Study On The Apoptosis Mechanism Of Functionalized AuNRs With Photothermal In Hep-2 Cell Line

Objectives:Cells can be maintain biological form of steady state by autophagy, while autophagy is dependent on the lysosome. We imagine using existing clinical similar single resistance drug as surface modification for AuNRs, to explore the EGFR receptor blocking in photothermal treatment of the tumor. Therefore, in this research we use the listed products- cetuximab as functional membranes for AuNRs、regard Hep-2 cell system as the research object、construct a model of EGFRmAb /AuNRs target cancer cells, it expect to enhance the effect of photothermal treatment of the tumor, to further research the regulating mechanism of autophagy/lysosomal pathway in the IMC-C225/AuNRs induce the laryngeal squamous cancer cells apoptosis.Methods:1. Observation of cell morphology by Inverted microscope;2. AuNRs’solar-thermal heating experiment;3. Cell proliferation by MTT;4. Testing protein level changes in the cell by Western Blot;5. Observation autophagy body by electron transmission microscope;6. Testing mRNA level changes in the cell by RT-qPCR.Results:1. The AuNRs with Hep-2 cells developObserve the cell morphology under a microscope, there is no obvious change between AuNRs/Hep-2 and normal Hep-2 cells. Determined by MTT method cell growth results shown: two kinds of cell in "S" type curve growth 1-7 day roughly, AuNRs shows no obvious cytotoxicity .2. Testing different concentrations of AuNRs’ photothermal Different concentrations of AuNRs after NIR irradiation exists obvious effect of optical (up to 36.5℃) and reach a certain temperature and constant after 3 min.3. Effects that different concentrations of AuNRs collocation with NIR irradiation on the cell vitality In Onmol/L、0.3nmol/L、0.6nmol/L、0.9nmol/L Hep-2/AuNRs four groups,NNIR group’s cell survival rates were 100%,98.47%,96.60%,98.47%; YNIR group cell survival rates were 55.56%, 39.69%, 35.39%, 39.69%, further compare NNIR group with YNIR cell survival rate, p<0.01, there is a high degree of statistical difference that NIR irradiation on cells have a significant effect.4. Effects that different concentrations of IMC-C225/AuNRs collocation with NIR irradiation on the cell vitalityUnder 5W, 6W, 7W and 8W irradiation intensity, Hep-2+0.3 nmol/L AuNRs group and Hep-2+IMC-C225/AuNRs group of cell survival rates were 87.37%,86.88%,84.95%,86.88% respectively and 95.20%,74.76%,54.49%,74.76%respectively, among them p of 5W group is meaningless, 6W group’s p<0.05, 7W group’s p<0.01, 8 w group’s p<0.01, differences were statistically significant,and with the increase of concentration of AuNRs , Hep-2 cell’s inhibition rate also increased. Results: IMC-C225/AuNRs, even under the condition of weak NIR irradiation, there is an obvious cell inhibitory effects of the cancer cells have a very strong lethality. Follow-up experiments we can choose 6W irradiation intensity, because in this condition, the thermal effect on cancer cell killing effect is obvious and easy to we record the result of the experiment.5. Effects that different concentrations of IMC-C225/AuNRs collocation with NIR irradiation on the cell vitality after join the autophagy inhibitorsUnder YNIR condition, Hep-2+3-MA+CQ+IMC/AuNRs group and Hep-2+PA+CA074 IMC/AuNRs group’s cell survival rates were 46.19% and 50.44%respectively, compared between the two group’s p<0.01,there are significant difference of statistical. Results: after joining lysosome autophagy inhibitors block the lysosome the protective effects of autophagy in cancer cells, so further enhance the IMC-AuNRs lethality of cancer cells.6. Testing protein level changes in the cell by Western Blot;Autophagy related proteins LC3II in YNIR group was obviously expressed,and increased with increasing concentration of gold nanorods; YNIR group compared with NIR group, p<0.05, there are significant difference of statistical.7. Testing mRNA level changes in the cell by RT - qPCRSelected six autophagy related genes’(ATG13、LC3、P62、ATG5、ULK1、VPS34D ) mRNA are rising as concentration of AuNRs are suppressed, and YNIR group’s autophagy related genes’ mRNA expression of the decline of more severe; NNIR group compared with YNIR group’s p<0.05, the difference was statistically significant.8. Observation autophagy body by electron transmission microscopeThree experiments are magnified 6000, 10000, and 30000 times to observe the cell morphology and internal structure, including the nucleus, mitochondria,lysosomes and endoplasmic reticulum. Found that each group’s to be in good condition, no obvious apoptosis phenomenon, some cells occur autophagy (find autophagy- ly so some). II and III groups of a small number of cells can be observed that are solid material (black substance of length to diameter less than 100 nmol/L) concentration enrichment in the mitochondria and lysosomes. And at the same magnification, the enrichment of the solid material in the III groups significantly greater than the number of cells in the two groups.Conclusions:1. NIR irradiation has a significant role for Hep-2.2. IMC-C225/AuNRs, under the condition of weak NIR irradiation, compared with the single use of nanometer gold rods significantly inhibits cell proliferation,strong killing effect on cancer cells .3. Blocked the the protective effects of autophagy in cancer cells after join the autophagy inhibitors, thus further enhance the IMC-C225/AuNRs lethality of cancer cells.4. Autophagy is involved in the process of functional AuNRs induce laryngeal squamous cancer cell death.