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12.05.2017 | Original Article | Ausgabe 6/2017

Cancer Chemotherapy and Pharmacology 6/2017

CYT-Rx20 inhibits ovarian cancer cells in vitro and in vivo through oxidative stress-induced DNA damage and cell apoptosis

Cancer Chemotherapy and Pharmacology > Ausgabe 6/2017
Yen-Yun Wang, Yuk-Kwan Chen, Stephen Chu-Sung Hu, Ya-Ling Hsu, Chun-Hao Tsai, Tsung-Chen Chi, Wan-Ling Huang, Pei-Wen Hsieh, Shyng-Shiou F. Yuan
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Electronic supplementary material

The online version of this article (doi:10.​1007/​s00280-017-3330-9) contains supplementary material, which is available to authorized users.



The β-nitrostyrene family has been previously reported to possess anticancer property. However, the biological effects of β-nitrostyrenes on ovarian cancer and the underlying mechanisms involved remain unclear. In the present study, we synthesized a β-nitrostyrene derivative, CYT-Rx20 3′-hydroxy-4′-methoxy-β-methyl-β-nitrostyrene), and investigated its anticancer effects and the putative pathways of action in ovarian cancer.


The effects of CYT-Rx20 were analyzed using cell viability assay, reactive oxygen species (ROS) generation assay, FACS analysis, annexin V staining, immunostaining, comet assay, immunoblotting, soft agar assay, migration assay, nude mice xenograft study and immunohistochemistry.


CYT-Rx20 induced cytotoxicity in ovarian cancer cells by promoting cell apoptosis via ROS generation and DNA damage. CYT-Rx20-induced cell apoptosis, ROS generation and DNA damage were reversed by thiol antioxidants. In addition, CYT-Rx20 inhibited ovarian cancer cell migration by regulating the expression of epithelial to mesenchymal transition (EMT) markers. In nude mice, CYT-Rx20 inhibited ovarian tumor growth accompanied by increased expression of DNA damage marker γH2AX and decreased expression of EMT marker Vimentin.


CYT-Rx20 inhibits ovarian cancer cells in vitro and in vivo, and has the potential to be further developed into an anti-ovarian cancer drug clinically.

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Supplementary Fig. 1 Chemical structures of the β-nitrostyrene derivatives CYT-Rx20, CYT-Rx21, CYT-Rx22, CYT-Rx44, CYT-Rx45, CYT-Rx46, and CYT-Rx47 (PDF 43 kb)
Supplementary Fig. 2a Cells were treated with CYT-Rx20 for 1 h and ROS level was determined by staining with H2DCFDA fluorescent dye and analysis by flow cytometry. b Cells were treated with various concentrations of CYT-Rx20 (2 μg/ml) for 24 h prior to the determination of DNA damage by neutral comet assay. c Effect of CYT-Rx20 on induction of γ-H2AX focus formation in ovarian cancer cells. After treatment with the indicated concentrations of CYT-Rx20 (2 μg/ml) for 24 h, cells were fixed and incubated with antibodies against γ-H2AX, followed by secondary antibodies conjugated with the fluorochrome FITC. Nuclei were stained with DAPI. After immunostaining, cells were viewed by fluorescence microscope (original magnification × 1000) (PDF 218 kb)
Supplementary Fig. 3 MDAH 2774, PA-1, and SKOV3 cells were treated with various concentrations of CYT-Rx20 for 24 h and then cell viability was assessed by the XTT colorimetric assay (PDF 31 kb)
Supplementary Fig. 4 a The body weights of the nude mice included in this study were measured every week. b Hematoxylin and eosin staining of the tissues from mice organs. The representative photographs were shown with ×200 magnification. Bar represents 200 μm (PDF 466 kb)
Supplementary material 5 (PDF 28 kb)
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