Study On The Anti-cancer Activity And Related Mechanisms Of HDF,A Compound With Patent

In the present study,we report the antimicrotubule activity of a novel farrerol derivative,6,8-dimethyl-7,4’-dimethoxy-5-hydroxyflavan（HDF）.Farrerol is used as antibechic and expectorant without microtubule-targeted activity.By the cell biology experiments,it is verified that HDF has the biological activity of inhibition tumor cell proliferation,influencing cell morphology,inhibiting cell migration,arresting cell cycle and inducing apoptosis.It was confirmed that HDF could inhibit microtubule dynamics and disturb the arrangement of microtubule in cells by means of microimaging,biochemistry and biophysics assays.Materials and methods:The effects of HDF on cancer cells were evaluated with human acute promyelocytic leukemia cell line HL-60,human lung carcinoma cell line A549 and human cervical carcinoma cell line HeLa cells.With cancer cells and tubulin purified from porcine brain,a series of cell biology,biochemistry and biophysics assays were conducted.（1）Cell proliferation was measured by MTT assay.（2）Scratch migration assay was used to measure the migration ability of A549 cells and HeLa cells.（3）Flow cytometry was used to identify the cell cycle and apoptosis of cancer cells treated by HDF.（4）Microtubule-targeted properties were studied through observing cytoskeleton networks by immunofluorescence assays.（5）Microtubule assembly assays and western blot measured the influence on tubulin polymerization.（6）The interaction betweenα/β-tubulin heterodimers and HDF was evaluated with isothermal titration calorimetry（ITC）.Results:（1）Experimental data indicated that HDF inhibited cell proliferation of HL-60,A549 and HeLa cells with IC₅₀ 1.3±0.2,1.5±0.6 and 0.9±0.1μM respectively.（2）HDF arrested cell cycles at G2/M phase and induced apoptosis.（3）Disruption of microtubule network was observed intracellular after treated with HDF.（4）Inhibition on microtubule assembly by HDF was identified in vitro.（5）Unlike colchicine,the positive control,ITC analysis suggested that there is no interaction between HDF and freeα/β-tubulin dimers.Conclusion:HDF presents potent anticancer effect in vitro with anti-proliferation,anti-migration,cell-cycle arrest and apoptosis inducing activities.The anticancer mechanism is that HDF inhibits assembly of microtubule.