Our data establish the strong growth inhibitory effect of P-ASA on human cancer cells. This effect (i) far exceeds that of aspirin, its parent molecule; (ii) appears to be generalized, and (iii) is mediated by changes in cellular redox homeostasis, which, through modulation of cell-signaling cascades, lead to a profound cytokinetic effect that engenders cell growth inhibition.
The cell lines used in this study originate from human colon, pancreatic, liver, breast and lung cancers and thus represent the major human cancers, which in 2006 accounted for ~54% of all new cases of cancer in the USA. Among them, the most sensitive to P-ASA was HepG2 (IC50
13.8 μM), whereas the least sensitive was the pancreatic cell line MIA-PaCa2 (IC50
113 μM). Thus, it is clear that the tissue of origin of these carcinoma cell lines has limited influence on their responsiveness to P-ASA.
The results from the colon cancer cell lines, the largest group of cell lines from a single organ that we studied, revealed that P-ASA's effect was not significantly affected by individual variations in cell lines. Rather, their IC50s fell within a relatively narrow range (14.3–67.6 μM), suggesting perhaps a broadly applicable mechanism of action.
An important finding was the uniform enhancement of the potency of P-ASA compared with conventional aspirin, whose precise IC50 could not be determined due to its limited solubility. In the 10 cell lines evaluated, the fold enhancement of potency ranged between >18 and >144, being on average >66.8. The reasons for the enhanced potency of P-ASA are not readily apparent, although they are clearly associated with the modification of aspirin's structure.
The growth inhibitory effect of P-ASA is brought about by a triple cell kinetic effect consisting of inhibition of proliferation, induction of apoptosis and necrosis, as well as the induction of cell cycle block at the G2/M phase. Specifically, P-ASA inhibited cell proliferation concentration-dependently (this inhibition reached 68% at 50 μM P-ASA) and induced both early and late apoptosis as well as pure necrosis. Treatment with P-ASA 25 μM increased the amount of apoptotic cells 5.5-fold and of necrotic cells 4-fold, compared with controls. The greatest increase concerned late apoptosis. The relative contribution of each effect to the overall growth inhibitory effect of P-ASA cannot be quantified precisely, but it appears that the induction of apoptosis is the dominant cytokinetic effect.
As we have previously suggested, the induction of ROS by a chemopreventive agent is a mechanistic event that is both significant and early (12
). This proved to be the case for P-ASA as well. As our data clearly demonstrate, P-ASA enhanced the intracellular levels of ROS assayed using a ‘general ROS probe’, which reacts with several individual species. Although ROS measured by this probe increased by 36.5% in response to P-ASA, the main redox effect of P-ASA was on the levels of superoxide anion in mitochondria. Superoxide anions detected in the entire cell by DHE did not change in response to P-ASA, and NO levels, which might have contributed to the DCFDA response, were also unchanged. The most significant ROS increase (112% over control) concerned the mitochondrial superoxide anion, indicating that mitochondria are the most important target of P-ASA. Indeed, mitochondrial superoxide anion plays a critical role in the initiation of apoptotic cell death and this is what we observed in response to P-ASA.
The intrinsic (mitochondrial) pathway of apoptosis is characterized by cytochrome c
release and activation of caspase 9 but not of caspase 8 (17
). This form of apoptosis can be triggered by increased ROS, which permeabilize the mitochondrial membrane and release proapoptotic factors from the mitochondrial intermembrane space into the cytosol (22
). Our data demonstrated that P-ASA activated caspase 9, while leaving caspase 8 intact. P-ASA increased mitochondrial ROS and led to the collapse of the mitochondrial membrane potential. Collectively, these findings establish that P-ASA targets the mitochondria and that this effect triggers the intrinsic apoptotic pathway. Further support that ROS are involved in the induction of cell death by P-ASA comes from the finding that the antioxidant agent NAC, which raises intracellular GSH levels, blocked both the rise of superoxide anion levels in the mitochondria and the induction of cell death by P-ASA.
The ROS levels in a cell represent the balance between ROS production and ROS inactivation by an intricate system of antioxidant mechanisms (23
). GSH, one of the most important antioxidant mechanisms in mammalian cells, responds directly to intracellular redox changes and is also used as a cofactor for antioxidant enzymes (15
). P-ASA depleted GSH stores in a concentration-dependent manner. That P-ASA may have increased ROS levels, at least in part, through its effect on GSH is evidenced by two manipulations of the system designed to affect the levels of GSH. First, BSO, which decreased intracellular GSH, reduced the IC50
of P-ASA for cell growth by more than half and second, supplementing the cells with NAC greatly attenuated the apoptotic effect of P-ASA. These effects are similar to those we obtained with nitroaspirin, a structurally similar compound, which depletes GSH by forming a conjugate with it (24
The increase in intracellular levels of ROS by P-ASA had important repercussions for the fate of the cancer cell. Our findings make it clear that ROS, elevated in response to P-ASA, modulated predominantly three signaling pathways: MAPK, COX-2 and NF-κB. MAPKs, a family of serine/threonine kinases, play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment (25
). MAPKs are required for specialized cell functions controlling cell proliferation, cell differentiation, as well as cell death and are deregulated in several malignancies including colon cancer (26
). We observed that P-ASA activated (by phosphorylation) p38 and JNK, while its effect on ERK1/2 was insignificant. It is noteworthy that the p38 and JNK ‘branches’ of the MAPK cascade are redox sensitive, whereas ERK, the one that remained unchanged, is not redox responsive (29
). AKT, which inhibits apoptosis and is frequently altered in various human malignancies (30
), was activated by P-ASA, albeit quite modestly. Interestingly, recent data suggest that antineoplastic compounds modulate this pathway and such effects may mediate their pharmacological activity (31
). This seems to also be the case for P-ASA. Indeed, P-ASA could exert part of its pharmacological effect through modulation of MAPKs. This is supported by our previous observations that nitroaspirin, a structurally related compound, modulated MAPKs and the cell growth inhibitory effect of nitroaspirin was prevented by MAPK inhibitors and by silencing the p38 and JNK MAPKs (27
Two signaling pathways that probably interact closely are COX and NF-κB, both regulate cell death and are of great importance to carcinogenesis (10
). In particular, NF-κB, a mediator of inflammatory responses, is now emerging as a link between inflammation and cancer (32
). P-ASA stimulated the expression of COX-2 and inhibited NF-κB signaling; the effect of P-ASA on both was redox dependent, as NAC reversed it. Although it is usually assumed that NF-κB activation induces COX-2, several other signaling cascades converge onto the cox-2
promoter, including Sp-1, c-MYB, activator protein-1, T-cell factor, cAMP responsive element and activator protein-2 (34
). Moreover, de Moraes et al.
) suggest two different scenarios to explain COX-2 regulation. In the ‘inflammatory scenario’, p53 activated by DNA damage, recruits NF-κB to activate COX-2, resulting in antiapoptotic effects that contribute to cell expansion in inflammatory precursor lesions. However, in a ‘constitutive proliferation scenario’, oncogenic stress due to activation of growth signaling cascades (e.g. those involving Wnt/β-catenin, K-ras or c-Myb) upregulates cox-2
independent of NF-κB to promote cancer progression (34
). In agreement with the above, we have recently shown that several chemopreventive agents increase ROS production leading to COX-2 overexpression (35
) and NF-κB inhibition (36
). These chemopreventive agents, including P-ASA, affect the thioredoxin system that is essential to maintain critical cysteine residues of the p50 and p65 subunits in their reduced form, which is required for NF-κB binding to its consensus sequence (36
). Thus, we postulate that P-ASA can activate COX-2, while inhibiting NF-κB binding.
Changes in cell signaling in response to pharmacological agents can be both extensive and complex. As we and others have pointed out (4
), it is often difficult to discern the relevance to the final pharmacological result of each pathway that has been changed. This appears to be the case with our data. Nevertheless, it is conceivable that one or more of the effects of P-ASA on these signaling pathways could be pivotal for its remarkable pharmacological action.
In conclusion, our data demonstrate that P-ASA (MDC-43), which targets several types of cancer, possesses broad anticancer properties. P-ASA is significantly more potent than conventional aspirin in inhibiting cancer cell growth. Underlying this growth inhibition appears to be changes in cellular redox homeostasis, which activate significant intracellular signaling pathways probably culminating in a major cytokinetic effect. These findings make it clear that P-ASA is a promising novel agent for the control of cancer that merits further evaluation.