Published: February 16, 2011 r2011 American Chemical Society 2040 dx.doi.org/10.1021/jf104231b | J. Agric. Food Chem. 2011, 59, 2040–2048 ARTICLE pubs.acs.org/JAFC Theaflavins Depolymerize Microtubule Network through Tubulin Binding and Cause Apoptosis of Cervical Carcinoma HeLa Cells Subhendu Chakrabarty, Amlan Das, Abhijit Bhattacharya, and Gopal Chakrabarti* Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India ABSTRACT: Here we studied the antiproliferative activity of theaflavins in cervical carcinoma HeLa cells by investigating their effects on cellular microtubules and purified goat brain tubulin. Theaflavins inhibited proliferation of HeLa cells with IC 50 value of 110 ( 2.1 μg/mL (p = < 0.01), caused cell cycle arrest at G 2 /M phase and induced apoptosis with alteration of expression of pro- and antiapoptotic proteins. Along with these antiproliferative activities, theaflavins act as microtubule depolymerizers. Theaflavins disrupted the microtubule network accompanied by alteration of cellular morphology and also decreased the polymeric tubulin mass of the cells. The polymerization of cold treated depolymerized microtubules in HeLa cells was prevented in the presence of theaflavins. In vitro polymerization of purified tubulin into microtubules was also inhibited by theaflavins with an IC 50 value of 78 ( 2.43 μg/mL (P < 0.01). Thus, disruption of cellular microtubule network of HeLa cells through microtubule depolymerization may be one of the possible mechanisms of antiproliferative activity of theaflavins. KEYWORDS: theaflavins, microtubules network, apoptosis, anticancer activity, cell cycle ’ INTRODUCTION Black tea is a promising agent for the chemoprevention of cancer. 1,2 Although epidemiological studies concerning the re- lationship between tea consumption and cancer risk have been inconclusive, 3 inhibition of carcinogenesis by black tea has been demonstrated in many animal models, including those involving cancers of the lung, skin, colon, esophagus and stomach. 1,2,4 Theaflavins (TF), the bioactive polyphenols of black tea, exert anticancer activity by inducing apoptosis. 5,6 Treatment with TF results in cell cycle arrest either in G 0 /G 1 phase 7,8 or in G 2 /M phase. 9 Theaflavins modulate pro- and antiapoptotic proteins 7,9 and inhibit growth and proliferation of cancer cells. 8 Tubulin, a heterodimeric (containing R and β subunits) protein, is polymerized to form microtubule, which has a versatile functions in cells, like cell division, maintenance of cell shape and structure, cell signaling and organelle transport. 10,11 Microtu- bules are dynamic in nature, and it was established that this dynamicity is responsible for functions of microtubules. Dynamic equilibrium of tubulin-microtubule is among the popular targets for anticancer therapy. Based on the mechanism of action of alternation of microtubule dynamics, drugs can be classified into two categories. One group of drugs, like paciltaxel and docetaxel, stabilizes microtubule polymer, and the other group, like vinblastine and vincristine, inhibits tubulin polymerization into microtubule. 12-14 All these microtubule inhibitors and various other tubulin binding agents perturb spindle microtubule function and arrest cell cycle at G 2 /M phase. 15-17 So far, all studies reported with TF were performed either in vivo or in cancer cells under culture conditions, but there is no report about the effect of TF on cellular cytoskeleton protein. In the present study, we examined the antiproliferative effects of TF against HeLa cells (cervical carcinoma cells) in relation to their ability to depolymerize cellular microtubule and perturb tubulin assembly, in vitro. We chose cervical carcinoma cells because there is no previous report on the effect of TF on cervical carcinoma although it is one of the most common forms of cancer in women. ’ MATERIALS AND METHODS Chemicals. Dulbecco’s minimal essential media (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin, and amphoter- icin B were purchased from HyClone, Logan, UT, USA. Human cervical carcinoma cells (HeLa) were obtained from National Centre for Cell Science, Pune, India. DAPI, mouse monoclonal anti-R-tubulin antibody (raised in mouse), anti-mouse rhodamine conjugated IgG antibody, anti-mouse HRP-conjugated IgG antibody, guanosine-5 0 -triphosphate (GTP), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), MgCl 2 , ethylene glycol tetraacetic acid (EGTA), and theaflavins were purchased from SIGMA, St. Louis, MO, USA. Annexin V-FITC apoptosis detec- tion kit was obtained from BD Biosciences, Franklin Lakes, NJ, USA. Other primary antibodies (against p53, Bax, Bcl-2, caspase-3) were purchased from Santa Cruz Biotechnology. The Bradford Protein estimation kit was purchased from Genei, India. All other chemicals and reagents were of analytical grade and purchased from Sisco Research Laboratories, India. Cell Culture. Human cervical carcinoma cells (HeLa) were main- tained in DMEM medium supplemented with 1 mM L-glutamine, 10% fetal bovine serum, 50 μg/mL penicillin, 50 μg/mL streptomycin and 2.5 μg/mL amphotericin B. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 . Cells were grown in tissue culture flasks until they were 80% confluent before trypsinization with 1 trypsin and splitting. The morphology of control and treated cells was observed by Olympus inverted microscope model CKX41. Received: November 1, 2010 Accepted: January 11, 2011 Revised: January 3, 2011