Pancreatic Cancer (PC) is a deadly disease, and only 4% of all patients with PC in the U.S. are expected to survive 5 years after diagnosis (Jemal et al., 2008
). The low cure rate in PC is in part due to the lack of an effective strategy for the prevention of tumor progression and/or treatment. Emerging evidence suggest that many dietary compounds exhibit beneficial effects toward the prevention of cancer (Sarkar and Li, 2003
; Sarkar and Li, 2004
; Sarkar and Li, 2006
). Although advances have been made in better understanding the biology of human cancers, much of the research focusing on cancer prevention using a variety of chemopreventive agents without any toxicity to normal cells remains to be clinically proven. In recent years combinatorial approach to cancer therapy or prevention is being widely recognized as an effective approach to maximize benefit. Recently, Nair et al
. reported combined inhibitory effects of green tea polyphenols and sulforaphane in human colon carcinoma cells (Nair et al., 2008
). In addition, combination therapy of gastrointestinal cancer using sulforaphane and dibenzoylmethane was recently reported (Shen et al., 2007
). The results from our laboratory have shown that the combination of isoflavone with reduced doses of chemotherapeutic agents could be much more effective compared to higher doses of chemotherapeutic agents in different types of cancers including prostate, breast, lung, and PCs, suggesting that enhanced anti-cancer activity could be achieved without any toxicity to normal cells (Li et al., 2005
; Sarkar and Li, 2006
). In the present study, we investigated whether the combinations of two dietary natural products (isoflavone and curcumin) could show greater degree of cell growth inhibition and induction of apoptosis in PC cell lines compared to single agents.
Our current data showed, for the first time, that the combined treatment of PC cells with isoflavone and curcumin caused significantly increased growth inhibition and apoptosis, suggesting that this strategy could be an effective approach for the killing of PC cells. In addition, our data provided mechanistic information supporting the concept of combination of two compounds, which exerts their pro-apoptotic effects by inhibiting Notch-1, resulting in the inactivation of NF-κB DNA-binding activity and the inactivation of their target genes.
Notch signaling plays important roles in maintaining the balance between cell proliferation, differentiation and apoptosis (Miele and Osborne, 1999
; Miele et al., 2006
; Miele, 2006
). The Notch gene is abnormally activated in many human malignancies. It has been reported that the Notch signaling is involved in PC cell survival and that Notch pathway components and Notch target genes are up-regulated in PCs (Miyamoto et al., 2003
; Wang et al., 2006a
; Wang et al., 2006d
). It has been known that Notch gene suppresses apoptosis and promotes cell proliferation through a growth factor mediated survival pathway. Nair et al
observed that Notch-1 induces cyclin D1 and CDK2 activity and inhibits p53 dependent apoptosis in cervical cancer cells through PI3K/AKT pathway (Nair et al., 2003
). Jang et al
have shown that Notch-1 expression regulates cell death through both apoptosis and cell cycle pathways in erythroleukemia cells with regulation of Bcl-xL, p21cip1/waf1
and the retinoblastoma protein Rb (Jang et al., 2004
). Based on our results, we speculate that one possible mechanism by which isoflavone and curcumin induce apoptosis is in part due to the down-regulation of Notch-1, which leads to the down-regulation of Notch-1 target genes, such as cyclin D1, Bcl-xL, resulting in the inhibition of cell proliferation, and induction of apoptosis. Indeed, we found that isoflavone and curcumin inhibited the expression of Notch-1. Moreover, the combined treatment was more effective, which could be attributed to the inactivation of Notch-1 signaling pathway as supported by greater degree of reduction in the levels of Notch-1 target gene expression (Cyclin D1, Bcl-2 and Bcl-xL) compared to either agent alone.
Notch-1 signaling pathway has been shown to activate NF-κB. Specifically, Notch-1 has been reported to strongly induce NF-κB promoter activity and induce the expression of several NF-κB subunits and NF-κB DNA-binding activity (Jang et al., 2004
; Oswald et al., 1998
; Wang et al., 2006a
). Levels of basal and stimulation-induced NF-κB activity were significantly decreased in mice with reduced Notch levels (Wang et al., 2004
). Constitutive levels of Notch activity are essential to maintain NF-κB activity in various cell types. It is generally accepted that Notch and NF-κB pathways are key regulators of numerous cellular processes such as proliferation, differentiation and apoptosis. In the present study, we found that isoflavone and curcumin inhibited NF-κB DNA-binding activity. We also found a greater degree of reduction in the DNA binding activity of NF-κB in PC cells in response to the combinatorial treatment compared to either agent alone, suggesting that these changes are partly responsible for the increased apoptosis or decreased survival of PC cells.
It is becoming increasingly clear that the blocking of multiple signaling pathways is an effective therapeutic approach for the prevention of disease progression and/or treatment of human cancers, including PC. Therefore, identification of inhibitors targeting multiple members of the signaling pathway is likely to provide a better therapeutic outcome in patients diagnosed with cancer, a disease caused by dysregulation of multiple and complex signaling pathways. Reddy et al
observed that cell growth inhibition in response to the combination of curcumin and EGFR inhibitor (EGFR Related Protein, ERRP) was significantly greater than that caused by either agent alone and this was associated with decreased activation (tyrosine phosphorylation) of EGFR, ErbB-2, ErbB-3, and/or IGF-1R. (Reddy et al., 2006
). Whereas curcumin inhibited constitutive activation of both EGFR and IGF-1R, ERRP decreased activation of EGFR, ErbB-2, and ErbB-3 but had no effect on IGF-1R (Reddy et al., 2006
). In addition, we have reported that isoflavone down-regulated the EGFR and Akt pathway (El-Rayes et al., 2006
) whereas curcumin inhibited COX-2 and EGFR expression and decreased Erk1/2 activity in PC cells (Lev-Ari et al., 2006
). Kotha et al
reported that resveratrol inhibits Src and Stat3 signaling and induces apoptosis of malignant PC cells containing activated Stat3 protein (Kotha et al., 2006
). Recently, Shankar et al
reported that EGCG caused growth arrest at G1 stage of cell cycle through regulation of cyclin D1, cdk4, cdk6, p21/WAF1/CIP1
, and induced apoptosis through generation of reactive oxygen species and activation of caspase-3 and caspase-9 (Shankar et al., 2007
; Shankar et al., 2008
). EGCG also inhibited the expression of Bcl-2 and Bcl-xL and induced the expression of Bax, Bak, Bcl-xS and PUMA in PC cells (Shankar et al., 2007
; Shankar et al., 2008
). Based on these results, we speculated that one possible mechanism by which four natural products, used in our study, inhibited cell growth to a greater degree is in part due to down-regulation of multiple signaling pathways such as Notch-1, Akt, EGFR, and NF-κB. However, further in-depth studies including clinical trials are needed to fully evaluate the value of isoflavone in combination with curcumin, resveratrol and EGCG for the prevention and/or treatment of human PC.
In Summary, our results demonstrate that exposure of PC cells to isoflavone together with curcumin caused a greater degree of cell growth inhibition than either agent alone. The effect of combinatorial treatment on the inhibition of growth and induction of apoptosis could be attributed to the inhibition of Notch-1 and NF-κB signaling pathways. Because neither isoflavone nor curcumin are known to exert toxic effects, the current combinatorial strategy using more than one natural product could potentially be a superior and nontoxic strategy for the prevention and/or treatment of PC.