Molecular Cancer Therapeutics
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Molecular Cancer Therapeutics 6, 1133-1142, March 1, 2007. doi: 10.1158/1535-7163.MCT-06-0639
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

Blockade of the phosphatidylinositol-3-kinase–Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated

Yusuke Fujiwara, Yoshihisa Hosokawa, Kazushi Watanabe, Susumu Tanimura, Kei-ichi Ozaki and Michiaki Kohno

Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan

Requests for reprints: Michiaki Kohno, Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. Phone: 81-95819-2417; Fax: 81-95819-2472. E-mail: kohnom{at}nagasaki-u.ac.jp.

Abstract

Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)–Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3ß, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated. [Mol Cancer Ther 2007;6(3):1133–42]

Introduction

Members of the class 1A phosphatidylinositol-3-kinases (PI3K) consist of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110), and catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate to yield phosphatidylinositol-3,4,5-trisphosphate. Phosphatidylinositol-3,4,5-trisphosphate functions as a membrane-bound second messenger by recruiting a subset of pleckstrin homology domain–containing proteins such as Akt and 3-phosphoinositide–dependent kinase 1 to the membrane, in which they become activated and initiate downstream signaling events. The abundance of phosphatidylinositol-3,4,5-trisphosphate is also determined by phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which specifically dephosphorylates phosphatidylinositol-3,4,5-trisphosphate to generate phosphatidylinositol-4,5-bisphosphate and thereby antagonizes the action of PI3K (1).

Members of the Akt family of serine-threonine kinases (Akt1, Akt2, Akt3) are important effectors of PI3K signaling (2). Akt is activated by phosphorylation on Thr308 by 3-phosphoinositide–dependent kinase 1 and subsequent phosphorylation on Ser473, most likely by the rictor-mTOR complex (3), and it plays a key role in protecting cells from various types of apoptotic stimuli by phosphorylating diverse downstream targets such as Forkhead family transcription factors, Bad, I{kappa}B kinase, MDM2, and procaspase-9. Akt also interacts, either directly or indirectly, with numerous other regulatory proteins including cyclin D, p21Cip1, glycogen synthase kinase (GSK)-3ß, and mTOR. The PI3K-Akt pathway has thus been implicated in the regulation of a wide variety of cellular processes including survival, proliferation, growth, and metabolism (1, 2).

Amplification of the genes for p110 or Akt, or mutation of the p85 gene has been detected in some human cancers (4). Deletion or mutation of the gene for PTEN, however, is relatively common in a wide spectrum of human cancers including glioblastoma and those of the thyroid, prostate, liver, and breast (5). These observations suggest that the PI3K-Akt signaling pathway contributes to tumorigenesis and that inhibition of this pathway is a potential strategy for cancer treatment. In particular, given that the PI3K-Akt pathway is an important regulator of survival during cellular stress, and that tumor cells often exist in intrinsically stressful environments (with limited nutrient and oxygen supplies and a low pH), specific blockade of this signaling pathway might be expected to have an optimal effect in combination with agents that increase cell stress, such as chemotherapeutic drugs or radiation (6).

In support of this notion, blockade of the PI3K-Akt pathway has been found to sensitize various tumor cell types to apoptotic cell death induced by a variety of anticancer drugs including etoposide (VP-16; refs. 7, 8), paclitaxel (7, 9), gemcitabine (7, 10), doxorubicin (8, 11), and sodium butyrate (12). However, the molecular mechanisms for such enhanced induction of tumor cell apoptosis by the combination of a PI3K-Akt inhibitor and anticancer agents have remained largely unknown. Inhibition of the PI3K-Akt pathway was shown to enhance the apoptosis-inducing effect of antimicrotubule agents by a mechanism that involves inhibition of mTOR function downstream of Akt (9), to enhance that of doxorubicin in a p53-dependent manner (11), and to enhance that of sodium butyrate through inactivation of mitogen-activated protein kinase and abrogation of p21Cip1 induction (12).

We now show that specific blockade of the PI3K-Akt pathway with chemical inhibitors markedly and selectively potentiates the apoptosis-inducing effect of microtubule-destabilizing agents in tumor cells in which this signaling pathway is constitutively activated. Inhibition of Akt-mediated phosphorylation of GSK-3ß and the consequent activation of this latter enzyme play an essential role in the synergistic proapoptotic effect of this combination of agents.

Materials and Methods

Drugs and Antibodies
Vincristine, cytosine ß-D-arabinofuranoside (Ara-C), paclitaxel, and PX-886 were obtained from Sigma-Aldrich (St. Louis, MO). VP-16, cisplatin, doxorubicin, and LY294002 were obtained from Wako Pure Chemical (Osaka, Japan), whereas Akt/protein kinase B signaling inhibitor-2 (API-2) and SB216763 were from Calbiochem (La Jolla, CA). TZT-1027 and navelbin were kindly provided by Teikoku Hormone, Mfg. (Kawasaki, Japan). Antibodies used included those to ß-actin, tau, {alpha}-tubulin, and acetylated {alpha}-tubulin were from Sigma-Aldrich; those to Akt, Ser473-phosphorylated Akt, Bad, Ser136-phosphorylated Bad, Asp175-cleaved caspase-3, caspase-9, and Ser9-phosphorylated GSK-3ß were from Cell Signaling (Beverly, MA); those to Bcl-2, GSK-3ß, and caspase-8 were from BD Biosciences (Bedford, MA); those to poly(ADP-ribose) polymerase and 14-3-3{xi} were from Santa Cruz Biotechnology (Santa Cruz, CA); and those to Ser199-phosphorylated tau were from BioSource International (Camarillo, CA).

Cell Culture
The human tumor cell lines A172 (glioblastoma), T98G (glioblastoma), LNCaP (prostate adenocarcinoma), ACHN (renal adenocarcinoma), T24 (bladder carcinoma), and WiDr (colon adenocarcinoma) as well as TIG-3 human diploid fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (13). Rat pheochromocytoma PC12 cells were maintained in DMEM supplemented with 10% horse serum and 5% fetal bovine serum. For experiments, PC12 cells cultured on collagen (type IV)–coated glass coverslips were incubated in serum-free medium [DMEM supplemented with bovine serum albumin (2 mg/mL), insulin (1 µg/mL), transferrin (2 µg/mL), 30 nmol/L of Na2SeO3, and 10 mmol/L of HEPES-NaOH (pH 7.4)] for 12 h, stimulated with ß-nerve growth factor (NGF; 20 ng/mL; Toyobo, Osaka, Japan) for 24 h to induce neuronal differentiation (14), and then treated with various reagents for 12 h.

Flow Cytometry
Cells exposed to various reagents were harvested by exposure to trypsin, fixed with ice-cold 70% ethanol, treated with RNase A (100 µg/mL; DNase-free; Sigma-Aldrich), and stained with propidium iodide (20 µg/mL; ref. 15). At least 1 x 104 cells were analyzed for DNA content with the use of a FACSCalibur flow cytometer and CellQuest Pro software (Becton Dickinson, San Jose, CA).

Immunoblot Analysis
Cell lysates were fractionated by SDS-PAGE and subjected to immunoblot analysis as described (14–16). Immune complexes were visualized with an enhanced chemiluminescence system (GE Healthcare Bio-Sciences, Piscataway, NJ) and were quantitated with Multi Gauge software, version 3.0 (Fuji Photo Film, Tokyo, Japan).

Analysis of DNA Fragmentation
Cells were lysed for 10 min on ice in TET lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA, 0.5% Triton X-100]. After centrifugation at 15,000 x g for 30 min at 4°C, lysates were treated with RNase A (100 µg/mL) for 60 min at room temperature, and DNA was then extracted and resolved by electrophoresis on a 2% agarose gel (15).

Immunofluorescence Microscopy
Cells grown on glass coverslips were exposed to various reagents, fixed in 3.7% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS before staining first with primary antibodies and then with Alexa Fluor 488–conjugated goat antibodies to mouse IgG (Molecular Probes, Eugene, OR), each at a dilution of 1:200 in PBS containing 2% bovine serum albumin and 0.05% Tween 20 (16). The cells were examined with an Axioskop 2 Plus fluorescence microscope (Carl Zeiss, Jena, Germany) for microtubule morphology.

Measurement of Cytosolic and Cytoskeletal Tubulin
Cells were scraped into MT-lysis buffer [20 mmol/L Tris-HCl (pH 6.8), 1 mmol/L MgCl2, 2 mmol/L EDTA, aprotinin (10 µg/mL), leupeptin (10 µg/mL), pepstatin (5 µg/mL), 2 mmol/L phenylmethylsulfonyl fluoride, 0.5% Nonidet P40] supplemented with 2 µmol/L of paclitaxel to maintain microtubule stability (17). Lysates were centrifuged at 20,000 x g for 30 min at 4°C to yield a supernatant (S fraction) containing soluble (cytosolic) tubulin, and a pellet (P fraction) containing polymerized (cytoskeletal) tubulin.

RNA Interference
The sequences 5'-CCGAGGAGAACCCAATGTTTCGTAT-3' (GSK-3ß, sense A) and 5'-CCGGAGCAAACCGTATTTGCAGTAT-3' (scrambled, A) as well as 5'-GGATAGTGGTGTGGATCAGTTGGTA-3' (GSK-3ß, sense B) and 5'-TACATCTCAACTAGACACCACCTCC-3' (scrambled, B) were designed to generate small interfering RNA duplexes specific for human GSK-3ß mRNA and corresponding scrambled oligoribonucleotides as controls (Stealth; Invitrogen, Carlsbad, CA). Subconfluent cultures of T98G cells was transfected with small interfering RNA (50 nmol/L) for 18 h with the use of the LipofectAMINE 2000 reagent (Invitrogen).

Statistical Analysis
Data are presented as means ± SD. Differences between means were analyzed by the two-tailed Student's t test. P < 0.05 was considered statistically significant.

Results

Blockade of the PI3K-Akt Pathway Potentiates Induction of Apoptosis by Microtubule-Destabilizing Agents in Tumor Cells with Constitutive Pathway Activation
Treatment of tumor cells with LY294002, a specific PI3K inhibitor (18), suppressed the activation of Akt in a concentration-dependent manner, with almost complete inhibition apparent at 10 µmol/L (Fig. 1A, insets ). Whereas the growth of WiDr cells, in which the PI3K-Akt pathway is not constitutively activated (11), was not affected by this agent, that of tumor cells in which this pathway is constitutively activated (T98G and T24 cells) was inhibited by LY294002 in a concentration-dependent manner (Fig. 1A). Flow cytometric analysis of cell cycle distribution revealed that 10 µmol/L of LY294002 induced a small increase (~7%) in the proportion of T98G (Fig. 1B) or T24 (data not shown) cells in G1 phase and a slight decrease in the proportion of those in S phase. However, LY294002 did not increase the proportion of cells with a fractional DNA content (cells in sub-G1 phase), which is a characteristic of apoptotic cell death (19).


Figure 1
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  		Figure 1. Selective potentiation of the death-inducing effect of vincristine by LY294002 in tumor cells. A, T98G, T24, or WiDr cells were incubated for the indicated times in the absence (Control) or presence of 3 or 10 µmol/L of LY294002 (LY). The cells were then harvested by exposure to trypsin, and viable cells were counted with a hemocytometer after staining with trypan blue. Points, means of values from triplicate dishes; bars, SD. Insets, lysates (20 µg protein) of cells treated with 10 µmol/L of LY294002 were subjected to immunoblot analysis with antibodies to Akt or to phospho-Akt. B, T98G cells incubated for the indicated times with the indicated concentrations of vincristine (VCR), paclitaxel (PTX), or Ara-C, in the absence (–LY) or presence (+LY) of 10 µmol/L of LY294002, were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. C, T98G or T24 cells were incubated for 48 h with the indicated concentrations of vincristine, paclitaxel, Ara-C, VP-16, cisplatin (CDDP), or doxorubicin (DXR), in the absence (–LY) or presence (+LY) of 10 µmol/L of LY294002. The cells were analyzed as in (B) and the proportion of cells with a fractional DNA content (sub-G1 phase) was determined. D, T98G cells incubated for various times in the absence or presence of 10 µmol/L of LY294002, paclitaxel (3, 5, or 10 nmol/L), or vincristine (3, 10, 30, or 100 nmol/L), as indicated, were analyzed for the proportion of cells in sub-G1 phase (left). T98G cells seeded in six-well plates (500 cells per well) were treated with the indicated concentrations of paclitaxel or vincristine, in the absence or presence of 10 µmol/L of LY294002 for 48 h, incubated for 6 d in fresh growth medium, fixed in 70% ethanol, and stained with 10% methylene blue; colonies of ≥50 cells were then counted (right). A and B, representative of three separate experiments. C and D, columns, means from three separate experiments, each done in duplicate; bars, SD; *, P < 0.05; **, P < 0.01 (two-tailed Student's t test).

 
The induction of apoptosis is essential for effective cancer chemotherapy. Given the antiapoptotic role of the PI3K-Akt pathway (6), we next examined whether specific blockade of this pathway sensitizes tumor cells to the cytotoxic action of chemotherapeutic agents by promoting apoptotic cell death. Each of the anticancer drugs examined (vincristine, paclitaxel, Ara-C, VP-16, cisplatin, and doxorubicin) induced the death of T98G and T24 cells, with the proportion of cells in sub-G1 phase increasing to between 40% and 80% after treatment of the cells for >48 h with optimal drug concentrations (Fig. 1D; data not shown). To examine the possible effect of LY294002 on death induction by these anticancer drugs, we treated the tumor cells with suboptimal concentrations of these agents, which increased the proportion of cells in sub-G1 phase to ~20% after 48 h (Fig. 1C). LY294002 (10 µmol/L) potentiated the induction of cell death by the antimicrotubule agents vincristine and paclitaxel in both T98G and T24 cells. In contrast, the effects of LY294002 on the cytotoxicity of Ara-C, VP-16, cisplatin, and doxorubicin were less pronounced or differed among tumor cell lines. Enhancement by LY294002 of the cytotoxic action of vincristine or paclitaxel, but not that of other anticancer agents such as VP-16 and cisplatin, has previously been described in human malignant glioma cells; the mechanism of this effect was not addressed, however (20). Although blockade of the PI3K-Akt pathway was previously shown to enhance doxorubicin-induced death of tumor cells, this effect is dependent on p53 function (11). Given that the p53 gene is mutated in both T98G and T24 cells, LY294002 failed to increase doxorubicin toxicity in these cells (Fig. 1C).

The enhancing effect of LY294002 on death induction by vincristine or paclitaxel was most pronounced at low concentrations of the latter drugs (Fig. 1D). Furthermore, the proportion of dead cells increased to ~70% when T98G (Fig. 1D) or T24 (data not shown) cells were treated with the combination of 10 µmol/L of LY294002 and 3 nmol/L of vincristine for 96 h. The effect of LY294002 on death induction by paclitaxel was less marked and became negligible after cells were treated with 10 µmol/L of LY294002 and 3 nmol/L of paclitaxel for ≥72 h. Such prominent and moderate enhancing effects of LY294002 on death induction by vincristine and paclitaxel, respectively, were confirmed by clonogenic cell survival assays (Fig. 1D). Therefore, in subsequent experiments, we focused on the effect of the PI3K-Akt pathway inhibition on the induction of cell death by microtubule-destabilizing agents.

LY294002 markedly enhanced cell death induction by 3 nmol/L of vincristine in tumor cells in which the PI3K-Akt pathway is constitutively activated (A172, LNCaP, T98G, T24) but not in those in which it is not (WiDr, ACHN; Fig. 1A, insets; ref. 11), or in TIG-3 diploid fibroblast cells (Fig. 2A ). Furthermore, blockade of the PI3K-Akt pathway not only by LY294002 but also by the Akt inhibitor API-2 (21) or the PI3K inhibitor PX-886 (22) increased the cytotoxicity of structurally different microtubule-destabilizing agents, including vincristine, TZT-1027, and navelbin (23), in T98G or T24 cells (Fig. 2B). These results indicated that specific blockade of the PI3K-Akt pathway enhances the induction of cell death by microtubule-destabilizing agents in tumor cells in which this signaling pathway is constitutively activated.


Figure 2
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  		Figure 2. Potentiation of the death-inducing effect of microtubule-destabilizing agents by blockade of the PI3K-Akt pathway in tumor cells in which this pathway is constitutively activated. A, TIG-3 diploid fibroblasts or WiDr, ACHN, A172, LNCaP, T98G, or T24 tumor cells were incubated with 10 µmol/L of LY294002 (LY), 3 nmol/L of vincristine (VCR), or both agents for 48 h, after which the proportion of cells in sub-G1 phase was determined by flow cytometry. B, T98G or T24 cells incubated with the indicated agents for 48 h were analyzed for the proportion of cells in sub-G1 phase. TZT, TZT-1027; Nav, navelbin. Columns, means from three separate experiments, each done in duplicate; bars, SD; *, P < 0.05; **, P < 0.01.

 
To characterize the cell death induced by the combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent, we examined several markers of apoptosis in T98G cells. Although dephosphorylation of Bad (which activates its proapoptotic activity) and phosphorylation of Bcl-2 (which inactivates its antiapoptotic activity; ref. 24) were induced by LY294002 (10 µmol/L) or vincristine (3 nmol/L) alone, these effects were more pronounced in the presence of both agents (Fig. 3A ). In addition, partial degradation of 14-3-3{xi} (a scaffolding protein that sequesters several proapoptotic proteins such as Bad and FKHRL1 after being phosphorylated by Akt; refs. 24, 25) was induced by the combination of LY294002 and vincristine. Furthermore, the cleavage of caspase-9 to produce its active 37-kDa fragment (indicative of activation of the mitochondrial pathway of apoptosis), the cleavage of caspase-3 (an effector caspase) to produce its active 19- and 17-kDa fragments (26), and the cleavage of poly(ADP-ribose) polymerase (an endogenous substrate of caspase-3; ref. 26) were detected in T98G cells treated with the combination of these agents for ≥24 h (Fig. 3B). The combination of LY294002 and vincristine also induced marked increases both in the proportion of cells negative for staining with propidium iodide and positive for staining with Annexin V (indicative of the early stage of apoptosis) by 18 h (data not shown), and in internucleosomal DNA fragmentation by 24 h (Fig. 3C). Although this drug combination induced the cleavage of caspase-8 to produce its active 43-kDa fragment (indicative of activation of the death receptor pathway; ref. 26), this effect was apparent only after 48 h (Fig. 3B). Collectively, these results suggested that the combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent induces apoptotic cell death in tumor cells primarily via the mitochondrial pathway.


Figure 3
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  		Figure 3. Induction of apoptosis in T98G cells by the combination of LY294002 and vincristine. A and B, cells were incubated in the absence (C) or presence of 10 µmol/L of LY294002 (LY), 3 nmol/L of vincristine (VCR), or both agents for the indicated times, after which cell lysates (30 µg protein) were subjected to immunoblot analysis with antibodies to the indicated proteins. Open arrowheads, phosphorylated forms of Bcl-2, a cleaved fragment (22 kDa) of 14-3-3{xi}, cleaved fragments (37, 35, and 17 kDa) of caspase-9, cleaved fragments (19 and 17 kDa) of caspase-3, a cleaved fragment (89 kDa) of poly(ADP-ribose) polymerase, or cleaved fragments (43 and 18 kDa) of caspase-8. ß-Actin was used as a loading control. C, cells incubated with the indicated concentrations of LY294002 and vincristine for 24 h were analyzed for DNA fragmentation by agarose gel electrophoresis and staining with ethidium bromide. The left lane contains DNA size markers (base pairs). Data are representative of three separate experiments.

 
Blockade of the PI3K-Akt Pathway Induces the Destabilization of Microtubules via GSK-3ß–mediated Phosphorylation of Microtubule-Associated Proteins
To reveal the molecular mechanism by which inhibition of the PI3K-Akt pathway promotes the induction of apoptosis by microtubule-destabilizing agents, we examined the possible effect of LY294002 on the structure and stability of cytoplasmic microtubules in tumor cells by immunostaining for {alpha}-tubulin (Fig. 4A ) as well as by measuring the relative amounts of polymerized versus soluble tubulin (Fig. 4B). Consistent with our observation that 3 nmol/L of vincristine alone did not exhibit death-inducing activity in T98G cells (Fig. 1D), it did not affect the structure or stability of microtubules in these cells (Fig. 4). In contrast, LY294002 (10 µmol/L) induced destabilization of cytoplasmic microtubules in T98G cells. Furthermore, this effect of LY294002 was markedly enhanced in the presence of 3 nmol/L of vincristine, resulting in extensive disruption of microtubules accompanied by a large decrease in the amount of polymerized tubulin and corresponding increase in the amount of soluble tubulin. The effect of the combination of these two agents was thus similar to that of treatment with a high concentration (30 nmol/L) of vincristine alone. Consistent with these observations, whereas 3 nmol/L of vincristine by itself did not affect the cell cycle distribution of T98G cells, its combination with LY294002 resulted in an increase in the proportion of cells in G2-M phase followed by accumulation of cells in sub-G1 phase, again similar to the effect of 30 nmol/L of vincristine alone (Fig. 1B).


Figure 4
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  		Figure 4. Destabilization of microtubules induced by blockade of the PI3K-Akt pathway in T98G cells. A, cells were incubated for 24 h in the absence (Control) or presence of 10 µmol/L of LY294002 (LY), 3 or 30 nmol/L of vincristine (VCR), or 30 nmol/L of paclitaxel (PTX) as indicated, and were then fixed and subjected to immunofluorescence analysis with antibodies to {alpha}-tubulin. Bar, 20 µm. Data are representative of three separate experiments. B, cells incubated as in (A) were lysed and fractionated into supernatant (S) and pellet (P) fractions. One-third of the respective fractions was subjected to immunoblot analysis with antibodies to {alpha}-tubulin (bottom), and the relative intensity of the {alpha}-tubulin bands was determined (top). Columns, means of values from three separate experiments, each done in duplicate; bars, SD; *, P < 0.05; **, P < 0.01.

 
The binding of microtubule-associated proteins (MAP) such as MAP-2, MAP-4, and tau induces the stabilization of microtubules (23, 27). Phosphorylation of these MAPs by GSK-3ß reduces their ability to bind and stabilize microtubules (27), whereas phosphorylation of GSK-3ß on Ser9 by Akt induces its inactivation (2). We therefore investigated the possible role of GSK-3ß–mediated phosphorylation of MAPs in the LY294002-induced destabilization of microtubules. To this end, we examined the phosphorylation of tau as a representative MAP in T98G glioblastoma cells. We analyzed the phosphorylation of tau on Ser199 because, although tau contains several Ser/Thr-Pro motifs that are phosphorylated by various kinases including GSK-3ß and Cdk5, phosphorylation of this residue has been shown to be mediated by GSK-3ß (28).

LY294002 inhibited the phosphorylation of GSK-3ß on Ser9 and induced the phosphorylation of tau on Ser199 in T98G cells (Fig. 5A ). Although 3 nmol/L of vincristine by itself did not inhibit GSK-3ß phosphorylation or induce tau phosphorylation, it enhanced the effect of LY294002 on tau phosphorylation, consistent with its enhancement of the microtubule-destabilizing effect of LY294002 (Fig. 4). Furthermore, the addition of 30 mmol/L of Li+ to suppress GSK-3ß activity (29), but not that of 30 mmol/L of K+, markedly inhibited both the phosphorylation of tau (Fig. 5A) and the destabilization of microtubules (Fig. 5B) induced by LY294002 alone or in combination with vincristine. In addition, Li+ inhibited the induction of cell death by the combination of LY294002 and vincristine in both T98G and T24 cells (Fig. 5C). SB216763, a specific inhibitor of GSK-3ß (30), also suppressed the induction of cell death by these agents in T98G cells. Vincristine at 30 nmol/L did not inhibit GSK-3ß phosphorylation or induce tau phosphorylation (Fig. 5A), and Li+ or SB216763 did not affect the cell death induced by this concentration of vincristine (Fig. 5C), indicating that cell death induced by high concentrations of vincristine is mediated by a mechanism independent of GSK-3ß function.


Figure 5
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  		Figure 5. Role of GSK-3ß–mediated phosphorylation of tau in microtubule destabilization induced by blockade of the PI3K-Akt pathway. A, T98G cells were incubated for 24 h in the presence of the indicated concentrations of LY294002 (LY), vincristine (VCR), or LiCl. Cell lysates (50 µg of protein) were then subjected to immunoblot analysis with antibodies to the indicated proteins. B, T98G cells were incubated for 24 h in the absence (Control) or presence of 10 µmol/L of LY294002 or 3 nmol/L of vincristine as well as in the presence of 30 mmol/L of LiCl or KCl, as indicated. They were then fixed and subjected to immunofluorescence staining with antibodies to {alpha}-tubulin. Bar, 20 µm. C, T98G or T24 cells were incubated for 48 h with the indicated additions, after which the proportion of cells in sub-G1 phase was determined by flow cytometry. D, T98G or A172 cells transfected with small interfering RNAs (A or B) specific for GSK-3ß mRNA or with the corresponding scrambled RNA duplexes were incubated for 24 h with LY294002 or vincristine as indicated. Cell lysates (50 µg of protein) were then subjected to immunoblot analysis with antibodies to the indicated proteins (left). Such transfected cells were also incubated for 48 h with LY294002 or vincristine, as indicated, after which the proportion of cells in sub-G1 phase was determined by flow cytometry (right). A, B, and D (left), representative of three separate experiments. C and D (right), columns, means of values from three separate experiments, each done in duplicate; bars, SD; *, P < 0.05; **, P < 0.01.

 
We then examined the effect of RNA interference–mediated depletion of GSK-3ß on the induction of apoptotic cell death by LY294002 and vincristine (3 nmol/L) in T98G and A172 cells. Immunoblot analysis revealed that transfection of both types of glioblastoma cells with small interfering RNAs (A or B) specific for GSK-3ß mRNA, but not with the corresponding control RNA duplexes, resulted in a pronounced reduction in the abundance of GSK-3ß as well as in the inhibition of the phosphorylation of tau induced by the combination of LY294002 and vincristine (Fig. 5D). Depletion of GSK-3ß by RNA interference also greatly inhibited the induction of apoptotic cell death by the combination of these agents. Together, these results indicated that the enhancing effect of LY294002 on the induction of apoptosis by microtubule-destabilizing agents in tumor cells is mediated, at least in part, by activation of GSK-3ß and consequent phosphorylation of tau, resulting in a decrease in its ability to bind and stabilize microtubules (27). The consequent destabilization of microtubules appears to markedly increase their sensitivity to microtubule-destabilizing agents such as vincristine, but not to microtubule-stabilizing agents such as paclitaxel (Fig. 1D).

The Combination of LY294002 and a Low Concentration of Vincristine Does Not Affect Microtubule Stability in Neuronal Cells
Microtubules not only form the mitotic spindle but also play a key role in intracellular transport of various organelles and vesicles, and are a primary determinant of cell morphology. These nonmitotic functions of microtubules predominate in cells of the nervous system, which no longer undergo cell division (31). The biological functions of microtubules in all cells are determined and regulated by the polymerization dynamics of these structures (23). Given that microtubules are abundant in nervous tissue and are essential for neuronal cell function, drugs that interfere with their polymerization dynamics might be expected to exhibit neuropathologic side effects. Indeed, the clinical utility of vincristine as a cancer chemotherapeutic agent is limited by the dose-dependent development of a peripheral neuropathy (32). We therefore examined the effect of the combination of 3 nmol/L of vincristine and 10 µmol/L of LY294002 on the stability of microtubules in neuronal cells. For this analysis, we used PC12 cells treated with NGF as a model of neuronal cells (33).

NGF induces the neuronal differentiation of PC12 cells, as manifested by neurite outgrowth (Fig. 6A ). Whereas a high concentration of vincristine (30 nmol/L) disrupted NGF-induced neurite outgrowth, LY294002, 3 nmol/L of vincristine, or the combination of LY294002 and 3 nmol/L of vincristine had no such effect. Microtubules are stabilized not only by association with various MAPs (23, 27) but also by posttranslational modifications of tubulin, including detyrosination at the COOH-terminus and acetylation of Lys40 of {alpha}-tubulin (34). Microtubules in the axons and dendrites of neurons are rich in acetylated {alpha}-tubulin; stabilization of these microtubules is essential for neuronal function (31). Immunostaining revealed that microtubules in the neurites of NGF-treated PC12 cells were rich in acetylated {alpha}-tubulin and that these microtubules were not affected by exposure of the cells to LY294002, 3 nmol/L of vincristine, or the combination of LY294002 and 3 nmol/L of vincristine (Fig. 6B). In T98G cells, whereas centrosome-associated microtubules were rich in acetylated {alpha}-tubulin, cytoplasmic microtubules were not. Although such "unstable" cytoplasmic microtubules were resistant to 3 nmol/L of vincristine alone, they were completely disrupted by exposure of the tumor cells to both LY294002 and 3 nmol/L of vincristine (Fig. 4A).


Figure 6
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  		Figure 6. Resistance of neuronal microtubules to the combination of LY294002 and a low concentration of vincristine. A, PC12 cells incubated in the absence (–) or presence of NGF (20 ng/mL) for 24 h were treated with 10 µmol/L of LY294002 (LY), 3 or 30 nmol/L of vincristine (VCR), or LY294002 and 3 nmol/L of vincristine, as indicated, for 12 h. The cells were then examined by phase-contrast microscopy. Bar, 20 µm. B, exponentially growing T98G cells or PC12 cells that had been treated with NGF (20 ng/mL) for 24 h and then exposed to 10 µmol/L of LY294002, 3 nmol/L of vincristine, or both agents for 12 h were fixed and subjected to immunofluorescence staining with antibodies to {alpha}-tubulin or to acetylated {alpha}-tubulin. Bar, 20 µm. All data are representative of three separate experiments.

 
Discussion

We have shown that, although specific blockade of the PI3K-Akt signaling pathway alone did not induce apoptosis, it markedly sensitized tumor cells to the induction of cell death by microtubule-destabilizing agents. This synergistic effect was restricted to tumor cells in which the PI3K-Akt pathway is constitutively activated. Specific inhibition of the PI3K-Akt pathway induces the activation (dephosphorylation) of GSK-3ß, which appears to be responsible for enhancement of the death-inducing effect of microtubule-destabilizing agents. GSK-3ß phosphorylates a variety of substrates including metabolic enzymes (such as glycogen synthase and pyruvate dehydrogenase), signaling proteins (such as IRS-1 and cyclins D1 and E), structural proteins (such as neurofilaments, MAP-2, and tau), and transcription factors (such as cAMP-responsive element binding protein, nuclear factor {kappa}B, p53, and ß-catenin; ref. 27). Our results now suggest that GSK-3ß–mediated phosphorylation of MAPs (such as tau) and the consequent reduction in their ability to bind and stabilize microtubules are responsible, at least in part, for the sensitization of tumor cells to microtubule-destabilizing agents conferred by inhibitors of the PI3K-Akt pathway. These observations likely explain why PI3K-Akt pathway inhibitors selectively enhance the death-inducing effects of microtubule-destabilizing agents.

Inhibitors of the PI3K-Akt pathway have previously been shown to enhance the induction of apoptosis by various agents such as VP-16, paclitaxel, and sodium butyrate (7–12). It has been proposed that such anticancer drugs induce an early and transient activation of Akt, and that suppression of this effect by PI3K-Akt inhibitors sensitizes tumor cells to the former agents (8). However, LY294002 has been shown to enhance the induction of cell death by anticancer drugs via mechanisms that do not involve inhibition of such Akt activation (9, 11, 12). In our experiments, cisplatin and VP-16, for example, each induced a slight (at most ~30%) and transient (lasting for 2 to 6 h) increase in the activity of Akt in T98G and T24 cells (data not shown). Under such conditions, administration of LY294002 potentiated the induction of cell death by cisplatin in T24 cells but not in T98G cells and it did not significantly affect the death-inducing efficacy of VP-16 in either tumor cell type (Fig. 1C). The mechanisms by which inhibition of the PI3K-Akt pathway enhances apoptotic cell death induced by these anticancer drugs thus remain to be determined.

Our unexpected finding that inhibitors of the PI3K-Akt pathway interfere with the polymerization dynamics of microtubules and thereby induce their destabilization suggested that the combination of such inhibitors and microtubule-destabilizing agents might also elicit neuropathologic side effects, given that microtubules, the functions of which are determined and regulated by their polymerization dynamics (23), are especially abundant in nervous tissue and are essential for neuronal cell function (31). Neuronal microtubules are stabilized by association with several MAPs as well as by posttranslational modifications of tubulin such as the acetylation of Lys40 of {alpha}-tubulin (34). Microtubules in the neurites of NGF-treated PC12 cells (a model of neuronal cells) were thus found to be rich in acetylated {alpha}-tubulin, whereas the cytoplasmic microtubules of T98G glioblastoma cells were not. Whereas the combination of LY294002 and a low concentration of vincristine induced the disruption of unstable cytoplasmic microtubules in T98G cells, it did not affect the "stable" microtubules in the neurites of NGF-treated PC12 cells. Thus, the combination of an inhibitor of the PI3K-Akt pathway and a low concentration of a microtubule-destabilizing agent might be expected to induce apoptotic cell death preferentially in tumor cells. Together, our results indicate that administration of such a drug combination is a promising chemotherapeutic strategy for the treatment of tumor cells in which the PI3K-Akt pathway is constitutively activated.

Footnotes

Grant support: Supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/16/06; revised 12/20/06; accepted 1/30/07.

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