NT, an important intestinal hormone for multiple physiologic functions in the GI tract, stimulates proliferation of normal intestine and NTR positive colorectal and pancreatic cancers through mechanisms that are not entirely understood (2
). In our present study, we show that NT selectively increases the expression and secretion of IL-8, a chemokine increasingly recognized as contributing to increased tumor invasion, progression and angiogenesis (16
). These findings identify a potentially important mechanism contributing to enhanced tumor growth by NT through the stimulation of multiple signaling pathways leading ultimately to the downstream regulation of tumor-secreted proteins such as IL-8. In addition, we find that the natural product curcumin, which has been evaluated as an anti-inflammatory and anti-tumor agent, blocked NT-mediated IL-8 gene induction, protein secretion and cell migration.
The predominant proliferative effects of NT appear to be mediated through the high-affinity NTR1 which is present in a number of colorectal, pancreatic and prostate cancers. (38
). We have used two non-peptide NTR antagonists to further demonstrate that the effects of NT are acting directly through the NTR1. Consistent with our findings, Zhao et al, (13
) demonstrated increased IL-8 promoter activity and protein secretion by NT in nontransformed human colonocytes stably transfected with NTR1. Therefore, our study using cancer cells with native NTR1 and the previous studies utilizing an artificially-derived cell line (13
), show that NT selectively regulates IL-8 activity through the NTR1. Since we (8
) and others (7
) have shown that SR48692 is nontoxic and effective in vivo
to block NT-mediated tumor growth, our findings have important implications in the possible development of novel therapeutic strategies for the treatment of human cancers based on NTR1 receptor blockade.
The signaling pathways and transcription factors regulating IL-8 expression have been described in different cell types in response to various stimuli. Similar to other physiological agents (eg, histamine, endothelin-1 and bradykinin), NT acts through Ca2+
/PKC, ERK activation and the ubiquitous AP-1 and NF-κB transcription factors to induce IL-8 gene expression in HCT116 cells. Most studies have shown that NF-κB plays the predominant role in IL-8 regulation. We found that both ERK-dependent AP-1 and ERK-independent NF-κB activation was necessary for NT-mediated IL-8 mRNA and secretion in HCT116 cells. Consistent with our findings, AP-1 and NF-κB co-operatively regulate IL-8 expression in other cell types. For example, the activation of both AP-1 and NF-κB was essential for IL-8 induction in human breast cancer cells in response to glutamine deprivation (40
), NF-κB-dependent AP-1 activity regulates VEGF expression (41
). Furthermore, ERK-dependent AP-1 activation synergizes with p65/NF-κB to stimulate IL-8 gene transcription in human epidermal carcinoma cells. Our findings suggest that either receptor blockade, or alternatively, an agent that inhibits both AP-1 and NF-κB activation would be required to block the effects of NT on IL-8 gene induction and protein secretion.
Curcumin, a component of curry spice turmeric, possesses anti-inflammatory and anti-cancer effects through the inhibition of NF-κB and AP-1 activation. We found that curcumin effectively inhibited NT-induced AP-1 and NF-κB induction and subsequent IL-8 gene expression and protein secretion in HCT116 cells. Similar to our findings, Nakamura et al (42
) found that curcumin decreased both AP-1 and NF-κB activation in prostate cancer cell lines. Moreover, curcumin markedly inhibited the activation of AP-1 and NF-κB DNA-binding induced by LPS, H2
or TNFα (43
). The inhibition of AP-1 and NF-κB by curcumin effectively suppressed IL-8 release in alveolar epithelial cells (43
) which corroborates our findings in HCT116 cells. IL-8, regulated by AP-1 and NF-κB transcription factors in various cancer cells, is becoming increasingly recognized as an important local factor in tumorigenesis and metastasis. The results from our study and those of others (22
) suggest that an important mechanism for the anti-tumor effects of curcumin may be through the suppression of chemokines induced by GI hormone or other stimuli. Moreoever, Zhao et al (13
) found that NT stimulates IL-8 secretion through NF-κB activation and plays a role in colonic inflammation, while numerous other studies showed that curcumin possesses anti-inflammatory effects through inhibition of NF-κB, and that colonic inflammation increases the risk for developing colorectal cancer (46
). Our findings further suggest that curcumin may be useful for colon cancer treatment as well as potential colon cancer prevention. In fact, curcumin is currently being evaluated in clinical trials as a cancer chemotherapeutic and chemopreventive agent for colorectal cancers (47
). Our findings provide additional evidence that curcumin may be beneficial in the suppression of certain chemokines (eg, IL-8) which play a role in tumor progression.
While a number of groups have confirmed that NT stimulates GI cancer cell growth and that curcumin suppresses the growth of various cancer types, few studies have analyzed the effect of NT or curcumin on colon cancer cell migration and/or metastasis. In our current study, we found that NT significantly increased HCT116 cells migration by ~3 fold and that curcumin blocked NT-induced HCT116 cell migration at a concentration (10 μM) which does not cause cell death as demonstrated in our current experiments and by Kang et al (44
). The increase in HCT116 migration by IL-8 treatment suggested that the effect of curcumin on NT-mediated HCT116 migration may be through the inhibition of IL-8. Numerous studies have shown that IL-8 can function as a motility factor for tumor cells, which is relevant to tumor invasion and metastasis. This concept was first shown in human melanoma cells (48
), and subsequently demonstrated in human colon carcinoma cells (49
). HCT116 migration was increased ~2-fold by IL-8 and ~3-fold by NT. We speculate that IL-8 is one of the motility factors in HCT116 cells which is stimulated by NT. In addition, NT has been shown to stimulate other pro-tumor factors, such as COX-2 (50
and matrix metalloproteinases (QD Wang, preliminary results) which may also contribute to the enhanced migration. Taken together, our current study has identified additional effects of NT which may enhance GI carcinogenesis. In addition to stimulating proliferation, NT can promote the induction and secretion of IL-8, a proinvasive factor and can increase tumor cell migration. The fact that curcumin, a natural product, can block these effects is appealing for future treatment strategies.
In summary, we show that NT, an intestinal hormone that is potently released by fat ingestion (4
), acts through its native NTR to stimulate Ca2+
/PKC, ERK/AP-1 and NF-κB pathways and ultimately increases expression and secretion of IL-8 and enhances colon cancer cell migration. These effects were blocked by either NTR1 antagonists or curcumin, a diet-derived chemopreventive and/or chemotherapeutic agent which blocks AP-1 and NF-κB induction. In addition to NT, other GI hormones (eg, bombesin, gastrin, GRP and substance-P) have been reported to stimulate expression of various cytokines/chemokines, such as IL-1β, IL-4, IL-6, IL-8, IL-12 and VEGF. Therefore, it is intriguing to speculate that GI hormones, released in response to dietary components, may enhance tumor growth and promote invasion through the increased expression and secretion of cytokines/chemokines and that these effects may be suppressed by curcumin. Our findings have important clinical ramifications since a majority of colorectal and pancreatic cancers possess receptors for various GI hormones, including NT.