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PH006, a novel and selective Src kinase inhibitor, suppresses human breast cancer growth and metastasis in vitro and in vivo

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Abstract

The central role of Src in tumor progression and metastasis has validated it as an attractive therapeutic target for the treatment of human breast cancer. The aim of this study was to identify potential Src kinase inhibitor, explore its activity, and mechanism of action in human breast cancer. A strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and high-throughput screening was adopted and a novel 6-hydrazinopurine-based inhibitor of c-Src kinase PH006 was obtained. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay. The binding mode between PH006 and Src was profiled by surface plasmon resonance approach and molecular simulation. The anti-proliferative activity was evaluated by Sulforhodamin B (SRB) and Colony formation. The anti-invasion and anti-migration activities were assessed by trans-well and wound healing assay. Results indicated that PH006 was an ATP-competitive Src inhibitor, which selectively inhibited c-Src with an IC50 of 0.38 μM among a panel of 14 diverse tyrosine kinases. PH006 potently inhibited c-Src phosphorylation and c-Src-dependent signal transduction, resulting in inhibition of cell proliferation, migration, and invasion in human breast cancer MDA-MB-231 cells. Further study demonstrated that the anti-proliferative activity of PH006 was ascribed to its capability to arrest cells in G1 phase, while its anti-motility activity was related to suppression of MMP2/9 and HGF secretion. Moreover, PH006 exhibited potent activity against tumor growth as well as metastasis of human breast cancer MDA-MB-435 xenograft beard in nude mice, which was accompanied with reduced Src/FAK signaling in tumor tissue. Taken together, PH006 is a novel selective inhibitor of c-Src and possesses potent activity against breast cancer growth and metastasis, which could be potentially developed as a lead candidate against breast cancers with elevated Src tyrosine kinase activity.

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Acknowledgments

The authors greatly appreciate Li-Juan Lu, Yong Xi, and Yan-Yan Shen for their technical assistance in the animal experiments to evaluate the in vivo anti-tumor activities of PH006. This study was supported by National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2009ZX09301-001), National Natural Science Foundation of China (30721005, 20721003 and 20872153), and Science and Technology Commission of Shanghai (07dz05906).

Conflicts of interest

No potential conflicts of interest were disclosed.

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Authors and Affiliations

  1. Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China

    Jin-gui Ma, Si-meng Chen, Yi Chen, Xian-liang Xin, Li-ping Lin, Jian Ding & Ling-hua Meng

  2. Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China

    He Huang & Hong Liu

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  1. Jin-gui Ma
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Correspondence to Hong Liu or Ling-hua Meng.

Additional information

Jin-gui Ma and He Huang contributed equally to this study.

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10549_2010_1302_MOESM1_ESM.eps

Fig S1. PH006 suppresses the adhesion, migration, and invasion of MDA-MB-435 cells. (A) PH006 inhibits cell adhesion. MDA-MB-435 cells were pretreated with 10 μM of PH006 for 30 min and allowed to adhere onto fibronection-coated 96-well plates for 1 h. Unattached cells were washed thrice with serum-free medium. Cells were stained with crystal violet and OD value at 540 nm was detected with a plate reader. Columns, mean; Bars, SD. (B) PH006 inhibits cell migration. Wound healing assay was applied to detect cell migration. MDA-MB-435 cells were treated with 10 μM of PH006 or vehicle for 48 h. Left, representative images of at least three independent experiments. Right, quantitative analysis of cell migration. Columns, mean of three experiments; Bars, SD. (C) PH006 inhibits cell invasion. Mitragel invasion chamber was used to detect cell invasion. MDA-MB-435 cells were treated with 10 μM of PH006 or vehicle for 48 h. Cells invading to the lower aspect of the Boyden chamber filter were stained and photographed. Left, representative images of at least three independent experiments. Right, quantitative analysis of cell invasion. Columns, mean of three experiments; Bars, SD (EPS 2076 kb)

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Ma, Jg., Huang, H., Chen, Sm. et al. PH006, a novel and selective Src kinase inhibitor, suppresses human breast cancer growth and metastasis in vitro and in vivo. Breast Cancer Res Treat 130, 85–96 (2011). https://doi.org/10.1007/s10549-010-1302-4

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