| Objective:According to the latest statistics,esophageal cancer is currently the seventh most common malignant tumor in the world and the sixth most deadly tumor-related disease.The two main histological types of esophageal cancer are esophageal squamous cell carcinoma?ESCC?and esophageal adenocarcinoma.In Asia,especially in China,esophageal squamous cell carcinoma is the main type of esophageal carcinoma.ESCC has become a major difficulty in cancer treatment due to its high aggressiveness and low five-year survival rate.Aryl hydrocarbon receptor?AHR?is a ligand-dependent transcription factor which was found in the research of environmental pollutants toxic effects on human and AHR plays an important role in environmental carcinogenic process.Studies have shown that AHR may participate in the regulation of the malignant biological behavior of various tumors,such as promoting the proliferation of tumor cells,inhibiting apoptosis,promoting migration and invasion and metastasis.However,researches on AHR regarding esophageal cancer are rarely reported and whether AHR has the same effect on promoting ESCC progression with a certain mechanism and whether AHR can become a therapeutic target of ESCC.Therefore,this study mainly investigated whether AHR had cancer-promoting effects at the genetic level,and after treatment of its modulator 3,3'-diindolylmethane?DIM?,whether effects of AHR could change at the pharmacological level.Finally,conducting an in vivo experiment again verified the functional effects of AHR on ESCC.Methods:1.Select 54 cases of patients who underwent esophageal squamous carcinoma resection in Department of Thoracic Surgery of The First Hospital of China Medical University in 2011-2013.The slides including paired tumor and para-tumor tissues were used for immunohistochemistry?IHC?staining of AHR.The IHC results were analyzed with clinical pathologic factors and overall survival time.At the same time,we tried to explore the expression levels of AHR through bioinformatics analysis in ESCC database;2.ESCC cell lines TE1 and KYSE150 were selected for AHR knockdown.CCK8,colony formation assay,EdU staining,flow cytometry,wound scratch assay and Transwell assay were used to verify the effect of AHR knockdown on cell proliferation,cycle,apoptosis,migration and invasion.Western Blotting?WB?was conducted for determining the underlying mechanism of aforementioned changes;3.After treatment of various concentrations of AHR modulator DIM,we tried to filter out the proper concentration and incubative time.CCK8 assay was used to detect changes of cell proliferation and verify whether DIM exerted its effects in an AHR-dependent manner.Colony formation assay was utilized to visualize the inhibitory effect of DIM on ESCC.Flow cytometry was performed to detect alterations of ESCC cell cycle and cell apoptosis after DIM treatment.Wound scratch assay and Transwell assay were performed to detect effects on capacities of ESCC migration and invasion.WB was conducted to test related mechanisms of these phenotypic changes;4.Explore whether AHR could mediate changes of COX2/PGE2/STAT3 pathway.After treatment with AHR knockdown and various concentrations of DIM,WB was performed to detect this pathway changes and verify the exact relationship between upstream and downstream.And after treatment of COX2 selective inhibitor celecoxib and prostaglandin E2?PGE2?,wound scratch assay and Transwell assay were used to determine the alterations of abilities of ESCC migration and invasion.WB was for visualizing related pathway changes;5.Tumor xenograft assay was conducted with nude mice to demonstrate that modulation of aryl hydrocarbon receptor by DIM could inhibit esophageal squamous cell carcinoma progression by repressing COX2/PGE2/STAT3 pathway.And IHC was used for staining relative gene expressions.Results:1.AHR was highly expressed in esophageal squamous cell carcinoma compared with adjacent tissues,and was associated with lymph node metastasis and clinical stage.Meanwhile,high AHR expression was related with poor prognosis.Bioinformatics analysis showed that AHR was indeed highly expressed in esophageal squamous cell carcinoma.2.After knocking down AHR gene,the proliferation of TE1 and KYSE150cell lines was inhibited and cell cycle was arrested in S phase.Cell apoptosis was promoted and abilities of cell migration and invasion were weakened.WB detection showed that the expressions of PCNA,Bcl2,MMP1,MMP2 and MMP9 were down-regulated,while the expressions of Bax and Cleaved Caspase 3 were up-regulated.3.After treatment with AHR modulator DIM,DIM inhibited the proliferation of ESCC cells in a dose-dependent and time-dependent manner,and the effect was obvious at48-hour with concentration of 40?mol/L.At the same time,DIM induced cell cycle arrested in G1phase and promoted cell apoptosis.Meanwhile,DIM could inhibited cell migration and invasion.WB detected down-regulated expressions of PCNA,Cyclin D1,Bcl2,MMP1,MMP2 and MMP9,while up-regulated expressions of Bax and Cleaved Caspase 3.4.DIM can inhibit the COX2/PGE2/STAT3 pathway by regulating AHR transcription,thus inhibiting the proliferation,migration and invasion of ESCC cells.5.After DIM treatment,the average volume of transplanted tumor was significantly lower than that of the control group,and the proliferation inhibition was obvious.Meanwhile,the results of IHC staining were the same as those of in vitro experiments.Conclusion:AHR is highly expressed in esophageal squamous cell carcinoma and is associated with lymph node metastasis,clinical staging and poor prognosis.After knocking down AHR gene,it inhibits the proliferation,migration and invasion of ESCC cells,and promotes cell cycle arrest and apoptosis.In vivo and in vitro experiments confirmed that DIM inhibits the progression of ESCC by repressing the COX2/PGE2/STAT3 pathway through modulating AHR. |
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