Project 2: Polyphenols Regulate Lipid inflammatory Processes in Pancreatic Cancer
Principle Investigator: Diane Harris, PhD
Co-Investigator: Guido Eibl, MD
Co-Investigator: Vay Liang W. Go, MD
Hypothesis: We hypothesize that polyphenolic compounds from green tea and Scutellaria baicalensis (SB):
(1) inhibit eicosanoid production in pancreatic cancer cells
(2) lower the risk of developing pancreatic cancer (preventive effect)
(3) reduce the growth and spread of established pancreatic cancer (therapeutic effect)
We will use metabolic tracer technology to evaluate the overall phenotypic effect of polyphenols on pancreatic cancer cells. The proposed studies will heavily utilize the Metabolomic, Phytonutrient, and Animal Model Cores of the Center.
Specific Aim 1: To determine if mixed extracts from green tea and SB inhibit cell proliferation in vitro and if this activity is mediated through the eicosanoid-generating pathways. It will be divided into three subaims.
a) To determine the effects of green tea, SB, and their isolated polyphenolic compounds (EGCG, EGC, ECG, and EC for green tea and baicalin, bacalein, and wogonin for SB) on pancreatic cancer cell proliferation, apoptosis, and cell cycle progression compared to vehicle control and two pharmaceutical controls (indomethacin and nordihydroguaiaretic acid (ADGA)). The primary objective is to determine if the extracts diminish cell proliferation in physiologically achievable doses. Secondary aims are to compare the response of the mixed extract to isolated polyphenolic compounds found in these extracts and pharmaceutical inhibitors and to see if the effect varies by cell phenotype in 6 different lines.
b) To evaluate the efficacy of green tea and SB polyphenols to inhibit eicosanoid production in cultured pancreatic cancer cells;
c) Determine the effects of green tea and SB extracts on the metabolic profile of pancreatic cancer cells using metabolic tracer technologies:
1. To characterize phenotypic changes using [1,2-13C2] glucose; and
2. To use isotopic labeled arachidonic acid and COX and LOX products as recovery standards to determine turnover of archidonic acid as well as quantitative analysis of eicosanoids.
Specific Aim 2: To evaluate the preventive effects of green tea and SB polyphenols on the development and progression of pancreatic cancer precursor lesions using a transgenic animal model. It will also include two secondary subaims to determine.
a) the relative level of COX and LOX expression and eicosanoid content (PGE2 and LTB4) in each microdissected PanIN relative to normal duct epithelium; and
b) the metabolic phenotype of each lesion in the progression from normal epithelium to high-grade PanIN and correlate to changes in eicosanoid production.
Specific Aim 3: To determine the therapeutic role of green tea and SB extracts on pancreatic cancer growth in vivo using xenograft animal models. Secondary subaims will be to determine relative levels of immunohistochemical markers of proliferation and apoptosis and COX and LOX expression plus PGE2 and LTB4 levels in tumor tissue
As stated in the original proposal Specific Aim 1a) is to determine the effects of SB and SB-derived polyphenols (wogonin, baicalin, and baicalein) on pancreatic cancer cell proliferation, apoptosis, and cell cycle progression. In the year 3 report, we demonstrated SB and SB-derived polyphenols decreased proliferation and stimulated apoptosis in pancreatic cancer (PaCa) cell. In BxPC-3 and MIA PaCa-2 cells, we have reported that baicalein induced cytochrome c release from mitochondria, caspase-3/7 activity, and cleavage of PARP and that the effect of baicalein is through mitochondria/caspase cascade. Therefore, we began to delineate the molecular mechanisms underlying the pro-apoptotic effects of baicalein in PaCa cells. First, we sought to determine the effect of baicalein on anti-apoptotic Bcl-2 family proteins expression. Baicalein decreased mRNA and protein expression of the anti-apoptotic Bcl-2 family protein Mcl-1 expression in a dose- and time-dependent manner, as shown by real-time PCR and Western blot. To delineate if Mcl-1 is essential for PaCa cell survival, Mcl-1 expression was Knocked-down by siRNA that resulted in marked cleavage of caspase-3, -7, and PARP and induction of apoptosis in BxPC-3 and MIA PaCa-2 cells. Baicalein also reduced Mcl-1 mRNA level by real time RT-PCR, suggesting that the effect of baicalein is mediated at least partly through inhibition of transcription. Over-expression of Mcl-1 greatly reduced the pro-apoptotic effect of baicalein. Mcl-1 has been shown to play a role in cancer cell resistance to the chemotherapeutic agent gemcitabine in other cancers. We tested the hypothesis that baicalein enhances gemcitabine sensitivity in PaCa cells. Panc-1 cells showed resistance to gemcitabine, while BxPC-3 was moderately sensitive as demonstrated by cell proliferation and the absence of apoptotic markers. Pre-treatment of cells for 24 h with baicalein, followed by treatment with both baicalein and gemcitabine for 48 hours increased sensitivity to gemcitabine in both cell lines. Cell proliferation was decreased and apoptosis increased at doses of gemcitabine lower than previously required to trigger cell death in these cell lines. In Panc-1 cells treated with baicalein, lower levels of Mcl-1 were observed. The decrease in Mcl-1 expression corresponded with the level of inhibition of cell proliferation. This study demonstrated that baicalein can enhance the sensitivity of pancreatic cancer cell lines to gemcitabine, possibly through baicalein’s reduction of Mcl-1 expression. All together, our results demonstrate that the pro-apoptotic effect of baicalein is mediated through a mitochondrial pathway via a reduction of the pro-survival function of Mcl-1 and thus enhanced gemcitabine sensitivity in PaCa cells. This study supports previous work that demonstrates the critical role of Mcl-1 in gemcitabine resistance and provides a novel phytochemical inhibitor of Mcl-1.
We also completed a metabolomic study of another panel of flavonoids — luteolin, resveratrol, and quercetin — against the metabolite flux-controlling properties of a synthetic targeted fatty acid synthase inhibitor drug C75 on multiple macromolecule synthesis pathways in pancreatic tumor cells using [1,2-13C2]-D-glucose as the single precursor metabolic tracer. MIA PaCa-2 pancreatic adenocarcinoma cells were cultured for 48 h in the presence of 0.1% DMSO (control), or 50 μM or 100 μM of each test compound, while intracellular glycogen, RNA ribose, palmitate and cholesterol as well as extra cellular 13CO2, lactate and glutamate production patterns were measured using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). The use of 50% [1,2-13C2]-D-glucose as tracer resulted in an average of 24 excess 13CO2 molecules for each 1000 CO2 molecule in the culture media, which was decreased by 29% and 33% (p<0.01) with 100 μM C75 and luteolin treatments, respectively. Extracellular tracer glucose-derived 13C-labeled lactate fractions (Sm) were between 45.52% and 47.49% in all cultures with a molar ratio of 2.47% M+1/Sm lactate produced indirectly by direct oxidation of glucose in the pentose cycle in control cultures; treatment with 100 μM C75 and luteolin decreased this figure to 1.80% and 1.67%. The tracer glucose-derived 13C labeled fraction (Sm) of ribonucleotide ribose was 34.73% in controls, which was decreased to 20.58% and 8.45% with C75, 16.15% and 6.86% with luteolin, 27.66% and 19.25% with resveratrol, and 30.09% and 25.67% with quercetin, respectively. Luteolin effectively decreased nucleotide precursor synthesis pentose cycle flux primarily via the oxidative branch, where we observed a 41.74% flux (M+1/Sm) in control cells, in comparison with only a 37.19%, 32.74%, or a 26.57%, 25.47% M+1/Sm flux (p<0.001) after 50 μM or 100 μM C75 or luteolin treatment. Intracellular de novo fatty acid palmitate (C16:0) synthesis was severely and equally blocked by C75 and luteolin treatments indicated by the 5.49% (control), 2.29% or 2.47% (C75) and 2.21% or 2.73% (luteolin) tracer glucose-derived 13C-labeled fractions, respectively. On the other hand there was a significant 192% and 159% (p<0.001), and a 103% and 117% (p<0.01) increase in tracer glucose-derived cholesterol after C75 or luteolin treatment. Only resveratrol and quercetin at 100 μM inhibited tracer glucose-derived glycogen labeling (Sm) and turnover by 34.8% and 23.8%, respectively. The flavonoid luteolin possesses equal efficacy to inhibit fatty acid palmitate de novo synthesis as well as nucleotide RNA ribose turnover via the oxidative branch of the pentose cycle in comparison with the targeted fatty acid synthase inhibitor synthetic compound C75. Luteolin is also effective in stringently controlling glucose entry and anapleurosis in the TCA cycle, while it promotes less glucose flux towards cholesterol synthesis than that of C75. Quercetin and resveratrol rather inhibit glycogen synthesis and turnover as their underlying mechanism of controlling tumor cell proliferation. Therefore the flavonoid luteolin controls fatty and nucleic acid syntheses as well as energy production with pharmacological strength, which can be explored as a non-toxic natural treatment modality for pancreatic cancer. This study will be a model for our subsequent metabolomic study of SB polyphenols.
Specific Aim 1b) is to evaluate the effect of SB polyphenols to inhibit eicosanoid production. There is strong evidence for an important role of COX-2 and COX-2 generated PGE2 during pancreatic tumorigenesis. In the year 3 report, we reported that baicalein reduced basal and arachidonic acid-stimulated PGE2 release by BxPC-3 cells, while incubation with exogenous PGE2 (1 µM) reversed the anti-proliferative effect induced by low concentrations of baicalein. PGE2 is formed by the isomerization of PGH2 by the action of three specific PGE2 synthases (PGES) and we showed that baicalein also inhibited mPGES-1 expression in BxPC-3 cells, which resulted in lower PGE2 levels, but had no effect on mPGES-2 and cPGES expression. We have shown the similar effects of both baicalein and wogonin in reducing PGE2 release and mPGES-1 expression in another COX-2 positive PaCa cells (HPAF II). Our data indicate that baicalein had a profound apoptotic and anti-proliferative effect in COX-2 positive pancreatic cancer cells through inhibiting PGE2 synthesis suggesting that inhibition of PGE2 synthesis represents a potential target for selection of effective anti-tumorigenic phytochemicals for prevention and/or adjuvant therapy in pancreatic cancer.
Specific Aim 3) is to determine the therapeutic role of SB extracts on pancreatic cancer growth in vivo using xenograft animal models. In the year 3 report, we reported the results from our preliminary bioavailability experiments that after feeding nude mice with AIN-76A diet containing 1% of SB extracts for 7 weeks. Plasma, liver, and pancreas were collected at the end of study and were subjected to HPLC analysis to determine levels of baicalein (BE), baicalin (BG), and wogonin (WO). In 1% SB fed mice, all three major SB compounds were detected most abundantly in liver (10 to 30 µM for BE and WO), and also in pancreas (1 to 3 µM). These results indicate that the appearance of SB compounds in plasma with SB feeding and the bioactive compounds of SB accumulate in the target tissue of interest such as liver and pancreas. We then conducted in vivo study using mouse xenograft model by injecting MIA PaCa-2 cells subcutaneously into the flank of nude mice. Mice were randomly divided to control and 1% SB diet (n=8 per group). SB feeding started 8 days prior to the cell inoculation and continued for 13 weeks. Animals were then sacrificed and tumor volumes and weights were determined, and tissues were harvested for measuring levels of baicalein, wogonin, oroxylin A, and their conjugates using HPLC. Tumor size from SB treated mice was significantly smaller (by 35%) than control diet mice, however, the tumor weight showed little difference between two groups. Further histopathological examinations, necrotic markers, and apoptotic markers will be investigated. The HPLC analysis showed that bacalin can accumulate in plasma, tumor xenografts, and pancreas, liver, lung, and other tissues in vivo.
While plasma contains predominantly conjugates of baicalein, wogonin, and oroxylin A, in all other organs tested and in tumor, both aglycones and conjugates were found. Our data can be used to predict the organ site that is likely beneficial to the SB treatment, and to provide important information on the interpretation of chemo-therapeutic efficacy of SB.
Specific Aim 1a) Baicalein still had an additional (albeit significantly less robust) effect on apoptosis in Mcl-1 knocked-down PaCa cells, suggesting alternative pathways (independent of Mcl-1) of apoptosis induction by baicalein. Further studies are necessary to delineate the role of other pro- and anti-apototic family proteins involved in baicalein-induced cell-death in PaCa cells, as well as documenting the metabolomic profile of cells treated with SB extract and SB polyphenols.
Specific Aim 1b) We plan to overexpress mPGES1 in BxPc-3 cells to block the baicalein effect on anti-proliferation and apoptosis. The effect of baicalein on other prostaglandins (PGI2, PGF2) and the expression/activity of PGE2-degrading enzyme 16-hydroxy-PGDH will be examined to understand the role of baicalein on shifting the balance of various prostanoid/eicosanoid production and its effect on PaCa cell proliferation and apoptosis.
Specific Aim 2 &3) We are in the process of examining the tissue/tumor slides collected from in vivo Xenograft studies for TUNEL staining and protein samples collected for apoptotic markers and pro-/anti-apoptotic family protein expression. We will conduct studies using the orthotopic xenograft model to study the therapeutic role of SB on in vivo pancreatic cancer proliferation and tumor formation. We also plan to evaluate the preventive effects of SB extracts on the development and progression of pancreatic cancer precursor lesions using a transgenic animal model.