QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.091363v1 316/1/25    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hundley, T. R. Articles by Rigas, B. Search for Related Content PubMed PubMed Citation Articles by Hundley, T. R. Articles by Rigas, B.

CELLULAR AND MOLECULAR

Nitric Oxide-Donating Aspirin Inhibits Colon Cancer Cell Growth via Mitogen-Activated Protein Kinase Activation

Division of Cancer Prevention, Department of Medicine, State University of New York, Stony Brook, New York

Received June 23, 2005; accepted September 14, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Nitric oxide-donating aspirin (NO-aspirin), representing a new concept in the development of more efficacious nonsteroidal anti-inflammatory drugs, consists of traditional aspirin bearing -ONO₂, which releases NO. Conventional aspirin prevents human colon cancer, but its toxicity precludes its application as a chemopreventive agent. NO-aspirin seems safer and in cultured cancer cells it is >1000-fold more potent than aspirin. To determine the mechanism by which NO-aspirin inhibits cell growth, we studied its effect on mitogen-activated protein kinase (MAPK) signaling in HT-29 human colon cancer cells. NO-aspirin stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 and Akt only marginally. The greatest increases in phosphorylation were seen in cJun NH₂-terminal kinase (JNK) and p38 MAP kinases, which were observed as early as 5 min and after 1 h of treatment, averaged more than 10-fold over control. The activation of JNK and p38 was accompanied by large increases in the phosphorylation of the downstream transcription factors cJun and activating transcription factor 2 (ATF-2). We used specific MAPK inhibitors, small interfering (siRNA) gene silencing methods, and dominant-negative cJun to determine the relevance of these phosphorylation events to the ability of NO-aspirin to inhibit colon cancer cell growth. Only the dual inhibitor of p38 and JNK and the use of combined siRNA silencing of p38 and cJun abrogated the ability of NO-aspirin to block cell growth. Our data indicate that NO-aspirin is dependent on both the p38 and the JNK MAP kinase pathways for its ability to inhibit the growth of colon cancer cells.

The mitogen-activated protein kinases (MAPKs) are a family of kinases that, in response to a variety of stimuli, transduce signals from the cell membrane to the nucleus, modulating gene transcription that leads to biological response (Pearson et al., 2001; Bode and Dong, 2004). MAPKs required for specialized cell functions controlling cell proliferation, cell differentiation, and cell death are deregulated in several malignancies, including colon cancer, and are presumed to be involved in their pathogenesis. Furthermore, recent data suggest that antineoplastic compounds modulate this pathway and such effects may mediate, at least in part, their pharmacological activity (Bode and Dong, 2004). The MAPK cascade, through sequential phosphorylations, diverges into selective activation of terminal kinases. There are three major groups of distinctly regulated MAPK cascades: ERK1/2, JNK, and p38 MAPK.

The two ERK isoforms, ERK1 and ERK2, target not only transcription factors but also membrane proteins (phospholipase A₂) and cytoplasmic kinases. How they affect cell function is not precisely known, but potentially important effects include the ERK1/2-dependent regulation of the AP-1 transcription factors and the phosphorylation of the Elk-1 by ERK1/2. The potential involvement of the ERK1/2 pathways in carcinogenesis is underscored by their enhanced activation in a variety of tumors (Platanias, 2003; Wada and Penninger, 2004), including colon cancer (Sun and Sinicrope, 2005). These cascades are likely involved in tumor cell survival and/or proliferation.

The JNK pathway, activated by chemical and radiation-induced stresses and by inflammatory cytokines, is involved in the regulation of cell proliferation and apoptosis (Manning and Davis, 2003). cJun and ATF-2 are substrates of its three isoforms, JNK-1, JNK-2, and JNK-3. Of the upstream JNK activators, mitogen-activated protein kinase kinase 4 is activated by environmental stress, whereas mitogen-activated protein kinase kinase 7 is activated by cytokines. Of the four known negative JNK regulators, nitric oxide is most relevant to the present work. JNK pathways show an interesting dichotomy, having displayed both an oncogenic and a pro-apoptotic function. In the former context, the JNK cascade seems to initiate cellular transformation, whereas its pro-apoptotic function seems to be required for the apoptosis induced by chemotherapeutic agents.

The p38 MAPK pathway (Engelberg, 2004), similar to some extent to the other two pathways, is involved in inflammation, cell growth, cell death, and cell differentiation. Activated by a similar array of stimuli, including proinflammatory mediators, growth factors, and environmental factors, it has a complex role in cancer, being, for example, able to both promote and suppress proliferation of leukemia cells.

Although MAPKs function, in general, as autonomous signaling modules, there is at times significant cross-talk between them. A not unusual result of such cross-talk is the simultaneous activation of two pathways, as is the case with p38 MAP kinase and the JNK pathway. The cellular context determines the outcome of such cross-talk.

The profound cell kinetic effect of NO-aspirin, encompassing cell proliferation, cell cycle, and cell death, combined with the role of MAPK cascades in these cellular events that are critical to carcinogenesis, prompted us to examine whether NO-aspirin acts, at least in part, by modulating this extensive signal transduction system. Our findings demonstrate that NO-aspirin indeed modulates these cascades and that its particular effect on the JNK signaling pathway is a pivotal event in its remarkable pharmacological action.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. NO-aspirin and its structural analogs (Kashfi et al., 2005) were as follows: para isomer [NCX-4040, 2-(acetyloxy) benzoic acid 4-(nitrooxy methyl) phenyl ester], meta isomer (NCX-4016, 2-(acetyloxy) benzoic acid 3-(nitrooxy methyl) phenyl ester], and denitrated NO-aspirin [2-(acetyloxy)benzoic acid 4-(hydroxymethyl-)phenyl ester]; they were gifts from Nicox S.A. (Sophia Antipolis, France). Antibodies against cJun, ATF-2, JNK, p38, ERK1/2, and Akt were from Cell Signaling Technology Inc. (Beverly, MA). Horseradish peroxidase-conjugated antibodies against -tubulin, mouse IgG, or rabbit IgG were from Calbiochem (San Diego, CA). MAPK inhibitors SB202190, SB203580, and PD98059, and wortmannin were from Calbiochem. The SP600125 MAPK inhibitor was from Alexis Biochemicals (Lausanne, Switzerland). Cell lines were from American Type Culture Collection (Manassas, VA), and cell culture reagents were from Mediatech (Herndon, VA).

Western Blots. Electrophoresis of cell lysates were performed on 10% SDS-polyacrylamide gel electrophoresis gels as described previously (Hundley et al., 2004); protein transfers were onto nitrocellulose membranes, which were stripped and reprobed when needed.

Cell Culture and Treatments. Cells were grown to 80% confluence in either McCoy's or RPMI 1640 medium (with 10% fetal calf serum, 1000 U/ml penicillin, and 1000 µg/ml streptomycin). For treatment with various reagents, 1.0 x 10⁶ cells were plated in wells on six-well plates (Corning Glassworks, Corning, NY). NO-aspirin and other reagents were incubated with cell monolayers in the presence of 1% dimethyl sulfoxide in culture medium. After incubation, cultures were placed on ice, the medium was collected, and cells were washed twice with phosphate-buffered saline. Lysis buffer (20 mM HEPES, 50 mM NaF, and 1 mM Na₃VO₄, pH 7.3, with 10% glycerol, 1% Triton X-100, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2.5 mM 4-nitrophenylphosphate) was added to cultures in wells. The floating cells from the cultures were pooled with the adherent cell lysates. Cell proliferation was assayed using the MTT colorimetric assay kit (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined using the BCA protein assay kit (Pierce Chemical, Rockford, IL).

Cell Transfections. –/–cJun in the TAM-67 expression vector was the kind gift of Dr. Z. Dong, Hormel Institute, Austin, MN (Huang et al., 1999). HT-29 cells were transfected with plasmid DNA 2 µg/well using Lipofectamine 2000 following the manufacturer's protocol (Invitrogen, Carlsbad, CA) and selected with G418 (Geneticin) at 600 µg/ml.

Statistical Methods. Continuous outcomes were compared among drug concentrations and, alternatively, between drugs at specified concentrations by using analysis of variance followed by Tukey's method for multiple pairwise comparisons (Miller, 1981). All tests were considered statistically significant at P < 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
NO-Aspirin Inhibits HT-29 Colon Cancer Cell Proliferation. Initially, we evaluated in our cell culture system two positional isomers of NO-aspirin, the meta and para isomer, denoting the position of the -CH₂ONO₂ group in the benzene ring with respect to the ester bond linking it to the conventional aspirin moiety. HT-29 colon cancer cells were treated either with one of these NO-aspirin positional isomers or with aspirin, and their growth was determined 24 h later. As seen in Fig. 1, all three compounds inhibited the growth of these cells, but their IC₅₀ values differed greatly: m- = 316 ± 12 µM (mean ± S.E.M., for this and all subsequent values), p- = 7.0 ± 3.0 µM, and aspirin = >5000 µM. Thus, both NO-aspirin isomers showed much enhanced potency compared with aspirin. However, the para isomer of NO-aspirin was the most potent; thus, our subsequent work was focused on the para isomer.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Effects of NO-aspirin and aspirin on cell growth. HT-29 colon cancer cell cultures were treated with various concentrations of para or meta NO-aspirin or aspirin for 24 h. Cell growth was assayed using the MTT cell proliferation assay. Inset shows the structures of para and meta NO-aspirin, with conventional aspirin within the box. Data are expressed as percentage of cell growth observed in untreated cultures. The results are the average of more than three experiments, and bars represent the S.E.M.

To establish an initial relationship between changes in the levels of activation of the various MAP kinases and the inhibition of cell proliferation produced by treatment with para NO-aspirin, lysates from HT-29 cell cultures treated for up to 24 h were analyzed by Western blotting for the phosphorylated forms of selected MAP kinases (Fig. 2).

View larger version (44K): [in this window] [in a new window]   Fig. 2. Activation of MAPK by NO-aspirin. HT-29 or SW-480 cells were incubated with NO-aspirin for up to 24 h before cultures were lysed, and the expression of proteins was determined by Western blotting. A, representative Western blots of HT-29 cell lysates probed for p-ERK1/2, p-Akt, total ERK1/2, total Akt, and -tubulin. B, graphic representation of averages of protein expression in HT-29 cell lysates. Concentrations of NO-aspirin for each time point are 1, 10, or 100 µM. Similar results were observed with SW-480 cell cultures. Calculations were determined as the fold increase over control band density minus background density. The results are the average of more than three experiments, and error bars represent the S.E.M.

Both ERK1/2 and Akt exhibited basal levels of phosphorylated proteins. The levels of phosphorylated ERK2 in response to 1 and 10 µM NO-aspirin fluctuate modestly over the 24-h observation period; however, there are no significant changes in its concentration. In contrast, significant increases in the phosphorylation of ERK2 are seen after 2 h of treatment with 100 µM NO-aspirin, reaching their maximum at 8 h (4.5-fold over baseline), returning to normal by 24 h. The phosphorylation of ERK1 followed an almost identical pattern of change as ERK2 (data not shown in the graph; Fig. 2B).

The phosphorylation of Akt followed a similar pattern to ERK1/2 for NO-aspirin (1–10 µM); phosphorylation levels also fluctuating modestly without a statistically significant change from baseline. In contrast to ERK1/2, at 100 µM NO-aspirin, the levels of phosphorylated Akt decrease progressively starting at 1 h and reach their nadir at 4 h (27% baseline), after which there is a progressive increase returning toward baseline values at 24 h.

NO-Aspirin Activates JNK and p38 MAP Kinases. In unstimulated HT-29 cells, phosphorylated JNK was essentially undetectable (Fig. 3). Within 30 min after treatment of these cells with NO-aspirin, two very strong bands of phosphorylated JNK appeared (Fig. 3); we determined the expression of these two bands individually. Occasionally, we detected a third band of higher molecular mass (>54 kDa), likely resulting from hyperphosphorylation of JNK1/2 (data not shown).

View larger version (108K): [in this window] [in a new window]   Fig. 3. Activation of MAPK by NO-aspirin. HT-29 or SW-480 cells were incubated with 1, 10, or 100 µM NO-aspirin for up to 24 h before cultures were lysed, and the expression of proteins was observed by Western blotting. Shown are representative Western blots of HT-29 cell lysates probed for p-JNK, p-p38, p-cJun, p-ATF-2, and the total expressions of JNK, p38, cJun, ATF-2, and -tubulin.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Activation of MAPK by NO-aspirin. Graphic representation of protein expression in HT-29 cell lysates. HT-29 cells were incubated with NO-aspirin for up to 24 h before cultures were lysed, and the expression of proteins was observed by Western blotting. Concentrations of NO-aspirin for each time point are 1, 10, or 100 µM. A, p-ATF-2. B, p-cJun. C, p-JNK2. D, p-JNK1. E, p-p38 (note compressed scale). Calculations determined as the fold increase over control band density minus background density. Results are the mean ± S.E.M. of more than three experiments.

NO-aspirin stimulated the phosphorylation of p38 in a similar manner to JNK. The lowest concentration (1 µM) had no appreciable effect, whereas higher concentrations did stimulate the formation of phosphorylated p38 markedly. Appreciable induction of phosphorylated p38 was evident even at 30 min, and it reached its maximum at 4 h: 100- and 200-fold over baseline for 10 and 100 µM, respectively. A rapid decline was evident at 8 h, and this dramatic effect had dissipated at 24 h. It is interesting to note that, as shown in Fig. 3, NO-aspirin induces a second band with slower electrophoretic mobility than phosphorylated p38 that was detected inconsistently.

NO-Aspirin Activates AP-1 Complex Transcription Factors. JNK and p38 MAP kinase signaling leads to gene activation via the activation of transcription factors of the AP-1 complex. JNK MAP kinase activates the transcription factors cJun and ATF-2, whereas p38 MAP kinase activates ATF-2. We examined whether NO-aspirin also activates and/or induces two transcription factors of the AP-1 complex, cJun and ATF-2 (Figs. 3 and 4).

Phosphorylation of cJun in response to NO-aspirin treatment of HT-29 cells is both time- and concentration-dependent. At the lowest concentration of NO-aspirin (1 µM), phosphorylation of cJun becomes clearly detectable at 1 h, remaining around the same low (but always significantly above the baseline value) levels throughout the 24 h of observation. When the cells were treated with 10 or 100 µM NO-aspirin, phosphorylated cJun was abundant as early as 30 min later, 10- and 20-fold higher than baseline, respectively. At 10 µM, the levels of phosphorylated cJun rise steadily to a maximum of about 25-fold at 8 h, returning to nearly baseline values at 24 h. In contrast, at 100 µM there is a rapid rise at 30 min, followed by a plateau at about 7-fold over baseline between 1 and 8 h, returning to about 2-fold over baseline values at 24 h.

Levels of phosphorylated ATF-2, detectable in untreated HT-29 cells, do not change appreciably in response to treatment with NO-aspirin (1 µM) over the period of observation. NO-aspirin (10 µM) increases the levels of phosphorylated ATF-2; maximal increase is noted at 1 h with a progressive decline to near baseline levels by 8 h. NO-aspirin (100 µM) also leads to maximal increase in the levels of phosphorylated ATF-2 at 1 h. Following this, levels drop to about 7-fold over baseline at 2 h and then increase to about 15-fold at 4 h, dropping to baseline by 24 h.

NO-Aspirin Does Not Inhibit Phosphatase Activity. One potential explanation for the increased levels of phosphorylated proteins that we observed in response to NO-aspirin treatment is that NO-aspirin inhibits phosphatase activity(ies), thus increasing their levels. To evaluate this possibility, we prepared protein lysates from NO-aspirin-treated cultures with and without phosphatase inhibitors. Lysates of HT-29 cells were collected in the presence or absence of Na₃VO₄ and 4-nitrophenyl-phosphate (both phosphatase inhibitors). As shown in Fig. 5, in the absence of phosphatase inhibitors, all six proteins that we studied were dephosphorylated. This was clear-cut in the case of cells treated with NO-aspirin at either 1 or 10 µM. However, some residual phosphorylation remained even in the absence of phosphatase inhibitors in cells treated with NO-aspirin at 100 µM. The reasons for this are not readily apparent. Notwithstanding, our findings indicate that, overall, NO-aspirin stimulated phosphorylation rather than inactivated their phosphatases.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Loss of phosphoprotein expression in the absence of phosphatase inhibitors. HT-29 cell cultures treated with NO-aspirin for 1 or 8 h were lysed with buffer containing or without phosphatase inhibitors, and the expression of phosphorylated proteins was determined by Western blotting.

As shown in Fig. 6, NO-aspirin studied at 1 to 100 µM inhibited HT-29 cell growth (IC₅₀ = 7.0 ± 3.0 µM). This effect was accompanied by concentration-dependent activation of MAP kinases, as already described; a modest activation of Akt was also noted. In contrast, both the denitrated derivative of NO-aspirin (10–500 µM) and aspirin (100–5000 µM) inhibited cell growth only modestly, never reaching IC₅₀ concentrations. Neither compound activated any of the six kinases that we studied. Although such correlational data do not necessarily prove that these kinases mediate the cell growth inhibitory effect of NO-aspirin, our finding is nevertheless consistent with this notion.

View larger version (53K): [in this window] [in a new window]   Fig. 6. Comparison of MAPK activation by NO-aspirin, denitrated NO-aspirin, or aspirin. A, HT-29 cells were incubated with drug for 1 h before cultures were lysed, and the expression of proteins was observed by Western blotting. B, HT-29 cell cultures were treated with drug for 24 h, and then cell growth was assayed using the MTT cell proliferation assay. Data are expressed as percentage of cell growth observed in untreated cultures. Results are the mean ± S.E.M. of more than three experiments. dn-NO-aspirin, denitrated NO-aspirin.

Of the five inhibitors, only SB202190 abrogated the inhibitory effect of NO-aspirin on cell growth; all others failed to substantially modify the growth inhibitor effect of NO-aspirin. As shown in Fig. 7, in the absence of NO-aspirin, SB202190 inhibited cell growth by 22% compared with control. At 10 µM NO-aspirin, cell growth was inhibited by 40% and pretreatment with SB202190 completely reversed this effect, restoring cell growth to that of untreated control cells. At 100 µM NO-aspirin, cell growth was inhibited by about 85%. SB202190 partially reversed this inhibitory effect, bringing cell growth to 35% of that of untreated cells; in other words, there were twice as many HT-29 cells when they were pretreated with the inhibitor. It is apparent that, at the higher NO-aspirin concentration, the cells are not fully responsive to the inhibitor; their commitment to cell death may be limiting their responsiveness.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Effect of various MAPK inhibitors on growth inhibitory effect of NO-aspirin. HT-29 cell cultures were incubated with inhibitors for 30 min before adding NO-aspirin to cultures, and incubation continued for 24 h. Inhibitors used were 25 µM PD98059 (inhibits mitogen-activated protein kinase kinase-ERK), 25 µM SB203580 (inhibits p38), 25 µM SP600125 (inhibits JNK1, JNK2, and JNK3), 25 µM SB202190 (inhibits p38/JNK), and 100 nM wortmannin (inhibits phosphoinositide 3-kinase). After 24 h, cell growth was assayed with the addition of MTT labeling solution for the MTT assay. Data are expressed as percentage of cell growth observed in untreated cultures. Results are the mean ± S.E.M. of more than three experiments. The target of each inhibitor is indicated in parentheses. Statistically significant differences from the corresponding control values are indicated by asterisks (*, P < 0.05; **, P < 0.01; **, P < 0.001); all other differences are not statistically significant.

Regarding the other four inhibitors, each one of them alone inhibited cell growth by about 40 to 50%. Attempts to use these inhibitors in combination gave uncertain results, likely because of greatly decreased cell proliferation by these kinase inhibitors in the absence of NO-aspirin.

In an effort to understand the underlying mechanism, we sought to assess the effect of these inhibitors on the expression of phosphorylated cJun, ATF-2, JNK, and p38, all key members of the JNK/p38 pathway. We focused on SB202190, which reversed the effect of NO-aspirin, and on SB203580 and SP600125, which did not reverse the effect. As can be seen in Fig. 8, compared with the effect of the combined p38/JNK inhibitor on the four proteins, the effect of the p38 inhibitor is much weaker, especially at 10 µM NO-aspirin. Furthermore, after 24 h of treatment with NO-aspirin, the combined p38/JNK inhibitor was still effective in inhibiting NO-aspirin-stimulated phosphorylation of cJun and ATF-2, whereas the p38 inhibitor was no longer effective (data not shown). In contrast, and with the exception of phosphorylated p38, the inhibitory effect of the JNK inhibitor on these proteins is stronger than that of the combined p38/JNK inhibitor. Of note, the JNK inhibitor activated p38 in the absence of NO-aspirin or at 1 µM NO-aspirin; at higher NO-aspirin concentrations, however, it attenuated the effect of NO-aspirin on the activation of p38. The activation of p38 by SP600125 has been reported recently (Vaishnav et al., 2003).

View larger version (40K): [in this window] [in a new window]   Fig. 8. Effect of MAPK inhibitors phosphoprotein expression. HT-29 cell cultures were incubated with inhibitors for 30 min before the addition of NO-aspirin to cultures, and incubation continued for 1 h. Inhibitors used were SB202190 (25 µM), SB203580 (25 µM), and SP600125 (25 µM). After 1-h incubation with NO-aspirin, cultures were lysed and the expression of proteins was observed by Western blotting.

View larger version (47K): [in this window] [in a new window]   Fig. 9. Inhibition of p38 expression with siRNA in HT-29 and SW-480 cells. HT-29 (A) and SW-480 (B) cells were transfected for 48 h with siRNA directed against p38 mRNA as described under Materials and Methods before Western blotting. siRNA-treated and wild-type cells were treated with NO-aspirin (0, 10, 25, or 100 µM).

In subsequent experiments, BxPc-3 (pancreatic carcinoma) cells were used for transfections of siRNAs directed at p38, cJun, JNK1, and JNK2; use of this cell line was necessitated because it was difficult to transfect HT-29 cells with these siRNAs. Similar to its effect on HT-29 and SW480, NO-aspirin also inhibited concentration dependently the growth of these cells (Fig. 10A). NO-aspirin activated JNK and p38 in a manner analogous to that observed in HT-29 cells (Fig. 10B). This effect was accompanied by phosphorylation of their dependent transcription factors cJun and ATF-2.

View larger version (48K): [in this window] [in a new window]   Fig. 10. Effect of NO-aspirin on growth and MAPKs in BxPC-3 pancreatic cancer cells. A, BxPc-3 pancreatic cancer cells and also HT-29 and SW480 colon cancer cells were treated with various concentrations of para NO-aspirin for 24 h. Cell growth was assayed using the MTT cell proliferation assay. Data are expressed as percentage of cell growth observed in untreated cultures. The results are the average of more than three experiments, and bars represent the S.E.M. B, representative Western blots of BxPc-3 cell lysates probed for p-JNK, p-p38, p-cJun, and p-ATF-2 expression; -tubulin was assayed as a loading control.

Transfections of BXPc-3 cells with siRNA directed at p38 or cJun alone failed to alter the effect of NO-aspirin on cancer cell growth. The combined insertion of cJun- and p38-directed siRNAs, however, inhibited significantly the effect on NO-aspirin on cell growth. As seen in Fig. 11, only when the expression of both cJun and p38 is attenuated in BxPc-3 cells is the effect of NO-aspirin on their growth reduced. Thus, these results support the observation seen with the combined pharmacological inhibition of these two pathways. Thus, based on these experiments it seems that both the JNK and the p38 MAPK pathways are required for the cell growth inhibitory effect of NO-aspirin.

View larger version (17K): [in this window] [in a new window]   Fig. 11. Effect of siRNA inhibition of cJun and p38 expression in BxPc-3 cells on growth inhibitory effect of NO-aspirin. BxPc-3 pancreatic cancer cells were transfected with siRNA directed against cJun and/or p38 mRNA. Forty-eight hours after transfection, cells were replated on 96-well plates and treated with NO-aspirin for 24 h before MTT assay was preformed. Data are expressed as percentage of cell growth of control. Statistically significant differences from the corresponding control values are indicated by asterisks (*, P < 0.05; **, P < 0.01); all other differences are not statistically significant.

To explore this mechanistic question further, we transfected HT-29 cells with a dominant-negative cJun (DNcJun) and assessed the ability of NO-aspirin to inhibit cell growth in its presence. Figure 12 shows typical results obtained with these experiments. Two of the transfectants were treated with various concentrations of NO-aspirin and compared with wild-type controls. The inhibitory effect on cell growth of the lowest concentration of NO-aspirin (10 µM) was almost completely reversed by the DNcJun. This reversal was progressively less pronounced as the concentration of NO-aspirin increased. These findings indicate that cJun plays a potentially critical role in mediating the cell growth inhibitory effect of NO-aspirin, especially at its lower concentrations.

View larger version (24K): [in this window] [in a new window]   Fig. 12. Effect of dominant-negative cJun on cell growth inhibitory effect of NO-aspirin. HT-29 cells were transfected to express –/–cJun (cannot be phosphorylated); after selection of colonies, cells were assayed for the ability of NO-aspirin to inhibit cell growth. Cells were treated with NO-aspirin for 24 h before MTT assay was preformed. Data are expressed as percentage of cell growth of control. Results are the mean ± S.E.M. of more than three experiments. Statistically significant differences from the corresponding control values are marked by asterisks (**, P < 0.01); all other differences are not statistically significant.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Traditional aspirin is a bona fide chemoprevention agent against colon cancer for which interventional studies have provided formal proof (Baron et al., 2003). The growth inhibitory effect of NO-aspirin is much stronger than that of aspirin, and this finding is in close agreement with reports from our laboratory (Williams et al., 2001; Kashfi et al., 2002) as well as those of others (Tesei et al., 2005). This enhanced effect of NO-aspirin is potentially very important and conceptually lies at the heart of its anticancer effect. NO-aspirin's anticancer effect encompasses both cancer prevention and cancer treatment; the latter is evidenced by the effect of NO-aspirin on already formed intestinal tumors in Min mice (Kashfi et al., 2005). Importantly, NO-aspirin has already entered clinical trials for colon cancer prevention.

As summarized in Fig. 13, we studied the effect of NO-aspirin on three individual pathways of the MAPK signaling cascade and on the Akt pathway that is involved in the regulation of apoptosis. NO-aspirin failed to activate Akt or ERK1/2 as evidenced by the absence of change of the levels of their phosphorylated forms, with the exception of ERK1/2 that was elevated only by NO-aspirin (100 µM) and only at 4 and 8 h.

View larger version (41K): [in this window] [in a new window]   Fig. 13. Action of NO-aspirin on MAPK signaling. Thickness of arrows indicates the relative effect of NO-aspirin on affected pathways.

NO-aspirin treatment of colon cancer cells activated JNK and p38 along with their respective downstream transcription factors, cJun and ATF-2. The activation of these two MAPKs was quantitatively pronounced. NO-aspirin stimulation of p38 seems biphasic, with an initial increase in phosphorylation occurring within the first hour of treatment, and a second much stronger increase at 4 h. These changes are accompanied by changes in the levels of phosphorylated ATF-2, which also display the weakly biphasic pattern of p38 phosphorylation.

The phosphorylation of both isoforms of JNK, JNK1 and JNK2, was increased rapidly in response to NO-aspirin, reaching essentially maximal levels by 30 to 60 min. Of note, the higher NO-aspirin concentration (100 µM) led to a more delayed and prolonged induction of phosphorylation of both JNK isoforms. Activation of phosphorylated cJun by the activated JNK was evident at 30 min. There was an interesting dichotomy here with respect to NO-aspirin concentrations. Whereas 10 µM NO-aspirin increased cJun phosphorylation progressively for the first 8 h, the 100 µM concentration increased the levels of phosphorylated cJun maximally at 30 min. Specific inhibition of p38 or JNK using appropriate inhibitors failed to block the effect of NO-aspirin on cell growth. In contrast, the dual inhibitor of p38 and JNK did block the effect of NO-aspirin on cell growth, suggesting that both these signaling proteins mediate the effect of NO-aspirin on cell growth.

This conclusion was, however, tentative because it was based on the use of pharmacological inhibitors, which always have the potential of unknown effects beyond the inhibition of their target proteins. The studies using either siRNA for gene silencing or the DNcJun were therefore needed for a thorough assessment of the contribution of p38 and JNK. Whereas transfections with siRNA directed at p38 or cJun alone failed to alter the effect of NO-aspirin on cancer cell growth, the combined silencing of cJun and p38 inhibited significantly the effect on NO-aspirin on cell growth. These findings, being in excellent agreement with the results obtained using pharmacological inhibitors, make essentially certain the conclusion that NO-aspirin needs to stimulate the p38 and JNK pathways to inhibit cell growth. The results with the DNcJun are consistent with this notion as well. The likely huge excess of nonphosphorylatable cJun occupied the AP-1 site blocking signal transduction via either the JNK or p38 pathway.

What remains unknown is the downstream mediators of NO-aspirin on cell growth. The two signaling pathways, p38 and JNK, that are so critical to the cell kinetic effect of NO-aspirin are known to modulate a range of molecules that ultimately determine cell renewal and/or cell death (Bode and Dong, 2004). Such effects include, for example, modulation of cell cycle or of members of the apoptotic pathways. Ongoing work attempts to decipher these interactions. We can, however, speculate that this effect is predominantly through the apoptosis pathway, which seems to be significantly affected by NO-aspirin (Kashfi et al., 2002).

In conclusion, NO-aspirin exerts a profound cell growth inhibitory effect on colon and other cancer cell lines by activating both the p38 and JNK MAPK signaling pathway. These findings have potentially important implications for understanding the mechanism of action of such a promising chemoprevention agent and also for the design of rational chemoprevention and perhaps chemotherapy approaches.

	Footnotes

 
This work was supported National Institutes of Health Grants CA92423 and CA92423-S2.

doi:10.1124/jpet.105.091363.

ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; NO, nitric oxide; NO-aspirin, nitric oxide-donating aspirin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, cJun NH₂-terminal kinase; AP-1, activator protein 1; ATF-2, activating transcription factor 2; MAP, mitogen-activated protein; SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) 1H-imidazole, HCl; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole; PD98059, 2'-amino-3'-methoxyflavone; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; p-, phosphorylated; siRNA, small interfering RNA; DN, dominant-negative.

Address correspondence to: Dr. Basil Rigas, Division of Cancer Prevention, Department of Medicine, Life Sciences Bldg. 06, SUNY at Stony Brook, Stony Brook, NY 11794-5200. E-mail: basil.rigas{at}sunysb.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Bak AW, McKnight W, Li P, Del Soldato P, Calignano A, Cirino G, and Wallace JL (1998) Cyclooxygenase-independent chemoprevention with an aspirin derivative in a rat model of colonic adenocarcinoma. Life Sci 62: PL367–PL373.[CrossRef]

Baron JA (2003) Epidemiology of non-steroidal anti-inflammatory drugs and cancer. Prog Exp Tumor Res 37: 1–24.[Medline]

Baron JA, Cole BF, Sandler RS, Haile RW, Ahnen D, Bresalier R, McKeown-Eyssen G, Summers RW, Rothstein R, Burke CA, et al. (2003) A randomized trial of aspirin to prevent colorectal adenomas. N Engl J Med 348: 891–899.[Abstract/Free Full Text]

Bode AM and Dong Z (2004) Targeting signal transduction pathways by chemopreventive agents. Mutat Res 555: 33–51.[Medline]

Engelberg D (2004) Stress-activated protein kinases-tumor suppressors or tumor initiators? Semin Cancer Biol 14: 271–282.[CrossRef][Medline]

Fiorucci S, Santucci L, Gresele P, Faccino RM, Del Soldato P, and Morelli A (2003) Gastrointestinal safety of NO-aspirin (NCX-4016) in healthy human volunteers: a proof of concept endoscopic study. Gastroenterology 124: 600–607.[CrossRef][Medline]

Huang C, Ma WY, Li J, Goranson A, and Dong Z (1999) Requirement of Erk, but not JNK, for arsenite-induced cell transformation. J Biol Chem 274: 14595–14601.[Abstract/Free Full Text]

Hundley TR, Gilfillan AM, Tkaczyk C, Andrade MV, Metcalfe DD, and Beaven MA (2004) Kit and FcRI mediate unique and convergent signals for release of inflammatory mediators from human mast cells. Blood 104: 2410–2417.[Abstract/Free Full Text]

Kashfi K, Borgo S, Williams JL, Chen J, Gao J, Glekas A, Benedini F, Del Soldato P, and Rigas B (2005) Positional isomerism markedly affects the growth inhibition of colon cancer cells by nitric oxide-donating aspirin in vitro and in vivo. J Pharmacol Exp Ther 312: 978–988.[Abstract/Free Full Text]

Kashfi K, Ryann Y, Qiao LL, Williams JL, Chen J, Del Soldato P, Traganos F, and Rigas B (2002) Nitric oxide-donating nonsteroidal anti-inflammatory drugs inhibit the growth of various cultured human cancer cells: evidence of a tissue type-independent effect. J Pharmacol Exp Ther 303: 1273–1282.[Abstract/Free Full Text]

Manning AM and Davis RJ (2003) Targeting JNK for therapeutic benefit: from junk to gold? Nat Rev Drug Discov 2: 554–565.[CrossRef][Medline]

Miller R (1981) Simultaneous Statistical Inference. Springer-Verlag New York Inc., New York.

Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, and Cobb MH (2001) Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22: 153–183.[Abstract/Free Full Text]

Platanias LC (2003) MAP kinase signaling pathways and hematologic malignancies. Blood 101: 4667–4679.[Abstract/Free Full Text]

Rice PL, Beard KS, Driggers LJ, and Ahnen DJ (2004) Inhibition of extracellular-signal regulated kinases 1/2 is required for apoptosis of human colon cancer cells in vitro by sulindac metabolites. Cancer Res 64: 8148–8151.[Abstract/Free Full Text]

Rigas B and Kashfi K (2004) Nitric-oxide-donating NSAIDs as agents for cancer prevention. Trends Mol Med 10: 324–330.[CrossRef][Medline]

Shiff SJ, Qiao L, Tsai LL, and Rigas B (1995) Sulindac sulfide, an aspirin-like compound, inhibits proliferation, causes cell cycle quiescence and induces apoptosis in HT-29 colon adenocarcinoma cells. J Clin Investig 96: 491–503.[Medline]

Sun Y and Sinicrope FA (2005) Selective inhibitors of MEK1/ERK44/42 and p38 mitogen-activated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells. Mol Cancer Ther 4: 51–59.[Abstract/Free Full Text]

Tesei A, Ulivi P, Fabbri F, Rosetti M, Leonetti C, Scarsella M, Zupi G, Amadori D, Bolla M, and Zoli W (2005) In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines. J Transl Med 3: 7.[CrossRef][Medline]

Testa JR and Bellacosa A (2001) AKT plays a central role in tumorigenesis. Proc Natl Acad Sci USA 98: 10983–10985.[Free Full Text]

Vaishnav D, Jambal P, Reusch JE, and Pugazhenthi S (2003) SP600125, an inhibitor of c-Jun N-terminal kinase, activates CREB by a p38 MAPK-mediated pathway. Biochem Biophys Res Commun 307: 855–860.[CrossRef][Medline]

Wada T and Penninger JM (2004) Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23: 2838–2849.[CrossRef][Medline]

Williams JL, Borgo S, Haspirinn I, Castillo E, Traganos F, and Rigas B (2001) Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) alter the kinetics of human colon cancer cell lines more effectively than traditional NSAIDs: implications for colon cancer chemoprevention. Cancer Res 61: 3285–3289.[Abstract/Free Full Text]

Williams JL, Kashfi K, Ouyang N, del Soldato P, Kopelovich L, and Rigas B (2004) NO-donating aspirin inhibits intestinal carcinogenesis in Min (APC(Min/+)) mice. Biochem Biophys Res Commun 313: 784–788.[CrossRef][Medline]

Yeh RK, Chen J, Williams JL, Baluch M, Hundley TR, Rosenbaum RE, Kalala S, Traganos F, Benardini F, del Soldato P, et al. (2004) NO-donating nonsteroidal antiinflammatory drugs (NSAIDs) inhibit colon cancer cell growth more potently than traditional NSAIDs: a general pharmacological property? Biochem Pharmacol 67: 2197–2205.[CrossRef][Medline]

This article has been cited by other articles:

				W. Zhao, G. G. Mackenzie, O. T. Murray, Z. Zhang, and B. Rigas Phosphoaspirin (MDC-43), a novel benzyl ester of aspirin, inhibits the growth of human cancer cell lines more potently than aspirin: a redox-dependent effect Carcinogenesis, March 1, 2009; 30(3): 512 - 519. [Abstract] [Full Text] [PDF]

				Y. Sun and B. Rigas The Thioredoxin System Mediates Redox-Induced Cell Death in Human Colon Cancer Cells: Implications for the Mechanism of Action of Anticancer Agents Cancer Res., October 15, 2008; 68(20): 8269 - 8277. [Abstract] [Full Text] [PDF]

				N. Ouyang, J. L. Williams, and B. Rigas NO-donating aspirin inhibits angiogenesis by suppressing VEGF expression in HT-29 human colon cancer mouse xenografts Carcinogenesis, September 1, 2008; 29(9): 1794 - 1798. [Abstract] [Full Text] [PDF]

				D. Maksimovic-Ivanic, S. Mijatovic, L. Harhaji, D. Miljkovic, D. Dabideen, K. Fan Cheng, K. Mangano, G. Malaponte, Y. Al-Abed, M. Libra, et al. Anticancer properties of the novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide in vitro and in vivo Mol. Cancer Ther., March 1, 2008; 7(3): 510 - 520. [Abstract] [Full Text] [PDF]

				S. V. Singh, S. Choi, Y. Zeng, E.-R. Hahm, and D. Xiao Guggulsterone-Induced Apoptosis in Human Prostate Cancer Cells Is Caused by Reactive Oxygen Intermediate Dependent Activation of c-Jun NH2-Terminal Kinase Cancer Res., August 1, 2007; 67(15): 7439 - 7449. [Abstract] [Full Text] [PDF]

				C. V. Rao, B. S. Reddy, V. E. Steele, C-X Wang, X. Liu, N. Ouyang, J. M.R. Patlolla, B. Simi, L. Kopelovich, and B. Rigas Nitric oxide-releasing aspirin and indomethacin are potent inhibitors against colon cancer in azoxymethane-treated rats: effects on molecular targets. Mol. Cancer Ther., June 1, 2006; 5(6): 1530 - 1538. [Abstract] [Full Text] [PDF]

				J. Antosiewicz, A. Herman-Antosiewicz, S. W. Marynowski, and S. V. Singh c-Jun NH2-Terminal Kinase Signaling Axis Regulates Diallyl Trisulfide-Induced Generation of Reactive Oxygen Species and Cell Cycle Arrest in Human Prostate Cancer Cells. Cancer Res., May 15, 2006; 66(10): 5379 - 5386. [Abstract] [Full Text] [PDF]

				N. Ouyang, J. L. Williams, G. J. Tsioulias, J. Gao, M. J. Iatropoulos, L. Kopelovich, K. Kashfi, and B. Rigas Nitric oxide-donating aspirin prevents pancreatic cancer in a hamster tumor model. Cancer Res., April 15, 2006; 66(8): 4503 - 4511. [Abstract] [Full Text] [PDF]

				J. J. Turchi Nitric oxide and cisplatin resistance: NO easy answers PNAS, March 21, 2006; 103(12): 4337 - 4338. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.091363v1 316/1/25    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hundley, T. R. Articles by Rigas, B. Search for Related Content PubMed PubMed Citation Articles by Hundley, T. R. Articles by Rigas, B.