Molecular Cancer Therapeutics Molecular Diagnostics in Cancer Therapeutic Development: Fulfilling the Promise of Personalized Medicine Tumor Immunology: New Perspectives
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by di Palma, A.
Right arrow Articles by Ricchi, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by di Palma, A.
Right arrow Articles by Ricchi, P.
Mol Cancer Ther. 2006;5:1318-1324
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Aspirin reduces the outcome of anticancer therapy in Meth A–bearing mice through activation of AKT-glycogen synthase kinase signaling

Antonella di Palma1, Giuseppe Matarese2, Vincenza Leone1, Tiziana Di Matola1, Fabio Acquaviva1, Angela Maria Acquaviva1 and Paolo Ricchi1

1 Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" and 2 Istituto di Endocrinologia ed Oncologia Sperimentale "G. Salvatore" del Consiglio Nazionale delle Ricerche, Università "Federico II," Naples, Italy

Requests for reprints: Angela Maria Acquaviva, Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Università "Federico II," via S. Pansini 5, 80131 Naples, Italy. Phone: 39-81-7463044; Fax: 39-81-7463252. E-mail: angacqua{at}unina.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aspirin displays, at millimolar concentrations, several mechanisms independent from its ability to inhibit cyclooxygenases. Occasionally, the mechanisms displayed in vitro have been clearly related to an effect of clinical relevance in vivo. An expanding literature has been focusing on the cytoprotective effect of aspirin in neurodegenerative disorders and the activation of AKT pathway in neuroprotection and induction of resistance to anticancer drugs. In this work, we tested the ability of aspirin to activate the AKT survival pathway in methylcholanthrene-induced fibrosarcoma cells (Meth A) transplanted into BALB/c nude mice and the clinical effect of aspirin cotreatment during etoposide (VP-16)–based anticancer therapy. We found that cotreatment with aspirin reduced VP-16-induced apoptosis and activated AKT in vitro and in vivo. In Meth A–bearing mice, aspirin administration also activated glycogen synthase kinase-3 and reduced the activity and the efficacy of anticancer therapy in VP-16 cotreated animals. Our data suggest that the antiapoptotic effect of aspirin operates in vivo through the activation of AKT-glycogen synthase kinase pathway causing a decrease in the outcome of VP-16-based therapy. These findings could have clinical relevance in treatment of human malignancies. [Mol Cancer Ther 2006;5(5):1318–24]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aspirin, a widely used nonsteroidal anti-inflammatory drug, is a nonselective inhibitor of cyclooxygenase (COX)-1 and COX-2 (1). High doses of salicylates (4–10 g/d), including sodium salicylate and aspirin, have been used to treat inflammatory conditions, such as rheumatic fever and rheumatoid arthritis. There are several actions displayed by high doses of aspirin independent from its ability to inhibit COXs. These include the following: (a) inhibition of the activation of nuclear factor-{kappa}B by prevention of the phosphorylation and degradation of the inhibitory subunit I{kappa}B (2), (b) modulation of gene transcription (3), and (c) modulation of several protein kinases and other molecular signaling pathways (4, 5). Several effects have been shown in vitro: aspirin treatment inhibits cell growth (6–8) and protects against a variety of toxic stimuli, such as H2O2, free radicals, hypoxia, and chemical toxins (9–15). Occasionally, the wide spectrum of molecular mechanisms and targets displayed in vitro has been clearly related to an effect with clinical relevance in vivo. For instance, high doses of salicylates can lower blood glucose concentrations by reducing signaling of the I{kappa}B kinase pathway (16). In addition, in an in vivo model of focal brain ischemia, aspirin has been recently found to be neuroprotective by inhibiting the sustained activation of extracellular signal-regulated kinase 1/2 pathway (17).

We have reported previously that millimolar doses of aspirin protected Caco-2 cells from apoptosis both induced by anticancer drugs treatment and serum deprivation (18, 19). We showed that these effects were determined by the ability of aspirin to activate dose-dependently the phosphatidylinositol 3-kinase (PI3K)/AKT signal transduction survival pathway in Caco-2 cells (19). In this study, we aimed to test the ability of aspirin to activate PI3K/AKT pathway in vitro and in vivo in methylcholanthrene-induced fibrosarcoma cells (Meth A) transplanted into BALB/c nude mice. We found that aspirin treatment activates AKT also in vivo, thus reducing the effect of anticancer drug and the survival of transplanted mice cotreated with aspirin with respect to animals treated with anticancer drugs alone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vitro Studies
Cell Growth and Culture. The transplantable sarcoma Meth A cells were kindly provided from Dr. Pramod Srivastava. Original tumor cell lines were induced by a single s.c. injection of 0.1 mg 3-methylcholantrene dissolved in 0.1 mL sesame oil. Meth A cells have a well-defined immunogenicity and typically produce either solid tumors or ascites when injected s.c. or i.p., respectively (20, 21). The cells were maintained by continuous propagation in the peritoneal cavity of BALB/c mice (6–7 weeks old). In addition, Meth A cells can routinely grown in 100-mm plastic dishes at 37°C in a humidified atmosphere of 5% CO2 in air in DMEM supplemented with 10% FCS, glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 µg/mL) and buffered with HEPES (20 mmol/L).

All experiments in vitro were done after one passage from peritoneal cavity. Aspirin (Sigma, Milan, Italy) was prepared as a 1 g/50 dL stock solution (111 mmol/L). The solution was buffered with Tris to obtain the final pH equal to that of control DMEM and prepared weekly; indomethacin (Sigma) was dissolved in DMSO and prepared as a 50 mmol/L stock solution. Etoposide (VP-16; 2 mg/mL) was obtained from Teva Pharma B.V. (Mijdrecht, the Netherlands) and prepared in its clinical formulation. 5,5-Dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU) was kindly provided by Merck Frosst (Kirkland, Quebec, Canada), dissolved in DMSO, and prepared as 10 mmol/L stock solution.

Apoptosis Detection. Apoptosis was evaluated by using Annexin V-FITC staining technique. Briefly, Meth A cells were collected and Annexin V-FITC stained by using a detection kit from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan) according to the manufacturer's instructions. Fluorescence analysis was done by a flow cytometer apparatus (Becton & Dickinson, Mountain View, CA) and the CellQuest analysis software. For each sample, at least 30,000 events were stored. Quadrant settings were based on the negative control. Each experiment was repeated at least thrice.

Western Blot Analysis. Phosphorylated AKT and total AKT were detected by using rabbit polyclonal antibodies from Cell Signaling Technology, Danvers, MA. Phosphorylated glycogen synthase kinase (GSK)-3{alpha}/ß (Ser21/Ser9) and GSK-3ß were detected by using rabbit polyclonal antibody and mouse monoclonal antibody from Cell Signalling. Cells were washed in cold PBS and lysed for 10 minutes at 4°C with 1 mL lysis buffer [50 mmol/L Tris (pH 7.4), 0.5% NP40, 0.01% SDS] containing complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatases inhibitor (1 mmol/L orthovanadate). Lysates were centrifuged at 12,000 x g for 15 minutes at 4°C and the supernatant was collected. Protein concentration in cell lysates was determined by Bio-Rad protein assay (Bio-Rad, Richmond CA), and 50 µg total protein from each sample was analyzed. Proteins were separated by a 12% SDS-PAGE and transferred on nitrocellulose membrane (Hybond-ECL nitrocellulose, Amsherman, Rainham, United Kingdom). After incubation with horseradish peroxidase–conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad) diluted 1:2,000 in PBS-0.2% Tween, the membranes were washed and protein bands were detected by an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Relative intensities of bands were quantified by densitometry with a desk scanner (Pharmacia Discovery System, Amersham Pharmacia Biotech) and RFLPrint software (PDI, New York, NY).

In vivo Studies
Female BALB/c mice (6–7 weeks old) were purchased from Charles River Laboratories (Milan, Italy). The research protocol was approved and mice were maintained in accordance to institutional guidelines of the University of Naples Animal Care and Use Committee. Mice were acclimatized at the University of Naples Medical School Animal Facility for 1 week before injection with cancer cells. All these animals received ~1 million Meth A tumor cells in 0.2 mL saline on day 0. VP-16 was used at 4 mg/kg, and aspirin was used at 15, 30, and 45 mg/kg. After 5 days, when established ascites were evident and animal weight started to increase, seven mice per group were treated i.p. at the indicated doses of aspirin alone or in combination with VP-16. Drugs were given (on days 1–3) for 3 days every 24 hours. Control group was injected with vehicle. All animals were injected i.p. with an equal volume of solvent as a control (300 µL). Then, mice were randomly divided into seven groups [1 control group, 1 VP-16-treated group, 2 aspirin-treated groups (15 and 45 mg/kg), and 3 aspirin and VP-16 cotreated groups], with each group containing seven animals. Of each group, six animals were observed daily to monitor their survival and their weight increase and one animal was killed 4 hours after completing treatment; ascitic fluid containing tumor cells was collected in PBS with proteases and phosphatases inhibitors and processed as described in Western Blot Analysis.

Statistical Analysis
Statistical comparisons were done using the Mann-Whitney U test and log-rank test. P < 0.05 was considered a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Aspirin Cotreatment on Anticancer Drug Responsiveness in Meth A Cells
Meth A cells were exposed for 72 hours at different concentrations of VP-16 and aspirin alone and in combination. Aspirin was used at 1, 2, and 3 mmol/L and VP-16 was used at 34 and 68 µmol/L. Apoptosis was evaluated through the analysis of cells positive for Annexin V staining at flow cytometry. Spontaneous apoptosis of Meth A cells in culture medium was in the average 10%. Treatment with aspirin up to 3 mmol/L concentration did not increase significantly apoptosis in Meth A cells with respect to basal level (Fig. 1, top ). The percentage of apoptosis increased up to 11% following treatment with 5 mmol/L aspirin (data not shown). Conversely, VP-16 at 34 and 68 µmol/L dose-dependently increased apoptosis from 26% to 46% (Fig. 1, top). In addition, the levels of apoptosis were significantly reduced (P < 0.05) for each concentration of VP-16 when given in the presence of all concentration of aspirin compared with single-agent treatment; Meth A cells cotreated with aspirin at 3 mmol/L were particularly resistant to VP-16-induced apoptosis (P < 0.002). Similar results were obtained when levels of apoptosis were evaluated with propidium iodide staining analysis (data not shown). These results indicated that VP-16 was an active anticancer agent in Meth A cells and that aspirin treatment reduced the ability of the drug to induce apoptosis.


Figure 1
View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. A, effect of 1 mmol/L (A1), 2 mmol/L (A2), and 3 mmol/L (A3) aspirin and 34 µmol/L (Vp34) and 68 µmol/L (Vp68) VP-16 treatments on apoptosis in Meth A cells. Cells were incubated with aspirin and VP-16 alone or in combination at the indicated concentrations for 72 h. B, effect of Indomethacin (50 and 100 µmol/L) and DFU (0,1, and 0.5 µmol/L) and VP (34 µmol/L). Cells were incubated with Indomethacin and DFU and VP34 alone and in combination for 72 h. Apoptosis was calculated as the percentage of cells positive at Annexin V-FITC as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD.

 
To evaluate whether the effects of aspirin cotreatment on anticancer drug responsiveness were also obtainable toward others anticancer agents, we tested the effect of aspirin treatment on cisplatin-induced apoptosis. Meth A cells were cotreated with aspirin at 1, 2, and 3 mmol/L and cisplatin at 2, 5, 10, and 20 µmol/L under the above-mentioned experimental conditions. Aspirin cotreatment dose-dependently reduced cisplatin-induced apoptosis for each concentration of cisplatin tested. The effect of aspirin on apoptosis was of the same range of magnitude of that observed in VP-16 and aspirin cotreated Meth A cells (data not shown).

To evaluate whether the cytoprotective effects of aspirin treatment on Meth A cells were dependent on the inhibition of COXs activity, we analyzed the effect of VP-16 cotreatments with DFU, a COX-2 selective inhibitor, and with indomethacin, a nonsteroidal anti-inflammatory drug that shows a COX-1/COX-2 ratio called the inhibition of COX isoform similar to that of aspirin. We used two different concentration of indomethacin and DFU both at doses well recognized to inhibit COX-1 and/or COX-2. Neither indomethacin at 50 and 100 mol/L nor DFU at 0.1 and 0.5 mol/L, respectively, reduced VP-16-induced apoptosis in Meth A cells (Fig. 1, bottom). These results, therefore, indicated that the cytoprotective action was unique to aspirin and was not shared by indomethacin.

Aspirin Treatment Activates AKT Protein
To show that aspirin-induced cytoprotective effect correlated with the activation of AKT in Meth A cells, we assayed AKT activation status following VP-16 treatment in the presence or absence of aspirin.

Lysates from cells treated with 1, 2, and 3 mmol/L aspirin alone or in the presence of VP-16 at 34 µmol/L for 72 hours were probed for phosphorylated AKT at Ser473. The bands were quantified and normalized to total AKT protein kinases, respectively.

As shown in Fig. 2 , aspirin treatment, but not VP-16 treatment, induced activation of AKT protein with respect to control cells. The relative densitometric analysis showed 7.4-, 9.5-, and 6.9-fold increase for AKT protein phosphorylation status following aspirin treatment at 1, 2, and 3 mmol/L, respectively (Fig. 2). Moreover, phosphorylation status of AKT protein were 2-, 3.5-, and 4.2-fold increased also in cells cotreated with 1, 2, and 3 mmol/L aspirin and VP-16, respectively, compared with untreated cells.


Figure 2
View larger version (47K):
[in this window]
[in a new window]
  		Figure 2. Effect of 1, 2, and 3 mmol/L aspirin and 34 µmol/L VP-16 treatment alone and in combination on (Ser437) phosphorylated AKT and total AKT expression in Meth A cells. Western blot analysis was done on protein lysates from cells cultured as described in Fig. 1. The immunoblots were stripped and reblotted with antibodies against total AKT. One of three separate experiments yielded similar results.

 
To further confirm the involvement of PI3K/AKT survival pathway, we evaluated the effect of the PI3K inhibitor LY294002 on the activation of AKT in aspirin-treated and cotreated Meth A cells. The use of LY294002 suppressed the activation induced by aspirin, restoring the basal level of AKT activation status (data not shown).

Effect of Aspirin Cotreatment on Anticancer Drug Responsiveness in Meth A–Bearing Mice
To investigate whether our in vitro finding occurred also in vivo, we evaluated the effect of aspirin and VP-16 treatments in nude mice injected i.p. with Meth A tumor cells.

BALB/c mice average starting weight was 24.75 g. Because the rapid growth of Meth A tumor cells induced ascites in control group, weight gain was used to monitor tumor burden in the animals. Figure 3 shows the percentage of increase in body weight with respect to initial weight in control group; after 5 days, when Meth A cells induced visible ascites, and after 14 days from the inoculum, an average 10% and 53% increase (38.2 g mean weight; data not shown) in weight was reached, respectively (Fig. 3). Later, surviving animals reaching a size not compatible with normal life and suffering for the great ascites developed were killed for bioethical reasons.


Figure 3
View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Effect of Meth A cells injection on body weight gain of BALB/c mice. Y axis, percentage of weight increase with respect to animal weight recorded at day 1 of inoculum. Columns, mean of triplicate experiments; bars, SD.

 
Treatments started after 5 days from inoculum; animals (six for each group) were treated i.p. with either aspirin (15 and 45 mg/kg) and VP-16 (4 mg/kg) alone or in combination with aspirin (at the dose of 15, 30, and 45 mg/kg) or with vehicle (control group). In case of coadministration, to mimic experimental condition obtained in vitro, aspirin and VP-16 were simultaneously injected at different sides of peritoneum.

During the first 7 days of treatment (days 1–7), when animal death was not yet observed, follow-up was based on the calculation of the percentage of increase in animal weight with respect to first day of treatment (Fig. 4 ); during the subsequent days (days 8–32), the effects of different treatments were evaluated recording animal survival and were displayed with Kaplan-Meier curves (Fig. 5 ).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. Effect of aspirin treatment at 15, 30, and 45 mg/kg and VP-16 (Vp) treatment (4 mg/kg) alone and in combination on percentage of increase in body weight. The percentage was calculated with respect to animal body weight recorded at day 1 of treatment. Treatments started after 5 d from i.p. injection of 2 x 106 Meth A sarcoma cells and body weight gain was monitored for 7 d. Points, mean of triplicate experiments; bars, SD. Representative experiment. Two separate experiments yielded similar results. Mann-Whitney U test was used to compare increase in body weight (%) at day 7 among different treatment groups.

 

Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Effect of VP-16 and aspirin treatment on survival of Meth A tumor-bearing mice. Five days following i.p. injection of 2 x 106 Meth A sarcoma cells, groups of mice were treated with aspirin at 15, 30, and 45 mg/kg or VP-16 at 4 mg/kg alone and in combination. Proportion of surviving animals in each group after 7 d from treatment as a function of days post-tumor challenge. The decrease in survival of aspirin at 30 and 45 mg/kg and VP-16 cotreated mice over VP-16-treated group was statistically significant at days 12 and 15, respectively, as determined by the log-rank test. Representative experiment. Two separate experiments yielded similar results.

 
Treatment with VP-16 completely arrested tumor growth and ascites generation, and the suppression of Meth A tumor cell growth was associated with an ~2% of reduction in body weight at days 5 to 7 (Fig. 4).

After 8 days from VP-16 treatment, 33.3% of animals died for causes not directly linked with weight increase (bleeding ascites and cachexia). In other 50% of animals, after a variable lag time from treatment, Meth A cells resumed a growth rate comparable with control, and animals were harvested for bioethical reason when they reached an average weight of 35 g (data not shown). In remaining 16.6% of animals, no tumor growth was detected until 25 days of observation (Fig. 5) and they remained tumor free for a period of observation of 40 days after treatment (data not shown). Therefore, although VP-16 treatment was not able to definitively cure almost all animals, tumor growth was arrested and mouse survival duration was significantly increased with respect to control (P < 0.001) for all the duration of follow-up.

Aspirin treatment as single agent at 15 and 45 mg/kg had no effect on animal body weight and survival with respect to the control group (Figs. 4 and 5); no precocious mortality for gastric toxicity was observed in the aspirin-treated groups. The combination of aspirin at 45 mg/kg and VP-16 did not significantly inhibit xenograft ascitic growth and did not produce any effects on animal survival compared with VP-16-treated group (P < 0.001), displaying a trend similar to control group (Figs. 4 and 5). These data suggest that the highest dose of aspirin abolished the activity and the efficacy of VP-16 in tumor-bearing mice.

The combination of aspirin at 30 mg/kg and VP-16 produced an increase in body weight of a lesser extent compared with that observed in control group. However, a more rapid increase in weight gain after 5 days of treatment was observed in cotreated animal when compared with animal treated with VP-16 alone (P < 0.002 at day 7; Fig. 4). After this lag of time, a statistically significant difference in survival was observed between these two groups of animals (Fig. 5); in fact, in group of cotreated animals, 33% of the animals died for causes not directly linked with weight increase, and after day 15 from treatment, no animal survived (Fig. 5). These results indicate that aspirin cotreatment at 30 mg/kg attenuates the activity and the efficacy of VP-16 although at a lesser extent than cotreatment at 45 mg/kg. Finally, in animals treated with the combination of aspirin at 15 mg/kg and VP-16, the body weight reduction was slightly minor (P < 0.02) compared with that observed in animal treated with VP-16 alone (Fig. 4). Similarly, a minimal but not statistically significant difference in survival probability was observed (Fig. 5). These results suggest that 15 mg/kg aspirin is able to reduce the activity but not the efficacy of VP-16-based therapy.

Aspirin Treatment Activates AKT-GSK Survival Pathway in Meth A–Bearing Mice
To analyze the molecular pathways associated with aspirin treatment in Meth A–bearing mice, one animal was harvested 4 hours after completing treatment and ascitic fluid containing tumor cells from previously considered experimental points was collected for biochemical evaluation. Consistently with our in vitro finding, treatment with aspirin at 15 and 45 mg/kg alone determined a 3.8- and 6.5-fold increase in activation status of AKT compared with control group, respectively (Fig. 6 ). In addition, aspirin cotreated groups showed higher levels of activation of AKT with respect to vehicle-treated group (Fig. 6).


Figure 6
View larger version (30K):
[in this window]
[in a new window]
  		Figure 6. A, effect of aspirin treatment (15 and 45 mg/kg) and VP-16 (4 mg/kg) alone and in combination on phosphorylated AKT and total AKT expression. Western blot analysis was done on protein lysates from ascitic cells collected from BALB/c mice 4 h after the end of each treatment. Immunoblots were stripped and reblotted with antibody against total AKT protein. B, effect of aspirin treatment (15 and 45 mg/kg) and VP-16 (4 mg/kg) alone and in combination on phosphorylated Ser9 GSK-3ß. The immunoblots were stripped and reblotted with antibody against total GSK-3 protein. Representative experiment. Three separate experiments yielded similar results.

 
Between several downstream targets of AKT, GSK-3 represents a convergence site of multiple signaling pathways involved in cell fate (22, 23). Phosphorylation at Ser21 (-isoform) or Ser9 (-isoform) of GSK-3ß by AKT leads to inhibition of its activity and reduces apoptosis. Inactivation of GSK-3 by AKT may thus contribute to antiapoptotic effects of PI3K/AKT signaling (23). Therefore, we evaluated the effect of different treatments on activation status of GSK-3.

As shown in Fig. 6B, phosphorylation status of GSK-3 in Ser9 was increased in aspirin-treated mice. The effect was of lesser extent with respect to that observed for AKT phosphorylation and persisted, at 45 mg/kg aspirin treatment, also in the presence of VP-16. These results indicate that aspirin activates the AKT-dependent survival pathway leading to increased phosphorylation of GSK-3 at Ser9.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Accumulating evidence suggests that wide spectrum of pharmacologic effect of aspirin might not only be dependent on COX inhibition. The mechanisms of action by which the drug exert its effect is still a matter of debate. Numerous pharmacologic and biochemical effects have been described. In fact, aspirin may target distinct molecular pathways in cells; however, these effects are often cell type specific and sometimes produces opposite effects (24).

An expanding literature has been focusing on the mechanism of action of aspirin as cytoprotective agent in neurodegenerative disorders. Aspirin and its metabolites have been shown to protect against cellular injury in neuronal and nonneuronal systems by preventing nuclear factor-{kappa}B nuclear translocation. More recently, the AKT pathway has been involved in neuroprotection in vivo and induction of resistance to anticancer drugs (25–28). In particular, several investigators have been focused on the involvement of AKT pathway in multidrug resistance (29). Although there is a great number of studies on the ability of aspirin to protect against toxic stimuli, there are no reports on the effect of aspirin on AKT survival pathway in vivo. We found previously the involvement of PI3K/AKT pathway in cytoprotective property of aspirin in cultured colon cancer Caco-2 cells (19). In this study, we used the fibrosarcoma Meth A cells as a system to evaluate aspirin interference with anticancer drug-induced apoptosis in vitro and in vivo. Our data showed for the first time that the antiapoptotic effects of aspirin are not cell type specific and operated in vivo through the activation of PI3K/AKT pathway. Our data showed that aspirin exposure, but not other COX-2 selective and nonselective inhibitors, prevented apoptosis induced by VP-16 and cisplatin in Meth A cells.

The in vitro data were consistent with previous results showing that aspirin protected cells from apoptosis through activation of PI3K/AKT. Because the AKT protein is the downstream effector of PI3K, further studies are needed to clarify at molecular levels the site(s) of aspirin interference with the PI3K survival pathway and the mechanism(s) responsible for PI3K activation. Preliminary data from our group indicate that aspirin is able to induce acetylation status of p85 regulative subunit of PI3K. It is possible to postulate that the structural change by acetylation increase the level of PIP3, as shown previously (19), and induce activation of PDK1 kinase.

Furthermore, the in vitro data reinforce the evidence coming from our and other previous experiments (6, 18, 19, 24) that high doses of aspirin slightly affected or may increase basal level of apoptosis but were protective from apoptosis induced by external toxic stimuli. This at the molecular level confirms the aspirin pleiotropic activity and suggests that the aspirin interference with different survival/death pathways determines a global prosurvival promoting effect.

One of the major purposes of this study was to evaluate whether combined treatment in vivo would produce effects that could be relevant in the clinical use. Our data indicated that aspirin as single agent did not affect Meth A cell growth while provided evidence that aspirin administration reduced in vivo the activity and the efficacy of VP-16 treatment in Meth A–bearing mice. Our data indicated also that in vivo aspirin operated in a dose-response fashion to reduce the outcome of anticancer treatment. This trend was evident behind the lowest dose and indicated that coadministration of minimum 30 mg/kg aspirin was required to produce a significant effect on animal survival. Furthermore, aspirin-induced increase of animal body weight and decrease of survival strictly correlates with the activation of AKT. Our results agree with previous data showing that AKT activation in vivo is a major factor in the resistance of tumor cells to apoptosis induced by chemotherapeutic drugs (29).

In addition, our in vivo data indicated that aspirin inhibited the activity of GSK-3, a key factor not only for survival and chemoresistance (30) but also for the attenuation of the cellular action of insulin (31). Because it has been shown that high-dose aspirin could improve glucose metabolism in humans (16) and that GSK-3 activity inhibitors may therapeutically benefit in treating insulin resistance and type 2 diabetes (32), the data of this report should encourage further evaluation of the in vivo mechanism for aspirin hypoglycemic effect.

Although an expanding literature is showing the role of nonsteroidal anti-inflammatory drugs used alone or in combination with conventional anticancer drugs and their ability to alter toxicity profile of anticancer drugs (33, 34), the use of aspirin in combination with anticancer drugs has never been investigated in vivo. In this study, we used doses of aspirin in vitro and in vivo approximately corresponding to that required for the treatment of arthritis and rheumatic fever. In fact, the clinical treatment of inflammation requires 2 to 5 g aspirin oral administration, which determines aspirin plasma level up to 1 to 3 mmol/L (35). In our experiments, the antiapoptotic effect of aspirin was observed starting at dose at least 10 times higher than those used for antiplatelet therapy (75 mg/d). Therefore, our finding could be of relevance in the clinical condition where treatment schedules involving both cytotoxic agents and high doses of aspirin are required, suggesting that cotreatment with aspirin could limit the outcome of anticancer therapy.

In conclusion, our data reinforce the emerging and wide observation coming from neuronal models (17) that aspirin operates also in vivo as cytoprotective agent. We also show that aspirin cytoprotective effect is mediated through the activation of AKT pathway. Because AKT pathway mediates survival against a wide variety apoptotic stimuli, it should be also verified the possible involvement of this pathway as mechanism by which aspirin exerts neuroprotection.


   Footnotes
 
Grant support: Italian Ministry of University and Research.

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 11/16/05; revised 3/11/06; accepted 3/24/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (part I). J Natl Cancer Inst 1998;90:1529–36.[Abstract/Free Full Text]
  2. Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I({kappa})B kinase-ß. Nature 1998;96:77–80.
  3. Xu XM, Sansores-Garcia L, Chen XM, et al. Suppression of inducible cyclooxygenase 2 gene transcription by aspirin and sodium salicylate. Proc Natl Acad Sci U S A 1999;96:5292–7.[Abstract/Free Full Text]
  4. Pillinger MH, Capodici C, Rosenthal P, et al. Modes of action of aspirin-like drugs: salicylates inhibit ERK activation and integrin-dependent neutrophil adhesion. Proc Natl Acad Sci U S A 1998;95:14540–5.[Abstract/Free Full Text]
  5. Dong Z, Huang C, Brown RE, Ma WY. Inhibition of activator protein 1 activity and neoplastic transformation by aspirin. J Biol Chem 1997;272:9962–70.[Abstract/Free Full Text]
  6. Ricchi P, Pignata S, Di Popolo A, et al. Effect of aspirin treatment on cell proliferation and differentiation of colon adenocarcinoma Caco-2 cells. Int J Cancer 1997;73:880–4.[CrossRef][Medline]
  7. Aas AT, Tonnessen TI, Brun A, Salford LG. Growth inhibition of rat glioma cells in vitro and in vivo by aspirin. J Neurooncol 1995;24:171–80.[CrossRef][Medline]
  8. Redondo S, Santos-Gallego CG, Ganado P, et al. Acetylsalicylic acid inhibits cell proliferation by involving transforming growth factor-ß. Circulation 2003;107:626–9.[Abstract/Free Full Text]
  9. Grilli M, Pizzi M, Memo M, Spano PF. Neuroprotection by aspirin and sodium salicylate through blockade of NF-{kappa}B activation. Science 1996;274:1383–5.[Abstract/Free Full Text]
  10. Kutuk O, Basaga H. Aspirin prevents apoptosis and NF-{kappa}B activation induced by H2O2 in HeLa cells. Free Radic Res 2003;37:1267–76.[CrossRef][Medline]
  11. Hsu CS, Li Y. Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention. Biochem Biophys Res Commun 2002;293:705–9.[Medline]
  12. Shi X, Ding M, Dong Z, et al. Antioxidant properties of aspirin: characterization of the ability of aspirin to inhibit silica-induced lipid peroxidation, DNA damage, NF-{kappa}B activation, and TNF-{alpha} production. Mol Cell Biochem 1999;199:93–102.[CrossRef][Medline]
  13. Chen N, Ma W, Dong Z. Inhibition of arsenite-induced apoptosis by aspirin. Anticancer Res 2001;21:3247–51.[Medline]
  14. Sairam K, Saravanan KS, Banerjee R, Mohanakumar KP. Non-steroidal anti-inflammatory drug sodium salicylate, but not diclofenac or celecoxib, protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats. Brain Res 2003;966:245–52.[CrossRef][Medline]
  15. Mattie MD, Freedman JH. Protective effects of aspirin and vitamin E ({alpha}-tocopherol) against copper- and cadmium-induced toxicity. Biochem Biophys Res Commun 2001;285:921–5.[CrossRef][Medline]
  16. Hundal RS, Petersen KF, Mayerson AB, et al. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 2002;109:1321–6.[CrossRef][Medline]
  17. Vartiainen N, Goldsteins G, Keksa-Goldsteine V, et al. Aspirin inhibits p44/42 mitogen-activated protein kinase and is protective against hypoxia/reoxygenation neuronal damage. Stroke 2003;34:752–7.[Abstract/Free Full Text]
  18. Ricchi P, Di Matola T, Ruggiero G, et al. Effect of non-steroidal anti-inflammatory drugs on colon carcinoma Caco-2 cell responsiveness to topoisomerase inhibitor drugs. Br J Cancer 2002;86:1501–9.[Medline]
  19. Ricchi P, Palma AD, Di Matola T, et al. Aspirin protects Caco-2 cells from apoptosis after serum deprivation through the activation of a phosphatidylinositol 3-kinase/AKT/p21Cip/WAF1pathway. Mol Pharmacol 2003;64:407–14.[Abstract/Free Full Text]
  20. Srivastava PK, DeLeo AB, Old LJ. Tumor rejection antigens of chemically induced sarcoma of inbred mice. Proc Natl Acad Sci U S A 1986;83:3407–11.[Abstract/Free Full Text]
  21. DeLeo AB, Shiku H, Takahashi T, John M, Old LJ. Cell surface antigens of chemically induced sarcoma of the mouse. J Exp Med 1976;146:720–34.
  22. Li M, Wang X, Meintzer MK, et al. Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3ß. Mol Cell Biol 2000;20:9356–63.[Abstract/Free Full Text]
  23. Loberg RD, Vesely E, Brosius FC III. Enhanced glycogen synthase kinase-3ß activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism. J Biol Chem 2002;277:41667–73.[Abstract/Free Full Text]
  24. Din FV, Dunlop MG, Stark LA. Evidence for colorectal cancer cell specificity of aspirin effects on NF{kappa}B signalling and apoptosis. Br J Cancer 2004;91:381–8.[Medline]
  25. Leinninger GM, Backus C, Uhler MD, et al. Phosphatidylinositol 3-kinase and Akt effectors mediate insulin-like growth factor-I neuroprotection in dorsal root ganglia neurons. FASEB J 2004;18:1544–6.[Abstract/Free Full Text]
  26. Yu X, Rajala RV, McGinnis JF, et al. Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17ß-estradiol-mediated neuroprotection. J Biol Chem 2004;279:13086–94.[Abstract/Free Full Text]
  27. Pommier Y, Sordet O, Antony S, et al. Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 2004;23:2934–49.[CrossRef][Medline]
  28. Hovelmann S, Beckers TL, Schmidt M. Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells. Br J Cancer 2004;90:2370–7.[Medline]
  29. Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 2004;428:332–7.[CrossRef][Medline]
  30. Beurel E, Kornprobst M, Blivet-Van Eggelpoel MJ, et al. GSK-3ß inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression. Exp Cell Res 2004;300:354–64.[CrossRef][Medline]
  31. Lochhead PA, Coghlan M, Rice SQ, Sutherland C. Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression. Diabetes 2001;50:937–46.[Abstract/Free Full Text]
  32. Plotkin B, Kaidanovich O, Talior I, Eldar-Finkelman H. Insulin mimetic action of synthetic phosphorylated peptide inhibitors of glycogen synthase kinase-3. J Pharmacol Exp Ther 2003;305:974–80.[Abstract/Free Full Text]
  33. Ricchi P, Zarrilli R, di Palma A, Acquaviva AM. Nonsteroidal anti-inflammatory drugs in colorectal cancer: from prevention to therapy. Br J Cancer 2003;88:803–7.[CrossRef][Medline]
  34. Dowd NP, Scully M, Adderley SR, Cunningham AJ, Fitzgerald DJ. Inhibition of cyclooxygenase-2 aggravates doxorubicin-mediated cardiac injury in vivo. J Clin Invest 2001;108:585–90.[CrossRef][Medline]
  35. Insel PA. The salicylates, in Goodman and Gilman's, the pharmacological basis of therapeutics. In: Hardman JG, Limbird LE, Molinoff RW, editors. McGraw-Hill Milan, Italy; 2001. p. 696–703.



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Zemskova, E. Sahakian, S. Bashkirova, and M. Lilly
The PIM1 Kinase Is a Critical Component of a Survival Pathway Activated by Docetaxel and Promotes Survival of Docetaxel-treated Prostate Cancer Cells
J. Biol. Chem., July 25, 2008; 283(30): 20635 - 20644.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by di Palma, A.
Right arrow Articles by Ricchi, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by di Palma, A.
Right arrow Articles by Ricchi, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online