Isolation And Identification Of Side Population Cells And The Effect And Mechanism Of TGF-β On Its Abundance In Human Gallbladder Cancer

PartⅠIsolation and identification of side population cells in a human gallbladder cancer cell line and in tumorsObjective:To isolate and characterize cancer stem-like side population cells from a human gallbladder cancer cell line GBC-SD the biological characteristics.Methods:SP cells were detected from GBC-SD and clinical human gallbladder cancer specimens by fluorescence-activated cell sorting (FACS). The properties of SP cells from GBC-SD, including invasiveness, chemoresistance and tumorigenicity, were determined in vitro or in vivo. The expression of four major drug transporters (ABCA2, MDR1, MRP1, and ABCG2) was detected by real-time PCR in either SP cells or non-SP cells. The surface marker of GBC-SD SP cells were detected by FACS with antibody staining.Results:SP cells accounted for 0.87% of viable cells, were more invasive than non-SP cells, expressed higher levels of ABC transporter mRNAs and showed higher resistance to chemotherapeutic drugs. SP cells were more tumorigenic than non-SP cells both in vitro and in vivo. SP cells regenerated SP and non-SP cells in proportions similar to the original population.7 Human gallbladder cancer specimens contained a minor SP subset.Conclusion:Human gallbladder cancers contain SP cells with stem cell characteristics.Part II The effect of TGF-β-induced epithelial to mesenchymal transition on side populationc cells in GBC-SDObjective:To investigate the effect of TGF-β-induced epithelial to mesenchymal transition (EMT) on side populationc cells in GBC-SD and provide an insight to explore the molecular mechanismMethods:To induce EMT in vitro, cells were allowed to adhere and then incubated with human recombinant TGF-β. Morphological alterations were examined by phase-contrast microscope and the expression of EMT-related protein and mRNA were detected by Western blotting and RT-PCR. The sensitivity to 3 chemotherapeutic drugs, including gemcitabine, mitoxantrone and epirubicin on GBC-SD underwent TGF-β-induced EMT was performed by MTT assay. Meanwhile, the percentage of SP cells and ABCG2 mRNA abuandance were examined by FACS and qRT-PCR, respectively.Results:These data showed that TGF-βcould induce GBC-SD morphological alteration from epithelial to mesenchymal, downregulate the expression of E-cadherin and upregulate the expression of vimentin in both protein and mRNA level. after 72 hours treated with 3 chemotherapeutic agents gemcitabine,mitoxantrone and epirubicin in different concentration to these transitioned GBC-SD, IC50 values for chemotherapeutic agents were significantly increased 8.1-fold,40-fold and 5.7-fold, compared to GBC-SD cells, respectively. Besides, ABCG2 mRNA expression and the abundance of SP cells were remarkedly increased in these transitioned GBC-SD than those in counterpart GBC-SD. When underwent the removal of TGF-β, these transitioned GBC-SD regained the epithelial phenotype.Conclusion:TGF-p-induced EMT increases resistance to chemotherapeutic agents by augmentment of an ABCG2-dependent SP, and allows to transform into stem-like cells in a human gallbladder cancer cell line GBC-SD.Part III The mechanism of side population cells upregulated by TGF-β-induced EMT in GBC-SDObjective:To investigate the function of Smad3 on side population cells upregulated by TGF-p-induced EMT in GBC-SD.Methods:GBC-SD cells were transfected with chemical synthesis samd3-siRNA (50nM), and Western blotting and FACS were performed to detect the expression of Smad3 and E-cadherin protein in GBC-SD with or without TGF-βand the percentage of SP cells respectively.Results:When GBC-SD was induced by EMT inducer TGF-β, Smad3 phosphorylation was enhanced in these transitioned cells. After transfecting Smad3-RNAi, the expression of Smad3 protein was reduced significantly. In combination of Smad3-RNAi with TGF-β, the percentage of SP cells was downregulated and the expression level of E-cadherin protein was upregulated compared to those EMT GBC-SD cells in the presence of TGF-β.Conclusion:TGF-P-Smad3-E-cadherin signaling pathway has an ability to augment the percentage of SP abundance, importantly, Smad3 palys a critical role in SP cells abundance in response to TGF-β-induced EMT.