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Mol Cancer Ther. 2006;5:1176-1182
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation

Jason Ramos, Mint Sirisawad, Richard Miller and Louie Naumovski

Pharmacyclics, Inc., Sunnyvale, California

Requests for reprints: Louie Naumovski, Pharmacyclics, Inc., 995 Arques Avenue, Sunnyvale, CA 94085. Phone: 408-215-3450; Fax: 408-328-3689. E-mail: lnaumovski{at}pcyc.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress–related proteins. MGd treatment of the follicular lymphoma–derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma–derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased ~2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation. [Mol Cancer Ther 2006;5(5);1176–82]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Motexafin gadolinium (MGd) is an expanded porphyrin molecule that disrupts redox-dependent pathways by targeting oxidative stress–related proteins (1). MGd treatment of A549 lung cancer and Ramos lymphoma cells has been found to induce up-regulation of metallothionein and zinc transporter genes, which leads to disruption of zinc ion homeostasis and redox stress (1, 2). MGd contains the paramagnetic Gd3+ ion in its central cavity and is detectable by magnetic resonance imaging (3, 4). Preclinical studies have shown that MGd localizes to tumor cells and enhances the efficacy of radiation and chemotherapy in tissue culture and in animal tumor models (4–7). Tumor-selective localization has also been confirmed in both animal models and human clinical trials using magnetic resonance imaging (4, 8–11). MGd is now in several clinical trials for the evaluation of its efficacy as a single agent and in combination with chemotherapy or radiation therapy for cancer treatment (12, 13).

Akt/protein kinase B is a serine/threonine kinase that acts in cell survival pathways to suppress apoptosis (14). After growth factor or stress signaling including oxidative stress, Akt is recruited to the plasma membrane and activated by phosphorylation (14, 15). Akt functions as an antiapoptotic factor through numerous mechanisms, including phosphorylation and inactivation of several proapoptotic factors such as Bad and caspase-9 (14). Akt also acts in survival pathways by promoting glycolysis and maintaining a physiologic mitochondrial membrane potential (16). Consistent with studies in tissue culture cells using overexpression and dominant-negative models, thymocytes and mouse embryonic fibroblasts derived from mice carrying a homozygous disruption of the akt1 gene are more susceptible to apoptosis induced by a wide variety of agents (17). Akt is initially phosphorylated and activated in response to proapoptotic agents such as cisplatin, vincristine, and etoposide but levels of phosphorylated Akt (pAkt) and Akt decrease as cells begin to die (18, 19). The decrease may be due to a combination of cleavage of Akt by caspases, ubiquitination and degradation by the proteasome, and/or down-regulation of mRNA levels (20–22).

Because MGd can target redox pathways and Akt is redox regulated, we examined the effects of MGd on total Akt and pAkt in two lymphoma-derived cell lines. Levels of pAkt increased after MGd treatment in both lines, but only in the cell line in which MGd induced apoptosis did levels of total and pAkt drop before cell death. Treatment of both cell lines with inhibitors of Akt phosphorylation in combination with MGd resulted in synergistic cytotoxicity, suggesting that the increase in pAkt after treatment with MGd plays a protective role. Cotreatment of cells with MGd and celecoxib or docetaxel, drugs which inhibit Akt phosphorylation, resulted in synergistic cytotoxicity. Our data show that MGd results in increased phosphorylation of Akt and that MGd can be used in conjunction with Akt kinase inhibitors to achieve synergistic cytotoxicity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
MGd (as a stock solution of 2 mmol/L in aqueous 5% mannitol) was directly added to cultures to the concentrations specified in the text and figure legends. The inhibitors of Akt phosphorylation, SH-5 and Akt inhibitor 1, were from Calbiochem (San Diego, CA). Caspase inhibitor Quinoline-Valine-Aspartic-CH2-O-Phenyl (Q-VD-OPh) was from Enzyme System Products (Aurora, OH).

Cell Lines and Growth Conditions
The HF-1 cell line was obtained from Ronald Levy, M.D. (Stanford University, Stanford, CA). The HF-1 cell line was derived from a patient with follicular lymphoma (23). The Ramos cell line was derived from a patient with Burkitt's lymphoma. Cells were grown in RPMI 1640 with 10% fetal bovine serum in a 5% CO2/air incubator at 37°C. For assays determining growth inhibition or apoptosis, cells were plated and treated with various concentrations of MGd, as specified in the text and figure legends, before analysis. The concentrations of MGd used in this study have been achieved in clinical trials (8, 10).

Analysis of MGd-Treated Cells
Cell numbers were determined using a Model Z2 counter (Beckman-Coulter, Miami, FL). Annexin V binding and propidium iodide exclusion were assayed with a FACSCalibur instrument (Becton Dickinson, San Jose, CA) using reagents from BioVision (Mountain View, CA) per protocol of the manufacturer.

For drug combination studies, cytotoxicity was evaluated after 2 days of treatment with MGd and various drug combinations (as described in the figure legends) using Annexin V staining. The data were then analyzed using the CalcuSyn program (Biosoft, Ferguson, MO) to determine the combination index (CI). CI > 1 indicates antagonism, CI = 1 additivity, and CI < 1 synergy.

Caspase-3 activity was assayed using the EnzChek Caspase-3 Assay Kit #2, which monitors the cleavage of a DEVD substrate (Molecular Probes, Eugene, OR). This activity assay also detects activated caspase-6, caspase-7, caspase-8, and caspase-10. Cell lysates were analyzed according to the protocol of the manufacturer except that catalase was added at 10 units/mL in the lysis buffer to degrade hydrogen peroxide that might potentially be produced by MGd in vitro because hydrogen peroxide can inactivate caspase-3 (24). For each cell line, measured fluorescence levels were normalized to fluorescence levels of nontreated cell lysates.

Cellular uptake of MGd was measured by flow cytometry using a 650-nm long pass filter to detect MGd fluorescence emission (peak at 760 nm) after excitation at 488 nm (25). Cells were washed once in Hanks solution and a "live-cell" gate was drawn based on forward/side scatter properties to determine MGd uptake in viable cells.

Western Blotting for Caspase Activation and Substrate Cleavage
Cells were lysed in triple-detergent lysis buffer [50 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1.0% NP40, supplemented with 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 2 mmol/L ß-glycerophosphate, and the COMPLETE protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN)] on ice for 10 minutes. After centrifugation at 10,000 x g for 10 minutes, protein concentration was quantitated in the supernatant and equal quantities of protein were resolved on the appropriate percentage SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). Gels were transferred to polyvinylidene difluoride membrane using a Semi-Dry Transfer Cell (Bio-Rad) and Western blotting was done using primary and antimouse and antirabbit secondary antibodies conjugated to Alexa Fluor 680 (Molecular Probes) and IRdye800 (Rockland Immunochemicals, Gilbertsville, PA), respectively. Antibodies to caspases and poly(ADP-ribose) polymerase (Cell Signaling Technologies, Beverly, MA) recognized the full-length and cleaved forms of their respective antigens. Antibodies against the Akt proteins recognized either the phosphorylated serine-473 (pAkt) or total fraction (Akt) of the protein, respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). All membranes were blotted with an anti-Hsc70 (Santa Cruz Biotechnology) antibody to control for loading and transfer. Bands were imaged and quantified in the linear range and normalized to Hsc70 using the Odyssey Infrared Imaging System (LICOR, Lincoln, NE).

Statistical Analysis
Data in figures are from representative experiments done in triplicate. Data are presented as mean ± SD. Asterisks are used to represent values that are statistically significantly different than the corresponding controls at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of Growth and Induction of Apoptosis by MGd in Lymphoma Cell Line HF-1
HF-1 and Ramos cells were treated in vitro with 50 µmol/L MGd for 5 days and cell numbers, Annexin V–positive cells, caspase-3 activity, and MGd uptake were measured. Both cell lines showed a delay in cell growth (Fig. 1A ) but only HF-1 cells underwent apoptosis as shown by an increase in Annexin V–positive cells and caspase activity (Fig. 1B and C). Drug uptake (as measured by FL3 channel fluorescence) was similar in both lines (Fig. 1D).


Figure 1
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  		Figure 1. MGd inhibits growth in HF-1 and Ramos cells but only induces apoptosis in HF-1. Cell lines were treated at 25,000 cells/mL with 50 µmol/L MGd or mannitol control for 5 d before analysis. A, normalized growth; B, Annexin V–positive cells; C, normalized caspase-3 activity; D, MGd uptake as assessed by FL3 fluorescence. E and F, Western blot analysis showing cleavage of caspase-9 and caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) during a 5-d time course in HF-1 (E) but not in Ramos (F). Hsc70 served as a loading control.

 
Western blotting of cells treated with MGd for 0 to 5 days revealed progressive cleavage of caspase-9 and caspase-3 and the caspase substrate poly(ADP-ribose) polymerase in HF-1 cells but not in Ramos cells (Fig. 1E and F).

Treatment with MGd Results in Increased Levels of pAkt
Treatment of FL5.12, a non-transformed growth factor–dependent murine hematopoietic cell line, with chemotherapy drug etoposide, cisplatin, or vincristine results in an initial increase in pAkt levels followed by a decrease as cells die (19). To determine if MGd resulted in a similar pattern of Akt phosphorylation in HF-1 cells, we evaluated MGd (50 µmol/L) modulation of Akt phosphorylation from 30 minutes to 48 hours. MGd initially induced an increase in pAkt, followed by a decrease to below baseline levels before cell death (Fig. 2A ). Levels of total Akt increased as levels of pAkt decreased (Fig. 2A).


Figure 2
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  		Figure 2. Phosphorylation status of Akt in MGd-treated cells. A, total and pAkt in MGd-treated HF-1; B, caspase inhibitor Q-VD-OPh blocks decrease in pAkt after MGd treatment of HF-1 for 48 h; C, total and pAkt in MGd-treated Ramos. Cells were treated with 50 µmol/L MGd and samples were taken at the indicated time points between 30 min and 48 h. Western blots were probed with antibody to total Akt, pAkt, and Hsc70 as a loading control. Total Akt and pAkt levels were normalized to Hsc70.

 
Decreased levels of Akt have been attributed to caspase cleavage in apoptotic cells although distinct fragments are not detected by Western blotting because the decrease in Akt can be blocked by caspase inhibitors and Akt can be cleaved by caspases in vitro (20–22). We determined if apoptosis was responsible for the decrease in pAkt observed in MGd-treated HF-1. Q-VD-OPh, an irreversible caspase inhibitor that is less toxic and more potent than other caspase inhibitors, inhibits MGd-induced apoptosis in HF-1 (26, 27). HF-1 cells were either untreated or treated with MGd, Q-VD-OPh, or MGd, along with Q-VD-OPh, for 48 hours. Western blotting revealed that pAkt decreased in MGd-treated cells but not in MGd/Q-VD-OPh treated cells (Fig. 2B). Total Akt was also elevated in Q-VD-OPh–treated cells compared with control cells possibly because Q-VD-OPh blocks spontaneous apoptosis in HF-1 (27). These data suggest that caspases activated during apoptosis may be responsible, at least in part, for the decrease in pAkt levels in MGd-treated HF-1.

Ramos cells, which do not undergo MGd-induced apoptosis, were treated with 50 µmol/L MGd and examined from 30 minutes to 48 hours to determine if MGd modulated Akt phosphorylation in these cells. Levels of pAkt were elevated up to 2-fold by 4 hours and did not return to baseline within 48 hours (Fig. 2C). Levels of total Akt, although somewhat variable, remained close to baseline (Fig. 2C).

MGd and Inhibitors of Akt Kinase Phosphorylation Display Synergistic Cytotoxicity
The increase seen in pAkt in HF-1 and Ramos cells detected early after MGd treatment might have a cytoprotective role and decrease the cytotoxic effects of MGd. To determine if the increase in pAkt has a protective role, we used two different inhibitors of Akt phosphorylation in combination with MGd. Akt inhibitor 1 binds to the pleckstrin homology domain of Akt, disrupting the translocation of Akt to plasma membrane and its ability to be phosphorylated and activated by membrane-bound phosphatidylinositol-dependent kinases (28). SH-5 inhibits the phosphorylation of Akt by inhibiting the upstream kinase phosphatidylinositol-3 kinase (29). HF-1 cells were treated with various doses of MGd or Akt inhibitor 1 or combinations of the two. Although MGd treatment resulted in an increase in pAkt levels and Akt inhibitor 1 resulted in a small decrease in pAkt levels, the combination resulted in a significant drop in pAkt levels after 2 hours of treatment (Fig. 3A ). To determine if the combination of MGd and Akt inhibitor 1 had synergistic cytotoxicity, HF-1 cells were treated with MGd, Akt inhibitor 1, and combinations of the drugs for 48 hours and Annexin V–positive cells were enumerated (Fig. 3B). A CI < 1 was observed, indicating that the drug combinations resulted in synergistic cytotoxicity (Fig. 3C). HF-1 cells were also treated with MGd, SH-5, and combinations of the two and analyzed by Western blotting for pAkt and apoptosis. The combination of MGd and SH-5 resulted in suppression of pAkt levels greater than either drug alone and enhanced cytotoxicity (data not shown). Calculation of the CI based on the Annexin data using the Calcusyn program revealed a CI < 1, consistent with synergy cytotoxicity (Fig. 3D).


Figure 3
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  		Figure 3. MGd synergizes with Akt inhibitor 1, an inhibitor of Akt phosphorylation, to kill HF-1 cells. A, Western blot of Akt, pAkt, and Hsc70 in MGd, Akt inhibitor 1, or combination drug–treated HF-1 cells. Cells were treated with control, MGd, Akt inhibitor 1, or combination of the drugs at concentrations shown in the figure and then analyzed by Western blotting after 2 h. B, Annexin V–positive cells 48 h after treatment of HF-1 with MGd, Akt inhibitor 1, or combinations. C, combination index plot showing a CI < 1, indicating synergy for the MGd/Akt inhibitor 1 combinations. D, combination index plot showing a CI < 1, indicating synergy for Annexin V values from MGd/SH-5 combinations. The following concentrations were used for the MGd/SH-5 combination: 25 µmol/L/2.5 µmol/L and 50 µmol/L/5 µmol/L.

 
To determine if inhibition of Akt phosphorylation was also synergistic with MGd in Ramos cells, we treated Ramos cells with various concentrations of MGd and Akt inhibitor 1, individually and in various combinations. After 2 hours of drug exposure, Western blotting showed that MGd alone resulted in slightly increased pAkt levels whereas Akt inhibitor 1 showed a dose-dependent decrease in pAkt levels (Fig. 4A ). Combined treatment with MGd and Akt inhibitor 1 resulted in even further suppression of pAkt levels (Fig. 4A). To determine if the combination of MGd and Akt inhibitor 1 had synergistic cytotoxicity, Ramos cells were treated with MGd, Akt inhibitor 1, and various combinations of the drugs for 48 hours and Annexin V–positive cells were enumerated (Fig. 4B). A CI < 1 was observed, indicating that the drug combinations resulted in synergistic cytotoxicity (Fig. 4C). Ramos cells were also treated with MGd, SH-5, and combinations of the two and analyzed by Western blotting for pAkt and apoptosis. The combination of MGd and SH-5 resulted in suppression of pAkt levels greater than either drug alone and enhanced cytotoxicity (data not shown). Calculation of the CI based on the Annexin data using the Calcusyn program revealed a CI < 1, consistent with synergy cytotoxicity (Fig. 4D). These studies show cytotoxic synergy between MGd and inhibition of Akt phosphorylation in both HF-1 and Ramos cells.


Figure 4
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  		Figure 4. MGd synergizes with Akt inhibitor 1, an inhibitor of Akt phosphorylation, to kill Ramos cells. A, Western blot of Akt, pAkt, and Hsc70 in MGd, Akt inhibitor 1, or combination drug–treated Ramos cells. Cells were treated with control, MGd, Akt inhibitor 1, or combinations of the drugs for 2 h and then analyzed by Western blotting. B, Annexin V–positive cells after treatment of Ramos cells with MGd, Akt inhibitor 1, or combinations for 48 h. C, combination index plot showing a CI < 1, indicating synergy for the MGd/Akt inhibitor 1 combinations in Ramos cells. D, combination index plot showing a CI < 1, indicating synergy for Annexin V values from MGd/SH-5 combinations in Ramos cells. The following concentrations were used for the MGd/SH-5 combination: 25 µmol/L/10 µmol/L and 50 µmol/L/20 µmol/L.

 
Synergistic Activity of MGd with Celecoxib or Docetaxel Correlates with Inhibition of Akt Phosphorylation
We hypothesized that MGd might synergize with other drugs that modulate pAkt. Celecoxib, a specific cyclooxygenase-2 inhibitor, has also been reported to inhibit the phosphorylation of Akt kinase (30–33). To determine if MGd or celecoxib perturbed pAkt levels, we treated Ramos cells with each drug or the combination for 2, 24, and 48 hours and probed for total Akt and pAkt. We noted a time-dependent decrease in the levels of pAkt in the combination treated cells (Fig. 5A-C ). With the combination of MGd and celecoxib, a small but reproducible difference was seen in levels of pAkt even at 2 hours, suggesting that this is an early event (Fig. 5A). The decrease in pAkt levels was associated with synergistic increases in cell death as shown by enhanced caspase-3 activity and Annexin V–positive cells in the MGd plus celecoxib–treated cells at 72 hours (Fig. 5D and E).


Figure 5
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  		Figure 5. MGd synergizes with celecoxib, a cyclooxygenase-2 inhibitor that also inhibits Akt phosphorylation, to kill Ramos cells. Ramos cells were treated with vehicle or MGd (50 µmol/L), celecoxib (25 µmol/L), or MGd/celecoxib for various times. Western blot analysis was with anti-Akt, anti-pAkt, and anti-Hsc70 antibodies at 2 (A), 24 (B), and 48 (C) h. D, normalized caspase-3 activity. E, Annexin V–positive cells. Total Akt and pAkt levels were normalized to Hsc70.

 
It is known that Akt mediates resistance to antimicrotubule agents and inhibition of Akt signaling results in sensitization to antimicrotubule agents (19, 34–36). We treated Ramos cells with various concentrations of MGd or the antimicrotubule agent docetaxel, separately and in combination, and assayed for total Akt and pAkt after 2 hours. Neither MGd nor docetaxel influenced levels of total Akt, but MGd resulted in a 25% to 50% increase in pAkt whereas docetaxel (50 and 75 nmol/L) resulted in decreased pAkt levels to ~50% of normal (Fig. 6A ). The combination of the two drugs (MGd 50 µmol/L and docetaxel 50 nmol/L) resulted in a dramatic decrease in pAkt levels to ~20% of normal (Fig. 6A). Cell death, determined by measuring Annexin V–positive cells at 48 hours, showed that the combination therapy killed substantially more cells than either agent alone (Fig. 6B). The combination of MGd and docetaxel was synergistic as indicated by CI < 1 (Fig. 6C).


Figure 6
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  		Figure 6. MGd synergizes with docetaxel, an antimicrotubule agent that inhibits Akt phosphorylation, to kill Ramos cells. A, Western blot of Akt, pAkt, and Hsc70 in MGd, docetaxel, or combination-treated Ramos. Cells were treated with control, MGd, docetaxel, or combinations of the drugs for 2 h and then analyzed by Western blotting. B, Annexin V–positive cells after treatment of Ramos cells with MGd, docetaxel, or combinations for 48 h. C, combination index plot from the Annexin V data showing a CI < 1, indicative of synergistic cytotoxicity between MGd and docetaxel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HF-1 and Ramos lymphoma cell lines both respond to MGd treatment with growth inhibition, but additionally, HF-1 cells also undergo apoptosis as shown by increased number of Annexin V–positive cells, caspase-3 activation, cleavage of caspase-9 and caspase-3 and the substrate poly(ADP-ribose) polymerase. Growth suppression in Ramos was not due to necrosis or a block in any specific phase of the cell cycle.1 Differences in drug uptake (as measured by FL3 fluorescence) do not explain the cellular responses because uptake was similar in each cell line.

In an effort to better understand the mechanisms that might be involved, we evaluated MGd-treated cells for phosphorylation of Akt, a kinase that is redox regulated and important in metabolism, survival, and apoptosis (14). In the HF-1 cell line in which MGd induces apoptosis and suppresses growth, treatment with MGd (30 minutes–48 hours) resulted in an initial increase in levels of pAkt but after 4 hours resulted in a decrease. The decrease in the levels of pAkt may be related to MGd-induced apoptosis in HF-1 because it could be blocked by caspase inhibition. Because Akt functions in survival pathways, the transient increase in pAkt levels during MGd treatment may represent a cellular adaptive response that could attenuate MGd-induced apoptosis in HF-1.

In the Ramos cell line in which MGd does not induce apoptosis but suppresses growth, MGd treatment resulted in an increase in pAkt levels by 30 minutes, which persistently remained elevated up to 48 hours. These results suggest that the persistent elevation of pAkt may account for the ability of the Ramos cell line to resist the proapoptotic effects of MGd. Therefore, we hypothesized that inhibitors of Akt phosphorylation (Akt inhibitor 1 and SH-5) would sensitize cells to the proapoptotic effects of MGd. For both HF-1 and Ramos cells, specific inhibitors of Akt phosphorylation suppressed the increase in pAkt in MGd-treated cells and synergistically enhanced cell killing by MGd as measured by Annexin V binding. These data suggest that Akt promotes resistance to MGd-induced apoptosis and show that MGd activity is enhanced by inhibition of Akt phosphorylation.

Based on the studies presented here with MGd and Akt inhibitors, we further hypothesized that MGd might also synergize with other drugs that down-regulate pAkt levels such as celecoxib (a cyclooxygenase-2 inhibitor) or docetaxel (an antimicrotubule agent). The ability of celecoxib to induce growth inhibition and apoptosis is most likely an "off-target" effect that may arise, at least in part, through inhibition of Akt phosphorylation via inhibition of 3-phosphoinosotide-dependent kinase 1, which seems to be a direct target of celecoxib (30, 32). We found that pAkt levels were decreased in MGd/celecoxib–treated Ramos cells and that this correlated with enhanced cell killing.

The cytotoxic activity of antimicrotubule agents, including docetaxel, is known to be mediated by the Akt pathway (19, 34–36). The combination of MGd with docetaxel resulted in a dramatic decrease in pAkt levels with synergistic cytotoxicity. This result is particularly interesting in view of the recent clinical data from Pandya and Phan (37), which report very promising tumor responses in patients with recurrent non–small-cell lung cancer treated with MGd and docetaxel. These responses were seen even in patients who had failed prior treatment with taxanes (37).

Our results suggest an important role for the Akt pathway in the response of cells to MGd. MGd treatment as a single agent results in an increase in pAkt levels that seem to be protective because MGd-induced cytotoxicity can be enhanced in combination with specific inhibitors of Akt phosphorylation. Additionally, synergistic cytotoxicity of MGd with celecoxib or docetaxel is associated with a decrease in pAkt levels, further highlighting the importance of the Akt pathway in modulating MGd function. Precisely how MGd modulates pAkt levels is currently unclear and under investigation but it may be through induction of oxidative stress, which can enhance the activity of kinases that phosphorylate Akt and attenuate the activity of the Akt phosphatase PTEN (38–42). Similar to what we have shown with MGd, up-regulation of pAkt by other agents that induce redox stress serves a protective role because abrogation of the response with inhibitors of Akt phosphorylation results in enhanced cell death (39, 41, 42). As expected for a protein that functions in a survival pathway, expression of constitutively activated myristoylated Akt or v-Akt inhibits hydrogen peroxide–induced cell death (42). The results reported here provide valuable insights for maximizing the therapeutic potential of MGd when used in combination with chemotherapy drugs that affect the phosphorylation status of Akt. Clinical trials based on these observations are in progress.


   Footnotes
 
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.

1 Unpublished observations. Back

Received 7/25/05; revised 2/ 1/06; accepted 3/ 6/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. A Dennis
Targeting Akt in Cancer: Promise, Progress, and Potential Pitfalls
Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 25 - 35.
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