Project 3: Flavonoids in Pancreatic Carcinogenesis & Angiogenesis
Principle Investigator: Joe O. Hines, MD
Co-Principle Investigator: Paul W. N. Lee, PhD
Research Assistant: Eliane Angst, MD
Collaborator: David Dawson, MD
Hypothesis: We hypothesize that flavonoids prevent the progression to pancreatic cancer, and that flavonoids may act as a chemotherapeutic in established pancreatic cancer.
Specific Aim 1: Determine the ability of flavonoids to prevent the progression of pancreatic intraepithelial neoplasia (PanIN) to invasive pancreatic ductal adenocarcinoma using a novel transgenic pancreatic cancer animal model.
Specific Aim 2: Assess the effect of flavonoids on immortalized human pancreatic cancer cell lines in an orthotopic xenograph model.
• tyrosine kinase inhibitor
• induces differentiation
• inhibits topoisomerase II
• inhibit angiogenesis
• combined with the TRAIL/Apo2L decreases pancreatic cancer cell proliferation
in vitro and tumor volume in vivo
• STAT3 is modulated by genistein in Panc-1 and MIA PaCA-2 and is inhibited at 10 _M
• increases growth inhibition and apoptosis induced by cisplatin, docetaxel, and
doxorubicin in PaCa cells
• in vivo, genistein shown to increase growth inhibition w/ gemcitabine or cisplatin
• genistein decreases CXCL8 and CXCL1
• flavonoid isomer of genistein, with hydroxylations at positions 5, 7, and 4’
• inhibits VEGF secretion with potency comparable to that of genistein
• inhibits hypoxia-inducible factor 1 (HIF-1) alpha
• inhibits TNF-alpha-induced IL-6 and CXCL8 production
• decreased primary tumor growth, increased apoptosis and prevented metastasis in
nude mice implanted with human pancreatic cells
• mitochondrial depolarization, cytochrome-c release followed by caspase-3 activation
• nuclear factor-kappa B activity is inhibited
• inhibit TNF-alpha-induced IL-6 and CXCL8 production (NFkB?)
Specific Aim 1, Preventive Model
Human Pancreatic Cancer Cells
Specific Aim 2, Treatment Model: orthotopic xenograph in nude mice
To date Project 3 has established the following findings:
- Therapeutic levels of apigenin and quercetin can be achieved in the plasma and pancreas of animals.
- Animal studies demonstrate that treatment with either quercetin or apigenin in the chow slows pancreatic cancer growth and metastasis.
- Metabolic profiling of pancreatic cancer cells treated with polyphenols demonstrates a clear altered metabolic signature.
- The combination of quercetin and the standard chemotherapeutic for pancreatic cancer, gemcitabine, results in an additive therapeutic effect compared to gemcitabine alone.
- Quercetin impairs cell proliferation by the inhibition of fatty acid synthesis and the induction of cell death.
- Apigenin induces pancreatic cancer apoptosis in vitro and in vivo.
- Apigenin-induced apoptosis in pancreatic cancer is mediated by transcription-independent p53 function via interactions with BclXL and PUMA.
During the past year the following new work has been completed:
Specific Aim I: Determine the ability of flavonoids to prevent the progression of pancreatic intraepithelial neoplasia (PanIN) to invasive pancreatic ductal adenocarcinoma using a novel transgenic pancreatic cancer animal model.
The LSL-KRASG12D mice are now 7 weeks into receiving either regular chow or 5% quercetin chow. The appropriate concentration of quercetin was confirmed in prior work (Figure 1). Control- and quercetin-fed animals will be sacrificed at 6, 9 and 12 months. Pancreata will be analyzed for the presence and extent of PanINs and invasive cancers as outlined below. At time of euthanasia, pancreata will be processed as described below for metabolic profiling, and angiogenic factors. Factors examined will be VEGFR2, VEGF, CXCR2, CXCR3, CXCL1, CXCL8, CXCL9, CXCL10, TSP-1, and MMP-9 protein, mRNA, and immunohistochemistry. Vascularity will be assessed. In addition, serum will be collected to measure levels of VEGF, CXCL1, CXCL8, CXCL9, CXCL10, and TSP-1.
Specific Aim II: Assess the effect of flavonoids on immortalized human pancreatic cancer cell lines in an orthotopic xenograph model.
Treatment with apigenin was associated with post-translational modification and subcellular trafficking of p53: Last year we had shown that treatment of pancreatic cancer cells (BxPC-3) with apigenin increased the ability of p53 to bind to promoter regions of targeted genes and thus partially restore the function of the mutant p53 protein. Follow-up work confirmed that nuclear extracts showed increased levels of p53 protein relative to cytoplasmic extracts peaking at 2 hours following apigenin treatment, indicating nuclear translocation of p53 (Figure 2). This occurred despite decreased overall expression of p53 (Figure 3).
Apigenin treatment induced the expression of p53-responsive proteins PUMA and p21: Apignenin treatment (25 uM) induced the expression of p21 and PUMA, consistent with our data showing nuclear translocation and increased DNA binding of p53 (Figure 4).
Apigenin influenced the interaction of p53 and PUMA with the anti-apoptotic BH-3 domain protein Bcl XL and induced binding between p53 and Bak: Immunopreciptiation of the anti-apoptotic Bcl-2 protein Bcl-XL showed there was dissolution of complexes between Bcl-XL/p53 and Bcl-XL/PUMA following apigenin treatment. Binding of p53 and PUMA with Bcl-XL effectively sequestered these molecules in the unstressed cells (Figure 5). Following liberation from Bcl-XL, p53 and PUMA both formed stable complexes with Bak.
TUNEL staining of tumor sections revealed more tumor cell apoptosis in apigenin-treated animals compared with control: Last year we reported that apigenin tumors were smaller in apigenin-fed mice. Subsequent TUNEL staining performed on tumor sections demonstrated increased signal indicating an increase in apoptotic cells for the treated tumors (Figure 6). Pancreatic cancer cells underwent apoptosis resulting in smaller tumors with higher levels of TUNEL staining. This data contributes additional support to a growing body of evidence indicating apigenin may hold promise as a chemotherapeutic for the treatment of pancreatic cancer.
Proposed mechanism of apigenin-induced apoptosis in pancreatic cancer cells (Figure 7): Based on these data and the data reported last year, we propose that apigenin induces dissociation of p53 and PUMA from sequestration by Bcl-XL allowing activation of Bak by both p53 and PUMA at the mitochondrial membrane leading to cytochrome c release and initiation of the caspase-driven mitochondrial cell death cascade. Release of p53 from Bcl-XL appears to have the added, secondary effect of nuclear translocation of p53 leading to increased transcription of p53 target genes, including PUMA. However, the transcriptionally-dependent function of p53 does not appear to be necessary for induction of apoptosis
Future directions and translational potential:
The main components of Aim II have been completed. We are working the metabolic core (Paul Lee) to complete the data regarding the metabolomic changes induced by apigenin and quercetin as outlined in the original project. The quercetin data has been assembled and the paper describing these results (outlined in the 2010 progress report) will soon be submitted. It does appear the apigenin, quercetin, and genestein have activity in established pancreatic cancer. These phytochemicals may find utility as additive remedies to established chemotherapeutic protocols, but will likely not be utilized as isolated single drug treatments.
The chemopreventive project in Aim I with apigenin has been initiated. This Aim is probably the most compelling with regard to the therapeutic implications. One might hypothesize that the use of phytochemicals to prevent the development of pancreatic cancer to be quite plausible. Given the time constraints of the current project, we anticipate completing only the apigenin chemopreventive project and not the other proposed phytochemicals.