Study On The Mechanism Of Doxorubicin Induced Epithelial-Mesenchymal Transition In Human Gastric Cancer Cells

Background and purpose. Metastasis is the main cause of cancer related death, yet it remains the most poorly understood component of cancer pathogenesis. The epithelial-mesenchymal transition is a cellular program by which epithelial cells convert to mesenchymal cells. This important process was initially recognized during several critical stages of embryonic development and has more recently been implicated in promoting carcinoma invasion and anti-cancer drug resistance. Chemotherapy is widely used in cancer treatment. A small population of cancer cells can acquire new traits of metastasis and drug resistance as consequences of internal and external stresses, which usually leads to chemotherapy failure. In this thesis, the human gastric cancer cells BGC-823were used as an objective of studying the effects and underlying mechanisms of doxorubicin, a widely used anti-cancer drug, induced EMT. We hope this study could provide new strategy for cancer treatment.Methods. BGC-823cells were treated with doxorubicin for48hours. Cell morphology changes were then observed under light microcopy. EMT-related molecular markers were evaluated by immunofluorescence, Western blot and RT-PCR. Cell migration ability was evaluated by Transwell and wound-healing assays. The induction of EMT by doxorubicin was investigated from three different levels:morphology, molecular expression and cell function. The activation of β-catenin signal pathway was evaluted by Western blot and RT-PCR. To further investigate the role of β-catenin signaling in doxorubicin induced EMT,β-catenin specific siRNA and indomethacin, a selective B-catenin inhibitor, was respectively added together with doxorubicin. After doxorubicin treatment, the survived cells were cellected and propogated for a long term. RT-qPCR and Western blot were used to check the expression of EMT-related cell markers. Four chromatin reprogramming moleculars expression, Oct4, Sox2, Klf4and c-myc, were also evaluated by RT-qPCR. Silenced by p300specific siRNA, the levels of EMT related moleculars were again evaluted by Western blot to confirm whether the maintence of EMT traits were depend on p300. Moreover, the colony forming ability of long term cultured drug survival cells was estimated by soft agar assay, while the doxorubicin tolerance was estimated by MTT assay. Results. After treatment with0.4μg/ml doxorubicin for48hours, the epithelial derived BGC-823cells converted into slim shape, the distance between cells was increased, and the arrangement of cells became irregular. Immunofluorescence, Western blot and RT-PCR assays showed that drug survival cells (DSC) expressed high levels of mesenchymal specific moleculars like Vimentin and Twist. However, the expression of E-cadherin in DSCs was significantly down-regulated. Transwell and wound-healing assays also proved that the migration ability of BGC-823was enhanced. Together, these results indicated that doxorubicin treatment induced EMT in BGC-823cells, treatment of doxorubicin increased the expression of β-catenin, and the transcription of Lefl, which indicated the activation of β-catenin signaling. Both indomethacin or B-catenin specific siRNA inhibited the doxorubicin-induced EMT and cell migration. These findings further suggested the critical role B-catenin signaling in doxorubicin-induced EMT. Down-regulation of β-catenin in response to treatment of doxorubicin plus indomethacin was propotional to doxorubicin concerntration. However, treatment with only indomethacin could enhance the expression of B-catenin, suggesting that other molecular(s) like PPAR-y was required for indomethacin to inhibit B-catenin. When the suvival cells of48hours doxorubicin treatment were cunltured for longer time, most cells died gradually, only minority population of cells survived and formed small clones. After several passage, these long term cultured DSC(1tDSC) still mainteined mesenchymal morphology, expressed high levels of Vimentin, Twist and B-catenin, suggesting that ltDSC hold EMT characteristic after several passages. By soft agar and MTT assays,1tDSCs were proved to have enhanced colony forming ability and doxorubicin tolerance. Moreover, in1tDSC, the increased transcription of chromatin reprogramming factors, Oct4and c-myc were confirmed by RT-qPCR. Histone acetyl transferase p300level also increased in1tDSC. After treatment with p300siRNA, the EMT characteristics disppeared in1tDSC, suggesting the important role of histone acetylation in mainteining of EMT in1tDSC.Conclusions. Doxorubicin treatment could induce EMT and enhance migration ability of BGC-823cells. β-catenin signaling played an important role in doxorubicin induced EMT.1tDSCs showed enhanced colony forming ability and were more resistant to doxorubicin. EMT phenotupe were maintained in1tDSCs after serial passages. Moreover,1tDSCs expressed high levels of chromatin reprogramming factors. Histone acetyl transferase p300was required for1tDSC to maintain EMT phenotype. These results indicated that epigenetic mechanism was involved in doxorubicin induced EMT. This study hold implication for better strategy of conquering metastasis and drug resistant in cancer treatment.