This study demonstrates that the combination of P-S and DFMO significantly prevents human colon cancer in a xenograft model. This effect is associated with reduced levels of a) polyamines, key regulators of cell proliferation; b) Trx-1 and TrxR, the two protein members of the thioredoxin system; and c) COX-2, a protein considered important in colon tumorigenesis and a target of chemopreventive agents. These molecular effects likely culminate in substantial inhibition of proliferation and induction of apoptosis, the net effect of which was the 70.9% reduction in xenograft growth by P-S/DFMO (Fig. 4D).
Agent safety is critical to chemoprevention, which by definition entails long-term (decades) of agent administration to often healthy individuals at risk of cancer. Neither DFMO nor P-S, even when combined, showed signs of toxicity, particularly gastrointestinal toxicity, a major limitation of conventional sulindac. Indeed, safety concerns have prompted efforts to devise chemopreventive protocols using the lowest possible dose of sulindac that maintains adequate efficacy, including the co-administration of a gastroprotective drug (24
). Our results are consistent with previous work indicating the preclinical efficacy of P-S (9
). For example, though 100% of mice receiving P-S (100 mg/kg/d) for 3 wks were alive and healthy, treatment with sulindac at an equimolar dose (66 mg/kg/d) resulted in 80% mortality (10
The chemopreventive efficacy of P-S/DFMO was stronger than that of either compound alone, and even though these effects only trended toward statistical significance, such lack of significance is probably due to lack of power. Nonetheless, we deem this finding as highly relevant. Thus, although this enhanced efficacy did not represent true pharmacological synergy, it is consistent with the notion that these compounds modulate the polyamine biosynthetic pathway synergistically. The growth inhibitory effect by P-S, DFMO and P-S/DFMO was associated with significantly decreased cell proliferation and increased apoptosis. Of the two, the effect on apoptosis was somewhat more pronounced. The end result of these two effects was significant suppression of xenograft growth.
The cytokinetic effect of the test compounds reflects the summation of changes in several cell signaling pathways. Analysis of cell signaling in response to P-S, DFMO, and P-S/DFMO revealed that at least three important pathways are modulated significantly by these two agents: polyamines, the thioredoxin system, and the eicosanoid pathway. Perhaps surprisingly, NF-κB does not seem to be part of this chemopreventive effect.
A strong relationship between polyamines and cancer is known to exist. However, our results make it clear that the relationship between each of the three polyamines and tumor growth inhibition is complex. None of them individually is significantly associated with tumor volume, though putrescine and spermidine are associated with cell proliferation, in keeping with their well-known role in cell renewal (3
). Putrescine and spermidine (the immediate metabolic product of putrescine) are associated with each other, whereas spermine, whose levels did not change in any of the study groups, seems to be differentially regulated by P-S and/or DFMO.
The most consistent and statistically strongest association with tumor volume concerned the thioredoxin system, most notably TrxR and, to a lesser degree, Trx-1. These two proteins are pivotal in regulating redox chemistry in the cell. Trx-1 reduces client proteins oxidized by reactive oxygen species under conditions of oxidative stress, itself undergoing oxidation during this process. TrxR reverses this, returning Trx-1 to its active (reduced) status. Under our experimental conditions, the apparently dominant effect was the significant suppression of TrxR and Trx-1 by P-S/DFMO, presumably precipitating a state of massive oxidative stress that leads to the death of HT-29 cells. We have previously proposed such a mechanism of chemopreventive action for P-S and other chemopreventive agents (26
). Adding complexity, there is a possible direct interaction between the thioredoxin system and polyamines. In MCF-7 human breast cancer cells, transfected Trx-1 decreased the expression and enzyme activity of spermidine/spermine N1-acetyltransferase and also lowered putrescine levels without changing those of spermine or spermidine (27
). Of interest, an inverse relationship (reduction of thioredoxin caused by polyamine depletion) has been reported in a different system (28
COX-2 levels were significantly suppressed by P-S/DFMO, but not by either one alone. The difference in the degree of suppression, especially by P-S compared to P-S/DFMO, was not great (31% vs
. 42%); in the case of P-S, however, its effect on COX-2 expression did not reach statistical significance. The relationship between the eicosanoid cascade and cancer prevention by P-S/DFMO is unclear. Contrary to expectations, Meyskens et al.
noted no change in PGE2
levels in the colonic mucosa of patients treated with sulindac/DFMO (29
). COX-2-independent effects in cancer prevention by NSAIDs have long been considered (30
). Nevertheless, the suppressed levels of COX-2 and the presumed accompanying suppression of PGE2
levels may have contributed to their chemopreventive effect.
The lack of any significant effect on NF-κB by our test compounds is rather surprising, especially since NF-κB is redox-sensitive and modulated by the thioredoxin system (21
). Multiple lines of evidence indicate that NF-κB is an important mediator of carcinogenesis; depending on biological context, it modulates proliferation and apoptosis (31
). Anticancer drugs, including P-S, inhibit NF-κB activation as part of their pharmacological activity (11
). This finding underscores the complexity of cell signaling pathways and suggests that the final outcome represents the balance of many competing processes.
In conclusion, our study demonstrates that P-S combined with DFMO is a promising drug combination for colon cancer prevention. P-S/DFMO has an intricate mechanism of action that extends beyond polyamines, involving emerging regulators of chemoprevention such as the thioredoxin system. The (preclinical) safety of P-S may enable it to overcome safety concerns regarding the clinically successful combination of conventional sulindac with DFMO. Given P-S's efficacy and apparent safety as well as its effective combination with DFMO, P-S deserves further evaluation for its role in colon cancer prevention.