| Background: Oral squamous cell carcinoma is one of the most common malignant tumor of oromaxillo-facial region,which constructs a serious threat to human life and health.Chemotherapy is one of the important means of oral squamous cell carcinomas sequence therapy,it can kill tumor cells effectively,prevent tumor recurrence and metastasis,but its application has been limited because of the side-effect on normal tissue and organ.Nanomaterias are widely applied to oncotherapy because they can load anticarcinogen or gene effectively and redue the side-effect of chemotherapy.Carbon dots are a kind of carbon nanomaterials emerging in recent years,which can be used as a nanocarrier,besides,they also have unique luminescent properties,these properties make carbon dots have tremendous application prospect in the field of tumor diagnosis and treatment including bioimaging and tumor chemotherapy.However,there are few studies on the killing effect of carbon dots on tumor cells,mainly focusing on the fluorescence characteristics of carbon dots and the application of nanocarriers.Carbon dots may be associated with the loaded drug or gene to play anticancer effect if they can directly kill the tumor cells.Many studies showed that nanomaterials can induce autophagy and apoptosis in tumor cells,then autophagy may have different effects on apoptosis.Therefore,it is possible to provide a new vision and strategy for the treatment of oral squamous cell carcinoma by studying the killing effect of carbon dots on oral squamous cell carcinoma KB cells and exploring the role of autophagy and apoptosis in this process.Objective: To explore the effect of ascorbic acid carbon dots on proliferation,autophagy and apoptosis of oral squamous cell carcinoma KB cells,to pinpoint the killing mechanism of the ascorbic acid carbon dots on KB cells,and to provide theoretical basis for the application of ascorbic acid carbon dots synergying with drug or gene loaded on oral squamous cell carcinoma chemotherapy in future.Methods: First the ascorbic acid carbon dots were synthesized by microwave method,then the shape and size of carbon dots were observed by transmission electron microscopy(TEM),and the size and potential of carbon dots were analyzed by dynamic light scattering.To detect the cellular uptake of KB cells to carbon dots,first we observed cell numbers with green fluorescence in various groups by fluorescence microscope,then analyzed by flow cytometry.MTT assay was performed to detect the inhibitory effect of carbon dots on KB cells proliferation,and colony formation assay was performed to detect the influence of carbon dots on KB cells colony formation ability.The intracellular autolysosome numbers were observed after AO staining by fluorescence microscope to evaluate the level of autopahgy,and Western Blot was performed to analyze the protein level of autophagy-ralated protein LC3 in various groups.The apoptosis rate was detected by flow cytometry after Annexin VFITC/ PI double staining.Results: According to TEM results,the size of ascorbic acid carbon dots was 5.36 nanometer average,and the lattice was 0.24 nanometer average.Dynamic light scattering results showed that the hydrodynamic size of carbon dots is 12.89 nanometer and the potential is +8.92 millivolt average.Fluorescence microscope observed that there was green fluorescence in cytoplasm after KB cells treated by 40 ?g/m L carbon dots for 2 hours,moreover flow cytometry results showed the cell numbers with green fluorescence was increased effectively after KB cells treated by 40 ?g/m L carbon dots for 4 hours(P<0.001)compared with blank control group,these results indicated ascorbic acid carbon dots could enter into KB cells.Compared with blank control group,the proliferation rates and colony formation ability were decreased obviously(P<0.01 and P<0.001)in 5?10?20?40 and 80 ?g/m L groups.Compared with blank control group,the numbers of cellular autolososome were increased in various experimental groups,however Western Blot results showed the protein level of LC3 II were markedly increased only in 40 ?g/m L group(P<0.05),suggested the carbon dots could induce autophagy in KB cells.Moreover,the apoptotic rate was also increased markedly in 40 ?g/m L carbon dots group(P<0.001),suggested that the carbon dots could induce apoptosis in KB cells.Compared with carbon dots group,the apoptotic rate reduced obviously in co-treated group with autophagy inhibitor 3-MA and carbon dots(P<0.05),suggested that the autophagy could promote cell apoptosis induced by carbon dots.Conclusions: The ascorbic acid carbon dots synthesized was spherical carbon nanomaterials,its average size was 5.36 nanometer and its average potential was +8.92 millivolt,and the ascorbic acid carbon dots could entry into KB cells.They could suppress the proliferation,weaken the colony formation ability,induce autophagy and apoptosis in KB cells.The autophagy induced by ascorbic acid carbon dots could promote the apoptosis of KB cells,suggested autophagy and apoptosis may play a synergistic effect in the process of the ascorbic acid carbon dots killing KB cells. |
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