Novel PI3K/Akt/mTOR signaling inhibitor, W922, prevents colorectal cancer growth via the regulation of autophagy

Abstract

W922, a novel PI3K/Akt/mTOR pathway inhibitor, exhibits efficient anti‑tumor effects on HCT116, MCF‑7 and A549 human cancer cells compared with other synthesized compounds. The present study aimed to investigate its anti‑tumor effects on colorectal cancer cells. A total, of seven different colorectal cell lines were selected to test the anti‑proliferation profile of W922, and HCT116 was found to be the most sensitive cell line to the drug treatment. W922 inhibited HCT116 cell viability and cell proliferation in vitro in concentration‑ and time‑dependent manners. Furthermore, W922 suppressed the tumor growth in a xenograft mouse model and exhibited low toxicity. The proteomic alterations in W922‑treated HCT116 cells were found to be associated with cell cycle arrest, negative regulation of signal transduction and lysosome‑related processes. W922 caused cell cycle arrest of HCT116 cells in G0‑G1 phase, but only triggered slight apoptosis. In addition, the PI3K/Akt/mTOR signaling proteins were dephosphorylated upon W922 treatment. It has been reported that inhibition of mTOR is relevant to autophagy, and the present results also indicated that W922 was involved in autophagy induction. An autophagy inhibitor, chloroquine, was used to co‑treat HCT116 cells with W922, and it was identified that the cell cycle arrest was impaired. Moreover, co‑treatment of W922 and chloroquine led to a significant population of apoptotic cells, thus providing a promising therapeutic strategy for colorectal cancer.

Keywords: W922; PI3K/Akt/mTOR inhibitor; colorectal cancer; cell cycle arrest; autophagy.

Figure 1

W922 inhibits tumor growth in vivo via the regulation of proliferation. (A) Chemical structure of W922. (B) Anti-proliferation effect of W922 on seven types of colorectal cancer cells after 24 h treatment (n=3) (C) W922 inhibited HCT116 cells in a concentration-dependent manner (n=3; one-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. 0 µM-24 h treatment; ^##P<0.01 vs. 0 µM-48 h treatment. (D) W922 time dependently inhibited HCT116 cells (n=3; one-way ANOVA followed by Dunnett's test. ^**P<0.01 vs. 0 h treatment. (E) Long-term cell viability was measured via clonogenic formation assay. HCT116 cells were seeded and treated with indicated concentrations of W922 for 2 weeks, and the number of the colonies were counted and quantified (n=3; one-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. 0 µM treatment. (F) W922 inhibited the growth of HCT116 solid tumors in vivo. The nude mice were transplanted with HCT116 cells and randomly grouped when tumor volume reached ~200 mm³. Then, the mice were treated with solvent, W922 or positive control (VS-5584) for 18 days, and the tumor volumes were measured every 3 days using a caliper. (G) Body weight of mice during treatment. (H) On the final day of treatment, the mice were sacrificed and the solid tumors were excised and weighed [n=3-5; two-way ANOVA (F and G) and one-way ANOVA (H) followed by Dunnett's test]. ^*P<0.05, ^**P<0.01 vs. solvent group. (I) Image of the excised tumors. MW, molecular weight.

Figure 2

W922 negatively regulates the cell cycle processes. (A) Volcano plot for protein abundances in HCT116 cells after W922 treatment. Red and green color points indicate significant up (FC >2)- and downregulated (FC <0.5) proteins, respectively. The lines represent P-values of 0.01 and 0.05 in two-tailed unpaired t-test. (B) Venn plot for proteins (P<0.05) in HCT116 cells after W922 treatment. (C) Heatmap of top 30 up (right)- and downregulated (left) proteins in W922 treatment. (D) Gene Ontology term enrichment of all significant downregulated proteins (FC <0.5; P<0.05) in W922 treated HCT116 cells. (E) Gene Ontology term enrichment of all significant upregulated proteins (FC >2; P<0.05) in W922 treated HCT116 cells. (n=3-5, unpaired Student's t-test). FC, fold change; Ctrl, control; BP, Biological process; MF, molecular function; CC, Cellular component; NR, negative regulation.

Figure 3

W922 inhibits HCT116 cell proliferation mainly by cell cycle arrest. (A) HCT116 cells were treated with W922 for 24 h, then the DNA contents were stained using PI and were analyzed via flow cytometry. The percentage of cells at G₀-G₁, S and G₂-M phases are presented. (n=3; G₀-G₁ phase was analyzed via one-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. 0 µM treatment. (B) HCT116 cells were treated with W922 for 24 h and the cell lysates were immunoblotted with antibodies against cyclin D1 and cyclin E1. (n=3; one-way ANOVA followed by Dunnett's test). ^*P<0.05, ^**P<0.01 vs. corresponding 0 µM. (C) Cells were treated with W922 for 24 or 48 h, and then were double stained with Annexin V-FITC and PI. Data were collected via flow cytometry (n=3; one-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. 0 µM of each time point. (D) HCT116 cells were treated with either DMSO (Ctrl) or 10 µM W922 for 48 h. The expression levels of cleaved caspase-3 (red) and ki67 (green) were visualized via cell immunofluorescence (magnification, ×20) and were quantified using ImageJ (n=3; unpaired Student's t-test). ^**P<0.01 vs. 0 µM treatment. Ctrl, control.

Figure 4

W922 suppresses the PI3K/Akt/mTOR signaling pathway. HCT116 cells were treated with (A) gradient concentrations of W922 for 24 h, or with (B) W922 at 10 µM for gradient time periods. Then, the cells were collected and lysed. Cell lysates were immunoblotted with antibodies against PI3K/Akt/mTOR-related total and p- proteins. (n=3; one-way ANOVA followed by Dunnett's test). ^*P<0.05, ^**P<0.01 vs. 0 µM or 0 h treatment. P-, phosphory-lated; p70S6K, p70S6 kinase; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1.

Figure 5

W922 induces autophagy in HCT116 cells. (A) HCT116 cells were treated with desired concentrations of W922 for 24 h. Collected cells were lysed and subsequently the lysates were immunoblotted with anti-LC3, anti-p-Beclin or anti-Beclin antibodies (n=3; one-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. 0 µM treatment. (B) HCT116 cells were treated with W922 for 24 h, fixed and stained with anti-LC3 antibody (green) and with DAPI (blue), magnification, ×20. Quantification on the right showed LC3 fluorescence determined by ImageJ (n=3). ^**P<0.01 vs. Ctrl. (C) Transfection efficacy detection. HCT116 cells were transfected with siATG5 or siRNA NC, and the expression of ATG5 was detected. (D) HCT116 cells were successfully transfected with siATG5 and the expression of LC3 after W922 treatment was measured via western blotting (n=3; one-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. Ctrl; ^##P<0.01 vs. W922 group. (E) Autophagic flux was examined via co-treatment of CQ, an autophagosome-lysosome fusion inhibitor. HCT116 cells were treated with W922 and/or CQ for 24 h and the expression of LC3 was determined via western blotting (n=3; unpaired Student's t-test). ^**P<0.01 vs. 10 µM CQ treatment; ^##P<0.01 vs. 20 µM CQ treatment. CQ, chloroquine; ATG5, autophagy related 5; Ctrl, control; siRNA, small interfering RNA.

Figure 6

Autophagy contributes to W922-induced cell cycle arrest. (A) HCT116 cells were pretreated with 50 µM CQ before 24 h-W922 treatment, and were stained with PI and analyzed using flow cytometry (n=3; G₀-G₁ phase was analyzed by two-way ANOVA followed by Dunnett's test). ^**P<0.01 vs. Ctrl; ^##P<0.01 vs. CQ single treatment. (B) Volcano plot for protein abundances in HCT116 cells pre-treated with 20 µM CQ before W922 treatment. (C) Gene Ontology term enrichment of top 20 up- and downregulated proteins in W922 and CQ co-treated HCT116 cells. (D) Heatmap of top20 up- (bottom) and downregulated (top) proteins in W922 and CQ co-treated HCT116 cells. (n=3-5, unpaired Student's t-test). (E) HCT116 cells were co-treated with 10 µM W922 and 50 µM CQ for 24 h, the apoptotic cells was detected via an Annexin V-FITC/PI double stain assay. (n=3; unpaired Student's t-test). ^**P<0.01 vs. 10 µM W922 treatment. CQ, chloroquine; Ctrl, control.