer cells. The technology of RT-PCR,Western blot and FCM detection get accordant result. Further observation finds that the gene interference can induce Hep-2laryngeal cancer cells apoptosis. This study suggests that EGFR might be involved in occurrence and development of laryngeal cancer. Using the RNAi technology to down-regulate the expression of EGFR can play a part of suppression effect on tumor, which is getting hope for exploring the new methods of anti-tumor targeting at the EGFR. PART II:Experimental study on the photothermal effect of AuNRs modificated by EGFRmAb functionally which inhibits proliferation of Hep-2cellsObjectives:Use EGFRmAb to modificate AuNRs functionally and obtain steady EGFRmAb/AuNRs conjugate reagent. Explore the mechanism of AuNRs coming into tumor cells. Find the warming law of photothermal conversion of AuNRs. Evaluate the biological safety of AuNRs. Observe the Hep-2cell proliferation inhibition made by photothermal of EGFRmAb/AuNRs through controlling the irradiation parameters of NIR laser.Methods:The EGFRmAb/AuNRs was characterized by transmission electron microscopy. Spectral analysis was made by UV-vis-NIR spectrometer. AuNRs and EGFRmAb/AuNRs were incubated with Hep-2cells respectively, and transmission electron microscope was used to observe AuNRs in cells. ICP-AES element analysis was used to know the amount of AuNRs coming into Hep-2cells mediated by EGFRmAb. After irradiating with NIR laser, thermometer was use to detect the temperature of solution with AuNRs under different conditions to know rise of temperature. MTT was used to detect the cytotoxicity of AuNRs at different concentrations. Trypan Blue Dye was used to observe the phenomenon of killing cancer cells by AuNRs photothermal. MTT was used to detect proliferation inhibition rate of Hep-2cell after different NIR laser irradiating AuNRs at different time points. Microscope was used to observe cellular form change. The effect of tumor suppression was compared between AuNRs group and EGFRmAb/AuNRs group.Results:(1) Characterization of the electron microscope shows that AuNRs in this experiment have good stability and dispersion, and particle size is uniform, which is49.81nm length and12.70nm diameter, Aspect ratio is3.92. UV-vis-NIR spectrometer test shows that AuNRs have double resonance absorption peaks, transverse peak at510nm, longitudinal peak at800nm. After modificated by EGFRmAb, the longitudinal resonance absorption peaks of AuNRs have a redshift from7to9nm, which does not change the original optical properties.(2) Electron microscopic observation shows that after incubating for30min AuNRs are seen on the membrane surface of Hep-2cell, which appears the swallow phenomenon. Incubating for3h AuNRs are seen in the lysosome in clumps status. Incubating for6h AuNRs exist in the cytoplasm, lysosome and mitochondria, and a small amount of AuNRs close to nuclear membrane. We do not find obvious organelles change of cellular shape and structure. The number of AuNRs in cells modificated by EFGRmAb functionally is more than non-modificated. Further ICP-AES element analysis shows that the number of modificated AuNRs in cells increases by48.14%when incubating for6h.(3) NIR laser irradiating can cause temperature rise quickly in AuNRs solution, and the range of temperature rise has correlation with concentration of AuNRs, power of NIR laser and containers/volume. This study also finds that, in room temperature of23℃, using3.0W/cm2power of NIR laser to irradiate the0.1nmol/L AuNRs solution (1ml/every hole,24hole boards) for4min leads to stable temperature rise (46℃), which is the ideal experimental condition.(4) MTT test shows that the cytotoxicity of AuNRs in concentration range from0.1nmol/L to2.0nmol/L is not obvious. After incubating with0.1nmol/L AuNRs conjugated by EGFRmAb, the cellular vitality is for95%.(5) The results of Trypan Blue Dye test show that all of Hep-2cells are dying at10min after irradiating the solution with5.2W/cm2power of NIR laser for8min. Further MTT detection shows that photothermal of AuNRs and AuNRs/EGFRmAb can obviously cause Hep-2cell proliferation inhibition with depending on the dose of laser power. NIR laser irradiation and EGFRmAb show no obvious proliferation inhibition for Hep-2cells. The proliferation inhibition of Hep-2cell is time dependence in the laser power at3.0W/cm2NIR. The inhibition rate is highest to69.24%at72h. Photothermal effect of AuNRs/EGFRmAb on Hep-2cell proliferation inhibition is obviously higher than non-modificated AuNRs, P<0.01.Conclusions:The original shape and optical properties of AuNRs modificated by EGFRmAb do not change significantly, but which can obviously increase the ability of AuNRs coming into Hep-2cells. This phenomenon may be relevant with the endocytosis mediated by EGFR. AuNRs in cell mainly exist in cytoplasm, lysosome and mitochondria. Irradiating with NIR laser AuNRs can quickly cause environmental temperature rise, which lead to cell proliferation inhibition or cell death. If controlling the irradiating power of NIR laser, cell proliferation inhibition is time dependence. Functional modification of EGFRmAb can obviously improve the suppression effect of AuNRs photothermal on tumor cell, but NIR laser and EGFRmAb have no cell proliferation inhibition. Since AuNRs in mitechondria, we assume that the cell proliferation inhibition may be the results of endogenous apoptosis induced by photothermal with AuNRs PART Ⅲ:Study on the apoptotic mechanism of Hep-2cell induced by photothermal with EGFRmAb/AuNRsObjectives:Control irradiation parameters of NIR laser to observe apoptotic phenomenon of Hep-2cell induced by AuNRs photothermal. Detect the protein molecules related with apoptosis of mitochondria pathway. Elaborate the molecular mechanism of apoptosis. Determine whether AuNRs modificated by EGFRmAb can obviously improve the apoptotic effects induced by photothermal targeting at Hep-2cell.Methods:The concentration of AuNRs was at0.1nmol/L. The irradiation power of NIR laser was at3.0W/cm2. The irradiation time was8min. Electron microscope was used to observe the ultrastructural changes and apoptosis of Hep-2cells after irradiation. Flow cytometry(FCM) was used to detect the cellular apoptosis and cycle at different time points with TUNEL/PI double dying method. Detect the positive expression rate of active Caspase-3, ROS levels and Ca2+concentration, positive expression rate of Bcl-2/Bax, decreasing rate of△ψm and positive expression rate of Cyt-c about Hep-2cell after irradiation at different time points.Results:(1)Electron microscopic observations show that NIR+AuNRs group and NIR+EGFRmAb/AuNRs group have visible cellular apoptosis after48h, but the form and structure of cells in EGFRmAb group and control group are normal.(2) Comparing with control group, the cells in NIR+AuNRs group and NIR+EGFRmAb/AuNRs group display obviously blocking phenomenon in G0/G1phase at24h and48h, and the S phase ratio decreases obviously, P<0.01, which has time dependence. At48h, comparing with AuNRs group,.the S phase ratio of cells in EGFRmAb/AuNRs group declines obviously, P<0.01.(3) Comparing with control group, cell apoptosis is obvious in NIR+AuNRs group and NIR+EGFRmAb/AuNRs group, P<0.01. At72h, the apoptosis rate of NIR+EGFRmAb/AuNRs is up to73.63%. The apoptosis rate of NIR+EGFRmAb/AuNRs group is higher than NIR+AuNRs group at the three time points (24h,48h,72h),P<0.05. The apoptosis rate of AuNRs group, NIR group and EGFRmAb group were not obvious at three time points, less than6%.(4) In NIR+AuNRs group and NIR+EGFRmAb/AuNRs group, the positive expression rate of active Caspase-3and Bax, cellular ROS levels, Ca2+concentration and decline rate of△ψm in cells increase, and positive expression rate of Bcl-2in cells reduce. By comparing between the two groups, the changes of above indexes about NIR+EGFRmAb/AuNRs group are more obvious,.P<0.05. The positive expression rates of Cyt-c in two observation groups increase significantly, P<0.01. At1h after irradiation, the positive expression of Cyt-c can be detected (highest at24h, and then gradually decline). The Cyt-c expression rise of NIR+EGFRmAb/AuNRs is more obvious at24h and48h than NIR+AuNRs group, P<0.01.Conclusions:If control the irradiation parameters of NIR laser in appropriate range, AuNRs photothermal can induce Hep-2cell apoptosis, which closely relate with the cell cycle arrest. ROS and Ca2+level rise,△ψm decrease,Cyt-c release,active Caspase-3rise and Bcl-2/Bax proportion decrease play an important role in the process of Hep-2cell apoptosis. It can be seen that the endogenous pathway mediated by mitochondria is needed for Hep-2cell apoptosis induced by AuNRs photothermal. EGFRmAb modification can improve the ability of AuNRs targeting at cells, which improves the apoptotic effect. Furthermore, EGFRmAb itself do not obviously take part in the occurrence of apoptosis. PART Ⅳ:Experimental study of photothermal therapy with EGFRmAb/AuNRs on the transplant tumor with laryngeal squamous cell cancer in nude miceObjectives:Observe the therapy effects on laryngeal transplant tumor and the apoptosis of tumor tissue cell in nude mice treated with AuNRs photothermal (local injection and systemic administration).Methods:Set up subcutaneous laryngeal transplant tumor nude mice models. AuNRs were injected into the tumor tissue. EGFRmAb/AuNRs were infused through tail vein. Irradiation parameters were same as the vitro studies in the third part. The temperature of skin near tumor was measured. Tumor size was measured with vernier caliper after irradiation. After two weeks at the end of experiment, drew the growth curve, euthanized the mice and cut off the tumor specimen. FCM was used to detect the apoptosis and necrosis of tumor cells. Results:After irradiating with NIR laser for3min, the skin temperature at tumor place was going to be stable. The temperature of skin in the group of AuNRs/local injection increased for15℃. The group of EGFRmAb/AuNRs increased for11℃. In two treatment groups, the growths of tumors were inhibited obviously. The speed of growth at each observation time points was significant slow in the two above groups. The therapy effect in the group of AuNRs/local injection after irradiation treatment was more obvious, P<0.05. All of systemic administration of EGFRmAb (Sigma, E3138) alone,small doses of NIR laser irradiation and systemic administration of AuNRs not modificated by EGFRmAb did not produce the suppression effect on tumor. Flow cytometry detection showed that there were apoptosis in the group of AuNRs/local injections and systemic administration of EGFRmAb/AuNRs,58.35%and30.46%respectively. The apoptosis and necrosis rates of AuNRs/local injection group were significantly higher than the systemic administration group of EGFRmAb/AuNRs, P<0.01.Conclusions:AuNRs with direct injection can suppress the growth of transplant tumors in mice. Systemic administration of EGFRmAb/AuNRs also shows treatment effect. Flow cytometry detection confirms that apoptotic phenomenon induced by photothermal is existing. The effect of animal experiments brings hope for photothermal therapy with EGFRmAb/AuNRs targeting at cancer. Due to the complexity of the environment in body, further study should be done.