Curcumin Binds To Microtubules And Triggers Apoptosis In HepG2Cells

Curcumin is a natural polyphenolic compound which is able to inhibittumorigenesis, proliferation and metastasis, but the exact mechanism of its action orthe identity of the target receptor is not completely understood. Microtubules arealways an established target of anticancer drug. If we destroy the microtubules ofcancer cells, it not only cannot get nutrients to be "starved to death"; but also unableto reorganized into the mitotic spindle to go to proliferation. Although theantiproliferative activity of curcumin has been reported, there are little reports aboutthe relationships between curcumin and microtubules or tubulin.In this paper, we exploited atomic force microscope (AFM), confocal laserscanning microscopy (CLSM), immunofluorescence, flow cytometry and othermethods to investigate the mechanism of curcumin binds to microtubules and triggersapoptosis in HepG2cells. This study includes three parts:(1) the effect of curcuminon apoptosis of HepG2cells;(2) curcumin targeted microtubules and inhibits HepG2cells migration;(3) inhibitory effect of curcumin on tubulin polymerization in vitro.We gained the following results:1. After HepG2cells were treated with different concentration curcumin for24h, theMTT results showed that the cell viability was decreased from(91.10±3.2%) to(10.84±4.0%), curcumin could efficiently inhibit the growth of HepG2cells in adoes-dependent manner but without cytotoxic effects to normal BRL cells. Flowcytometry quantitatively detected that curcumin treatment resulted in a dosedependent accumulation of HepG2cells in G2/M phase, and the early apoptosis ratewas from (1.7±0.24%) up to (60.1±9.27%). AFM analysis including cell topographyand cell-membrane ultra-structure showed that cells began to changed into round andcell surface also became rougher. Besides, the nucleuses and chromatin also appearedcondensation, margination and fragmentation after treated with curcumin. Theseresults show that curcumin can effectively induce apoptosis of HepG2cells.2. Used immunofluorescence and imaged by CLSM to investigate the changes ofmicrotubules. After being treated with curcumin for24h caused dose-dependent depolymerization of microtubules network in HepG2cells. At thesame time, AFM imaging also observed that curcumin could make the cytoskeletondepolymerized into small pieces or particles. All of these changes inhibited themigration of HepG2cells. CLSM combined with flow cyometry qualitative andquantitative analysis found that curcumin was able to up-regulate the expression ofpp2a in HepG2cells, the expression of PP2A was decreased from (90.7%±12.2%) to(2.5±1.2)%. These results suggested that pp2a participated in the process ofmicrotubules assembly, treatment with curcumin resulted cells cannot form themitotic spindle to go to proliferation and migration, ultimately achieved the goal ofantitumor.3. To further study the effects of curcumin on tubulin polymerization, we establishedan experiment of tubulin polymerization-depolymerization in vitro. Tubulinpolymerization is a temperature sensitive event, optimal polymerization occurs at37°C, and depolymerization at4°C. Curcumin had the capacity to hinder tubulinself-assembly in a dose-dependent manner which is similar to the effect of colchicine.In addition, CD spectra analysis suggested that curcumin has obvious effect on thesecondary structure of tubulin.In a word, curcumin as a potential anti-cancer drug, which could destroymicrotubule dynamic balance, inhibit cell proliferation and induce apoptosis ofHepG2cells. We have established that tubulin is one of the targets of curcumin, whichwell provide some help to develop new microtubule targeted drugs and cancertreatment.