Identification of DW532 as a novel anti-tumor agent targeting both kinases and tubulin
Abstract
Ting PENG1, #, Jian-rui WU2, #, Lin-jiang TONG1, Meng-yuan LI1, Fang CHEN2, Yi-xin LENG2, Rong QU1, Kun HAN1, Yi SU1, Yi CHEN1, Wen-hu DUAN2, *, Hua XIE1, *, Jian DING1, *
1Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Aim: 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (DW532) is one of simplified analogues of hematoxylin that has shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities.
Methods: In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to evaluate the anti-angiogenesis efficacy.
Results: DW532 inhibited EGFR and VEGFR2 in vitro kinase activity (the IC50 values were 4.9 and 5.5 μmol/L, respectively), and suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 (1 and 10 μmol/L) induced G2/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mps1, the two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells. Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo.
Conclusion: DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer agent.
Keywords: anti-cancer agent; hematoxylin; combretastatin; tyrosine kinase; EGFR; VEGFR; tubulin; cell cycle; spindle assembly checkpoint; anti-angiogenesis
The authors acknowledge funding from the National Natural Science Foundation of China (No 20272078) and the Shanghai Municipal Science and Technology Commission (No 03J.C.14083).
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail jding@simm.ac.cn (Jian DING);
hxie@simm.ac.cn (Hua XIE);
whduan@simm.ac.cn (Wen-hu DUAN)
Received 2014-02-25 Accepted 2014-04-07
Keywords:
1Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Aim: 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (DW532) is one of simplified analogues of hematoxylin that has shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities.
Methods: In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to evaluate the anti-angiogenesis efficacy.
Results: DW532 inhibited EGFR and VEGFR2 in vitro kinase activity (the IC50 values were 4.9 and 5.5 μmol/L, respectively), and suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 (1 and 10 μmol/L) induced G2/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mps1, the two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells. Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo.
Conclusion: DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer agent.
Keywords: anti-cancer agent; hematoxylin; combretastatin; tyrosine kinase; EGFR; VEGFR; tubulin; cell cycle; spindle assembly checkpoint; anti-angiogenesis
The authors acknowledge funding from the National Natural Science Foundation of China (No 20272078) and the Shanghai Municipal Science and Technology Commission (No 03J.C.14083).
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail jding@simm.ac.cn (Jian DING);
hxie@simm.ac.cn (Hua XIE);
whduan@simm.ac.cn (Wen-hu DUAN)
Received 2014-02-25 Accepted 2014-04-07