Access Type

Open Access Dissertation

Date of Award

12-1-2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Nutrition and Food Science

First Advisor

Smiti Gupta

Abstract

Adenocarcinoma of pancreas is recognized for its poor prognosis, as it progresses asymptomatically and is rarely diagnosed at early stage. According to American cancer society pancreatic cancer, it has lowest survival rate in all cancer types, with less than 6% five year survival rate. Surgical resection is only possible for 20% of diagnosed cases and current chemotherapy has only 15% response rate. Even the most favored drug, Gemcitabine, increases survival time only by a few months, depending on the stage at diagnosis. Some bioactive food components or nutraceuticals have shown chemopreventive and chemotherapeutic effects without added side effects. Garcinol is a polyisoprenylated benzophenone derivative from Garcinia Indica fruit extract. Garcinol has shown dose dependent favorable response in pancreatic cancer cell lines, alone and in combination with Gemcitabine. In this study, we investigated the invivo effects of dietary Garcinol in a transgenic mouse model of Pancreatic Cancer (PaCa). This model is considered to be the animal model which best mimics development of human PaCa. Based on invitro data from our lab we hypothesized that Dietary Garcinol treatment will slow down the progression of Pancreatic Cancer in Kras and p53 transgenic Pancreatic Cancer mouse model. We had three specific aims. Specific aim 1) To investigate the invivo response of dietary Garcinol on PaCa animal models and to monitor the tumor progression with MRI. We also ruled out the possibility of toxicity of dietary Garcinol by blood smears and fore stomach H& E slides. Additionally we did detailed histological investigation of Intraepithelial neoplasia lesions in the pancreatic tissue collected at sacrifice. Specific aim 2, To investigate the mouse pancreatic and liver tissue, for response to the dietary Garcinol at molecular level. We studied changes in the micro RNA profile of Garcinol treated vs. non-treated animals and investigated the response of dietary Garcinol on tumor promoter target genes of miRNAs from different molecular pathways, in pancreas and a common metastatic liver tissue. Specific aim 3, To investigate the differences in Urinary Metabolomic profiles of Garcinol treated mice compared to non-treated mice and Target analysis to identify metabolites responsible for differences. Methods, Six to eight week old male KPC mice (kras and P53 conditional mutant) were divided into 4 groups( KC- cancer control; KGr- 0.05% Garcinol diet; KGm-Gemcitabine injected; KGG- Garcinol+Gemcitabine), littermates without mutation served as non- cancer controls were divided into 2 groups( CC- isocaloric diet, CG- Garcinol diet) . Mice were fed 0.05% Garcinol added diet or isocaloric diet (Dyets- Bethlehem, PA). Mice were individually housed. Body weight were measured twice/week from week 1 to week 6. Two groups received Gemcitabine weekly injections (100mg/kg- intraperitoneal injections) up to 5 week. MRI T2 weighted image scans were conducted on 7T scanner (ClinScan, Bruker, Karlsruhe, Germany) in week 1 and week 5 of the study. Urine samples were collected in week 2, 4 and 6 for metabolomic analysis. Mice were euthanized in week six of the study and tissues were collected. Blood smear slides were prepared at the time of sacrifice and after drying were fixed in 95% Ethanol. The smears were stained with Wright Giemsa Stain and observed under the microscope (Nikon Eclipse 80i). Pancreatic tissues was fixed in 10% neutral buffered formalin and then transferred to 70 % ethanol before staining. H&E stained slides were prepared and observed under microscope (Nikon Eclipse 80i). Specific immuno histostaining (IHC) with S100P and DPC4/SMAD4 was performed to confirm the findings. MicroRNA array analysis was conducted and some miRNA were validated by RTPCR. Some of the target genes of miRNA identified to be differently expressed were investigated by RTPCR. Nuclear Magnetic resonance spectroscopy was performed on Varian-500S. Simca P+ and CHENOMX NMR suite were used for Metabolomic analysis of urinary spectra. Results, Dietary Garcinol arrests the Progression of Pancreatic Cancer invivo in KRAS and p53 pancreas- specific mutant mouse model. All the mice in Garcinol group survived the 6 week study, with 75% survival in KGm and KGG. We observed tumor reduction in KGr, KGm and KGG groups. There was no toxicity observed to the blood in blood smears with Garcinol treatment, NK and NKT cell were found to be in higher percentage in KGr group compared to KC group. No toxicity to Garcinol was observed in fore stomach H&E slides, additionally we observed reduced papilloma formations in KGr group compared to KC group. Reduction in total number of Pancreatic intraepithelial neoplasia (mPanIN) was observed in both Garcinol treated groups with lowest number of mPanIN 3 in KGG group indicating slowed progression of mPanIN 1 to mPanIN 3. Histology findings were confirmed with specific immuno-histostaining with S100P and DPC4/SMAD4 antibodies. MicroRNA microarray data showed down regulation of multiple tumor promotor miRNA and up regulation of several tumor suppressor miRNA with Garcinol treatment. Lower expression levels of miRNA 23a and higher expression levels miRNA 451a after Garcinol treatment were validated by RTPCR. Relative mRNA expressions levels of MMP9, CCND1 and Notch1 were found to be decreased in KGr and KGG groups compared to KC group in pancreatic tissue samples. Relative expression levels of CCND1 were decreased in KGr and KGG groups in Liver tissue samples. MMP9 relative expression levels showed reduction in Garcinol treated groups KGr group. Relative expression levels of Bcl2 were found to be decreased in KGr, KGm and KGG groups. Urinary metabolomics profiles from week 6 Garcinol treated group were closer to non- cancer and week 2 of non-treated KC group. Target analysis with CHENOMX identified Taurine, Tartrate and Phenylacetate to be in higher concentrations in Garcinol treated group compared to non-treated group and Allantoin was found to be lower in Garcinol treated group compared to KC non-treated group. This invivo dietary Garcinol study has highlighted anti-cancer potential of this bioactive food component. Our data indicates that dietary Garcinol retarded pancreatic cancer progression in transgenic pancreatic cancer mice. Garcinol has shown pleotropic effects, targeting multiple tumor related pathways. Further investigation of molecular pathways identified can lead to its incorporation as a chemotherapeutic agent in a clinical trial.

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