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Novel reversible selective inhibitor of CRM1 for targeted therapy in ovarian cancer.

Liu X, Chong Y, Liu H, Han Y, Niu M - J Ovarian Res (2015)

Bottom Line: This effect was clearly reversible in the majority of the cells, whereas the inhibitory effect of LMB could not be reversed.Mechanistically, S109 treatment increase the expression of the cyclin-dependent kinase inhibitor p21, while it reduced the expression of cell cycle promoting proteins, Cyclin D1 and Cyclin B.CRM1 level itself was also down-regulated following S109 treatment.

View Article: PubMed Central - PubMed

Affiliation: Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu, China.

ABSTRACT

Background: Ovarian cancer represents the most fatal type of gynecological malignancies. Unfortunately, there are still no effective targeted treatment strategies for ovarian cancer. Overexpression of CRM1 has been correlated with poor prognosis of patients with ovarian cancer.

Aim: In this study, we investigated the antitumor effects of a novel reversible inhibitor of CRM1 in ovarian cancer cells.

Methods: The effects of S109 on proliferation was detected by CCK-8, EdU, clonogenic assay. The protein expression were determined by Western blot. The subcellular localization of RanBP1 was analyzed by immunofluorescence microscopy assay.

Results: We demonstrated that S109 could induce nuclear accumulation of RanBP1, a canonical biomarker for CRM1 inhibition. This effect was clearly reversible in the majority of the cells, whereas the inhibitory effect of LMB could not be reversed. Our data reveal that treatment with S109 results in decrease in proliferation and colonogenic capacity of ovarian cancer cells by arresting cell cycle. Mechanistically, S109 treatment increase the expression of the cyclin-dependent kinase inhibitor p21, while it reduced the expression of cell cycle promoting proteins, Cyclin D1 and Cyclin B. CRM1 level itself was also down-regulated following S109 treatment. Furthermore, the nuclei of cells incubated with S109 accumulated tumor suppressor proteins (Foxo1, p27 and IκB-α). More importantly, Cys528 mutation of CRM1 abolished the ability of S109 to block proliferation of ovarian cancer cells.

Conclusions: Together, our study identifies CRM1 as a valid target in ovarian cancer and provides a basis for the development of S109 in ovarian cancer.

No MeSH data available.


Related in: MedlinePlus

S109 inhibits proliferation and colony formation of SKOV-3 cells. a Representative EdU analysis of cell proliferation after S109 treatment. b Quantitativeresults of EdU incorporation assay. c S109 inhibits the colony formation of SKOV-3 cells. d Quantitative results of clonogenic 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 < 0.05)
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Fig3: S109 inhibits proliferation and colony formation of SKOV-3 cells. a Representative EdU analysis of cell proliferation after S109 treatment. b Quantitativeresults of EdU incorporation assay. c S109 inhibits the colony formation of SKOV-3 cells. d Quantitative results of clonogenic 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 < 0.05)

Mentions: To further confirm the effects of S109 on cell proliferation, EdU fluorescence staining and clonogenic assays were performed. S109 treatment resulted in a significant reduction of the mean percentage of positive proliferative cells compared with control group (Fig. 3a and b). At 2 μM of S109, the proliferation was reduced to about 50.6 %. Since CCK-8 and EdU assay is a short term assay for proliferation and does not effectively convey cell survival, we performed a cell survival clonogenic assay to evaluate the long term effect of CRM1 inhibition. Figure 3c and d also show a dose dependent inhibition of clonogenic potential by S109 in SKOV-3 cells. The results from the clonogenic assay were consistent with the CKK-8 and EdU assay data shown suggesting that S109 inhibited cell growth in ovarian cells.Fig. 3


Novel reversible selective inhibitor of CRM1 for targeted therapy in ovarian cancer.

Liu X, Chong Y, Liu H, Han Y, Niu M - J Ovarian Res (2015)

S109 inhibits proliferation and colony formation of SKOV-3 cells. a Representative EdU analysis of cell proliferation after S109 treatment. b Quantitativeresults of EdU incorporation assay. c S109 inhibits the colony formation of SKOV-3 cells. d Quantitative results of clonogenic 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 < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465006&req=5

Fig3: S109 inhibits proliferation and colony formation of SKOV-3 cells. a Representative EdU analysis of cell proliferation after S109 treatment. b Quantitativeresults of EdU incorporation assay. c S109 inhibits the colony formation of SKOV-3 cells. d Quantitative results of clonogenic 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 < 0.05)
Mentions: To further confirm the effects of S109 on cell proliferation, EdU fluorescence staining and clonogenic assays were performed. S109 treatment resulted in a significant reduction of the mean percentage of positive proliferative cells compared with control group (Fig. 3a and b). At 2 μM of S109, the proliferation was reduced to about 50.6 %. Since CCK-8 and EdU assay is a short term assay for proliferation and does not effectively convey cell survival, we performed a cell survival clonogenic assay to evaluate the long term effect of CRM1 inhibition. Figure 3c and d also show a dose dependent inhibition of clonogenic potential by S109 in SKOV-3 cells. The results from the clonogenic assay were consistent with the CKK-8 and EdU assay data shown suggesting that S109 inhibited cell growth in ovarian cells.Fig. 3

Bottom Line: This effect was clearly reversible in the majority of the cells, whereas the inhibitory effect of LMB could not be reversed.Mechanistically, S109 treatment increase the expression of the cyclin-dependent kinase inhibitor p21, while it reduced the expression of cell cycle promoting proteins, Cyclin D1 and Cyclin B.CRM1 level itself was also down-regulated following S109 treatment.

View Article: PubMed Central - PubMed

Affiliation: Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu, China.

ABSTRACT

Background: Ovarian cancer represents the most fatal type of gynecological malignancies. Unfortunately, there are still no effective targeted treatment strategies for ovarian cancer. Overexpression of CRM1 has been correlated with poor prognosis of patients with ovarian cancer.

Aim: In this study, we investigated the antitumor effects of a novel reversible inhibitor of CRM1 in ovarian cancer cells.

Methods: The effects of S109 on proliferation was detected by CCK-8, EdU, clonogenic assay. The protein expression were determined by Western blot. The subcellular localization of RanBP1 was analyzed by immunofluorescence microscopy assay.

Results: We demonstrated that S109 could induce nuclear accumulation of RanBP1, a canonical biomarker for CRM1 inhibition. This effect was clearly reversible in the majority of the cells, whereas the inhibitory effect of LMB could not be reversed. Our data reveal that treatment with S109 results in decrease in proliferation and colonogenic capacity of ovarian cancer cells by arresting cell cycle. Mechanistically, S109 treatment increase the expression of the cyclin-dependent kinase inhibitor p21, while it reduced the expression of cell cycle promoting proteins, Cyclin D1 and Cyclin B. CRM1 level itself was also down-regulated following S109 treatment. Furthermore, the nuclei of cells incubated with S109 accumulated tumor suppressor proteins (Foxo1, p27 and IκB-α). More importantly, Cys528 mutation of CRM1 abolished the ability of S109 to block proliferation of ovarian cancer cells.

Conclusions: Together, our study identifies CRM1 as a valid target in ovarian cancer and provides a basis for the development of S109 in ovarian cancer.

No MeSH data available.


Related in: MedlinePlus