The Polymerization-Promoting Effect Of Wangzaozin A,An Ent-Kaurane Diterpene,on Tubulin

Microtubule is always a hot target for research and development of anti-cancer drug.The destruction of the drug on the microtubules of the cells can prevent the cells from correctly forming the mitotic spindle and cause inhibition of cell proliferation to achieve anticancer effects.It has been found that the variety of microtubule-targeted drugs,including paclitaxel,vinblastine and colchicine.These drugs have not only shown significant value in cancer treatment,but also made important contributions to the functional study of intracellular microtubule and the interpretation of the molecular mechanisms of tubulin assembly.It is important significance to further explore new microtubule-targeted drugs for the development of new anticancer drugs and to research the function and assembly mechanism of microtubule as molecular probes.Our lab previously separated a kind of terpenoid compound named wangzaozin A from Labiatae living in Gansu.And it was found that the compound had the effect of stabilizing cellular microtubule and promoting intracellular microtubule polymerization.Therefore,it is extremely possibly a kind of potential microtubule target drug.In the study,the influence of the compound wangzaozin A on the assembly of microtubule was studied in cellular system and extracellular system through selecting HeLa,HUVEC cell lines and purified tubulin protein as the research materials by means of immunofluorescence,flow cytometry,Western blot technology,and other experimental ways.The results andconclusions as follows:(1)The effect of different concentrations of wangzaozin A on cell growth was detected by MTT assay.It was found that 0.2-2.0 μmol/L wangzaozin A had significant growth inhibition on HeLa cells.Flow cytometry analysis showed that wangzaozin A could result cell cycle arrest at G2/M phase,but not induce apoptosis(Annexin V/PI double-staining detection).(2)It could be seen from immunofluorescence technique with microscopic observation that microtubule network area increased(by 39.6%compared to cell microtubule area of the control group),microtubule density increased and microtubule end bundled were treated by 0.8-1.4 μmol/L wangzaozin A for 24 hours in HeLa,HUVEC cell,which resulted in apparent increase of fluorescence staining intensity of cell microtubule(increased by 3 times compared to that of the control group).The group treated by 0.8 nmol/L Paclitaxel also had the same phenomenon And the further Western blot experiment showed that 0.8-1.4 μmol/L wangzaozin A did not change the expression of intracellular tubulin protein,only increased polymerization microtubule.These results showed that wangzaozin A could stabilize intracellular tubulin protein assembly and promote polymerization.(3)After HeLa cells were treated by 0.2-1.4 μmol/L wangzaozin A for 24 hours and by 5-15μmol/L wangzaozin A for 3 hours.Afterwards,it was treated at low temperature(4℃)for 15 minutes.The observation found that the cell microtubules of the control group were almost completely observed.Depolymerization,microtubule network disappears,microtubules accumulate in the perinuclear to form bright spots,while wangzaozin A treatment cells have less microtubule depolymerization,although the microtubule network has a lower density compared to untreated cells,but remains Complete;at the same time,it was observed that low temperature could not cause the depolymerization of microfilaments and keratin fibers in the cells.The changes of microfilaments and keratin fiber skeletons in the wangzaozin A treatment group were consistent with those in the control group,and there was no significant difference;these results indicated that wangzaozin A could protect and resists the depolymerization of microtubules caused by low temperature.(4)Through extracellular tubulin polymerization(37 ℃)-depolymerization(4℃)experiments,the results showed that the addition of wangzaozin A significantly promoted tubulin polymerization in a concentration-dependent manner at 37℃.The promotion effect of 10 pmol/L wangzaozin A and μmol/L paclitaxel was comparable.(5)Binding kinetics analysis of the tubulin paclitaxel binding site,wangzaozin A can exchange the fluorescein-binding paclitaxel derivative Flutax-2 from the tubulin binding site,and wangzaozin A has a concentration of Flutax-2 exchange.Dependence,the substitution efficiency of Flutax-2 at μmoUL wangzaozin A was the highest(97.8%).In summary,the compound wangzaozin A can promote microtubule polymerization of HeLa and HUVEC cell,change the dynamics process of tubulin protein,and have the same promoting effect under the in vitro condition Moreover,it is also shown that wangzaozin A can bind to the paclitaxel site of tubulin protein and play an important role in promoting tubulin protein polymerization similar to paclitaxel.The above conclusion indicates that wangzaozin A can be used as a potential anti-microtubule drug,wangzaozin A inhibits cell proliferation and arrests cell cycle by disrupting the dynamic balance of microtubules.It is important value for R&D of microtubule-targeted therapeutic and the study of microtubule assembly as molecular probes.