Báo cáo khoa học: Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells ppt

Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells Mithu Banerjee, Parminder Singh and Dulal Panda Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai, India Keywords apoptosis; BubR1; combination study; delayed mitosis; dynamic instability Correspondence D Panda, Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India Fax: +91 222 572 3480 Tel: +91 222 576 7838 ⁄ 7770 E-mail: panda@iitb.ac.in (Received 14 April 2010, revised 21 May 2010, accepted 24 June 2010) doi:10.1111/j.1742-4658.2010.07750.x In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule–kinetochore attachment and disturbed the mitotic spindle structure Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint In addition, curcumin treatment increased the metaphase ⁄ anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53 Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules Introduction Curcumin, a natural product found in the rhizome of Curcuma longa, is emerging as an important anticancer agent on account of its manifold clinical applications [1–5] Although the phase I clinical trial of curcumin for the prevention of colon cancer has already been completed (clinicaltrials.gov Identifier: NCT00027495), clinical trials to determine its efficacy in the treatment of rectal cancer (clinicaltrials.gov Identifier: NCT00745134), advanced pancreatic cancer (clinicaltri- als.gov Identifier: NCT00094445), colorectal cancer (clinicaltrials.gov Identifier: NCT00973869) and multiple myeloma (clinicaltrials.gov Identifier: NCT00113841) are currently in progress In addition, the potential of curcumin to reduce the symptomatic side effects of chemoradiation in patients suffering from non-small cell lung cancer (clinicaltrials.gov Identifier: NCT01048983) is under clinical investigation Curcumin has also entered into a phase II clinical trial for Abbreviations CI, combination index; FITC, fluorescein isothiocyanate; PI, propidium iodide FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS 3437 Curcumin suppresses microtubule dynamics M Banerjee et al advanced pancreatic cancer [2] and a phase III clinical trial in combination with gemcitabine and celebrex for the treatment of metastatic colon cancer [2] Curcumin inhibits tumor growth in animal models [3] Further, the uptake of high doses of curcumin in both animals and humans has been found to be nonhazardous and relatively nontoxic [4,5] Curcumin has also been found to be an effective stress reliever and neuroprotective agent [6] Curcumin has been shown to inhibit the proliferation of several types of cancer cells in culture, including pancreatic, cervical, colon and breast cancer [7–15] It arrests the cell-cycle progression of human pancreatic cancer cells (BxPC-3) and glioma cells (U251) at the G2 ⁄ M phase of the cell cycle [7,8] and has been shown to affect the progression of MCF-7 cells through the G2 ⁄ M phase [9] Curcumin treatment caused an increase in the G0 ⁄ G1 phase of the cell population implying apoptosis in MCF-7 cells [10] Curcumin is found to induce apoptosis in several cell lines [1,7,10] It stimulated Bax-mediated p53-dependent apoptosis in MCF-7 cells [10] Curcumin promotes the action of certain drugs by overcoming chemoresistance [11] It overcomes P-glycoprotein-mediated multidrug resistance in multiple cell lines [1] The migration and invasion of human lung cancer cells are also inhibited by curcumin [1] Curcumin has been suggested to inhibit cell proliferation by diverse mechanisms [1,12–16]; however, the primary mechanism by which inhibition occurs remains obscure Recently, curcumin has been found to bind to purified tubulin, to inhibit tubulin polymerization in vitro and to depolymerize microtubules in HeLa and MCF-7 cells in culture [12] In addition, curcumin has been shown to perturb the microtubule spindle structure [12,13] and to stimulate micronucleation in MCF-7 cells [13] In 32D cells, curcumin has also been shown to affect the activity of the chromosomal passenger complex, resulting in multipolar chromosome segregation promoting mitotic catastrophe [14] Moreover, curcumin induced mitotic catastrophe in Ishikawa and HepG2 cancer cells, indicating that it might perturb microtubule assembly dynamics [15] Dynamic microtubules are the key structural elements in mitotic spindle formation and they orchestrate chromosome distribution during the cell division [17,18] In this study, we found that curcumin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells At low effective proliferation inhibitory concentrations, curcumin inhibited microtubule dynamics in MCF-7 cells without causing a significant depolymerization of microtubules However, high concentrations (‡ · IC50) of curcumin 3438 were found to depolymerize both the interphase and mitotic microtubules in MCF-7 cells Curcumin treatment perturbed the mitotic spindle network in MCF-7 cells, activated the mitotic checkpoint and delayed mitotic progression We present several lines of evidence indicating that curcumin inhibits cell proliferation by inhibiting microtubule dynamics The results suggest that tubulin is one of the major targets for the antiproliferative activity of curcumin Results Curcumin inhibited the proliferation of MCF-7 cells and induced apoptosis Consistent with previous studies [9,10,12], curcumin was found to inhibit the proliferation of MCF-7 cells in a concentration-dependent manner (Fig 1A) For example, 20 and 40 lm curcumin inhibited the proliferation of MCF-7 cells by 70% and 93%, respectively, and the half-maximal inhibition of proliferation (IC50) was determined to be 16 ± 0.3 lm MCF-7 cells were either treated with the vehicle or different concentrations of curcumin for 48 h Vehicle-treated MCF-7 cells did not display Annexin V and propidium iodide (PI) staining, although a population (46%) of cells treated with 12 lm curcumin stained positive for Annexin V alone, showing that the cells were at the early stage of apoptosis (Fig 1B) Cells treated with 24 lm curcumin showed greater numbers (71%) stained with Annexin V (Fig 1B) At a still higher curcumin concentration (36 lm), cells were stained with both Annexin V and PI, indicating late apoptosis (Fig 1B) Further, curcumin treatment strongly increased the nuclear localization of p53 and p21 in MCF-7 cells (Fig 1C,D) For example, and 24% of MCF-7 cells showed nuclear localization of p53 (Fig 1C), whereas and 33% of cells showed nuclear localization of p21 in the absence and presence of 24 lm curcumin, respectively (Fig 1D) Curcumin disrupted the mitotic spindle network inducing formation of monopolar spindles and depolymerized microtubules in MCF-7 cells MCF-7 cells were incubated without or with 24 and 36 lm curcumin for h Curcumin treatment strongly depolymerized mitotic spindle microtubules (Fig 2A) However, it did not induce significant depolymerization of the interphase microtubules after h of incubation, suggesting that curcumin exerted a stronger depolymerizing effect on the microtubules of the mitotic cells than those of the interphase cells (data not FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS M Banerjee et al Curcumin suppresses microtubule dynamics A B C D Fig Curcumin inhibited the proliferation of MCF-7 cells and induced cell death (A) MCF-7 cells were treated with different concentrations of curcumin for one cell cycle and the inhibition of cell proliferation was determined by the sulforhodamine B assay (B) Curcumin induced apoptosis in MCF-7 cells MCF-7 cells were incubated with 0.1% dimethylsulfoxide (control) and different concentrations (12–36 lM) of curcumin for 48 h and then stained with Annexin V ⁄ PI Scale bar, 10 lm Curcumin (24 lM) treatment increased the nuclear accumulation of p53 (C) and p21 (D) in MCF-7 cells Scale bar, 10 lm DNA Merge B Control Tubulin 15 25 min 15 25 Curcumin Curcumin 36 μM Curcumin 24 μM Control A Fig Curcumin-perturbed mitotic spindle structures of MCF-7 cells (A) MCF-7 cells were incubated without or with 24 and 36 lM of curcumin for h Microtubules are shown in red and the nucleus in blue Scale bar, 10 lm (B) Curcumin suppressed the reassembly of the colddepolymerized mitotic spindle microtubules The upper and lower panels show growth kinetics of spindle microtubules in the absence or the presence of 36 lM curcumin Scale bar, 10 lm FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS 3439 Curcumin suppresses microtubule dynamics M Banerjee et al shown) Consistent with a previous study [12], curcumin was found to induce significant depolymerization of both the interphase and mitotic microtubules of MCF-7 cells after 24 h of incubation (Fig S1A,B) In the control population, 70% and 30% of the mitotic cells were found to be bipolar and monopolar, respectively, whereas 85% of the mitotic cells were monopolar in the presence of 24 lm curcumin, suggesting that curcumin induced the formation of monopolar spindles The effect of curcumin on the polymerized mass of microtubules in MCF-7 cells was analysed by western blotting The ratio of polymeric ⁄ soluble tubulin was found to be 2.44 ± 0.77 in the absence of curcumin, and 1.97 ± 0.20, 1.56 ± 0.17 (P < 0.03) and 1.32 ± 0.15 (P < 0.01) in the presence of 12, 24 and 36 lm curcumin, respectively, indicating that curcumin depolymerized microtubules in MCF-7 cells (Fig S1C) A B Curcumin inhibited the reassembly of mitotic microtubules in MCF-7 cells MCF-7 cells were synchronized in the M phase of the cell cycle by treating with 1.3 lm nocodazole for 20 h Nocodazole treatment completely depolymerized the spindle microtubules Nocodazole was removed and the cells were incubated with fresh media in the absence or presence of 36 lm curcumin on ice for 30 Subsequently, cells were incubated at 37 °C Spindle microtubules in control cells reassembled within 25 to form normal mitotic spindle; the spindle microtubules of curcumin-treated cells did not reassemble (Fig 2B) The results showed that curcumin inhibited reassembly of the mitotic spindle microtubules Curcumin suppressed the dynamic instability of individual microtubules in live MCF-7 cells Consistent with previous reports [19,20], microtubules in control MCF-7 cells were found to be highly dynamic (Fig 3A) Low concentrations of curcumin (5 and 12 lm) noticeably dampened the dynamic instability of the individual microtubules in live MCF-7 cells (Fig 3B,C) Curcumin treatment reduced the rate and extent of both growing and shortening events (Table 1) For example, 12 lm curcumin reduced the rates of shortening and growing phases by 39% and 19%, respectively, and reduced the extent of the growing and shortening phases by 60% and 65%, respectively Like several other tubulin-targeted agents such as benomyl, estramustine, epothilone B and paclitaxel 3440 C Fig Curcumin suppressed dynamic instability of individual microtubules in live MCF-7 cells Life-history traces of individual microtubules in MCF-7 cells in the absence (A) and presence of (B) lM curcumin and (C) 12 lM curcumin, respectively [19–22], curcumin also strongly increased the time that microtubules spent in the pause state, neither growing nor shortening detectably, and decreased the time microtubules spent in the growing or shortening phases Curcumin (12 lm) increased the time spent in the pause state from 28.9% (control) to 71.6% Further, curcumin (12 lm) altered both the time- and length-based transition frequencies of the interphase microtubules in MCF-7 cells The dynamicity (dimer exchange per unit time from the ends of microtubules) was reduced by 50% and 72% in the presence of and 12 lm curcumin, respectively FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS M Banerjee et al Curcumin suppresses microtubule dynamics Table Effects of curcumin on the dynamic instability parameters of the interphase microtubules in MCF-7 cells Twenty-five microtubules were measured for each condition Data are given as mean ± SD Control )1 Growth rate (lmỈmin ) Growth length (lm) Growth time (min) Shortening rate (lmỈmin)1) Shortening length (lm) Shortening time (min) Pause time (min) % Time spent in growing % Time spent in shortening % Time spent in pause Dynamicity (lmỈmin)1) Rescue frequency (eventsỈmin)1) Catastrophe frequency (eventsỈmin)1) Rescue frequency (eventsỈlm)1) Catastrophe frequency (eventsỈlm)1) 14.7 2.4 1.2 23.5 3.3 0.53 0.72 47.5 22.4 28.9 12.6 7.7 2.1 0.35 0.24 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lM curcumin 2.9 1.1 0.3 10.4 1.9 0.18 0.28 9.8 8.3 11.6 4.6 3.4 0.70 0.20 0.12 12.1 1.12 0.55 18.2 2.1 0.43 1.43 22.7 18.7 59.5 6.3 9.9 0.59 0.67 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± a 2.6 0.5b 0.23a 5.7a 1.1a 0.19 0.35a 8.8a 7.9a 14.0a 3.0a 3.5a 1.1a 0.35a 0.27a 12 lM curcumin 11.9 0.97 0.33 14.4 1.14 0.28 1.82 13.5 11.6 71.6 3.50 12.74 1.50 0.99 1.08 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.9a 0.3a 0.15a 4.95a 0.51a 0.11a 0.36a 7.3a 4.7a 13.8a 1.99a 2.50a 0.96a 0.42a 0.55a P < 0.0001; bP < 0.001 a Effects of curcumin on cell-cycle progression It was previously reported that curcumin treatment markedly increased the number of MCF-7 cells in metaphase [13] Because curcumin suppressed microtubule dynamic instability (Fig and Table 1), we examined whether it could inhibit mitosis The number of cells in mitosis in the absence or presence of different concentrations of curcumin was determined by Hoechst 33258 staining of the chromosomes Only 2.6 ± 1.6% of the control (vehicle-treated) cells were found to be in mitosis, whereas 3.2 ± 0.2%, 5.0 ± 0.1% and 6.2 ± 1% (P < 0.0001) of the cells were found to be in mitosis in the presence of 12, 24 and 36 lm curcumin, respectively If curcumin caused a delay in mitosis, it might lead to an increase in the metaphase ⁄ anaphase ratio The metaphase ⁄ anaphase ratio was calculated to be 0.43 ± 0.06 and 1.88 ± 0.40 (P < 0.0001) in the absence and presence of 24 lm curcumin, supporting the idea that curcumin could prolong the duration of metaphase Further, MCF-7 cells were synchronized in the M phase of the cell cycle by nocodazole treatment for 20 h Nocodazole-blocked cells were washed with fresh medium and subsequently incubated in medium without and with curcumin Flow cytometry analysis demonstrated that nocodazole-induced mitotic arrest was gradually released over time for control cells For example, the percentage of cells in mitosis was 87%, 53% and 16% in nocodazole-treated control flask immediately, and and h after release of the nocodazole block However, in the presence of curcumin, the percentage of cells in the mitotic phase was 83% and 81% after and h of block release Thus, treatment of cells with curcumin significantly delayed release of the mitotic block (Fig 4A) However, flow cytometric analysis of the cell cycle using PI staining showed that there was no significant cell-cycle block after 24 h of curcumin treatment (Fig S2) Microtubule inhibitors are known to induce mitotic block by activating the spindle assembly checkpoint proteins [20,23,24] It has been suggested that a compound may drive the cells towards delayed mitosis through activation of spindle checkpoint proteins such as BubR1 [23] and Mad2 [24] Nocodazole, a wellknown inhibitor of mitosis, led to the accumulation of Mad2 and BubR1 at the kinetochores (Fig 4B,C) Similar to the action of nocodazole, curcumin treatment also activated Mad2 and BubR1 in MCF-7 cells (Fig 4B,C) Curcumin exhibited antagonism with paclitaxel, but an additive effect with vinblastine for inhibition of MCF-7 cell proliferation Curcumin, paclitaxel and vinblastine inhibited MCF-7 cell proliferation with median inhibitory doses of 15 ± lm, 40 ± nm and 17 ± 10 nm (Fig S3A–C) Curcumin (8 lm) and paclitaxel (2 nm) inhibited proliferation of MCF-7 cells by 26% and 13%, respectively, when used alone, whereas their combination inhibited proliferation by 9% The combination index (CI) for the combination of lm curcumin and nm paclitaxel was found to be 3.1 ± 1.5 The proliferation of MCF7 cells was inhibited by 22% and 24% in the presence of and nm vinblastine, respectively, whereas in FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS 3441 Curcumin suppresses microtubule dynamics B 100 200 300 400 500 200 400 600 8001000 A 20 40 60 80 100 120 C 20 40 60 80 100 120 0 80 160 240 320 80 160 240 320 M Banerjee et al 30 60 90 120 150 30 30 60 90 120 150 80 160 240 320 100 200 300 400 500 0 20 40 60 80 100 120 60 90 120 150 Fig Curcumin treatment delayed mitotic progression in MCF-7 cells (A) MCF-7 cells were incubated with 1.3 lM nocodazole Nocodazole was washed off with fresh medium Cells were incubated in the absence or presence of 35 lM curcumin for and h and then stained with PI DNA content of the cells was quantified by flow cytometry Nocodazole and curcumin treatment activated Mad2 (B) and BubR1 (C) in MCF-7 cells MCF-7 cells were incubated with nocodazole (500 nM) and curcumin (36 lM) for 24 h and cells were then stained with Mad2 and BubR1 antibodies Scale bar, 10 lm combination with lm curcumin, these concentrations of vinblastine inhibited proliferation by 44% and 51%, respectively The CI values for the combination of lm curcumin with and nm of vinblastine were estimated to be 0.92 ± 0.23 and 0.97 ± 0.19, respectively A CI value < indicates a synergistic effect, indicates an additive effect and > indicates an antagonistic effect [25,26] The results suggested that curcumin was antagonistic to paclitaxel, whereas it displayed an additive effect with vinblastine in inhibiting MCF-7 cell proliferation Curcumin affected the localization of the kinesin protein Eg5 Because curcumin produced monopolar spindles in MCF-7 cells, we examined the effect of curcumin on the localization of Eg5, a motor protein that plays an essential role in bipolar spindle formation [27,28] In control cells, Eg5 was localized throughout the bipolar spindle and remained concentrated at the spindle poles (Fig 5A) Consistent with a previous study [27], monastrol (50 lm) was found to induce monopolar spindle formation (Fig 5B) In monastrol-treated cells, Eg5 mainly localized to the pole of the monoastral spindle 3442 and also diffused all along the monoastral microtubules (Fig 5B) In the presence of 24 lm curcumin, Eg5 primarily remained confined to the pole of the monopolar spindles Some Eg5 also delocalized along the microtubules of the monopolar spindles (Fig 5C) Discussion In this study, we have provided several lines of evidence indicating that the antiproliferative mechanism of action of curcumin involves the perturbation of microtubule dynamics Brief incubation of curcumin with MCF-7 cells produced a noticeable depolymerizing effect on the mitotic microtubules of MCF-7 cells and also inhibited the assembly of cold-depolymerized spindle microtubules indicating that curcumin perturbs microtubule assembly in cells Further, similar to the effects of several other microtubule-targeted drugs such as benomyl [19], estramustine [20], epothilone [21] and paclitaxel [22] on microtubule dynamics, curcumin was also found to reduce the dynamic instability of individual microtubules in live MCF-7 cells Curcumin treatment caused defective chromosome alignment in the mitotic spindles and the cells eventually died via the p53-dependent apoptotic pathway Curcumin was FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS M Banerjee et al A Curcumin suppresses microtubule dynamics Tubulin Eg5 DNA Tubulin + Eg5 Tubulin + Eg5 + DNA B C Fig Localization of Eg5 in control and curcumin-treated MCF-7 cells Cells were treated without and with curcumin for 24 h, fixed, and co-immunostained with a-tubulin (green), Eg5 antibody (red) and DNA was stained with Hoechst 33258 (A) In control mitotic cells, Eg5 remained mainly concentrated at the poles of the bipolar spindle and to some extent delocalized along the spindle microtubules (B) In the presence of 50 lM monastrol, monopolar spindles were formed Eg5 localized mainly at the pole of the monopolar spindle and remained diffused along the microtubules in the overlayed image (C) Curcumin at a concentration of 24 lM induced monopolar spindle formation In the overlain image the Eg5 localized to the centre of the monopolar spindle and also remained dispersed over the microtubules Scale bar, 10 lm found to bind to purified tubulin and to perturb microtubule assembly in vitro [12] The results together indicated that curcumin inhibits MCF-7 cell proliferation by targeting microtubules The plus-end-directed motor Eg5 (kinesin spindle protein) plays an important role in proper chromosome separation and the formation of a proper bipolar spindle [27,28] Similar to the action of monastrol [27], curcumin also induced monopolar spindle formation in association with the perturbation of Eg5 localization in MCF-7 cells, indicating that curcumin may inhibit Eg5 function and thereby induce monopolar spindle formation Curcumin might inhibit the binding of Eg5 to microtubules and perturb the movement of Eg5 over the microtubules leading to abnormal spindle formation Alternatively, curcumin might directly interact with Eg5 and inhibit its function Effects of curcumin on the progression of the cell cycle Curcumin increased the metaphase ⁄ anaphase ratio and slowed the release of mitotic block in nocodazolesynchronized MCF-7 cells, indicating that it can delay cell-cycle progression at mitosis However, it failed to induce substantial mitotic block in MCF-7 cells In several cases, higher concentrations of microtubuletargeted agents are required to inhibit cell-cycle progression at mitosis than are required to inhibit the proliferation [29–32] In a KB ⁄ HeLa (human cervical epitheloid carcinoma) cell line, a derivative of benzylidene-9(10H)-anthracenone gave an IC50 value of 0.09 lm for the inhibition of cell proliferation, whereas 50% arrest in the G2 ⁄ M phase occurred in the presence of 0.205 lm of compound [29] The anthracenone derivative caused cell-cycle arrest in a K-562 cell line at 0.3 lm, whereas its IC50 in the same cell line was 0.02 lm In smooth muscle cells, 68.6% of the cells were arrested in the G2 ⁄ M phase at 100 nm concentration of LY290181 (IC50 of inhibition of cell proliferation being 20 nm) [30] In human non-small cell lung carcinoma cells A549, low concentrations of paclitaxel (3-6 nm) inhibited cell proliferation without causing mitotic arrest [31] Moreover, treatment with a low concentration of paclitaxel induced abnormal cell formation without the G2 ⁄ M block [32] A 50% inhibition of cell growth after 72 h incubation required 3.4 nm paclitaxel and 9.5 nm discodermolide [32] These concentrations were closer to that required for aneuploidy induction rather than mitotic arrest [32] FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS 3443 Curcumin suppresses microtubule dynamics M Banerjee et al Thus, the induction of abnormal mitosis and aneuploidy is dependent on the drug mechanism and the concentration of the drug used [31,32] Several microtubule-targeted agents are known to activate checkpoint proteins and to arrest cells in mitosis [20,33–35] The checkpoint proteins accumulate in the kinetochoric region after detecting a flaw in kinetochore–microtubule attachment or reduced tension at the kinetochores [24] For example, nocodazole enhances the accumulation of Mad2 and BubR1 to the kinetochores and induces mitotic arrest (Fig 4B,C) [36] Several inhibitors of microtubule dynamics were found to delay G2 ⁄ M transition [37] The ability of a compound to activate spindle checkpoint proteins may sometimes lead to delayed mitosis [38] Conditions that perturb proper kinetochore–microtubule attachment may cause checkpoint protein translocation and the affected cells may be held back from progressing further in the cell cycle, leading to a delay in mitosis [38] Curcumin was found to perturb microtubule–kinetochore attachment and also activated the mitotic checkpoint, resulting in delayed mitosis A delay in mitosis has been shown to induce apoptosis in cancer cells [39] Curcumin treatment enhanced the nuclear accumulation of p53 in MCF-7 cells An alteration in expression of the tumor suppressor gene p53 is known to induce apoptosis in several types of cells [40–42] It has been suggested that p53 is transported into the nucleus through the microtubule network [40,41] Compounds that stabilize microtubule dynamics have been suggested to promote p53 translocation to the nucleus [19,41] Several antimitotic drugs have been found to induce apoptosis by inhibiting microtubule assembly dynamics [43] Curcumin suppresses the dynamic instability of microtubules, therefore, it may enhance nuclear translocation of p53 through the stabilized microtubule track Curcumin in combination with vinblastine, a microtubule depolymerizing agent, inhibited cell proliferation in an additive fashion However, it antagonized the action of paclitaxel, a compound that promotes microtubule assembly; supporting the idea that curcumin inhibits cell proliferation by targeting microtubules The results also indicated that curcumin may be used in combination with microtubule depolymerizing agents such as vinblastine to improve the efficacy and reduce the toxic dose of the drug It has been found that an oral intake of curcumin is not toxic to humans up to 8000 mgỈday)1 for months [44] Moreover, curcumin (C3 ComplexÔ, Sabinsa Corp., East Wind3444 sor, NJ, USA) in single oral doses up to 12 000 mg was found to be well tolerated in healthy volunteers [45] Therefore, the concentrations of curcumin used in this study are expected to be within tolerable doses It has been suggested that less potent dietary compounds can enhance the effect of a more potent and toxic drug by lowering its toxicity level [46,47] Therefore, combination between two such drugs can provide superior clinical efficacy than a single drug alone [46] Materials and methods Reagents Curcumin, sulforhodamine B, fetal bovine serum, BSA and G418 were purchased from Sigma (St Louis, MO, USA) Annexin V and PI were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) All other reagents were of analytical grade Cell culture MCF-7 cells, human breast carcinoma cells, were grown in minimum essential medium (HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum, 2.2 gỈL)1 sodium bicarbonate, along with 1% antibacterial and antimycotic solution containing streptomycin, amphotericin B and penicillin and 10 lgỈmL)1 of human insulin at 37 °C in a humidified atmosphere of 5% CO2 [48] Curcumin stock solution was prepared in 100% dimethylsulfoxide and different concentrations of curcumin were added to the culture medium (dimethylsulfoxide was £ 0.1% v ⁄ v) 24 h after seeding Dimethylsulfoxide (0.1%) was used as a vehicle control Cell proliferation assay and mitotic index calculation The effect of curcumin on the proliferation of MCF-7 cells was determined by sulforhodamine B assay [49] For mitotic index calculation, MCF-7 cells were seeded at a density of 1.0 · 105 cellsỈmL)1 on poly(l-lysine)-coated glass coverslips followed by treatment with curcumin for 24 h [20] The coverslips were centrifuged in a Labofuge 400R cytospin (Heraeus, Hanau, Germany) for 10 (1200 g at 30 °C) and fixed with 3.7% formaldehyde for 30 at 37 °C The cells were permeabilized with methanol and stained with Hoechst 33258 The number of cells in mitosis and interphase were counted using the Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) At least 800 cells were counted for each set and the experiment was repeated three times The numbers of cells at the metaphase and anaphase stages of the cell cycle were calculated for both the control and curcumin-treated cells FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS M Banerjee et al Immunofluorescence microscopy and transfection MCF-7 cells (0.6 · 105ỈmL)1) were seeded on glass coverslips in 24-well plates for 24 h and incubated with different concentrations of curcumin for another 24 h The cells were fixed with 3.7% formaldehyde at 37 °C, and immunostained as reported earlier [48] Cells were stained with the following primary antibodies: mouse monoclonal anti(a-tubulin IgG) (1 : 300) from Sigma, rabbit polyclonal anti-(a-tubulin IgG) (1 : 300) from Abcam (Cambridge, MA, USA), mouse monoclonal anti-p53 IgG (1 : 300), mouse monoclonal anti-p21 IgG (1 : 300) purchased from Santa Cruz (Santa Cruz, CA, USA), mouse anti-BubR1 IgG (1 : 500) from BD Biosciences (San Jose, CA, USA) rabbit anti-Mad2 IgG (1 : 300), mouse monoclonal antiEg5 IgG (1 : 800) from Abcam (Cambridge, MA, USA) The secondary antibodies used were Alexa 568-conjugated sheep anti-(mouse IgG) (1 : 400) purchased from Molecular Probes (Eugene, OR, USA), fluorescein isothiocyanate (FITC)-conjugated anti-(mouse IgG) (1 : 400) and FITCconjugated anti-(rabbit IgG) (1 : 400) from Sigma The nucleus was stained using lgỈmL)1 of Hoechst 33258 (Sigma) The slides were observed under an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) using a 40 · objective The images were captured using CoolSNAP-Pro camera image-pro plus software 4.0 (Media Cybernetics, Bethesda, MD, USA) was used for image acquisition and processing MCF-7 cells were transfected with EGFP– a-tubulin plasmid, as described previously [20] and the stably transfected MCF-7 cells were maintained in the presence of the antibiotic G418 Annexin V ⁄ propidium iodide staining MCF-7 cells were grown in the absence and presence of different concentrations of curcumin for 48 h and were stained with Annexin V ⁄ PI, as reported previously [20,48] The manufacturer’s protocol was used for staining the cells using an Annexin V apoptosis detection kit (Santa Cruz Biotechnology) and processed for microscopy [20,48] The cells exhibiting positive Annexin V and PI staining were seen under microscope using the FITC and PI fluorescence, differential interference contrast microscopy was used for visualizing total number of cells Cell-cycle analysis MCF-7 cells were grown in the absence and presence of 25 and 35 lm curcumin for 24 h The cells were first fixed in 70% ethanol, washed with NaCl ⁄ Pi and then incubated with 50 lgỈmL)1 PI containing lgỈmL)1 RNase for h at °C The DNA content of the cells was quantified using a flow cytometer (FACS Aria; Becton Dickinson, San Jose, CA, USA) Curcumin suppresses microtubule dynamics MCF-7 cells were treated without and with 1.3 lm nocodazole for 20 h Nocodazole was washed off with fresh media The cells were incubated without or with curcumin for and h, and then stained with PI The effect of curcumin on the kinetics of the release of the mitotic block was examined in a flow cytometer and the data were analysed using the modfit lt program (Verity Software, Topsham, ME, USA) Effect of curcumin on the reassembly of cold-depolymerized mitotic microtubules MCF-7 cells (0.5 · 105ỈmL)1) were seeded on glass coverslips for 24 h and then incubated with 1.3 lm nocodazole for 20 h Nocodazole was removed by washing with fresh medium Cells were then incubated without or with 36 lm curcumin on ice for 30 Subsequently, cells were transferred to 37 °C and the assembly of microtubules was followed by fixing the cells after every The microtubule network was visualized by staining the fixed cells with anti-a-tubulin Ig The DNA was stained with Hoechst 33258 Western blot analysis The effect of curcumin on the polymeric mass of microtubules in the cells was analysed by western blot, as described previously [20] The protein concentrations of the polymeric and the soluble fraction were determined by the Bradford method [50] The polymeric and the soluble tubulin fractions were run on SDS ⁄ PAGE and electroblotted on poly(vinylidene difluoride) membranes The membranes were probed with mouse monoclonal anti-(a-tubulin IgG) (1 : 1000) and alkaline phosphatase-conjugated secondary anti-(mouse IgG) (1 : 5000) (Sigma) The band intensities were calculated using image j software Effects of curcumin on the dynamic instability of individual microtubules in MCF-7 cells The effects of curcumin on the dynamic instability of the interphase microtubules in MCF-7 cells were determined as described previously [20,51] Briefly, MCF-7 cells having stably transfected green fluorescent protein–a-tubulin were grown on glass coverslips for 24 h Cells were then incubated in the absence or presence of and 12 lm curcumin for an additional 24 h The coverslips were transferred to glass-bottomed dishes (Prime BioScience, Pandan Loop, Singapore) containing media without phenol red and were maintained at 37 °C on a warm stage Time-lapse imaging of microtubules was carried out using an FV-500 laser scanning confocal microscope (Olympus, Tokyo, Japan) with a 60 · water immersion objective The images were acquired at s intervals for a maximum duration of using fluoview software (Olympus, Tokyo, Japan) The FEBS Journal 277 (2010) 3437–3448 ª 2010 The Authors Journal compilation ª 2010 FEBS 3445 Curcumin suppresses microtubule dynamics M Banerjee et al plus end of microtubules was tracked using image j software Life-history traces were obtained by plotting the length of individual microtubules against time Length changes of ‡ 0.5 lm for a minimum of two data points were considered as growth or shortening excursions and a change < 0.5 lm in length was considered as a pause state A transition from a shortening to a growth or pause state is called a rescue, whereas the transition from a growth or pause state to a shortening state is defined as a catastrophe [51] Twenty-five microtubules were analysed for each experimental condition Statistical significance was calculated using the Student’s t-test CI determination MCF-7 cells were incubated either separately with curcumin, paclitaxel and vinblastine or in combination with curcumin and vinblastine or paclitaxel for one cell cycle The combination index was calculated using the Chou and Talalay method [26,52], with the help of the following equation CI ¼ Dị1=Dxị1 ỵ Dị2=Dxị2 where (D)1 and (D)2 are the concentrations of drug and drug used in combination, which produce a particular effect, (Dx)1 and (Dx)2 are the concentrations of the drugs that produce similar effect when used alone The concentration of curcumin which produced a particular effect was calculated from 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(Sigma) The band intensities were calculated using image j software Effects of curcumin on the dynamic instability of individual microtubules in MCF-7 cells The effects of curcumin on the dynamic instability. .. off with fresh media The cells were incubated without or with curcumin for and h, and then stained with PI The effect of curcumin on the kinetics of the release of the mitotic block was examined... with vinblastine in inhibiting MCF-7 cell proliferation Curcumin affected the localization of the kinesin protein Eg5 Because curcumin produced monopolar spindles in MCF-7 cells, we examined the