Abstract
BackgroundItraconazole has been proved therapeutically effective against a variety of human cancers. This study assessed the effect of itraconazole on the Hedgehog (Hh) pathway and proliferation of human gastric cancer cells.MethodsCCK-8 assay and colony formation assay were used to assess the effects of itraconazole on proliferation of gastric cancer cells. The expression of Hh signaling components in gastric cancer cells treated with itraconazole was evaluated by reverse-transcription polymerase chain reaction, immunoblotting and dual luciferase assay. Tumor xenograft models were used to assess the inhibitory effect of itraconazole on the proliferation of gastric cancer cells in vivo.ResultsItraconazole could remarkably inhibit the proliferation of gastric cancer cells. When in combination with 5-FU, itraconazole significantly reduced the proliferation rate of cancer cells. Furthermore, itraconazole could regulate the G1-S transition and induce apoptosis of gastric cancer cells. Hh signaling was abnormally activated in human gastric cancer samples. In vitro, studies showed that the expression of glioma-associated zinc finger transcription factor 1 (Gli1) was decreased at both transcriptional and translational levels after treatment with itraconazole. Dual luciferase assay also indicated that itraconazole could inhibit the transcription of Gli1. In vivo studies demonstrated that monotherapy with itraconazole by oral administration could inhibit the growth of xenografts, and that itraconazole could significantly enhance the antitumor efficacy of the chemotherapeutic agent 5-FU.ConclusionsHh signaling is activated in gastric tumor and itraconazole can inhibit the growth of gastric cancer cells by inhibiting Gli1 expression.
Highlights
Itraconazole has been proved therapeutically effective against a variety of human cancers
The results demonstrated that itraconazole could inhibit proliferation of gastric cancer cells by inhibiting glioma-associated zinc finger transcription factor 1 (Gli1) expression
Itraconazole inhibits proliferation of gastric cancer cells and enhances the chemotherapeutic response of 5-FU in vitro and in vivo In order to verify the potential anti-cancer properties of itraconazole, we firstly tested whether it could inhibit the proliferation of gastric cancer cells
Summary
Cell culture and reagents Two gastric cancer cell lines, MKN45 (KCLB 80103) and AGS (ATCC CRL 1739), were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (ScienCell) and 1% penicillin-streptomycin sulfate (Gibco). Ten μl CCK-8 solution from Cell Counting Kit 8 (CCK-8, Dojindo, Japan) was added to each well and the plate was incubated at 37 °C for an additional 1 h. Different concentrations of itraconazole solution and DMSO were added to the medium after cells adhered. Colonies were fixed with 75% methanol and stained with 1% crystal violet (BIO BASIC INC) Those cell clusters containing more than 50 cells under a microscope were considered as colonies. Dual luciferase assay MKN45 and AGS cells grown in 24-well plates were cotransfected with 10 ng of pRL-TK and 200 ng Gli, Smo promoter-driven luciferase reporter vector (Gli1-pGL3, Smo-pGL3) or the luciferase reporter plasmid pGL3-Basic vector with FuGene transfection reagent (Promega). The cells were harvested 48 h after transfection and luciferase assays were performed using the dual luciferase assay kit (Promega) according to the manufacturer’s instructions. A two-sided p-value of
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