Epidemiological and clinical studies, as well as experimental studies, suggest that NSAIDs are effective agents for cancer prevention. COX-2 is overexpressed in both animal and human pancreatic cancers, and several preventive and therapeutic strategies revolve around COX-2 as an important molecular target for chemoprevention [31
]. Hence, NSAIDs and COX-2-selective inhibitors have been widely tested for cancer prevention, including that of pancreatic cancer. For example, Funahashi et al. found that the selective COX-2 inhibitor nimesulide delays the progression of pancreatic cancer precursor lesions in the KrasG12D
]. However, the gastrointestinal toxicities and cardiovascular risks accompanying these agents have prompted a search for novel approaches/agents devoid of unwanted side effects but with similar or higher efficacies. In the present study, we have demonstrated the chemopreventive effects of NO-aspirin against formation of PanIN lesions and their progression to PDAC in p48Cre/+
mice. This mouse model captures many significant features of human pancreatic cancer development including aspects of histology, molecular biology, and tumor biology and has been well justified for use in chemoprevention studies [8,29
]. The delay of PanIN progression to PDAC in the p48Cre/+
mice shows the potential of NO-aspirin to be an effective chemopreventive agent. We did not observe a dose dependency. In fact, the lower dose of NO-aspirin appeared to be more protective than the higher dose. The reasons for the lack of better inhibitory effects at higher doses needs further investigation.
Results also suggest that NO-aspirin delays the progression from PanIN-1 and PanIN-2 lesions to PanIN-3 and PDAC (). Because only PanIN-3 are classified as carcinoma in situ
, delaying the progression toward PDAC suggests a major impact on cancer development. Of great interest has been the observation that the long-term administration of NO-aspirin is not associated with overt toxicity. Similar to the observations described here, we have shown previously that an epidermal growth factor receptor (EGFR) inhibitor and atorvastatin inhibit Kras-activated development of PanINs and PDAC [8,29
]. This is the first report on the effect of NO-aspirin against pancreatic cancer in a mutant Kras mouse model. However, tumor efficacy effects of NO-aspirin are not new; previously, NO-aspirin was tested for efficacy against chemically induced pancreatic tumors in a hamster model [33
] and colon cancers in rats [34
]; both studies supported the chemopreventive property of this agent. Furthermore, low-dose NO-aspirin inhibited spread of the carcinoma by 97% (), suggesting that this agent may block invasiveness within the pancreas and further metastasis to nearby organs. The bioassay protocol using PDAC as an end point limited the comparative analysis of overall survival and rate of metastasis in control diet and treatment groups. However, loss of 20% of the mice on the control diets but not in the treated groups supports the notion that NO-aspirin may increase survival by limiting metastasis.
An indirect insight into the mechanism underlying the cell kinetic changes induced by NO-aspirin is provided by our study of the expression of p21WAF1/CIP1
]. p21 is activated by p53 to produce cell cycle arrest in response to DNA damage. p21WAF1/CIP1
blocks cell cycle progression, both by acting as a general inhibitor of cyclin dependent kinase (Cdk)/cyclin complexes and by inhibiting DNA replication by binding to PCNA, a subunit of DNA polymerase. We observed a significant increase in p53 and p21 expressions in the low-dose-treated NO-aspirin and reduced expression of tumor promoting markers cyclin D1, β-catenin, COX-2, PCNA, and iNOS. Aspirin and NO-aspirin treatments of colon cancer cells activated the p38 [mitogen-activated protein kinase (MAPK)] pathway along with their respective downstream transcription factors such as c-Jun and activating transcription factor 2 (ATF-2) [36,37
]. Aspirin-induced p38 activation precedes rapid ubiquitin-dependent cyclin D1 degradation [37
]. Similarly, we observed a dose-dependent increase in p38 and a decrease in cyclin D1 mRNA expression in this study, suggesting that NO-aspirin causes inhibition of cyclin D1 through the p38 MAPK pathway ().
We have previously reported that NO-NSAIDs including NO aspirin have strong inhibitory activity against colon carcinogenesis associated with suppression of COX, iNOS, and β-catenin levels in colon tumors [34
]. Here, NO-aspirin showed a good suppression of iNOS, COX-2, and β-catenin protein expressions (, D–F
, , and ) corresponding to inhibition of pancreatic tumorigenesis, thereby confirming the previous results in colon cancer. The profound effects of NO-aspirin on cell proliferation, cell cycle, and cell death, combined with the role of MAPK cascades in these cellular events that are critical to carcinogenesis, confirm our findings that NO-aspirin acts, at least in part, by modulating this extensive signal transduction system. Thus, it is apparent that NO-aspirin exerts effects on multiple molecular targets in pancreatic cancer, perhaps more than the present study has revealed. The actual contribution of each of these changes to the chemopreventive effect of this agent remains to be determined. As argued elsewhere regarding the mechanism of action of this compound [19
], the choice between mechanistic dominance (one pathway is sufficient for the effect) and mechanistic redundancy (effects on multiple pathways are required) is unresolved and our present study underscores this point.
In summary, our data indicate that NO-aspirin has significant potential for pancreatic cancer prevention and possibly for treatment.