(C) HCT-15 cells were treated with LMB or S109 at the indicated concentrations for 2?h (left panel). S109 to block nuclear export and inhibit the proliferation of colorectal malignancy cells. AM211 Interestingly, S109 decreased the CRM1 protein level via proteasomal pathway. We argue that reversible CRM1 inhibitors but not irreversible inhibitors can induce the degradation of CRM1, because the dissociation of reversible inhibitors of CRM1 changes the conformation of CRM1. Taken together, these findings demonstrate that CRM1 is usually a valid target for the treatment of colorectal cancer AM211 and provide a basis for the development of S109 therapies for colorectal malignancy. has not yet been investigated. For the first time, we herein statement our investigation of the effect of a novel reversible CRM1 inhibitor, S109, on colorectal malignancy. AM211 S109, a derivative of CBS9106, could block the function of CRM1 followed by the degradation of CRM1. Furthermore, we also found that S109 inhibits cell proliferation and invasion and induces cell cycle arrest in colon cancer cells. These data show that S109 is usually a promising drug for the treatment of colorectal cancer. Results S109 inhibits the proliferation and colony formation of colorectal malignancy cells To assess the effects of S109 on growth the inhibition of colon cancer cells, HCT-15 and HT-29 cells were treated with S109, and cell viability was estimated using a CCK8 assay. As shown in Fig.?1B, S109 induced a marked decrease in cell viability in a dose-dependent manner compared with the control group. The estimated IC50 values ranged from 1.2 or 0.97?M in HCT-15 or HT-29 cells. To confirm the anti-proliferative activity of S109, we also tested the rates of cell proliferation by EdU fluorescence SPP1 staining. S109 treatment resulted in a significant reduction of the mean percentage of proliferating cells compared with the control group (Fig.?1C and ?and1D).1D). HCT-15 cells exposure to 2 and 4?M S109 reduced the proliferation to approximately 59.84% and 32.75%, respectively. These data suggest that S109 can significantly inhibit the viability of colorectal malignancy cells. Open in a separate window Physique 1. S109 suppresses cell proliferation and colony formation of colorectal cells. (A) Chemical structure of S109. (B) Cell growth inhibition curves of S109 treatment. HCT-15 and HT-29 cells were treated with vehicle (0.1% DMSO) or different concentrations of S109 for 72?hours. Cell viability was measured by CCK-8 assay. (C) Representative EdU analysis of cell proliferation after S109 treatment. (E) S109 inhibits the colony formation of HCT-15 cells. (G) Representative photographs of invading HCT-15 cells during a 36-hour incubation with S109. (D, F and H) Quantitative results of EdU incorporation assay, clonogenic assay and invading cell figures, respectively. The percentage of proliferative cells or colony formation were normalized to that of the control group. All data are offered as the imply SEM of 3 replicates (* 0.05, ** 0.01). A clonogenic assay was performed to elucidate the long-term effects of S109 on AM211 cell proliferation. Fig.?1E and 1F show the dose dependent inhibition of clonogenic potential by S109 in HCT-15 cells. Compared with the control group, the colony formation markedly decreased by 58.46%, 83.15% and 91.41% in response 1, 2, and 4?M treatment, respectively. Taken together, these results provide unequivocal proof of the potential of S109 as a new anticancer drug. To examine the ability of S109 to prevent the invasion of colorectal malignancy cells, we conducted invasion assay. The results showed that S109 induced a dose-dependent decrease in invasion (Fig.?1G and 1H). Exposure of HCT-15 cells to 0.5 and 1?M S109 decreased the fraction of invading cells by 44.58% and 67.24%, respectively. The results clearly show that S109 treatment decreases the invasiveness of malignancy cells compared to the untreated control. S109-induced G1 arrest is usually associated with a change in the expression of multiple cell cycle regulators We then analyzed the cell cycle to examine the effect of S109 on colorectal malignancy cell cycle progression. The cell cycle distribution of HCT-15 cells was determined by propidium iodide staining after treating cells with either DMSO control or S109 for 24?h. As shown in Fig.?2A and 2B, the HCT-15 cells were arrested at G1 phase of the cell cycle in response to treatment with S109, as evidenced by an increase in the G1 portion from 46.1% in the control cells to 71.3% in S109-treated cells. In addition, a significant decrease in the S phase populations compared with the control group was also observed. We next examined whether S109 modulates cell cycle regulatory proteins to induce G1 arrest. Treatment with S109 enhanced the expression levels of cell cycle inhibitory proteins p27 and p53. Moreover, the expression levels of Cyclin B1, Cyclin D1 and CDK4 were dose-dependently reduced (Fig.?2C). Taken together, these results suggest that S109 induces cell.