Screening the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2 kinase

Abstract

Objective: To screen and evaluate the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 phosphatase.

Methods: The affinity chromatography purified glutashione-S-transferase/Cdc25A phosphatase fusion protein and Cdc2/cyclin B from the extracts of starfish M phase oocytes are used as the cell cycle-specific targets for screening the antimitotic constituents. We tested 9 extracts isolated from the Chinese medicinal herbs and vegetables including the agents currently used in cancer treatment by measuring the inhibition of Cdc25A phosphatase and Cdc2 kinase activity. The antitumor activity of the extracts was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry.

Results: Cdc25A inhibitory activity and antitumor activity are detected in the extracts isolated from three Chinese medicinal herbs Agrimona pilosa; Herba solani lyrati; Galla chinesis.

Conclusion: We found three extracts isolated from Chinese medicinal herbs have potential inhibitory activity of Cdc25 phosphatase using a highly specific mechanism-based screen assay for antimitotic drug discovery.

Fig. 1

Active constituents of Chinese medicinal herbs inhibit the activity of recombinant Cdc25 phosphatase. (a) The Cdc2/cyclin B kinase was purified from oocyte extracts by affinity chromatography on p9CKShs1-Sepharose beads are used as the cell cycle-specific targets for screening the antimitotic constituents. Cdc2 kinase activity inhibition was assayed as described under “MATERIALS AND METHODS”. [32P] Phosphate incorporation in histone H1 was measured by direct counting; (b) The Cdc25A phosphatase is inhibited by active constituents of Chinese medicinal herbs. GST-Cdc25A phosphatase was exposed to various concentrations of the extracts and assayed as described under “MATERIALS AND METHODS”. Shown is an average of three determinations. The letter of each curve represents same extract as indicated in Table 1

Fig. 2

Active constituents of Chinese medicinal herbs inhibit tumor cell proliferation The growth inhibition of K562 cells by the potential Cdc25 phosphatase inhibitors was evaluated by MTT assay as described under “MATERIALS AND METHODS”. The treatment of Agrimona pilosa (a); Galla chinesis (b) and Herba solani lyrati (c) at various concentrations for 48 h, the cell growth was significantly inhibited. The values are presented as mean±SD obtained from triplicate samples

Fig. 2

Active constituents of Chinese medicinal herbs inhibit tumor cell proliferation The growth inhibition of K562 cells by the potential Cdc25 phosphatase inhibitors was evaluated by MTT assay as described under “MATERIALS AND METHODS”. The treatment of Agrimona pilosa (a); Galla chinesis (b) and Herba solani lyrati (c) at various concentrations for 48 h, the cell growth was significantly inhibited. The values are presented as mean±SD obtained from triplicate samples

Fig. 2

Active constituents of Chinese medicinal herbs inhibit tumor cell proliferation The growth inhibition of K562 cells by the potential Cdc25 phosphatase inhibitors was evaluated by MTT assay as described under “MATERIALS AND METHODS”. The treatment of Agrimona pilosa (a); Galla chinesis (b) and Herba solani lyrati (c) at various concentrations for 48 h, the cell growth was significantly inhibited. The values are presented as mean±SD obtained from triplicate samples

Fig. 3

Active constituents of Chinese medicinal herbs inhibit cell cycle progression Human leukemia K562 cell line was used for cell cycle analysis by flow cytometry as described under “MATERIALS AND METHODS”. After 24 h of treatment with the extracts at concentrations of 10, 100 and 500 μg/ml, the effect of the active constituents of Agrimona pilosa; Herba solani lyrati and Galla chinesis on cell cycle progression is evaluated by detection of G2M arrest