Monica Chen

The flavonoid quercetin inhibits pancreatic cancer growth in vitro and in vivo

Objectives The flavonoid quercetin holds promise as an antitumor agent in several preclinical animal models. However, the efficacy of oral administration of quercetin in a pancreatic cancer mouse model is unknown. Methods The antiproliferative effects of quercetin alone or in combination with gemcitabine were tested in 2 human pancreatic cancer cell lines using cell count and MTT assays. Apoptosis was evaluated by flow cytometry. Tumor growth in vivo was investigated in an orthotopic pancreatic cancer animal model using bioluminescence. Quercetin was administered orally in the diet. Results Quercetin inhibited the growth of pancreatic cancer cell lines, which was caused by an induction of apoptosis. In addition, dietary supplementation of quercetin attenuated the growth of orthotopically transplanted pancreatic xenografts. The combination of gemcitabine and quercetin had no additional effect compared with quercetin alone. In vivo quercetin caused significant apoptosis and reduced tumor cell proliferation. Conclusions Our data provide evidence that oral administration of quercetin was capable of inhibiting growth of orthotopic pancreatic tumors in a nude mouse model. These data suggest a possible benefit of quercetin in patients with pancreatic cancer.

Evidence for activation of mutated p53 by apigenin in human pancreatic cancer.

Pancreatic cancer is an exceedingly lethal disease with a five-year survival that ranks among the lowest of gastrointestinal malignancies. Part of its lethality is attributable to a generally poor response to existing chemotherapeutic regimens. New therapeutic approaches are urgently needed. We aimed to elucidate the anti-neoplastic mechanisms of apigenin-an abundant, naturally-occurring plant flavonoid-with a particular focus on p53 function. Pancreatic cancer cells (BxPC-3, MiaPaCa-2) experienced dose and time-dependent growth inhibition and increased apoptosis with apigenin treatment. p53 post-translational modification, nuclear translocation, DNA binding, and upregulation of p21 and PUMA were all enhanced by apigenin treatment despite mutated p53 in both cell lines. Transcription-dependent p53 activity was reversed by pifithrin-α, a specific DNA binding inhibitor of p53, but not growth inhibition or apoptosis suggesting transcription-independent p53 activity. This was supported by immunoprecipitation assays which demonstrated disassociation of p53/BclXL and PUMA/BclXL and formation of complexes with Bak followed by cytochrome c release. Treated animals grew smaller tumors with increased cellular apoptosis than those fed control diet. These results suggest that despite deactivating mutation, p53 retains some of its function which is augmented following treatment with apigenin. Cell cycle arrest and apoptosis induction may be mediated by transcription-independent p53 function via interactions with BclXL and PUMA. Further study of flavonoids as chemotherapeutics is warranted.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Increases Prostaglandin E2 in Pancreatic Tumors

Objectives: Prostaglandin E2 (PGE2) is a product of cyclooxygenase (COX) and PGE synthase (PGES) and deactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH). Down-regulation of PGDH contributes to PGE2 accumulation in lung and colon cancers but has not been identified in pancreatic cancer. Methods: Normal human pancreatic and tumor-matched tissues, as well as MiaPaCa-2 and BxPC-3 cell lines, were assessed for COX-2, microsomal PGES-1, PGDH, and snail homolog 1 (SNAI1) and SNAI2 expressions by real-time polymerase chain reaction and Western blotting and PGE2 by enzyme-linked immunosorbent assay. Results: Normal tissues exhibited low COX-2 messenger RNA (mRNA) and protein expressions and high PGDH mRNA and protein expressions and PGE2 levels at 13 pg/mg of protein. In contrast, tumor tissues exhibited high COX-2 mRNA and protein expressions and low PGDH mRNA and protein expressions and PGE2 levels at 32 pg/mg of protein. Tumor tissues exhibited significantly elevated expressions of SNAI2 mRNA and protein but not SNAI1 because SNAI1 and SNAI2 reportedly down-regulate PGDH expression. The COX-2-positive BxPC-3 but not the COX-2-negative MiaPaCa-2 treated with 100-nmol/L PGE2 induced phosphorylated extracellular signal-related kinase that was blocked by the mitogen-activated protein kinase kinase inhibitor U0126, demonstrating the ability of PGE2 to activate ERK. Conclusions: These results suggest that enhanced PGE2 production proceeds through the expressions of COX-2 and microsomal PGES-1 and down-regulation of PGDH by SNAI2 in pancreatic tumors.Abbreviations: COX-2 - cyclooxygenase 2, MEK - mitogen-activated protein kinase kinase, MAPK - mitogen-activated protein kinase, ERK - extracellular signal-related kinase, EGF - epidermal growth factor, PKC - protein kinase C, PG - prostaglandin, PGDH - 15-hydroxyprostaglandin dehydrogenase, mPGES - microsomal prostaglandin E synthase

Apigenin inhibits NNK-induced focal adhesion kinase activation in pancreatic cancer cells.

Objectives Tobacco-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates &bgr;-adrenergic receptor (&bgr;-AR) signaling through Src/focal adhesion kinases (FAKs)/mitogen-activated protein kinase to modulate proliferation, migration, and survival. Apigenin (4′, 5, 7-trihydroxyflavone) is reported to attenuate proliferation and migration of cancer cells. This study was designed to determine the effects of apigenin on NNK-induced procarcinogenesis using human pancreatic cancer cells BxPC-3 and MIA PaCa-2, which express &bgr;-AR. Methods Proliferation and migration were assessed by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and scratch assays. &bgr;-AR, FAK/mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) expression and activation were assessed by Western blotting and real-time polymerase chain reaction. Results 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone caused a dose- and time-dependent increase in BxPC-3 and MIA PaCa-2 cell proliferation that was inhibited by propranolol or apigenin. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone also stimulated a time-dependent increase in FAK and ERK activation that was suppressed by propranolol or apigenin. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone–enhanced gap closure at 24 hours was prevented by either propranolol or apigenin. Conclusion Apigenin suppressed the effects of NNK on pancreatic cancer cell proliferation and migration that are mediated through the &bgr;-AR and its downstream signals FAK and ERK activation. These findings suggest a therapeutic role for this natural phytochemical in attenuating the procarcinogenic effects of NNK on pancreatic cancer proliferation and migration.

Abstract 62: Vitamin D3 induces 15-Hydroxyprostaglandin Dehydrogenase to suppress pancreatic cancer cell proliferation

BACKGROUND: Excess prostaglandin E2 (PGE2) mediates growth and survival of several cancers, including prostate and pancreatic cancers (PaCa). 15-Hydroxyprostaglandin dehydrogenase (PGDH) catalyzes PGE2 to an inactive 15-keto metabolite. We have previously shown that PGDH is down-regulated in human PaCa, contributing to accumulated levels of PGE2 that can subsequently feed back to suppress further PGDH expression. In contrast, PGDH expression has been reportedly up-regulated by vitamin D3 (VD3) in prostate cancer cells but has not been confirmed in pancreatic cancer cells. AIM: The study focuses on whether VD3 up-regulates PGDH expression in pancreatic cancer cells. METHODS: Human PaCa cell lines BxPC-3, HPAF-II and MIA PaCa-2 were treated with VD3 were analyzed for vitamin D receptor (VDR), cyclooxygenase-2 (COX-2) and PGDH expression by RT-PCR. Protein extracts were analyzed by Western blotting for VDR, COX-2, PGDH, SNAI1, SNAI2 and GAPDH expression. Cellular proliferation was analyzed by MTT assay. PGE2 was measured by ELISA. RESULTS: Dose-dependent treatment with VD3 at 1000 nM resulted in a maximal decrease in proliferation by 1.3 ± 0.1-fold in BxPC-3, 1.7 ± 0.3-fold in HPAF-II expressing elevated VDR and 1.1 ± 0.3-fold in MIA PaCa-2. VD3 at 10 nM resulted a time-dependent increase in PGDH mRNA expression by 3.8 ± 0.3-fold in BxPC-3 and 7.7 ± 0.6-fold in HPAF-II, whereas expression remained unchanged in MIA PaCa-2. Conversely, COX-2 mRNA expression decreased by 0.5 ± 0.1-fold in BxPC-3 and 0.6 ± 0.1-fold in HPAF-II, but not in MIA PaCa-2. PGDH protein expression increased at 96 and 120 hrs following treatment with VD3 in BxPC-3 and HPAF-II whereas COX-2 protein expression decreased in HPAF-II cells but was abolished by 96 and 120 hrs in BxPC-3 cells. PGDH protein expression was increased at 120 hrs in MIA PaCa-2 following addition of VD3 but COX-2 protein expression in this cell line was not detected. VD3 decreased the PGDH transcriptional regulators SNAI1 protein expression by 120 hrs in BxPC-3 and HPAF-II cells but not MIA PaCa-2 whereas SNAI2 decreased in all cell types by 96 hrs. Knockdown of SNAI by siRNA at 100 nM in HPAF-II resulted in a 1.3 ± 0.3-fold decrease proliferation and 1.4 ± 0.1-fold decrease in PGE2 production. Combined treatment with 100 nM of siSNAI and 10 nM of VD3 did not result in synergistic decrease in proliferation or PGE2 production. CONCLUSION: PaCa cells expressing vitamin D receptors responded to VD3 treatment with a corresponding increase in PGDH coupled with a decrease in COX-2 expression. VD3 or siSNAI resulted in a decrease in cellular proliferation and PGE2 production but the combined treatment did not augment further decreases, suggesting that the decrease by VD3 could be mediated through SNAI regulation of PGDH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 62. doi:1538-7445.AM2012-62

OA04.02. Mechanisms of growth inhibition of pancreatic cancer by omega-3 polyunsaturated fatty acids

Purpose Omega-3 polyunsaturated fatty acids (PUFAs) are widely considered health promoting. We have previously reported that the omega-3 PUFA eicosapentaenoic acid (EPA) inhibits growth of pancreatic cancer (PaCa) cells in vitro and in vivo. However, the mechanism underlying the effects of EPA in PaCa cells is unknown. Methods