Korean J Physiol Pharmacol.  2016 Mar;20(2):161-168. 10.4196/kjpp.2016.20.2.161.

CRM1 inhibitor S109 suppresses cell proliferation and induces cell cycle arrest in renal cancer cells

Affiliations
  • 1Insititute of Nervous System Diseases, Xuzhou Medical College, Xuzhou 221002, Jiangsu, China.
  • 2Blood Disease Institute, Xuzhou Medical College, Xuzhou 221002, Jiangsu, China. msniu24@hotmail.com
  • 3Department of Hematology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu, China.
  • 4Brain Hospital, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu, China.
  • 5Dalian Center for Disease Control and Prevention, Dalian 116002, Liaoning, China.

Abstract

Abnormal localization of tumor suppressor proteins is a common feature of renal cancer. Nuclear export of these tumor suppressor proteins is mediated by chromosome region maintenance-1 (CRM1). Here, we investigated the antitumor eff ects of a novel reversible inhibitor of CRM1 on renal cancer cells. We found that S109 inhibits the CRM1-mediated nuclear export of RanBP1 and reduces protein levels of CRM1. Furthermore, the inhibitory eff ect of S109 on CRM1 is reversible. Our data demonstrated that S109 signifi cantly inhibits proliferation and colony formation of renal cancer cells. Cell cycle assay showed that S109 induced G1-phase arrest, followed by the reduction of Cyclin D1 and increased expression of p53 and p21. We also found that S109 induces nuclear accumulation of tumor suppressor proteins, Foxo1 and p27. Most importantly, mutation of CRM1 at Cys528 position abolished the eff ects of S109. Taken together, our results indicate that CRM1 is a therapeutic target in renal cancer and the novel reversible CRM1 inhibitor S109 can act as a promising candidate for renal cancer therapy.

Keyword

Cell cycle; CRM1; Nuclear export; Renal cancer; S109

MeSH Terms

Active Transport, Cell Nucleus
Cell Cycle Checkpoints*
Cell Cycle*
Cell Proliferation*
Cyclin D1
Kidney Neoplasms*
Tumor Suppressor Proteins
Cyclin D1
Tumor Suppressor Proteins

Figure

  • Fig. 1 S109 inhibits ovarian cancer growth and nuclear export of RanBP1.(A) Structure of S109. (B) Cell growth inhibition curves of S109 treatment for 72 h. 786-O cells were treated with vehicle (0.1% DMSO) or different concentrations of S109 (0.04, 0.2, 1, 5, 25, and 50 µM) for 72 h. Cell viability was measured by CCK-8 assay. All the data are presented as mean±SEM in three repeats (*p<0.05). (C) S109 inhibits nuclear export of RanBP1. Cells were treated with indicated doses of S109 for 2 h. Fixed cells were stained for RanBP1 and DAPI and analyzed by fluorescence microscopy. (D) S109 reduces expression level of CRM1 protein in 786-O cells. Cells were treated with indicated doses of S109 for 24 h, and the whole cell lysates were analyzed by Western blot. (E) S109 reduces expression level of CRM1 protein in OS-RC-2 cells. Cells were treated with indicated doses of S109 for 24 h, and the whole cell lysates were analyzed by Western blot.

  • Fig. 2 The binding of S109 to CRM1 is reversible.(A) 786-O cells were treated with 0.1% DMSO, LMB (5 nM), and S109 (1 µM, 2 µM) for 2 h, followed by immunofluorescence staining. The localization of RanBP1 was observed. (B) Reversible effect of S109 on the localization of RanBP1. Cells were incubated with indicated doses of S109 or LMB. After 2 h, the drugs were washed out and fresh medium was added. Cells were incubated for 2 h and then analyzed by fluorescence microscopy.

  • Fig. 3 S109 inhibits proliferation and colony formation of 786-O cells.(A) Representative EdU analysis of cell proliferation after S109 treatment. (B) Quantitative results of EdU incorporation ass ay. (C) S109 inhibits the colony formation of 786-O cells. (D) Quantitative results of clonoge nic assay. The percentage of proliferative cells or colony formation were normalized to that of the control group. All the data are presented as mean±SEM in three repeats (**p<0.01).

  • Fig. 4 S109 induces cell cycle arrest and nuclear accumulation of tumor suppressor proteins.(A) 786-O cells were exposed to 2 µM of S109 for 24 h. Cells were harvested, stained with propidium iodide and analyzed by flow cytometry. (B) 786-O were treated with S109 at the indicated concentrations for 24 h. Cells were then harvested and subjected to Western blot analysis. The expression levels of p53, p21, and Cyclin D1 were measured. (C) 786-O cells were treated with S109 at the indicated concentrations for 24 h. The nuclear proteins were extracted and subjected to immunoblot analysis. (D) OS-RC-2 cells were treated with S109 at the indicated concentrations for 24 h. The nuclear proteins were extracted and subjected to immunoblot analysis.

  • Fig. 5 Cys528 mutation of CRM1 abolished the ability of S109 to inhibit nuclear export.(A) Identification of stable CRM1-WT or CRM1-C528S over-expression 786-O cell lines. Western blot analyses the expression level of exogenous CRM1. (B) Growth inhibition assay in wild type and C528S mutant cells. Cells were seeded in 96-well plates and incubated with S109 at indicated concentrations for 72 h. Growth inhibition was analyzed by the CCK-8 assay. All the data are presented as mean±SEM in three repeats (*p<0.05). (C) Nuclear export inhibition assay in wild type and C528S mutant cells. Cells were seeded in 96-well plates and incubated with S109 at indicated concentrations for 2 h. The subcellular localization of RanBP1 was analyzed.


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