Quercetin Abrogates Taxol-Mediated Signaling by Inhibiting Multiple Kinases

Experimental Cell Research 270, 1–12 (2001) doi:10.1006/excr.2001.5326, available online at http://www.idealibrary.com on

Quercetin Abrogates Taxol-Mediated Signaling by Inhibiting Multiple Kinases Maria Marone,* Giuseppina D’Andrilli,* Nandita Das,* Cristiano Ferlini,* Sukalyan Chatterjee* ,† ,1 and Giovanni Scambia* ,1 *Laboratory of Anti-neoplastic Pharmacology, Department of Obstetrics and Gynecology, Catholic University, Rome, Italy; and †Instituto Gulbenkian de Ciencia, Oetras, Portugal S.C. dedicates this paper to his Babukaku for everything.

INTRODUCTION Cell cycle block in G 2/M initiates apoptosis, but the mechanism of this signaling cascade are largely unknown. The microtubule-perturbing agent Taxol has multiple effects on this signaling pathway and is a potent inducer of apoptosis. The specific pathways activated by low, clinically relevant concentrations of the drug are still largely unknown and are dependent on cell type and drug concentration. In this work, we have investigated why HeLa cells respond to Taxol by undergoing complete apoptosis, whereas MCF-7 cells remain in an intermediate phase with reduced death. Three phases were distinguished in these apoptotic pathways. The initial phase characterized by cellular detachment is followed by a second phase which includes the onset of apoptotic morphology, and p38 and Bcl-2 phosphorylation. These two phases are common to both cell lines. HeLa cells then proceed to the third and final execution phase, which culminates in death, whereas MCF-7 cells do not progress. Interestingly, the isoflavonoid Quercetin, a known general kinase inhibitor and an antioxidant, was able to prevent the onset of Taxol-induced cellular detachment and to protect from cell death. Moreover, it blocked Taxolinduced phosphorylation of p38 and Bcl-2, and prevented a Taxol-induced change in relative mobility of the apoptosis signal-regulating kinase 1 (Ask1). Our data elucidate the signaling pathways activated by Taxol at low clinically relevant concentrations. © 2001

Apoptosis is a mechanism of programmed cell death (PCD) that plays a decisive role in both normal development and disease [1, 2]. PCD is the outcome of a signaling cascade whose initiating stimuli originate within the cell or from the external environment, whose signaling and regulation can occur at multiple levels from the membrane to the final instruments of destruction [3, 4]. Signals impinge mostly upon pathways initiating from the mitochondria that finally converge at the level of the Bcl-2 family members [5, 6] and eventually the caspases, the ultimate executioners of cell death [7]. The discrete specificity of the signaling cascades is determined in the early steps prior to converging to the later death-blowing event. What these signaling cascades are and how they achieve specificity are still open questions. Microtubules interact with the molecular components of signal transduction pathways either directly or through microtubule-associated proteins, which act as a scaffold for the regulation of signal transduction. Microtubule-associated proteins and microtubule pertubing agents (Taxol, colchicine, menedione, and nocodazole) have multiple effects on signaling pathways, indicating that this dynamic polymer might be a key element in signal transduction [8]. Microtubule perturbation triggered by oxidants or other agents affects protein complexes that are essential for intracellular trafficking, determination of cell polarity, and interorganelle communication. Taxol (paclitaxel) is an antimitotic agent used extensively as an antineoplastic drug for the treatment of certain types of cancers with reasonable efficacy [9]. Taxol and its derivatives stabilize cytoskeletal microtubules by binding to ␤-tubulin and preventing depolymerization and consequently blocking the cell cycle in the G 2/M phase, causing mitotic arrest at metaphase [10, 11]. A number of diverse effects have been ascribed to Taxol besides the specific G2/M block, ranging from mitochondrial damage and

Academic Press

Key Words: Taxol; Quercetin; apoptosis; caspases; signaling; p38; Ask1.

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Address correspondence and reprint request to either Giovanni Scambia, Department of Obstetrics and Gynecology, Catholic University, L.go Gemelli 8, 00168 Rome, Italy. Fax: ⫹39-06-35508736. E-mail: [email protected] or Sukalyan Chatterjee, Instituto Gulbenkian de Ciencia, Rua da Quinta Grande, 6; Apart. 14, PT2781, Oeiras Codex; Portugal. Fax: ⫹351 21 4407970. E-mail: [email protected]. 1

0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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induction of early response genes, activation of various stress kinase pathways involved in cell proliferation, and death, but the specific mechanisms which trigger Taxol-induced apoptosis are still largely unknown [10]. Another significant level of modulation of apoptosis is in post-translational modifications of pro- and antiapoptotic proteins of the Bcl-2 family in response to death and survival signals. These proteins can be regulated by phosphorylation, oligomerization, proteolytic cleavage, and, in certain cases, revised cellular localization [12]. The kinases which phosphorylate some of these proteins, and the tentative signal transduction pathways have been identified at very high Taxol concentration [13–15]. In particular, apoptosis induced by Taxol and other Taxanes has been shown to correlate with Bcl-2 phosphorylation [16, 17], but although putative kinases responsible for phosphorylating Bcl-2 have been identified, an established correlation with cell death is still lacking. Different stress-activated kinase pathways can lead to cell death and have been shown to be modulated by high concentrations of Taxol [15, 18, 19]. The recently identified apoptosis signalregulating kinase (Ask1) is a MAPK kinase kinase (MPKKK) that can activate the SEK1-JNK and MKK3/ MKK6-p38 signaling cascades [20] and has been shown to be central in stress-induced apoptosis [20, 21] and in Bcl-2 phosphorylation [22]. Although a vast literature is available on Taxol, most of the studies use a very high concentration (ⱖ0.5 ␮M), conditions under which striking effects can be observed, which may include nonspecific general stress effects [10]. Moreover, in the perspective of clinical relevance we believe it important to study doses applicable to therapeutic management. In this work we have studied microtubule polymerization-mediated apoptosis using Taxol at low, clinically relevant concentration, as the microtubule-blocking drug. Using HeLa and MCF-7 cells as the main systems, we have established that microtubule block-induced apoptosis occurs in three distinct phases. Furthermore we show that Quercetin, an isoflavonoid, prevents Taxol-mediated apoptosis. We have delineated the pathway involved in cell death and have also identified the mechanism of action of Quercetin in preventing Taxolinduced cell death. MATERIAL AND METHODS Tissue culture. The breast cancer cell line MCF-7 and the cervical cancer cell line HeLa were maintained in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL-Life technologies, Karlsruhe, Germany), 1⫻ nonessential amino acids (Gibco BRL-Life Technologies), and 100 ␮/ml kanamycin (Gibco BRL-Life Technologies). HeLa cells were seeded at 1 ⫻ 10 5 cells/ml, MCF-7 cells at 2 ⫻ 10 5 cells/ml, 24 h prior to treatment. For measurement of phosphorylation levels of p38, ERK, and JNK, cells

were preincubated for 24 h in FBS-free medium, to reduce background phosphorylation. Drugs. Paclitaxel was solubilized in DMSO, at a final concentration of 10 mM. LY294002 was purchased from Calbiochem (La Jolla, CA), SB 202190 and PD 98059 from New England Biolabs (Beverly, MA). Quercetin, L-buthionine-[S,R]-sulfoximine (BSO), N-acetylcysteine (NAC), and dithiothreitol (DTT) were purchased from Sigma. Control cells were treated with the same amount of vehicle alone. Assays for cell viability. Cell viability was assayed mostly with the colorimetric Cytotox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI), which quantitatively measures endogenous lactate dehydrogenase (LDH). Cells were grown in 24-well plates, each point in duplicate, and the assay was performed according to the manufacturer’s protocol. Values reported are the mean of three experiments, performed on duplicate wells, and are expressed as percentage of untreated control. Alternatively, cell viability was measured with the standard Trypan blue exclusion method by counting blue and clear cells. Data were expressed as percentages of control and are the mean of three experiments. DNA fragmentation. Genomic DNA was extracted according to a standard protocol [23]. About 10 ␮g of DNA per sample was electrophoresed on 2% ethidium bromide-stained agarose gel. Images of the gels were acquired with a Cohu CCD camera. Caspase-3 activity assay. Caspase-3 activity was measured with the ApoAlert CPP32/caspase-3 assay kit (Clontech, Palo Alto, CA), based on spectrophotometric detection, at 400 nm, of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA. The assay was performed according to the manufacturer’s protocol. Antibodies. The following antibodies were used: mouse anti-human bcl-2 (clone 124, Dako, Glostrup, Denmark), rabbit anti-human Ask-1 (H-300) (Santa Cruz Biotechnology, Santa Cruz, CA). PhosphoPlus p42/44, SAPK/JNK, and p38 antibody kits from New England Biolabs. Western blots. Cell pellets were lysed as described earlier [24]. Total proteins were separated by SDS–PAGE and electroblotted onto polyvinylidene fluoride membranes (PVDF, Millipore Co., Bedford, MA) and processed as described earlier [24]. Detection was performed with the DAB kit (Vector Labs, Burlingame, CA). Alternatively, following incubation with a goat anti-rabbit or goat antimouse HRP-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA), detection was performed with the ECL Plus system (Amersham International, Buckinghamshire, UK) and the blots were exposed to X-AR-5 OMAT Kodak films. Cell cycle analysis by flow cytometry. Cells were plated at a concentration of 10 5 cells/ml in the specific medium supplemented as above. At various times (4 to 24 h) after beginning of the treatment with Taxol and/or Quercetin, cells were harvested and nuclei isolated and stained using a solution containing 0.1% Na ⫹ citrate, 0.1% NP-40, 4 mM EDTA, and 50 ␮g/ml propidium iodide (PI) as DNA dye [25]. Nuclei were incubated in staining solution for at least 12 h at 4°C. Flow cytometric DNA analysis was performed by acquiring a minimum of 2 ⫻ 10 4 nuclei with a Facscan flow cytometer (Becton Dickinson, San Jose, CA). DNA fluorescence was collected in linear mode and pulse signal processing was used to set a doublet discrimination gate. Cell cycle analysis was performed using a Multicycle software package (Phoenix, San Diego, CA). Image analysis and quantification. Images of the ethidium bromide-stained agarose gels, X-ray films and Western blotting PVDF membranes were acquired with a Cohu CCD camera and quantification of the bands was performed by Phoretix 1 D (Phoretix International Ltd., Newcastle upon Tyne, UK).

QUERCETIN PREVENTS TAXOL-INDUCED DEATH

RESULTS

Inhibition of Cell Growth and Cell Death Induction by Taxol A block in the G 2/M phase of the cell cycle is known to induce apoptosis; however, the mechanisms responsible for this outcome are still an enigma. As a paradigm to study apoptosis induced by G 2/M block we have used Taxol at low, clinically relevant doses (10 –50 nM) which is known to induce cell death by apoptosis in a number of different systems. Taxol caused cell death in the breast cancer cell lines MCF-7 and MDA, in the ovarian cancer cell lines A2780 and 2008, and in the cervical cancer cell line HeLa with varying degrees of efficacy. MCF-7 and 2008 cells proved to be less sensitive to Taxol, whereas HeLa and A2780 cells were more sensitive (data not shown). We chose two extremes of the spectrum of response, namely HeLa and MCF-7, as our model systems. HeLa cells clearly undergo apoptosis between 20 and 48 h, whereas MCF-7 cells are more resistant to Taxol (Fig. 1A). Morphological analysis showed that 4 – 8 h from the start of treatment, cells round up and over time detach. By 24 h HeLa cells showed explicit signs of apoptosis, which became massive by 48 h, whereas only a minor percentage of MCF-7 cells became clearly apoptotic (Fig. 1A). HeLa cells responded in a clear time- and dose-dependent manner: cell death, measured by LDH production, was massive by day 2 (45% viability with 50 mM Taxol, 25% with 100 nM Taxol) and increased even further by day 4 (28 and 10% with 50 and 100 nM Taxol, respectively) (Fig. 1B). In the initial phase of rounding up, response of MCF-7 cells was comparable to that of HeLa cells (84.7 and 64.9% viability with 50 and 100 nM Taxol, respectively, at day 2), but exposure for longer than 2–3 days did not cause any dramatic effects. At day 3 viability was down to 67.4 and 47.4% with 50 and 100 nM Taxol, respectively, and it did not decrease any further at day 4 (Fig. 1B). Cell viability, assayed by trypan blue exclusion, shown in Fig. 1C, mirrored this observation. A conspicuous DNA ladder was evident in HeLa after 24 h of Taxol treatment, whereas no DNA fragmentation was visible in MCF-7 cells up to 96 h, in a Taxol concentration ranging from 10 to 100 nM. On the contrary, when exposed to 250 ␮M H 2O 2, both cell lines produced a clear DNA ladder within 24 h (Fig. 1D). These data (Fig. 1) describe and clearly establish a difference in response of the two cell lines to Taxol challenge. An early and striking morphological effect of Taxol is cellular rounding up. Interestingly, when the round floating cells, evident after 24 h of treatment with Taxol, were replated in the absence of the drug, the vast majority of MCF-7 cells were still alive and able to reattach and proliferate, HeLa cells did so with a much

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lower efficiency (Fig. 2A). As shown in Fig. 2B, Taxol induced caspase-3 activity in HeLa cells but not in MCF-7 cells. On the other hand, both caspase-8 and caspase-9, which have also been shown to be activated by Taxol in different systems, were activated following Taxol treatment in both cell lines (data not shown). The different outcome of Taxol treatment may be explained by the fact that MCF-7 cells lack a functional caspase-3 gene and, as a consequence, fail to undergo the canonical phases of apoptosis when exposed to staurosporine, which is known to induce apoptosis through caspase-3 [26]. Our results in Fig. 1C establish that caspase-3 activation is necessary for Taxol-mediated DNA fragmentation, as has been shown with other apoptosis models. On the other hand, it is clear from Fig. 1C that H 2O 2-induced DNA fragmentation in MCF-7 cells is independent of caspase-3 activity. The ovarian cancer cell line A2780, which expresses a functional caspase-3, showed clear morphological signs of apoptotic cell death accompanied by DNA laddering (data not shown). A number of studies have provided evidence that phosphorylation of Bcl-2 is a determining biochemical event in the pathway of Taxol-mediated apoptosis [16, 17]. Following 24 h of treatment with Taxol, the detached, floating cells were collected separately from the adherent cells, and we observed that bcl-2 was phosphorylated mostly in the round, detached cells and only slightly in the treated but still adherent cells (Fig. 2C). Taxol-induced bcl-2 phosphorylation is only a transitory phenomenon which peaks at 24 h and disappears by 48 h in both cell lines (data not shown). It is to be noted that at this time point cell death is extensive in HeLa, whereas most of the MCF-7 cells are still alive. Thus a direct correlation of bcl-2 phosphorylation and cell death is lacking in these systems. Quercetin Abrogates Effects of Taxol The pentahydroxyflavone Quercetin markedly prevented the early morphological changes involving the cellular rounding up typically observed on Taxol treatment, as shown in Fig. 3A. The effect was consistent with Quercetin used in the concentration range of 5 to 20 ␮M. Although at the highest concentration of 20 ␮M Quercetin was slightly cytotoxic per se (in treatments longer than 24 h), in the time frame of 6 –24 h its effect on cell morphology was still marked. Protection from Taxol with no toxic effects was detected at the lower concentration (5 ␮M). After a 20-h treatment with 10 nM Taxol about 70 –90% cells were detached and floating, whereas when Quercetin was included with Taxol, this number was drastically reduced to close to zero in both cell lines. Furthermore, as shown in Fig. 3B, Quercetin at a concentration of 15–25 ␮M, completely abolished the Taxol-induced phosphorylation of Bcl-2,

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FIG. 1. Cell death induced by Taxol in HeLa and MCF-7 cells. (A) Micrographs of the two cell lines at different time points. Apoptosis accompanied by membrane blebbing and later shrinking is very evident in HeLa cells, whereas minor blebbing with no other major morphological change is evident in MCF-7 cells (B) Time course and dose dependence of Taxol effect on cell viability measured by LDH production assay. (C) Time course of cell viability following treatment with 50 nM Taxol. (D) Ethidium bromide stained agarose gel of DNA of HeLa and MCF-7 cells.

an effect consistent in HeLa, MCF-7 (Fig. 3C), and A2780 cells (data not shown). While known as a general kinase inhibitor [27, 28], Quercetin is also a pow-

erful antioxidant [29, 30, 31] and could therefore interfere with the Taxol pathway to death at a number of steps.

QUERCETIN PREVENTS TAXOL-INDUCED DEATH

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FIG. 2. Cellular rounding up and detachment induced by Taxol. (A) Cells which detached after 24 h of treatment with Taxol treatment were replated in fresh medium with no drug, whereas reattachment efficiency was very low for HeLa cells, most MCF-7 cells were able to reattach and grow. Treatment with hydrogen peroxide had the same deadly effect on both cell lines. (B) Caspase-3 activity in HeLa and MCF-7 cells at 24 and 48 h under different Taxol concentrations. (C) Western blot for bcl-2 in Taxol-treated cell. Cells were separated according to their adhesiveness. Adherent are the cells which did not detach nor round up, floating are the detached cells which were freely floating in the medium, wash are the rounded cells which were still mildly attached to the culture flask and came off with a PBS wash. Bcl-2 phosphorylated forms (P) are clearly evident in the floating and washed off cells, corresponding to the round cells. Adherent cells showed a minor degree of phosphorylation.

Anti-oxidants or Inhibitors of MAP Kinases Do Not Block Taxol-Mediated Apoptosis In order to understand whether the protective effects shown by Quercetin on Taxol-induced death could be due to its antioxidant properties, we used a number of

antioxidants in combination with Taxol. HeLa and MCF-7 cells were treated with N-acetyl cysteine (NAC), dithiothreitol (DTT) (Fig. 4A) or ␤-mercaptoethanol (data not shown) and then assayed for cell viability. No significant protection from Taxol-induced cell death was observed on either of the two cell lines.

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FIG. 3. Combined effects of Taxol and Quercetin. (A) Micrographs of HeLa and MCF-7 cells treated for 20 h with 50 nM Taxol, 15 ␮M Quercetin, or both. (B) Western blot for bcl-2. Cells were exposed to the drugs for 24 h.

In fact, NAC and DTT mildly potentiated cell death in HeLa cells. The antioxidant glutathione monoethyl ester (GME) in the range of 100 ␮M to 1 mM, had no significant effect on either cell lines (data not shown). BSO, a prooxidant that functions by depleting glutathione, when used in the range of 5 to 500 ␮M did not significantly change the survival in combination with Taxol (Fig. 4A). Our results indicate that in these cell lines, Taxol-induced cell death is not effected by redox changes and Taxol probably does not cause intracellular redox changes in these cell lines. A number of reports show that Taxol at high doses (500 nM–1 ␮M) activates a number of kinase cascades, specially the MAP kinases, although with different specificity in different systems. We addressed this by investigating whether the inhibitors for specific steps of some of the signal cascades may render Taxol (50 nM) ineffective on HeLa and/or MCF-7 cells. SB202190 (an inhibitor of p38), PD98059 (an inhibitor of MEK1 and -2), and LY294002 (an inhibitor of PI-3-kinase) were used in combination with Taxol. None of these inhibitors was effective in pre-

venting the characteristic rounded morphology due to Taxol-mediated microtubule bundling and G2/M block (data not shown). They also failed to prevent cell death and actually potentiated Taxol-induced death in HeLa, with minor effects on the viability of MCF-7 cells (Fig. 4B). Genistein, a general tyrosine kinase inhibitor also had no significant effect on morphology or viability (data not shown). Furthermore, the specific inhibitor for PI-3 kinase LY294002 had no protective effect on cell viability, but rather caused a fivefold decrease in cell viability in HeLa cells (Fig. 4B). Quercetin is a known inhibitor of PI-3 kinase [31], but our data indicate that although PI-3 kinase may be involved in Taxol-induced cell death, it is not a key component in Quercetin protection. Kinase Cascades Involved in Taxol-Induced Cell Death Taxol induced p38 phosphorylation in both cell lines (Fig. 5A). Intriguingly, Quercetin treatment in combination with Taxol abolished this phosphorylation com-

QUERCETIN PREVENTS TAXOL-INDUCED DEATH

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cetin mildly upregulated ERK1 in MCF-7 cells and not in HeLa cells (Fig. 5) which could be a nonspecific effect with no significant consequences on the biology of the system under consideration. As shown in Fig. 5 we observed no drastic effect on JNK, a protein reported to be very susceptible to change under stress, at both 4 and 24 h of Taxol treatment. Cell Cycle Analysis

FIG. 4. Effect of BSO, NAC, and DTT on Taxol-treated HeLa and MCF-7 cells. (B) Effect of SB (SB202190), PD (PD98059), and LY (LY294002) on Taxol-treated HeLa and MCF-7 cells.

pletely, in both cell lines. Since inhibition of p38 using its specific inhibitor SB202190 did not block apoptosis at the concentration tested, it is clear that other kinases, besides p38, are involved in the Taxol-induced cell death. It is of interest that none of the other MAP kinases in the known signal transduction pathways (JNK and ERK) underwent any drastic change (Fig. 5A). It has been reported recently that phosphorylation of Bcl-2 is a decisive step in G 2/M block-related apoptosis and that Ask1, a stress response kinase, may be the cognate kinase for Bcl-2 [22, 32]. We investigated this possibility by enquiring whether Quercetin had any effect on Ask1 and/or blocked its kinase activity on its known substrates, p38 and JNK. As we show in Fig. 5, Ask1 undergoes a drastic mobility shift, which may be due to phosphorylation, when HeLa are treated with 50 nM Taxol. When Quercetin was also included, the shift was completely abolished. In MCF-7 cells we observe a less striking shift in the conditions tested. Ask1 may have alternate functions/substrates, other than Bcl-2 phosphorylation, to promote apoptosis. It is possible that Quercetin pleiotropically blocks other kinases but protects cell death by inhibiting Ask1. Quer-

It is well established that Taxol, by binding to tubulin, blocks the mitotic spindle and thus causes a specific block in the G 2/M phase of the cell cycle. On the contrary, Quercetin effect on the cell cycle is poorly understood. It has been shown that Quercetin can cause a cycle arrest in different phases of the cell cycle, depending on the cell line in analysis [33, 34]. Propidium iodide staining and DNA analysis by flow cytometry showed that Quercetin had varying effects in our model systems. As shown in Table 1, after 24 h of exposure to Quercetin, HeLa cells were blocked mostly in the S phase, whereas MCF-7 cells were blocked mostly in G 2/M. The ovarian cancer cell line A2780, in which Quercetin also showed the same protective effect from Taxol challenge, was blocked in the G 1 phase (data not shown). In HeLa cells, when Quercetin was combined with Taxol, the effect of Taxol on the cell cycle was overridden, as is seen in Table 1: the pronounced block in the S phase has the predictable consequence that no cells entered G 2/M. On the other hand, the combined effects of Quercetin and Taxol produced an enhanced block in G 2/M in MCF-7 cells. Hence, the effects of Quercetin on the cell cycle cannot explain its ubiquitous protective effect. Moreover, whereas Quercetin-induced cell cycle block was evident at 24 h or longer, even a concentration as high as 20 ␮M had no significant effect on the cell cycle at earlier time points, that is when its protective effects have already become evident (Table 2). A lower Quercetin concentration (5 ␮M), competent in preventing Taxol-induced morphology changes, had no significant effect on the cell cycle even at 24 h (Table 1). Our data indicate that Quercetin-induced disturbance of the cell cycle is a late phenomenon visible only at high concentration and is thus likely to be distinct from its morphological and protective effect on Taxol. DISCUSSION

It is well established that the perturbation of microtubules and consequent apoptosis induced by Taxol involve hyperphosphorylation of Bcl-2 [16, 17, 35]. However, little is known regarding the proximal steps leading to this phosphorylation event. In this study, we show that signaling induced by Taxol-mediated microtubule-polymerization leading to apoptosis occurs in

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FIG. 5. Effect of Taxol on kinase pathways. (A) Western blots for p38, ERK1 and -2, and SAPK/JNK. The phosphorylated and total forms of these kinases were detected with specific antibodies. Samples were collected after 24 h treatment. (B) Western blot for Ask1, detected with a single antibody which recognizes all forms of the protein.

three broad but distinct phases and that the downstream signaling culminating in cell death can vary from cell type to cell type. In the early apoptotic phase, treatment with Taxol causes a rounding up and detachment of all cell lines observed. The morphology progressively changes for HeLa cells and becomes clearly apoptotic by 24 – 48 h, whereas morphologically, MCF-7 cells do not deteriorate further beyond the initial rounding up. On the contrary, both cell lines undergo complete apoptosis when treated with H 2O 2. Measurement of cell viability

under Taxol challenge shows that the early-detached cells can reattach upon replating in the absence of Taxol and grow identical to untreated control cells, albeit with less efficiency for HeLa cells than for MCF-7 cells. Hence, it is possible to retrieve cells from this early event. After longer treatments (48 –72 h) HeLa are mostly unable to reattach and not viable, whereas MCF-7 fail to progress significantly further down the death pathway subsequent to the morphological change, and remain mostly viable. In this second phase, starting at 24 h, Taxol treatment induces Bcl-2

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QUERCETIN PREVENTS TAXOL-INDUCED DEATH

TABLE 1 Distribution of Cells in the Different Phases of the Cell Cycle, by Propidium Iodide Staining and Flow Cytometric Analysis after 24 Hours of Treatment HeLa

Control Tax 10 nM Tax 50 nM Quercetin 20 ␮M Quercetin 20 ␮M ⫹ Taxol 10 nM Quercetin 20 ␮M ⫹ Taxol 50 nM

MCF-7

G1

S

G 2/M

G1

S

G 2/M

59.4 39.3 4.3 47.4 22.1 61.8

28 39.2 18.2 46.7 77.9 38.2

12.6 21.5 77.6 5.9 0 0

54.1 58.8 17.3 38.9 24.1 26.9

42 32.7 20.6 35.9 33.7 3.4

3.8 8.5 62.1 25.2 42.2 69.7

Note. The numbers indicate the percentage of cells in each phase of the cycle.

phosphorylation in both cell lines, an effect that is lost at later time points. Bcl-2 phosphorylation has been identified as a mechanism of inactivation which could lead to apoptosis [36]. Recently, it has been proposed that Bcl-2 phosphorylation may actually correlate with M-phase rather than directly with entry into apoptosis [37, 38]. In our systems, Bcl-2 phosphorylation occurs in all cell lines analyzed, but an apparent direct correlation with susceptibility to cell death is lacking. This phosphorylation event may be required, but it is not determining for the later phases of apoptosis. Phosphorylation of Bcl-2 could also be a consequence of the first apoptotic phase and be required for further downstream signaling which is deficient in MCF-7 cells. The observation that HeLa and MCF-7 cells have differential sensitivity to Taxol is again confirmed in the third and final “execution” phase when HeLa cells undergo complete cell death in a caspase-3-dependent dependent manner, and MCF-7 cells remain resistant to cell death. Our data show that MCF-7 cells remain blocked in the second apoptotic phase, with rounded morphology and phosphorylated Bcl-2, but with a

TABLE 2 Cell Cycle Analysis by Propidium Iodide Staining and Flow Cytometry Following Treatment with 20 ␮M Quercetin for 4, 8, and 16 Hours HeLa Time 4 Hours Control Quercetin 20 ␮M 8 Hours Control Quercetin 20 ␮M 16 Hours Control Quercetin 20 ␮M

MCF-7

G1

S

G 2/M

G1

S

G 2/M

49.4 58.0

41.1 33.1

9.5 8.9

37.7 41.5

43.2 41.8

19.1 16.7

56.5 55.2

30.0 36.6

13.5 8.2

44.4 41.0

39.7 37.8

15.9 21.2

53.1 48.9

35.5 41.5

11.4 9.6

54.0 38.3

42.5 36.7

3.5 25.0

higher percentage of viable cells than HeLa. Intriguingly, Taxol also activates the initiator caspases-9 and -8 in both cell lines. Presumably, the apoptotic cascade goes down via one of these caspases [39] and is then blocked in MCF-7 cells, which lack functional caspase-3. Thus, MCF-7 cells remain mostly viable, whereas in HeLa cells, the cascade is completed through caspase-3-mediated cell death. A recent work provided evidence that compensatory pathways of caspase activation may be activated in the absence of caspase-3 [39]. Since viability remained high under all conditions tested in MCF-7 cells, we can deduce that the defect in caspase-3 functionality in MCF-7 cells cannot be completely overcome and that caspase-3 activation remains one of the major pathways to death induced by Taxol. Signaling cascades have been shown to be promiscuous and a single signaling protein or a cascade is perfectly amenable to serve in diverse physiological functions like mitogenic signaling, proliferation, and/or apoptosis. Our results clearly show that Taxol-mediated apoptosis is independent of MAP kinase signal transduction pathway in the concentration range of Taxol we used. This is in apparent contradiction with previous published works which showed that MAP kinases are activated by Taxol, especially JNK, and that these MAP kinase pathways can be associated with phosphorylation of Bcl-2 [14, 15, 32, 40]. This apparent contradiction can be explained by the drastic difference in the concentration of Taxol used by the respective groups. We have strictly maintained Taxol concentrations clinically and pharmacologically relevant and have avoided using very high doses. At very high doses cells may undergo pleiotropic stress and multiple pathways can be activated that may not be relevant and/or specific. It has been shown recently that oxidative stresses at varying levels activate different cellular signal transduction systems dependent on the dose and time of treatment of the agent [39]. The mild activation of ERK we observed in MCF-7 cells may be the outcome

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of such a nonspecific stress event and may not play a significant physiological function. Recent work by Mackeigan et al. [40], suggested that the block of the ERK pathway with specific inhibitors used in combination with Taxol to enhance apoptosis, could be studied as a new anticancer strategy. Our data provides evidence that this idea can potentially be applied to other kinase inhibitors, as those of p38 and PI-3, but can function only in systems in which Taxol pathway to death is not compromised (such as by caspase-3 deficiency). The isoflavonoid Quercetin, which is a broad inhibitor of protein kinases and an antioxidant [27–30], prevents the occurrence of the rounded cell phenotype observed early on treatment with Taxol. This effect appears to be specific for this pathway since no protection was observed from H 2O 2-mediated cell death in either cell line. We have shown that a number of antioxidant agents could not protect from Taxol-induced cell death, indicating that Quercetin may act through a different mechanism. Moreover, Quercetin inhibited Taxol-induced Bcl-2 and p38 phosphorylation, which allows us to hypothesize that its action may be due to inhibition of kinase(s) that play a role in microtubule polymerization-induced apoptosis. We have observed that Bcl-2 does not become phosphorylated on addition of Taxol to isolated mitochondria, indicating that Taxol-induced phosphorylation of Bcl-2 is due to a nonmitochondrial kinase or needs other cytosolic signaling proteins (unpublished observation). A putative kinase downstream of Taxol-mediated bundling of microtubules, G 2/M block and apoptosis may be involved. Recently, it has been shown in Jurkat cells that the stress-associated kinase Ask1 is downstream of Taxol-induced signal transduction [32] and that the Ask1/JNK pathway is responsible for normal cell cycle-related and Taxol-induced phosphorylation of Bcl-2 [22]. Nevertheless, in this latter work Yamamoto et al. were able to block Bcl-2 phosphorylation only by expressing combined dominant negative mutants of Ask1/JNK/MKK7, whereas the single mutants were ineffective. We have shown that none of the MAP kinase inhibitors used were competent to prevent Taxolinduced cell death. It is evident that Taxol-mediated G 2/M block activates multiple stress pathways and hence, as discussed in the paper mentioned earlier, the cells in this phase of the cell cycle are more susceptible to apoptosis. Intriguingly, Taxol induces a modification of Ask1 which alters its relative mobility and which is blocked by Quercetin, a shift that is more pronounced in HeLa, the cell line more prone to Taxol-induced cell death. We have not characterized this modification as yet, but the result is provocative. Our data show that Quercetin not only affects Ask1 and abrogates phosphorylation of Bcl-2 and of p38, but also prevents cell death. We believe that Ask1 in combination with other

factors plays a role in Taxol-induced cell death and Quercetin could negate the effects of Taxol by acting at multiple, and/or independent steps. It is possible that Quercetin functions by inhibiting also other kinases that still need to be identified. Recently, it has been shown that PKA plays a prominent role in paclitaxelinduced phosphorylation of Bcl-2 and apoptosis [41]. We have treated HeLa and MCF-7 cells with the general phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (concentration range 10 nM to 100 ␮M), 8-brcAMP, a cAMP analog (concentration range 1–50 ␮M), Forskolin, an adenylate kinase activator (1–100 ␮M), and observed no cytotoxic effects. It is possible that the PKA pathway is not relevant in our system or microtubule polymerization-mediated cell death is the outcome of multiple signaling pathways. In conclusion, by using Taxol at clinically relevant doses, which can be reached in vivo without toxic effects, we have shown that, in our systems, p38 and Ask are involved in Taxol-induced apoptosis, but not the stress kinase JNK. We have shown that Ask1 is posttranslationally modified under Taxol treatment but the nature of this modification is still unknown. Quercetin, a general kinase inhibitor, blocks this modification suggesting that this could be a phosphorylation event although it cannot be definitively stated at this point. The effect on Ask1 is more pronounced in HeLa than in MCF-7 indicating a possible difference in the signaling responses of the two cell lines correlating well with our other data with the two cell lines. This would suggest that Ask1-independent pathways might also mediate p38 and Bcl-2 phosphorylation. The block that Quercetin caused on Ask1 mobility shift, p38 and Bcl-2 phosphorylation, is indicative of the association of these proteins in one or more interconnected pathways. Moreover, this block was correlated with prevention of apoptosis, thus establishing a clear link between the Ask1/p38/Bcl-2 pathway(s) and Taxol-induced cell death. Ask1 has been shown to be regulated by phosphorylation on Ser 81 under oxidative stress which attenuates the activation of p38 [42]. Our data indicate that the posttranslational modification of Ask1 we observed activates p38 and is abrogated by Quercetin. It is possible that another site other than Ser 81 may be involved in Taxol signaling with differing effects on p38. At present we cannot rule out other forms of modification of Ask1 besides phosphorylation. In light of the central role of Ask1 in stress-mediated signaling it may be more than speculative to say that it may have complex and multiple levels of regulation yet to be elucidated. We thank Willy van de Greef for continuous support and for revising the manuscript and Enzo Maniccia for help with the computational analyses.

QUERCETIN PREVENTS TAXOL-INDUCED DEATH

KKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90 –90.

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Received December 27, 2000 Revised form received May 30, 2001 Published online September 11, 2001

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