Here, we got benefit of our set up 5-FU resistant HCT-116R and DLD-1R cell lines to examine regorafenibs efficiency against these cells

Here, we got benefit of our set up 5-FU resistant HCT-116R and DLD-1R cell lines to examine regorafenibs efficiency against these cells. confirmed that ectopic appearance of miR-34a suppressed cell proliferation, migration and invasion in various cancer cells, which could also contribute to drug resistance in breast cancer by targeting a variety of oncogenes [6]. However, the role and mechanism of miR-34a in the regulation of colon cancer stem-like cells is far from being completely elucidated at present. It was our goal to investigate the potential efficacy of regorafenib on cancer stem-like cells in this study. We first established two colon cancer cell lines resistant to fluorouracil by acclimatization. These 5-FU resistant colon cancer cells exhibited enhanced tumorigenic phenotypes including CD44+ and side-population cells, increased colony and tumor sphere formation. These observations were associated with the elevated expression of stemness markers such as Nothc1, WNT1 and -catenin. Importantly, we showed that the treatment of regorafenib in these 5-FU resistant Regorafenib monohydrate cancer cells suppressed the aforementioned tumorigenic phenotypes and stemness markers. The combination of regorafenib and 5-FU Regorafenib monohydrate synergistically suppressed colon cancer viability both in vitro and in vivo. Finally, regorafenib treatment was mechanistically associated with the increased level of a tumor suppressor, miR-34a. Thus, this study provides novel and important mechanistic explanations underlying regorafenibs ability to treat chemo-refractory colon cancer and the combination of regorafenib and 5-FU regimen may provide an Rabbit Polyclonal to IRX3 improved treatment efficacy. Methods Chemicals and reagents Regorafenib (BAY 73C4506, catalog No. S1178) and Fluorouracil (5-FU, catalog No. S1209) were purchased from SelleckChem. The primary and secondary antibodies used for western blotting and immunohistochemical experiments were all purchased from Cell Signaling Technology unless otherwise specified. Generation of 5-FU resistant cell lines Human colon cancer cell lines, HCT-116 and DLD-1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and the cells were maintained under the conditions accordingly. To generate 5-FU resistant cells, both HCT-116 and DLD-1 cells were initially exposed to 5-FU (5?M 72?h) and the survived cells were subsequently passaged and maintained under the same culture conditions for at least 20 more passages. The resultant 5-FU-acclimatized cells were termed HCT-116R and DLD-1R (R as 5-FU resistant line). Side population (SP) and tumor sphere assays We performed the side population (SP) assay to identify and quantify the cancer stem-like and/or drug resistant cancer cells. SP cells are defined as a sub-population of cells with high expression of ATP-binding cassette transporters (ABCG2) and the ability to exclude Hoechst 33,342 nuclear dye [7]. We used FACSAria? technology platform to determine and compare the SP cells in HCT-116, HCT-116R, DLD-1 and DLD-1R cells. Cells were first labeled with Hoechst 33342 dye (2.5?g/mL) for 30?min at 37?C. The control cells were treated with verapamil (50?M, Sigma-Aldrich). Propidium iodine (PI) 1?g/mL served to identify dead cells. After identification and cell sorting, SP cells were cultured under stem cell conditions: serum-free of HEScGRO medium, N2 supplement (Invitrogen, Carlsbad, CA), 10?ng/mL human recombinant bFGF (Invitrogen), and 10?ng/mL EGF (Invitrogen) in ultra-low attachment CoStar plates (Corning, NY). Tumor spheres were measured and those ?200?m were counted as a tumor sphere forming unit. The data calculated for the number and size of the tumor spheres is the average of three independent experiments. Cell viability test Sulforhodamine B (SRB) dye (Sigma-Aldrich, Chemie GmbH, Munich, Germany) was used to test the Regorafenib monohydrate effects of selective inhibitors on cell growth and viability of SP cells. The regorafenib were dissolved in dimethyl sulfoxide (DMSO) before diluting with growth medium to a final DMSO concentration of 0.05%. The HCT-116R and DLD-1R cells were seeded into 96 well plates in growth medium at 3000 cells/well. After 24?h the medium was replaced with fresh growth medium containing the regorafenib. The cells were incubated for another 48?h. The cells were fixed with trichloroacetic acid (TCA) by gently adding 50?L TCA (50%) to each well to a final TCA concentration of 10% with subsequent incubation for 1?h at 4?C. The plates were then washed 5 times with tap water and air dried. The dried plates were stained with 100?L of 0.4% (w/v) SRB prepared in 1% (v/v) acetic acid for 10?min at room temperature. The plates were rinsed quickly 4 times with 1% acetic acid to remove unbound dye, and then air dried until no moisture was visible. The bound dye was solubilized in 20?mM Tris-base (100?L/well) for 5?min on a shaker. Optical densities were read on a microplate reader (Molecular Devices, Sunnyvale, CA) at 562?nm. SDS-PAGE and western blotting HCT-116R and DLD-1R cells Regorafenib monohydrate were lysed and prepared using ReadyPrep Protein Extraction Kit (Bio-Rad, Hercules, CA) according to the vendors instructions. Total cell lysates (50?g) were separated by.

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