5-FU is one of the most prescribed drugs in clinical oncology for colo-rectal cancer patients [18
], however side effects are often reported, leading to compliance failure. The intestinal barrier function may suffer from 5-FU cytotoxic insults in various ways, including mucosal inflammatory reactions, intestinal epithelial barrier disruption, and bacterial translocation [20
The data described here are in accordance with previous work from Soares et al
., who followed 5-FU-treated rats (1°, 3°, 5°, 15° and 30° day) and showed marked leukopenia, MPO activity, inflammatory cell infiltration, villus shortening, and crypt destruction in the duodenal lamina propria on the third day post-5-FU challenge [5
Interestingly, we found a marked crypt mitotic arrest in 5-FU treated mice, which was ameliorated by COG 133 peptide only in wild-type but not ApoE knock-out mice, as shown by our findings of crypt mitotic index. This may be caused by a disrupted growth factor signaling, since we had shown impaired intestinal adaptations during catch-up growth following re-feeding in severe undernourished ApoE knock-out mice, with poor intestinal IGF-1 expression [21
Our data suggest that ApoE mimetic peptide may modulate inflammation by increasing NF-κB expression in recovered epithelia from duodenal crypts, therefore may be also activating the NF-κB anti-inflammatory pathway. NF-κB signaling has been found to improve epithelial migration after intestinal injury [22
], and has been shown to be associated with cyclin 1 expression [23
], both factors that could improve crypt recovery from 5-FU epithelial damage. Although, COG 133 peptide could also enhance intestinal mucosal repair in ApoE knock-out mice, the NF-κB immunolabeling was less increased than in the wild-type mice following ApoE-peptide treatment. Further studies are warranted to examine this effect in more depth, especially addressing growth factors involved in mucosal restitution and their regulation by ApoE peptides.
Recent studies in rats demonstrated that 5-FU administration (150 mg/kg i.p.) significantly increased MPO activity (275%) in the jejunum, as compared with unchallenged controls [24
]. In addition, increased concentrations of TNF-α and IL-1β with involvement of platelet-activating factor were found in the rat duodenum [25
]. In order to assess inflammatory responses to 5-FU challenge, we measured ileal cytokines and MPO levels. Our findings also showed marked increases in intestinal MPO activity on the 3rd day after 5-FU challenge (450 mg/kg i.p), effects that were abrogated in mice treated with 3 μM COG133 peptide, suggesting a reduction of the MPO-producing inflammatory cell infiltrates by the peptide.
In our study, we detected increased levels of IL-1β and TNF-α and their transcripts (and reduced IL-10 levels) and increased expression of the inflammatory mediator iNOS mRNA in the proximal small intestine among 5-FU challenged Swiss mice, findings that were partially abrogated by ApoE mimetic peptide treatment, supporting an anti-inflammatory role of the ApoE mimetic peptide in the 5-FU-driven intestinal mucositis. This finding of ApoE modulatory action on pro-inflammatory cytokines is reinforced by reports of a pro-inflammatory state in mice lacking the APOE gene [26
] and poor survival of these animals in models of sepsis [27
]. Studies by Lynch and colleagues have demonstrated an anti-inflammatory effect of the ApoE peptide, COG 133, when administered intra-peritoneally to LPS-challenged (i.p.) mice, leading to significant reductions in serum TNF-α after 1 and 3 hours. Such anti-inflammatory effect was also found in the brain tissue of these mice, with reductions in the TNF-α mRNA expression 3 hours after LPS injection [8
]. Furthermore, an anti-LPS role is also postulated in studies using human recombinant ApoE. ApoE- enriched emulsion can shift bacterial LPS from liver Kupffer cells to hepatocytes, therefore reducing LPS-derived endotoxemia (binds at ratio of 1:2 LPS molecules) [28
]. These findings suggest that the benefit of ApoE mimetic peptide we observed may have been through blocking effects of bacterial (or LPS) translocation following 5-FU-induced intestinal barrier leakage.
Regarding mediators that might be altered by ApoE mimetic peptides, TNF-α and IL-1β are important pro-inflammatory cytokines activated by NF-κB pathway, which is up-regulated during the pathogenesis of 5-FU-induced mucositis [30
]. Interestingly, the ApoE COG-112 mimetic peptide was found to inhibit NF-κB signaling and downstream pro-inflammatory cytokines in Citrobacter rodentium
-induced colitis in mice [32
]. In this model, authors also found reductions in tissue neutrophil infiltration, corroborating our findings of lower MPO levels (as a neutrophil marker) in intestine with ApoE peptide treatment. In addition, IL-1 receptor antagonist was found to reduce intestinal toxicity of 5-FU in mice, suggesting that IL-1 peptides are key players in the intestinal inflammatory response. Our findings with NF-κB suggest that although this signaling pathway is involved in the inflammatory response, it may be important for crypt recovery following 5-FU-induced epithelial damage.
Our findings that ApoE deficient mice show higher TUNEL-labeled cells, more inflammation and more blunted villi than wild-type controls, reinforce the role of ApoE in protecting the intestinal mucosa against 5-FU-induce intestinal mucositis.
In our in vitro studies, however we only find improvements in the number of Ki67-labeled IEC-6 cells with ApoA-I peptide using a high treatment dose (20 μM). However, increased cell numbers were found with COG 133, suggesting that the peptide increased cell proliferation before Ki67-stained mitoses occurred. On the other hand, we found beneficial effects of COG 133 in lower doses using the WST assay, a method that measures the mithocondrial-formazan product seen in viable cells. These differences may relate to the time periods assessed, still under blunting mitotic effects following 5-FU treatment [34
]. In addition, WST measurements show stronger proliferative effects of COG 133 in glutamine free media (rather than standard media with glutamine) in IEC-6 cells following 5-FU challenge, suggesting that the peptide has a potential biological effect under catabolic disease conditions when glutamine is more likely deprived.
In order to compare the ApoE mimetic peptide effect on cell migration and apoptosis, we also conducted in vitro experiments with ApoA-I (D-4F), which we found causing similar benefits to those seen with the ApoE peptide. Thus, both may affect a common downstream pathway. However, both COG 133 and the D-4F peptides are amphipathic alpha helices. Thus, the other possibility is that they can cross plasma membranes and enter the cell cytoplasm and have similar effects in a receptor-independent fashion.
Noteworthy is that the intestine is a major site of action of this peptide [35
]. Recently, a novel anti-inflammatory mechanism of ApoE-mimetic peptides was identified, with inhibition of SET, a protein capable of increasing endogenous PP2A phosphatase activity, which reduces levels of phosphorilated kinase signaling and inflammatory cytokines [36
]. SET is also involved in neuronal apoptosis due to amyloid precursors [37
]. If the same mechanism is operating in the IEC-6 cell apoptotic machinery, the inhibition of SET by ApoE mimetic peptide would be anti-apoptotic, as seen in our flow cytometry studies after 5-FU challenge. Increased TNF-α-induced apoptosis would be also counterbalanced by the anti-inflammatory actions of the peptide, possibly mediated by a fine regulation of serine-threonine kinase 1 (RIP-1) ubiquitinization and caspase signaling [38
]. At this point, the exact mechanism of the anti-apoptotic property of the ApoE COG 133 remains unclear.
In contrast to anti-proliferative findings of COG 112 and COG 133 peptides in lymphocyte cultures after myelin oligodendrocyte glycoprotein (MOG) exposure in a murine model of multiple sclerosis [11
], our in vitro
data in crypt IEC-6 cells in addition to the observation of improved crypt histology in treated mice suggest a beneficial role of the peptide in improving cell viability and crypt cell renewal following 5-FU challenge
Previous studies have shown that mucositis is not only a manifestation of tissue change, but also with systemic effects, with elevation of proinflammatory cytokines in serum, such as TNF-α, IL-1β, and IL-6 of 5-FU-treated patients, in association with the chemotherapy toxicity without any improvements in its anti-tumor activity [39
Colton et al
. demonstrated an APOE allelic-distinct effect on microglial nitric oxide production in the transgenic mouse brain [40
]. A significant increase in NO production was found in isolated macrophages from human APOE4 targeted transgenic mice by activation of cationic amino acid transporters (CAT) with up-regulated innate immune system [41
]. Therefore, a possible downregulation of CAT-1 transporters during a pro-inflammatory state of the intestinal mucositis would contribute to lower NO generation and thus milder inflammatory cytokine production.
Studies from Hoane et al
. in models of brain injury have demonstrated improvements in cognitive tests following ApoE peptide treatment in rats. There was a dose-dependent peptide (COG 1410) effect, with the best dose found of 0.8 μg/kg, in studies showing recovery following traumatic brain injury [42
]. While the mechanism of this benefit is unclear, it may involve an anti-inflammatory effect like that which we have seen.