Podofilox suppresses gastric cancer cell proliferation by regulating cell cycle arrest and the c-Myc/ATG10 axis

Abstract

Gastric cancer (GC) is a malignancy for which effective therapeutic drugs are limited. Podofilox exhibits antitumor effects in various types of cancer; however, whether it may inhibit GC growth remains unknown. The aim of the present study was to investigate the role of podofilox in GC. Cell Counting Kit-8, colony formation and cell cycle assays were used to detect the role of podofilox on cellular proliferation and the cell cycle, respectively. A microarray was used to detect the transcriptional changes induced by podofilox in GC cells. The results of the present study demonstrated that podofilox inhibited GC cell proliferation and colony formation. The half maximal inhibitory concentration of podofilox in AGS and HGC-27 cells was 2.327 and 1.981 nM, respectively. In addition, treatment with podofilox induced G₀/G₁ cell cycle arrest. Molecular analysis based on microarray data demonstrated that podofilox altered the expression levels of genes involved in the cell cycle, c-Myc and p53 signaling. Autophagy-related 10 (ATG10), which was highly expressed in GC tissues, was also downregulated by podofilox, as demonstrated by the results of the microarray analysis and immunoblotting. To determine the involvement of ATG10 in GC, ATG10 was knocked down in GC cells by small interfering RNA, which suppressed the proliferation and colony formation of GC cells compared with those observed in the control-transfected cells. Taken together, the results of the present study suggested that podofilox may inhibit GC cell proliferation by preventing the cell cycle progression and regulating the c-Myc/ATG10 signaling pathway.

Keywords: apoptosis; autophagy; cell cycle; gastric cancer; podofilox.

Figure 1

Podofilox inhibits the proliferation of HGC-27 and AGS cells. (A and B) Cell viability was assessed in (A) AGS and (B) HGC-27 cells following incubation with podofilox (0-80 nM) for 48 h. (C and D) Cell proliferation curve was analyzed following incubation with vehicle or 3.4 nM podofilox at the indicated time points. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. vehicle.

Figure 2

Podofilox inhibits cell colony formation. (A) AGS and (B) HGC-27 cells were incubated with vehicle or 3.4 nM podofilox for 7 days, and cell colony formation was assessed. ^**P<0.01 and ^***P<0.001 vs. vehicle.

Figure 3

Podofilox inhibits cell cycle progression of HGC-27 and AGS cells. (A and B) The cell cycle was analyzed in (A) AGS and (B) HGC-27 cells following incubation with vehicle or 3.4 nM podofilox for 48 h. Left, representative images; right, quantification analysis. ^*P<0.05 and ^**P<0.01 vs. vehicle.

Figure 4

Microarray analysis of dysregulated genes following treatment with podofilox. (A) Heatmap of dysregulated genes in AGS cells treated with vehicle or podofilox. A total of 566 genes were upregulated, and 713 genes were downregulated. Green, downregulated genes; red, upregulated genes. (B) KEGG and HALLMARK pathway enrichment analyses of cell cycle, c-Myc and p53 signaling pathways. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5

Western blot analysis of dysregulated genes following treatment with podofilox. Immunoblots of HK2, PKM2, p53, c-Myc, ATG10 and GAPDH following incubation with vehicle or 3.4 nM podofilox for 48 h are presented on the left, and the quantitative analysis is presented on the right. ^*P<0.05 and ^**P<0.01 vs. vehicle. HK2, hexokinase 2; PKM2, pyruvate kinase M1/2; ATG10, autophagy-related 10.

Figure 6

Downregulation of ATG10 inhibits cell proliferation and colony formation. (A) The expression levels of ATG10 in gastric cancer and normal tissues were analyzed using The Cancer Genome Atlas database. (B) Immunoblotting results of ATG10 and GAPDH in siCtrl-, siATG10-1- and siATG10-2-transfected HGC-27 and AGS cells (left) and the quantitative analysis (right). (C and D) Cell proliferation curves were analyzed in siCtrl-, siATG10-1- and siATG10-2-transfected cells. (E and F) Colony formation was assessed in siCtrl-, siATG10-1- and siATG10-2-transfected HGC-27 and AGS cells. (E) Images of colonies and (F) quantified data. ^**P<0.05 and ^***P<0.001 siATG10-1 vs. siCtrl; ^##P<0.01 and ^###P<0.001 siATG10-2 vs. siCtrl. si, small interfering RNA; Ctrl, negative control; ATG10, autophagy-related 10.