Cancer Therapy Vol 3, 407-418, 2005

Synergistic augmentation of vincristine-induced cytotoxicity by phosphatidylinositol 3-kinase inhibitor in human malignant glioma cells: evidence for the involvement of p38 and ERK signaling pathways

Research Article

Daniel R. Premkumar, Beth Arnold, John Mathas, and Ian F. Pollack*

Department of Neurosurgery, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute Brain Tumor Center Pittsburgh, Pennsylvania 15213, USA

__________________________________________________________________________________

*Correspondence: Ian F. Pollack, M.D., F.A.C.S., F.A.A.P., Department of Neurosurgery, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA15213, USA.; Phone: 412-692-5881; Fax: 412-692-5921; E-mail: ian.pollack@chp.edu

Key words: Synergy, vincristine, microtubule inhibiting agents, PI3K, glioma, MAPK, apoptosis, cell cycle

Abbreviations: 3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H Tetrazolium, (MTS); bovine serum albumin, (BSA); c-Jun NH₂-terminal kinase/stress activated protein kinase, (JNK/SAPK); cyclin dependent kinase, (CDK); dimethyl sulfoxide, (DMSO); extracellular signal-regulated kinase kinase, (MEK); extracellular signal-regulated kinase, (ERK); fetal bovine serum, (FBS); fraction affected, (Fa); microtubule inhibiting agent, (MIA); mitogen activated protein kinase, (MAPK); phenazine methosulfate, (PMS); phenyl methylsuphonyl fluoride, (PMSF); phosphatase and tensin homologue deleted on chromosome ten, (PTEN); phosphate-buffered saline, (PBS); phosphatidylinositol 3’-kinase, (PI3K); poly (adenosine diphophate-ribose) polymerase, (PARP); sodium dodecyl sulfate-poly acrylamide gel electrophoresis, (SDS-PAGE)

This work was supported by NIH grant P01NS40923 and a grant from the Wichmann Foundation.

Received: 23 March 2005; Revised: 20 May 2005

Accepted: 06 July 2005; electronically published: July 2005

Summary

Microtubule-interfering agents, such as vincristine, are widely used for the treatment of cancer, and are included in many treatment regimens for childhood brain tumors. Anticancer properties of vincristine have been attributed in part to interference with microtubule assembly, impairment of mitosis, and cytoskeletal changes, with additional effects on mitogen-activated protein kinase signaling and caspase activation. Because malignant gliomas commonly have dysregulation of PI3K/Akt signaling, which can promote cell survival and potentially limit the activity of conventional chemotherapeutic agents, we questioned whether phosphatidylinositol 3’-kinase inhibitor (PI3K) inhibition with LY294002 could potentiate the efficacy of vincristine in a panel of glioma cell lines versus normal astrocytes. We therefore examined the effects of vincristine and the PI3K inhibitor, LY294002, alone and in combination, on cell survival, signal transduction and apoptosis. Simultaneous exposure to these inhibitors significantly induced cell death, and inhibited proliferation and clonogenicity of a series of glioma cell lines at concentrations that had little or no independent activity. Quantitative analysis revealed that enhancement by LY294002 of vincristine-induced cytotoxicity was synergistic, leading to pronounced caspase activation and increased sub-G₁ peak on cell cycle analysis at concentrations that had no significant effects on non-neoplastic cells. The enhanced cytotoxicity of this combination was associated with significant activation of p38 MAPK signaling. Pre-treatment with either SB203580 or z-VAD.fmk, selective inhibitors of p38 MAPK and caspase signaling, respectively, abrogated the apoptotic response to the combination of LY294002 and vincristine. Taken together, these findings demonstrate that PI3K/Akt inhibition can potentiate the effects of vincristine, and that the combination of molecularly targeted therapies and conventional agents could provide a potent strategy to treat patients with malignant gliomas.

I. Introduction

Glioblastoma multiforme (GBM) are highly malignant tumors of the central nervous system. They are characterized by rapid growth, extensive vascularization and poor prognosis (Nagane et al, 1997; Hanahan and Weinberg 2000; Maher et al, 2001). Even with radiation therapy and chemotherapy, the prognosis remains poor, with survival usually less than 1 year from the time of diagnosis. There is considerable evidence demonstrating a role for phosphatidylinositol 3-kinase (PI3K) signaling in oncogenic transformation, cancer progression, and resistance of cancer cells to cytotoxic therapies. The tumor suppressor gene PTEN, possessing a lipid phosphatase activity, negatively regulates PI3-kinase activity by dephosphorylation of PtdIns P3 leading to reduction in Akt activity. Genetic and biochemical evidence suggests that activation of PI3K or inactivation of PTEN by mutation or deletion play a major role in glial tumorigenesis (Maehama and Dixon 1999; Tamura et al, 1999). Deregulated PI3K signaling also provides an attractive target for therapy and pharmacological inhibitors of this pathway are already in early clinical trials (Di Cristofano and Pandolfi 2000).

Microtubule-interfering agents (MIAs) are widely used for the treatment of cancer (Joel 1996; Wall 1998; Gidding et al, 1999). The anticancer properties of MIAs have been attributed in part to interference with microtubule assembly, impairment of mitosis, and cytoskeletal changes (Wang et al, 1999). There is growing evidence that MIAs have multiple cellular targets. For example, MIAs elicit differential effects on mitogen activated protein kinase (MAPK) family signaling pathways (Stone and Chambers 2000; Mabuchi et al, 2002) and influence gene expression (Subbaramaiah et al, 2000). Furthermore, MIAs cause growth arrest, and induce caspase activation, degradation of PARP, and apoptosis in neoplastic cells (Stone and Chambers 2000). Vincristine is an MIA that is widely used to treat patients with malignant disease, including those with brain tumors (Kellie et al, 2004). However, many tumor cell lines are resistant to clinically achievable concentrations of this agent, which may in part reflect a resistance to undergoing apoptosis, as a result of dysregulated PI3K/Akt signaling. Accordingly, a potential strategy for enhancing the antitumor efficacy of vincristine involves combining this agent with targeted disruption of PI3K/Akt-mediated survival pathways. Previous studies by Shingu et al (2003) have suggested the potential synergy of microtubule inhibiting agents, such as vincristine, and PI3kinase inhibition. In this report, we demonstrated the differential efficacy of this approach in a panel of glioma cell lines versus normal astrocytes and fibroblasts and identified the involvement of p38 and ERK in mediating the synergistic effects of combining vincristine and PI3K inhibition with LY294002. Our results suggest that multiple pathways are important for cell survival in malignant glioma cells and identify a role for activation of p38 and inhibition of ERK in the augmentation of vincristine-induced cytotoxicity by PI3K inhibition.

II. Materials and Methods

A. Cell Culture

The established malignant glioma cell lines U87, T98G, A172, and human pulmonary fibroblasts were obtained from the American Type Culture Collection. Human astrocytes and human cerebellar astrocytes were obtained from ScienCell Research Laboratories, San Diego, CA. LN18, LN-Z308, and LN-Z428 were generously provided by Dr. Nicolas de Tribolet. U87, T98G and human pulmonary fibroblasts were cultured in growth medium composed of minimum essential medium supplemented with sodium pyruvate and non-essential amino acids; A172, LN18, LN-Z308, and LN-Z428 in α-minimal essential medium supplemented with L-glutamine; human astrocytes in Astrocyte Growth Medium (ScienCell Research Laboratories). All growth media contain 10% fetal calf serum, L-glutamine, ribonucleosides, deoxynucleosides, 100 IU/ml penicillin, 100mg/ml streptomycin and 0.25 mg/ml amphotericin (Life Technologies, Inc., Bethesda, MD). Cells were grown in 75-cm² flasks at 37^oC in a humidified atmosphere with 5% carbon dioxide and were subcultured every 4 to 7 days by treatment with 0.25% trypsin in Hanks’ balanced salt solution (Life Technologies, Inc.).

B. Cell proliferation and cytotoxicity assay

Cells (5 X 10³/well) were plated in 96-well microtiter plates (Costar, Cambridge, MA) in 100 ml of growth medium. After an overnight attachment period, cells were exposed for 3 days to varying concentrations of vincristine with or without 5mM LY294002. Control cells received vehicle alone (DMSO). All studies were performed in triplicate and repeated at least three times independently. After the 3-day treatment period, cells were washed in inhibitor-free medium and the number of viable cells was determined using a colorimetric cell proliferation assay (CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI), which measures the bioreduction of the tetrazolium compound MTS (3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium) by dehydrogenase enzymes of metabolically active cells into a soluble formazan product, in the presence of the electron coupling reagent PMS (phenazine methosulfate) (Riss TL 1992).

To perform the assay, 20ml of combined MTS/PMS solution containing 2mg/ml MTS and 150mmol/L PMS in buffer (0.2g/L KCL, 8.0g/L NaCl, 0.2g/L KH₂PO, 1.15g/L Na₂HPO₄, 133 mg/ml CaCl₂.2H₂0, 100 mg/ml MgCl₂.6H₂0, pH7.35) was added to each well and then after 1 h of incubation at 37^oC in a humidified 5% CO₂ atmosphere, absorbance was measured at 490nM in a microplate reader. Triplicate wells with predetermined cell numbers were subjected to the above assay in parallel with the test samples to normalize the absorbance readings; this also provided internal confirmation that the assay was linear over the range of absorbance and cell numbers measured.

To assess cellular toxicity, 2.5 X 10⁵ cells were seeded in 60-mm Petri dishes and on the following day, treated with selected concentrations of inhibitors or vehicle. Cells were harvested, stained with trypan blue, and counted using a hemacytometer. All samples were tested in triplicate. Viable (trypan blue-excluding) and dead cell numbers were plotted as a function of inhibitor concentration.

C. Clonogenic growth assay

A more direct assessment of the effect of different inhibitor concentrations on cell viability was performed using a clonogenic assay. For these studies, 250 cells were plated in 6-well trays in growth medium and, after an overnight attachment period, were exposed to selected inhibitor concentrations or vehicle for 24h. The medium was aspirated and cells were washed with inhibitor-free medium. Cells were allowed to grow for an additional 2-week period. Colonies of a diameter of approximately 2 to 4 mm were counted directly. All studies were performed in quintuplicate.

D. Western blotting analysis

Treated and untreated cells were washed once in cold phosphate-buffered saline (PBS) and lysed in buffer containing 30mM Hepes, 10%glycerol, 1%Triton X-100, 100mM NaCl, 10mM MgCl₂, 5mM EDTA, 2mM Na₃VO₄, 2mM b-glycerophosphate, 1mM PMSF, 1mM AEBSF, 0.8mM Aprotinin, 50mM Bestatin, 15mM E-64, 20mM Leupeptin, 10mM Pepstatin A. After lysing on ice for 15 minutes, protein samples were collected from the supernatant after centrifugation of the samples at 12,000 X g for 15 min, and protein was quantified using Protein Assay Reagent (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE and electrotransferred onto a nylon membrane (Invitrogen, Carlsbad, CA). The blots were blocked with 2% BSA in Tris-buffered saline (TBS)-Tween 20 (0.1%) at room temperature for 1h and probed with the appropriate dilution of primary antibody overnight at 4^oC. The blots washed three times in TBS-Tween 20 for 15 min and then incubated with a 1:1500 dilution of horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA) in TBS-Tween 20 at room temperature for 1h. After washing three times in TBS-Tween 20 for 15 min, the proteins were visualized by Western Blot Chemiluminescence Reagent (Cell Signaling Technology). Where indicated, the blots were reprobed with antibodies against b-actin (Sigma, St.Louis, MO) to ensure equal loading and transfer of proteins.

The primary antibodies, such as extracellular signal related kinase 1/2 (ERK 1/2) and phospho-p44/42 ERK (Thr²⁰²/Tyr²⁰⁴), p38 and phospho-p38 MAPK (Thr¹⁸⁰.Tyr¹⁸²), JNK and phospho- SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵), Akt and phospho Akt (Ser⁴⁷³), CDK4, CDK6, Cyclin D1, Cyclin D3, p27Kip, phospho-Cdc2, cleaved caspase 3, cleaved PARP, phospho-Bcl-2, Bcl-2-x_L, and Bax were obtained from Cell Signaling Technologies.

E. Inhibition of p38 kinase and caspases

T98G cells were seeded 24 h before treatment. Cells were pretreated with z-VAD.fmk (a broad spectrum caspase inhibitor) or SB203580 (a specific p38 kinase inhibitor) in the culture media 60 min prior to treatment with vincristine or LY294002 or the combination of both for 9 h. The cells, after washing twice with phosphate buffered saline, were lysed and Western immunoblot analysis was performed using cleaved caspase 3 specific antibody as described above.

F. Cell cycle analysis

Analysis of DNA content of cells by flow cytometry was performed as described elsewhere (Gesbert et al, 2000). Briefly, T98G cells grown exponentially to 40-50% confluency were exposed to vincristine and/or LY294002, or vehicle (DMSO), harvested at the indicated time, washed briefly in ice-cold PBS, and fixed in 70% ethanol. DNA was stained by incubating the cells in PBS containing propidium iodide (50 mg/ml) and RNase A (mg/ml) for 60 min at room temperature, and fluorescence was measured and analyzed using a Becton Dickinson FACScan and the Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

G. Statistical analysis

To define IC₅₀ concentrations and to characterize synergistic effects between the agents, a commercially available software program was used (Calcusyn; Biosoft, Ferguson) (Chou and Talalay 1984).

III. Results

A. Inhibition of cell proliferation by vincristine

To characterize the interaction between the chemotherapeutic agent vincristine and the PI3-kinase inhibitor LY294002, a panel of human glioma and normal cells were cotreated with varying concentrations of vincristine and 5mM LY294002, a concentration well below IC₅₀ in human glioma cell lines (Premkumar et al manuscript submitted), and then cell proliferation was measured using an MTS assay. As shown in Figure 1A, vincristine inhibited the growth of glioma cells in a dose-dependent manner. At the concentrations examined, there were no significant effects on the normal cells, such as human astrocytes, human cerebellar astrocytes, and human fibroblasts (data not shown). The observed inhibitory effect of vincristine was further potentiated by LY294002. To determine whether this potentiation was due to additive or synergistic interactions, we performed concentration-effect and isobologram analyses. The data were then applied to determine the combination index (CI) which provides a semiquantitative assessment of the presence of additive, synergistic or antagonistic interactions at different effect levels (Chou and Talalay 1984). The combination index is 1 for additive interactions, greater than 1 for antagonistic interactions, and less than 1 for synergistic interactions. Figure 1B illustrates plots of the CI versus fraction affected (Fa) for vincristine and LY294002. The combination of vincristine and LY294002 produced a synergistic inhibition, based on the observation that the CI was substantially less than 1.

In order to confirm the specificity towards tumor cells, we compared the effect of vincristine and LY294002 alone or in combination on human glioma (U87 and T98G) and normal cells (human cerebellar astrocytes and human fibroblast). Cells were treated with 1nM vincristine or 5mM LY294002 or the combination of both for 3 days and cell proliferation was assessed by MTS assay. Exposure to 5mM LY294002 alone was minimally toxic to these cells (about 10% reduction of cell proliferation versus control), whereas 1nM vincristine had no significant growth inhibitory effect in U87, human cerebellar astrocytes and human fibroblasts (Figure 2A). However, the combination of vincristine and LY294002 significantly reduced cell proliferation in glioma cells (by 60 and 70% compared to control in U87 and T98G, respectively), whereas the combination had little effect on normal cells (25 and 15% reduction from control in human cerebellar astrocytes and human fibroblasts, respectively). These results indicate that combination of vincristine and LY294002 works very efficiently in tumor cells compared with non-tumorigenic human astrocytes and fibroblasts.

The cytotoxic effect of vincristine and LY294002 was further confirmed using a clonogenic assay. U87 and T98G cells were treated with varying concentrations of vincristine for 1 day, medium was aspirated, and cells were washed with inhibitor-free medium. Cells were allowed to grow for an additional 2-week period. There was a dose-dependent decrease in colony forming ability due to vincristine and the IC₅₀ was 0.776 and 1.583nM for U87 and T98G respectively (Figure 2B). Vehicle or 5mM LY294002 alone had no significant effect on clonogenicity of either U87 or T98G cells (data not shown), but the combination of LY294002 with varying concentrations of vincristine significantly reduced their colony forming potential (Figure 2B).

B. Vincristine and LY294002 cooperate to induce a sub-G₁ fraction on cell cycle analysis of human glioma cell lines

As our previous results indicated that human glioma cells are sensitive to the antiproliferative properties of vincristine, we performed a more detailed analysis of the effect of the drug on the cell cycle. We performed a time course of this effect by analyzing the DNA profile in T98G cells exposed to 1 nM of vincristine using propidium iodide staining and flow cytometry. As shown in Figure 3A, as early as 12h after treating the cells with 1nM of vincristine the percentage of cells in the G₁ phase had increased to 61%, versus 39% in untreated control cells (Figure 3A). 5mM LY294002 was shown to induce 71, 10, and 18% G₁, S and G₂/M fractions, respectively after 72h.

Apoptotic cells undergo DNA fragmentation and therefore display a sub-G₁ (<2N) DNA content. Although neither vincristine nor LY294002 independently induced a substantial sub-G₁ fraction at the above concentrations, the combination of vincristine and LY294002 displayed a significant increase in cell death, revealed by the presence of a pronounced sub-G₁ fraction on flow cytometry. T98G cells were shown to exhibit 32, 4 and 6% G₁, S and G₂/M phase fractions, respectively after 72 h (Figure 3B) when the cells were exposed concomitantly to vincristine (1nM) and LY294002 (5mM), whereas the fraction of cells with <2N DNA content (sub-G₁) was 55%. This indicates that the decreased cell proliferation observed after treating with vincristine and LY294002 is at least partly due to induction of apoptosis.

Given the striking combinatorial effects of vincristine and PI3K inhibition on cell proliferation and colony forming ability of glioma cells, we questioned whether this

Figure 1. Growth inhibition of human glioma cell lines by vincristine and PI3K inhibitor. (A) Logarithmically growing glioma cell lines were incubated with or without varying concentrations of vincristine with or without 5mM LY294002 for 3 days. The relationship between the compounds and cell numbers was assessed semiquantitatively by spectrophotometric measurement of MTS bioreduction in T98G, A172, LN308 and LN428, established malignant human glioma cell lines. Points represent the mean of three measurements ± standard deviation. There was a dose-dependent reduction in cell growth and addition of 5mM LY294002 potentiated the vincristine-induced toxicity. No significant inhibition was detected in control cells treated with equivalent concentrations of vehicle (DMSO) or 5 mM LY294002 alone. (B) Graphs showing concentration-response plots of inhibition (expressed as fraction affected) versus combination index. Four established human glioma cell lines (A172, LN308, LN18 and LN428) were exposed to varying concentrations of vincristine and LY294002 at a fixed molar ratio (1:3000) for 3 days. Each point was derived from triplicate measurements of cell numbers as assessed by an MTS-based colorimetric assay. The data were then used to calculate the combination index (CI) using commercially available software (Calcusyn; Biosoft), which provides a semiquantitative assessment of the presence of additive, synergistic, or antagonistic interactions at different effect levels. The CI is substantially less than 1 for the combination of vincristine and LY294002, indicating synergistic interactions.

combination would have comparable effects on cell cycle regulatory proteins. Accordingly, we examined the effects of these agents, alone and in combination, on several intermediates that play critical roles in glioma cell cycle progression. T98G cells were therefore seeded at subconfluency, treated with vincristine, LY294002, or the combination of both, and the effects on protein expression levels were assessed. Results from Western blot analysis showed that the combination of vincristine and LY294002 had no significant effect on phospho-cdc2, CDK4, CDK6, Cyclin D1, p21WAF and p27Kip compared to controls or each agent individually. However, coadministration of vincristine and LY294002 resulted in a modest but discernible reduction in Cyclin D3 (Figure 3C), suggesting that a reduction in Cyclin D3 expression could be responsible for the observed G₁ arrest and increased apoptosis.

C. Vincristine down regulates ERK, Akt and activates p38 MAP kinase

To establish whether vincristine induced selective effects on different signaling pathways, the phosphorylation status of ERK, p38, JNK/SAPK and Akt was evaluated in control and vincristine-treated T98G cells by Western immunoblot analysis using respective phosphospecific antibodies. A vincristine concentration (50 nM) well above the IC₅₀ was used to optimally demonstrate the time course of the effects observed. High levels of the phosphorylated forms of both ERK1 and 2 and Akt were detected in untreated control cells and treatment with 50nM vincristine produced a gradual decrease in these levels in a time-dependent manner (Figure 4A). Conversely, cells exposed to vincristine exhibited a time-dependent increase in phosphorylated p38. Quantification of the results revealed that phospho p38 was maximally stimulated to about 9 fold at 9 h and reduced to basal level after 24h (Figure 4A). Phosphorylated JNK/SAPK was not detected in the treated cells (data not shown).

Figure 2. Vincristine and LY294002 preferentially inhibit growth and colony formation of glioma cell lines. (A) Logarithmically growing glioma cell lines (T98G and U87), normal cells human cerebellar astrocytes (HAC), and human fibroblasts (HF) were incubated in media (C) with 1nM vincristine (V) or 5µM LY294002 (LY) or the combination of vincristine and LY294002 (V+LY) for 3 days and cell numbers were assessed by MTS assay. Points represent the mean of three measurements ± standard deviation. The combination of vincristine and LY294002 significantly reduced cell proliferation of glioma cells compared to normal cells. (B). Graph showing the relationship between colony counts (± standard deviation) and concentration of the inhibitors. Human glioma cell lines, U87 and T98G were exposed to varying concentrations of vincristine with or without LY294002 (5mM) for 24 h. On the following day, the media was changed and complete media was added and cells were grown for additional 14 days in the absence of inhibitors. Colonies were then counted. Points represent the mean of two experiments ± standard deviation.

Figure 3. Cell cycle analysis of vincristine-treated T98G cells. (A) Exponentially growing T98G cells were exposed to 1nM vincristine for the indicated times, harvested, fixed, and DNA-stained with propidium iodide. DNA content of the cells was obtained by flow cytometry, and representative histograms for the indicated times are shown with values for each phase of the cell cycle. (B) Asynchronous T98G cells, grown to 40-50% confluency, were exposed to vincristine (1nM) or LY294002 (5mM) or the combination of both for 72h. Control cells received DMSO. DNA content (%) of cells was obtained by flow cytometry. (C) Cell lysates were obtained from T98G cells treated with vincristine (1nM) or LY294002 (5mM) or the combination of both for 72h, and 50mg of total protein from the cell lysates were resolved in polyacrylamide-SDS gels. Proteins were analyzed by Western immunoblotting using indicated antibodies as described in “Materials and Methods” and detected by enhanced chemiluminescence. Control cells received DMSO. Levels of ß-actin are indicated and served as a control to ensure equal protein loading per lane.

Figure 4. Vincristine downregulates ERK and upregulates p38 MAPK. Logarithmically growing T98G cells were incubated for designated intervals in the presence of 50nM vincristine (A) or in vincristine (1nM) or LY294002 (5mM) or the combination of both (B). The cells were lysed, and proteins were separated by SDS-PAGE and probed with a phosphospecific ERK 1, 2 antibody, which recognizes phosphorylated (Thr²⁰² and Tyr²⁰⁴) ERK MAP kinase. Activation of p38 and Akt was assessed using phosphospecific p38 (Thr¹⁸⁰ and Tyr¹⁸²) and phosphospecific Akt (Ser⁴⁷³) antibodies, respectively; then the blots were stripped and reprobed with total ERK, p38 or Akt.

To assess potential interactions between vincristine and LY294002, T98G cells were exposed to each of these agents alone or in combination for varying durations and the cell lysates were probed with phosphospecific ERK, Akt and p38 antibodies. Vincristine (1nM) or LY294002 (5mM) each had very little effect on phosphorylated ERK1/2 even after 3 days of exposure (Figure 4B), whereas combined exposure to these agents resulted in a significant (as early as 3h) to complete (after 24h) inhibition of phosphorylated ERK. Combined exposure to these agents resulted in significant to complete inactivation of pAkt after 24h. To determine the impact on the p38 activation, T98G cells were exposed to each agent individually or in combination. Incubation with 1nM vincristine induced phosphorylation of p38 at 6h and this was abolished after 24h, whereas coincubation with LY294002 further potentiated the p38 activation, which persisted over the ensuing 72h.

D. Vincristine and LY294002-induced activation of caspases and PARP. Caspases are aspartate-specific cysteine proteases activated by cleavage of their inactive pro-caspase forms, which function as important intermediates in apoptotic signaling. We examined the involvement of caspases and the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), a target of caspase cleavage in the apoptotic effects of vincristine and LY294002. T98G cells were treated with vincristine (1nM), LY294002 (5mM) or the combination of both for varying durations and the apoptotic cleavage of caspase 8, caspase 9, caspase 3 and PARP was assessed by Western analysis using specific antibodies. Cells incubated with vincristine or LY294002 did not show any activation of caspases or PARP cleavage (Figure 5A), whereas the combination of vincristine and LY294002 significantly increased the expression of the active forms of caspases 8, 9, 3 and PARP (Figure 5A, B). Activation of cleaved caspase 3 and PARP were seen as early as 6h after treatment and increased with longer durations of exposure. The degree of caspase activation observed with the combination of 1 nM vincristine and 5 mM LY294002 was comparable to that observed with 10-fold higher concentrations of vincristine administered alone (Figure 6C). This effect was not associated with changes in expression of Bcl-2, Bcl-x_L, or Bax (Figure 5C). Importantly, non-neoplastic human astrocytes showed no activation of caspase 3 or PARP even after 3 days of treatment (Figure 5D).

E. Vincristine and LY294002-induced Activation of caspase 3 is inhibited by SB203580 and z-VAD.fmk

To confirm the role of p38 signaling and caspase activation in mediating the glioma-specific cytotoxicity of the combination of vincristine and LY294002, we examined the effect of selective inhibition of these pathways, using SB203580, which prevents p38 activation, and z-VAD.fmk, a broad-spectrum caspase

Figure 5. Vincristine and LY294002 induces caspases and PARP activation in T98G cells. Logarithmically growing T98G cells were incubated for designated intervals in the presence of 1nM vincristine with or without LY294002 (5mM). The cells were lysed, and proteins were separated by SDS-PAGE and probed with specific antibodies which recognize the cleaved products of caspase-3 (C. caspase) or PARP (C. PARP) (A) as described in “Materials and Methods”. B. T98G glioma cell line (B and C) and normal human astrocytes (D) were treated as above for 3 days and the proteins were separated and probed with indicated antibodies, analyzed by Western immunoblot and detected by enhanced chemiluminescence. Control cells received DMSO.

inhibitor. Cells were pretreated with caspase and p38 inhibitors in the culture media 60 min prior to treatment with vincristine or LY294002 or the combination of both for 9 h. Western immunoblot analysis was performed using cleaved caspase 3 specific antibody and cell viability was assessed by trypan blue exclusion analysis in parallel. Vincristine induced caspase 3 activation which was further potentiated when vincristine was combined with LY294002 (Figure 6A, B). On the other hand active caspase 3 was not detected in the cells pretreated with SB203580 or z-VAD.fmk. Neither LY294002 nor SB203580 had a significant effect on cell survival. In contrast, exposure to 10nM vincristine resulted in 31% cell death and combination of 10mM LY294002 and 10nM vincristine resulted in a further increase in cell death (to about 60%). Pretreating the cells with p38 inhibitor, SB203580 or z-VAD.fmk significantly reduced the vincristine and LY294002-induced cell death (to 12 and 10% respectively). This suggests that the inhibition of p38 activation by vincristine and LY294002 protects the cells by preventing caspase 3 activation and progression towards apoptosis.

IV. Discussion

Cancer progression has been suggested to involve the loss of cell cycle checkpoint controls that regulate the passage through cell cycle. Checkpoints are control mechanisms that ensure the proper timing of cell cycle events and monitor the integrity of the DNA (Hartwell and Weinert 1989). At high concentrations, MIAs arrest cells in mitosis by triggering the mitotic checkpoint, a series of biochemical reactions that ensure proper attachment of chromosomes to the mitotic spindle before cells enter anaphase (Rudner and Murray 1996; Amon 1999; Burke 2000). In the present study, we have shown that LY294002, an inhibitor of PI3K/Akt kinase, interacts synergistically with low concentrations of vincristine, a chemotherapeutic MIA to induce both G1 arrest and apoptosis. Cells treated with vincristine demonstrated G₁ arrest at low drug concentrations, whereas co-administration of LY294002 produced a substantial sub-G₁ fraction, suggesting that the combination treatment induced apoptosis at concentrations of vincristine and LY294002 that produced minimal independent cytotoxicity.

The sensitivity of cells to apoptosis-inducing stimuli appears to be dependent on the balance between apoptosis-inducing signals and survival signals (Berra et al, 1998; Murillo et al, 2001; Shingu et al, 2003). Several lines of evidence implicate an important role for PI3K/Akt pathways in tumorigenesis and suppression of apoptosis (Datta et al, 1997; Datta et al, 1999; Katso et al, 2001). Akt represents a major downstream target of PI3K and is linked to a wide variety of antiapoptotic functions (Datta et al, 1999; Nicholson and Anderson 2002). Active Akt prevents apoptosis by a variety of mechanisms, including phosphorylation of Bad, caspase-9, Forkhead transcription factors and IkB kinase(Datta et al, 1997; Cardone et al, 1998; Vanhaesebroeck and Alessi 2000; Gelfanov et al, 2001).

Figure 6. Vincristine-induced toxicity is blocked by the inhibition of p38 MAPK. Logarithmically growing T98G cells were incubated in the presence of SB203580 (10mM) or z-VAD.fmk (100mM) 60 min prior to vincristine (10nM) or LY294002 (10mM) or the combination of both for 9h. Equal amounts of proteins (50mg) were separated by SDS-PAGE and probed with caspase-3 antibody which recognizes the cleaved products of caspase-3 (A) as described in “Materials and Methods”, and detected by enhanced chemiluminescence. Control cells received DMSO. (B) In parallel, at the end of the incubation period, the dead and viable cell numbers were determined by trypan-blue exclusion assay. For each analysis, at least 1000 cells were evaluated. The values represent the mean ± standard deviation for 2 separate experiments performed in triplicate.

Conversely, inhibition of Akt signaling may potentiate apoptosis in response to conventional chemotherapeutic agents as well as other growth signaling inhibitors (Ng et al, 2000; O'Gorman et al, 2000; Hu et al, 2002; Shingu et al, 2003). Microtubule stabilizing agents such as paclitaxel and docetaxel and microtubule-disrupting drugs such as vincristine, vinblastine and colchicines, interfere with the microtubule-related functions in signaling and gene expression (Kumar 1981; McNally 1996; Haldar et al, 1997; Saunders and Limbird 1997; Jordan and Wilson 1998; Subbaramaiah et al, 2000), and have antimitotic and apoptosis-inducing activity (Donaldson et al, 1994). Blagasklonny et al (Blagosklonny et al, 1997) and Poruchynsky et al (Poruchynsky et al, 1998) showed that exposure to MIAs may promote apoptosis by phosphorylating Bcl-x_L, Bcl-2 and related family members or by post-translational modifications in the proteins that interfere with anti-apoptotic functions. These findings led to the concept that Bcl-2/Bcl-x_L phosphorylations represent key steps in the cell death pathway induced by microtubule disruption. Although we found that vincristine as a single agent or in combination with LY294002 produced no change in expression of Bcl-2, Bcl-x_L, and Bax in glioma cell lines, we used substantially lower concentrations (1nM versus 100 nM or more) (Poruchynsky et al, 1998; Srivastava et al, 1998). Nonetheless, our observations suggest that alternate mechanisms operate to enhance apoptosis in glioma cells, and that the augmentation of vincristine-induced cytotoxicity by LY294002 was not due to increased expression of Bax or decreased expression of antiapoptotic regulatory proteins like Bcl-2 or Bcl-x_L.

Previously, MIAs have been observed to induce p38, JNK and ERK MAPK activities in various cell lines (Lee et al, 1998; Schmid-Alliana et al, 1998; Wang et al, 1998; Moos et al, 1999). In this report, we demonstrate that the combination of PI3 kinase inhibition and vincristine results in concomitant down-regulation of ERK and activation of p38 MAP kinase in glioma cells, with minimal effects on JNK activity. This contrasts with observations in other tumor cell lines that MIAs activate JNK/SAP kinase (Wang et al, 1998; Shtil et al, 1999; Stone and Chambers 2000), although is consistent with recent findings that the microtubule stabilizer taxol activates p38 kinase in MCF-7 cells (Shtil et al, 1999). These disparate findings suggest that there may be differential regulation of various MAP kinase family members in response to microtubule inhibition in different human tumor cell lines. Thus, the synergistic augmentation of vincristine-induced cytotoxicity by LY294002 and the ability of a cell to die or survive may dictated by a critical balance between ERK and the p38 pathway.

Taken together, our observations support and extend the observations of Shingu et al (2003), which suggested the potential synergy of microtubule inhibiting agents, such as vincristine, and PI3kinase inhibition in glioma cells. In this report, we demonstrated the differential efficacy of this approach in a more extensive panel of glioma cell lines versus normal astrocytes and fibroblasts, and identified the involvement of p38 and ERK in mediating the synergistic effects of combining vincristine and PI3K inhibition with LY294002. These results highlight the involvement of several signaling molecules in determining the susceptibility of human glioma cells to vincristine-induced apoptosis. The observation that inhibition of a major mediator of survival signals, PI3 kinase, can notably sensitize cells to cytotoxic drug-induced apoptosis, is a finding which may be of significant therapeutic importance, given the current interest in developing selective inhibitors of this target. We conclude that the combination of molecularly targeted therapies and conventional agents may provide a potent strategy to treat patients with malignant gliomas.

References

Amon A (1999) The spindle checkpoint. Curr Opin Genet Dev 9, 69-75.

Berra E, Diaz-Meco MT, and Moscat J (1998) The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway. J Biol Chem 273,10792-10797.

Blagosklonny MV, Giannakakou P, el-Deiry WS, Kingston DG, Higgs PI, Neckers L, Fojo T (1997) Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res 57, 130-135.

Burke DJ (2000) Complexity in the spindle checkpoint. Curr Opin Genet Dev 10:26-31

Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318-1321.

Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22, 27-55.

Datta SR, Brunet A, Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13, 2905-2927.

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241.

Di Cristofano A, Pandolfi PP (2000) The multiple roles of PTEN in tumor suppression. Cell 100, 387-390.

Donaldson KL, Goolsby GL, Kiener PA, Wahl AF (1994) Activation of p34cdc2 coincident with taxol-induced apoptosis. Cell Growth Differ 5, 1041-1050.

Gelfanov VM, Burgess GS, Litz-Jackson S, King AJ, Marshall MS, Nakshatri H, Boswell HS (2001) Transformation of interleukin-3-dependent cells without participation of Stat5/bcl-xL: cooperation of akt with raf/erk leads to p65 nuclear factor kB-mediated antiapoptosis involving c-IAP2. Blood 98, 2508-2517.

Gesbert F, Sellers WR, Signoretti S, Loda M, Griffin JD (2000) BCR/ABL regulates expression of the cyclin-dependent kinase inhibitor p27Kip1 through the phosphatidylinositol 3-Kinase/AKT pathway. J Biol Chem 275, 39223-39230.

Gidding CE, Kellie SJ, Kamps WA, de Graaf SS (1999) Vincristine revisited. Crit Rev Oncol Hematol 29, 267-287.

Haldar S, Basu A, Croce CM (1997) Bcl2 is the guardian of microtubule integrity. Cancer Res 57, 229-233.

Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100, 57-70.

Hartwell LH, Weinert TA (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629-634.

Hu L, Hofmann J, Lu Y, Mills GB, Jaffe RB (2002) Inhibition of phosphatidylinositol 3'-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Cancer Res 62, 1087-1092.

Joel S (1996) The comparative clinical pharmacology of vincristine and vindesine: does vindesine offer any advantage in clinical use? Cancer Treat Rev 21, 513-525.

Jordan MA, Wilson L (1998) Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr Opin Cell Biol 10, 123-130.

Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD (2001) Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17, 615-675.

Kellie SJ, Koopmans P, Earl J, Nath C, Roebuck D, Uges DR, De Graaf SS (2004) Increasing the dosage of vincristine: a clinical and pharmacokinetic study of continuous-infusion vincristine in children with central nervous system tumors. Cancer 100, 2637-2643.

Kumar N (1981) Taxol-induced polymerization of purified tubulin. Mechanism of action. J Biol Chem 256, 10435-10441.

Lee LF, Li G, Templeton DJ, Ting JP (1998) Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). J Biol Chem 273, 28253-28260.

Mabuchi S, Ohmichi M, Kimura A, Hisamoto K, Hayakawa J, Nishio Y, Adachi K, Takahashi K, Arimoto-Ishida E, Nakatsuji Y, Tasaka K, Murata Y (2002) Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel. J Biol Chem 277, 33490-33500.

Maehama T, Dixon JE (1999) PTEN: a tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol 9, 125-128.

Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, DePinho RA (2001) Malignant glioma: genetics and biology of a grave matter. Genes Dev 15, 1311-1333.

McNally FJ (1996) Modulation of microtubule dynamics during the cell cycle. Curr Opin Cell Biol 8, 23-29.

Moos PJ, Muskardin DT, Fitzpatrick FA (1999) Effect of taxol and taxotere on gene expression in macrophages: induction of the prostaglandin H synthase-2 isoenzyme. J Immunol 162, 467-473.

Murillo H, Schmidt LJ, Tindall DJ (2001) Tyrphostin AG825 triggers p38 mitogen-activated protein kinase-dependent apoptosis in androgen-independent prostate cancer cells C4 and C4-2. Cancer Res 61, 7408-7412.

Nagane M, Huang HJ, Cavenee WK (1997) Advances in the molecular genetics of gliomas. Curr Opin Oncol 9, 215-222.

Ng SSW, Tsao MS, Chow S, Hedley DW (2000) Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells. Cancer Res 60, 5451-5455.

Nicholson KM, Anderson NG (2002) The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 14, 381-395.

O'Gorman DM, McKenna SL, McGahon AJ, Knox KA, Cotter TG (2000) Sensitisation of HL60 human leukaemic cells to cytotoxic drug-induced apoptosis by inhibition of PI3-kinase survival signals. Leukemia 14, 602-611.

Poruchynsky MS, Wang EE, Rudin CM, Blagosklonny MV, Fojo T (1998) Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 58, 3331-3338.

Riss TL MR (1992) Comparison of MTT, XTT and a novel tetrazolium compound MTS for in vitro proliferation and chemosensitivity assays. Mol Biol Cell 3, 184.

Rudner AD, Murray AW (1996) The spindle assembly checkpoint. Curr Opin Cell Biol 8, 773-780.

Saunders C, Limbird LE (1997) Disruption of microtubules reveals two independent apical targeting mechanisms for G-protein-coupled receptors in polarized renal epithelial cells. J Biol Chem 272, 19035-19045.

Schmid-Alliana A, Menou L, Manie S, Schmid-Antomarchi H, Millet MA, Giuriato S, Ferrua B, Rossi B (1998) Microtubule integrity regulates src-like and extracellular signal-regulated kinase activities in human pro-monocytic cells. Importance for interleukin-1 production. J Biol Chem 273, 3394-3400.

Shingu T, Yamada K, Hara N, Moritake K, Osago H, Terashima M, Uemura T, Yamasaki T, Tsuchiya M (2003) Synergistic augmentation of antimicrotubule agent-induced cytotoxicity by a phosphoinositide 3-kinase inhibitor in human malignant glioma cells. Cancer Res 63, 4044-4047.

Shtil AA, Mandlekar S, Yu R, Walter RJ, Hagen K, Tan TH, Roninson IB, Kong AN (1999) Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells. Oncogene 18, 377-384.

Srivastava RK, Srivastava AR, Korsmeyer SJ, Nesterova M, Cho-Chung YS, Longo DL (1998) Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 18, 3509-3517.

Stone AA, Chambers TC (2000) Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells. Exp Cell Res 254, 110-119.

Subbaramaiah K, Hart JC, Norton L, Dannenberg AJ (2000) Microtubule-interfering agents stimulate the transcription of cyclooxygenase-2. Evidence for involvement of ERK1/2 AND p38 mitogen-activated protein kinase pathways. J Biol Chem 275, 14838-14845.

Tamura M, Gu J, Tran H, Yamada KM (1999) PTEN gene and integrin signaling in cancer. J Natl Cancer Inst 91, 1820-1828.

Vanhaesebroeck B, Alessi DR (2000) The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 346 Pt 3, 561-576.

Wall ME (1998) Camptothecin and taxol: discovery to clinic. Med Res Rev 18, 299-314.

Wang LG, Liu XM, Kreis W, Budman DR (1999) The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review. Cancer Chemother Pharmacol 44, 355-361.

Wang TH, Wang HS, Ichijo H, Giannakakou P, Foster JS, Fojo T, Wimalasena J (1998) Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. J Biol Chem 273, 4928-4936.

Ian F. Pollack