Primary Study On Autophagy And Cisplatin Resistance Induced By Areca Nut Extract In Oral Squamous Cell Carcinoma Cells:Roles Of Reactive Oxygen Species/AMPK Signaling

Oral squamous cell carcinoma(OSCC)is one of the common malignancies in the head and neck.In many OSCC pathogens,betel nut chewing has a significant correlation with the increased risk of OSCC,especially in the Indian and Southeast Asian countries,including some provinces in mainland China.However,the effect of chewing betel nut on the chemosensitivity of OSCC still lacks the corresponding study.Autophagy is an intracellular adaptive response that maintains energy balance under various stresses,such as hypoxia,starvation,ischemia/reperfusion,and so on.Although autophagy is still controversial in the treatment of OSCC,autophagy has become a potential anticancer target in cancer prevention and treatment.Reactive oxygen species(ROS)can cause genomic instability by inducing DNA damage.ROS can induce autophagy and autophagy can reduce oxidative damage in contrary.ROS can also be related to tumor stem cell and chemotherapy resistance.The areca nut extract can induce ROS elevation in cancer cells and normal oral epithelial cells,and induce autophagy by ROS.However,the potential molecular mechanism of ROS and autophagy-induced cisplatin resistance induced by areca nut extract is not clear.Adenosine monophosphate-activated protein kinase(AMPK)plays an important role in energy metabolism,which can also be triggered by ROS signaling;AMPK activation down-regulates mTOR activation,whereas mTOR is a key negative regulatory signal that inhibits autophagy.In this study,it was proved that areca nut extract could induce the autophagy of OSCC for a long time and could be involved in the resistance of cisplatin in OSCC cells by autophagy,and ROS and AMPK/mTOR signaling pathway played a key role in this process.Based on the results of this study,it is intended to provide potential optimization strategies for chemotherapy in OSCC patients with chewing betel nut.Part Ⅰ:Effects of low dose of areca nut extract on intracelluar reactive oxygen species(ROS)and autophagy level in oral squamous cell carcinomaObjective:To investigate the changes of intracellular ROS and autophagy levels after long-time stimulation with low concentrations of areca nut extract,and to explore the relationship between ROS and autophagy caused by areca nut extract.At the same time,we studied expression of autophagy specific protein LC3 in OSCC primary tissue caused by chewing betel nut.Material and methods:The effects of areca nut extract on cell viability of OSCC cell line and its relationship with the concentration were studied.The appropriate concentration of areca nut extract was used to stimulate the oral squamous cell carcinoma cell line.The expression of autophagy-related proteins in OSCC cells was detected by Western Blot.The ROS level in OSCC cells was detected by fluorescence microscopy and cell flow cytometry.Flow cytometry was used to detect intracellular MDC staining levels.And the relationship between ROS and autophagy was studied by using ROS scavenger NAC pretreatment to detect autophagy-related protein and MDC staining intensity.Eighty-two OSCC patients treated with cisplatin preoperatively were enrolled in this study.Forty-two OSCC patients without chewing betel nut habit were used as controls.Forty patients with OSCC with chewing betel nut history were examined.And to explore the relationship between the sensitivity of cisplatin and the expression of LC3 in OSCC patients.Results:1.CCK-8 cell viability assay and Annexin V-FITC apoptosis assay showed that the effect of areca nut extract on the viability of oral squamous cell carcinoma,it was revealed that a time and dose-dependent toxic effect when the concentration of betel nut was greater than 8μg/mL.When the concentration of areca nut extract was 1-4μg/ml,the cell viability and apoptosis rates were not significantly different from those in the control group.2.Effects of long-term non-toxic doses of areca nut extract on ROS and autophagy levels in oral squamous cell carcinoma:Effects of 3μg/mL areca nut extract were used on Cal-27 and Scc-9 cells for 6 days,intracellular DCF-DA fluorescence proved that areca nut extract stimulation can lead to increased ROS levels.According to the expression of autophagy-related proteins and the level of MDC staining,it was proved that areca nut extract promoted the level of autophagy in OSCC cells.According to the DCF-DA/MDC staining and the results of Western Blot,the pretreatment of NAC(4mM)with active oxygen scavenger can effectively reduce the level of MDC staining and decrease the autophagy-related protein LC3-Ⅱ and Atg5-Atg12,whereas SQSTM1/p62 expression was elevated.3.The results of immunohistochemical staining showed that the distribution of autophagosomes was expressed in the cytoplasm of OSCC tissue cells in the form of spots.LC3 was expressed higher in the primary tissues of OSCC patients with chewing betel history,also the expression of LC3 was significantly higher in cisplatin-insensitive patients than in cisplatin-sensitive group.Conclusion:Based on the above results,we found that low concentration areca nut extract(1-4μg/ml)had no direct toxic effect on cells.After 6 days of stimulation with low concentration(3μg/ml)of areca nut extract in OSCC cells,intracellular ROS and autophagy levels were significantly increased,and the level of autophagy was dependent on the ROS signal.Part Ⅱ:Study on AMPK/mTOR Signaling Pathway in Autophagy induced by Areca nut Extract in OSCC CellsObjective:Study on the effects of AMPK/mTOR pathway on autophagy with low toxicity and chronic stimulation in OSCC cells induced by areca nut extract,which were investigated by a variety of experimental techniques.Material and methods:The expression of AMPK/mTOR pathway-related protein was detected by Western Blot and immunofluorescence technique.The expression of AMPK/mTOR/S6 pathway and autophagy-related protein was detected by Western Blot after ANE treatment.And the expression of AMPK/mTOR/S6 pathway and autophagy-related protein was also detected after pretreated with AMPK pathway inhibitor Compound C.The autophagy specific marker LC3 was quantitatively analyzed by immunofluorescence technique in OSCC cells under different treatment conditions.The confocal fluorescence microscopy was observed and photographed.At the same time,autophagy was detected by transmission electron microscopy to further verify the effect of Compound C pretreatment on the autophagy induced by areca nut extract.The expression level of p-AMPK/p-mTOR was detected by immunohistochemistry using the clinical specimens(40 cases)of OSCC patients with chewing betel history.And the relationships between p-AMPK/p-mTOR with autophagy-specific marker LC3 were analyzed by Spearsman correlation test in tissue samples.Results:1.The effect of low toxicity areca nut extract on the expression of AMPK/mTOR pathway-related protein in oral squamous cell carcinoma cells:Western Blot and immunofluorescence showed that the areca nut extract could up-regulate p-AMPK expresssion,whereas the expression level of p-mTOR/p-S6 were also decreased.2.Effects of Compound C pretreatment on AMPK/mTOR pathway and autophagy level:Compound C pretreatment could effectively reduce the level of autophagy in oral squamous cell carcinoma cells caused by areca nut extract,it was revealed that LC3-Ⅱ was lower than that of the only areca nut extract gourp,while p62 was higher than that of only areca nut extract group.At the same time,Compound C could inhibit the activation of AMPK,which affected the phosphorylation of mTOR and S6 level.Quantitative analysis of autophagy-specific marker LC3 by cellular immunofluorescence technique:Compound C pretreatment can be used to reduce the autophagy effects before the administration of areca nut extract.TEM results showed that the number of autophagosomes was significantly higher in Cal-27 cells after stimulation with areca nut extract.Similarly,AMPK inhibitor Compound C could reduce the autophagy activity in Cal-27 cells according to autophagosomes detected by TEM.3.The relationship between the expression level of p-AMPK/p-mTOR and LC3 in tissue samples:The clinical specimens(40 cases)of patients with OSCC caused by chewing betel nut were detected by immunohistochemical staining:the expression level of LC3 in the tissue samples was negatively linearly correlated with the level of p-mTOR staining(Y = 567.3-48.9X,r2 = 0.2641,p = 0.0007).The level of p-AMPK staining was positively correlated with LC3 expression(Y＝91.7 + 66.2X,r2 = 0.3735,p<0.0001).Conclusion:The areca nut extract can induce autophagy through the AMPK/mTOR pathway.Part Ⅲ:In vitro study on correlations between ROS/AMPK mediated autophagy induced by areca nut extract and cisplatin resistanceObjective:We assume that ROS/AMPK pathway-mediated autophagy may be associated with cisplatin resistance,and this study aims to demonstrate that areca nut extract is involved in cisplatin resistance in vitro.Material and methods:The cell viability of Cal-27 and Scc-9 under different treatment conditions was measured by cell viability assay.The number of apoptosis in Cal-27 and Scc-9 cells under various treatments was detected used by Annexin V-FITC Apoptosis Detection Kit.The expression of cl-PARP and cl-caspase3 in cisplatin-stimulated Cal-27 cells was detected by Western blot.Results:1.The effect of cisplatin on the viability of cells after ANE pretreatment in OSCC cells:Compared with the control group,the treatment group with areca nut extract showed higher cell viability after cisplatin exprosure.Annexin V-PI double staining showed that cisplatin-induced apoptotic cells were significantly decreased in OSCC cells treated with areca nut extract,suggesting that OSCC stimulation with areca nut extract could increase cell viability when stimulated with cisplatin.2.The relationship between autophagy and cisplatin on OSCC cytotoxicity mediated by ANE:The results of cell viability and apoptosis test showed that,during the stimulation of OSCC cells with areca nut extract,using 3-MA and NAC treatment can significantly increase the toxicity of cisplatin to OSCC cells by effectively blocking autophagy.Compound C also significantly reversed the drug resistance of cisplatin in Cal-27 cells treated with ANE.3.The expression of cl-PARP and cl-caspase3 in Cal-27 cells treated with betel nut was significantly higher than that of control cells.NAC and Compound C pretreatment could partially down regulate the cl-PARP and cl-caspase3 protein expression,seperately.Conclusion:ROS/AMPK-mediated autophagy caused by areca nut extract could be involved in the reduction of cisplatin toxicity,and the expression of cl-PARP and cl-caspase 3 could be related with decreased cisplatin toxicity.Taken together,our results suggest that the low toxic areca nut extract can lead to elevated levels of ROS and autophagy in OSCC cells,while areca nut extract can reduce the toxicity of cisplatin by inducing autophagy.The ROS and AMPK/mTOR signaling pathways play key roles in this process.