Normal Pancreatic Duct Epithelial Cells Research Articles

Migratory Activity of CD105+ Pancreatic Cancer Cells Is Strongly Enhanced by Pancreatic Stellate Cells

CD105 expression correlates with prognosis for several cancers. However, its significance in pancreatic cancer is unclear.We analyzed CD105 expression in resected pancreatic cancer tissue and pancreatic cancer cell lines, compared the properties of CD105(+) and CD105(-) cells using quantitative RT-PCR and migration assays, and evaluated the relationship between CD105(+) cells and pancreatic stellate cells (PSCs).Immunohistochemistry showed that the frequency of CD105 expression was higher in pancreatic cancer than that in normal tissue(8% vs 0%, respectively). In flow cytometry, CD105 was expressed in pancreatic cancer cells, whereas weak CD105 expression was detected in normal pancreatic ductal epithelial cells. Quantitative RT-PCR showed that E-cadherin mRNA expression was suppressed and vimentin mRNA was overexpressed in CD105(+) cells (P < 0.05). Migration of CD105(+) cancer cells was strongly enhanced (more than that of CD105(+) cells) in coculture with PSCs (P < 0.05). CD105 expression did not correlate to clinicopathologic characteristics or the Kaplan-Meier survival analysis.Suppression of an epithelial marker and over expression of a mesenchymal marker suggest that epithelial-mesenchymal transition is induced in CD105(+) pancreatic cancer cells. CD105(+) pancreatic cancer cell migration is strongly enhanced by PSCs, suggesting that these cells play a role in the pancreatic cancer microenvironment.

Expression of major histocompatibility complex class I-related chain A/B (MICA/B) in pancreatic carcinoma

Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind to the immunoreceptor NKG2D and play an important role in mediating cytotoxicity of NK and T cells. Release of MIC molecules from the cell surface is thought to constitute an immune escape mechanism of tumor cells and thus could be associated with more aggressive course of tumor growth. In this study, we investigated the expression of MICA/B in ductal pancreatic carcinoma and serum in relation to tumor stage, differentiation and survival. MICA/B expression in tumor tissues and sera from patients with pancreatic cancer were analyzed by immunohistochemical staining (IHC), western blotting and ELISA, respectively. MICA/B expression was present in 17 of 22 (77%) of the tumors but not in normal pancreatic ductal epithelial cells. Poorly differentiated tumors showed more pronounced MICA/B expression compared to differentiated tumors, but did not correlate significantly to other tumor characteristics. MICA/B-negative tumors displayed significantly lower incidence of lymph node metastases (p<0.01), and less mortality within 3 years following resection (p<0.02). In conclusion, tissue levels of MICA/B expression were elevated in pancreatic cancer cells without elevated levels in serum, despite well-recognized acute phase reactants in serum. Poorly differentiated tumors showed high MICA/B expression, which was related to extended tumor lymph node metastases and less frequent long-term survival.

Autophagy and enhanced chemosensitivity in experimental pancreatic cancers induced by noninvasive radiofrequency field treatment

Patients with pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options and poor response to the standard gemcitabine (GCB)-based chemotherapy. In the current study, the authors investigated the feasibility of noninvasive short-wave radiofrequency (RF) electric fields to improve the cytotoxic effect of GCB on PDAC cells and determined its mechanism of action. The cytotoxicity of RF alone and in combination with GCB was studied in vitro on normal pancreatic human pancreatic ductal epithelial cells and different PDAC cell lines by flow cytometry, and in vivo on ectopic and orthotopic human PDAC xenograft models in mice. The mechanism of RF activity was studied by Western blot analysis and immunohistochemistry. Toxicity was determined by histopathology. Exposure of different PDAC cells to 13.56-megahertz radio waves resulted in a substantial cytotoxic effect, which was accompanied by the induction of autophagy but not apoptosis. These effects of RF were found to be absent in normal cells. Excessive numbers of autophagosomes in cancer cells persisted 24 to 48 hours after RF exposure and then declined. The addition of a subtoxic dose of GCB to RF treatment inhibited the recovery of cancer cells from the RF-induced autophagy and enhanced the cytotoxic effect of the latter on cancer cells. The treatment of PDAC in situ in mice with the combination of noninvasive RF and GCB was found to have a superior antitumor effect compared with the use of RF or GCB alone, yet there was no evidence of systemic toxicity. Noninvasive RF treatment induced autophagy but not apoptosis in cancer cells and demonstrated potential as an enhancer of chemotherapy for treating patients with pancreatic cancer without toxicity to normal cells.

Expression of microRNA‐218 in human pancreatic ductal adenocarcinoma and its correlation with tumor progression and patient survival

Our aim was to analyze clinicopathologic and prognostic values of microRNA (miR)-218 in pancreatic ductal adenocarcinima (PDAC). TaqMan quantitative RT-PCR was used to determine the expression of miR-218 in human PDAC cells and tissue samples. The association of miR-218 expression with clinicopathologic variables was analyzed. Kaplan-Meier survival analysis was performed to analyze the association of miR-218 expression with recurrence-free survival or overall survival of patients. Univariate and multivariate Cox regression analyses were performed. The relative level of miR-218 in PDAC cells was significantly lower than that in normal human pancreatic duct epithelial cell line. Also, the mean level of miR-218 in PDAC tissues was significantly lower than that in normal pancreatic tissues. Statistical analyses indicated that low miR-218 expression was closely associated with poor tumor differentiation, advanced tumor stage, higher incidence of lymph node metastasis, and tumor recurrence. Kaplan-Meier survival analyses showed that patients with low miR-218 expression had lower recurrence-free and overall survival than those with high miR-218 expression. Univariate and multivariate Cox regression analyses showed that miR-218 might be an independent prognostic factor. Reduced miR-218 in PDAC tissues was correlated with tumor progression, and might be an independent poor prognostic factor for patients.

Pharmacological Reversal of Histone Methylation Presensitizes Pancreatic Cancer Cells to Nucleoside Drugs: In Vitro Optimization and Novel Nanoparticle Delivery Studies

We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines by increased apoptosis. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies.

Specific protein kinase C makes tight junctional differences between pancreatic cancer cells and normal pancreatic duct epithelial cells

s / Pancreatology 13 (2013) S1–S80 S17 down-regulation of Bcl-2 or Bcl-xL protein expression had a much weaker effect on PaCa apoptosis. Surprisingly, simultaneous knock-down of BclxL and Mcl-1 induced apoptosis strongly. However, simultaneous knockdown of Bcl-xL and Bcl-2 or Mcl-1 and Bcl-2 had no additive effect. The apoptosis inducing effect of simultaneous knock-down of Bcl-xL and Mcl1 is associated with Bax activation, mitochondrial cytochrome c release, and caspase cascade. Furthermore, the apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was confirmed in animal model. Conclusions: These data suggest Bcl-xL and Mcl-1 as a potential therapeutic target for PaCa treatment and may help to develop novel therapeutic strategies for PaCa cells.

Abstract A30: Inhibiting histone methylation as an epigenetic therapeutic approach for pancreatic cancer

Abstract We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies. Citation Format: Rajgopal Govindarajan. Inhibiting histone methylation as an epigenetic therapeutic approach for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A30.

Triterpenes from Poria cocos suppress growth and invasiveness of pancreatic cancer cells through the downregulation of MMP-7

Poria cocos is a medicinal mushroom that is widely used in traditional Asian medicine. Here, we show that a characterized mixture of triterpenes extracted from P. cocos (PTE) and three purified triterpenes: pachymic acid (PA), dehydropachymic acid (DPA) and polyporenic acid C (PPAC) suppress the proliferation of the human pancreatic cancer cell lines Panc-1, MiaPaca-2, AsPc-1 and BxPc-3. Moreover, the most effective compound, PA, only slightly affects the proliferation of HPDE-6 normal pancreatic duct epithelial cells. The anti-proliferative effects of PTE on BxPc-3 cells are mediated by the cell cycle arrest at G0/G1 phase. DNA microarray analysis demonstrated that PTE significantly downregulates the expression of KRAS and matrix metalloproteinase-7 (MMP-7) in BxPc-3 cells. In addition, PTE and PA suppress the invasive behavior of BxPc-3 cells. The inhibition of invasiveness by PTE and PA was associated with the reduction of MMP-7 at the protein level and the role of MMP-7 further confirmed by the gene silencing of MMP-7 which also suppressed the invasiveness of BxPc-3 cells. In conclusion, triterpenes from P. cocos demonstrate anticancer and anti-invasive effects on human pancreatic cancer cells and can be considered as new therapeutic agents in the treatment of pancreatic cancer.

Abstract 4233: MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer.

Abstract Aberrant DNA hypermethylation, the most well defined epigenetic changes in cancer, is associated with inappropriate gene silencing and this feature is utilized to search for tumor-specific DNA methylation biomarkers. Methyl-CpG binding domain (MBD) proteins (MBPs) can elicit the repressive potential of methylated DNA and play a major role in gene silencing mechanisms. Therefore, if the genes governed by MBPs are specifically reactivated, it should be possible to uncover them. We developed a method termed “methyl-CpG targeted transcriptional activation (MeTA)” that employs a fusion gene comprised of the MBD from MBD2 and the NFκB transcriptional activation domain. Microarray coupled with MeTA (MeTA-array) provides not only the information about methylated genes but also the one about transcriptional repression in a single experiment. We first applied either MeTA or a conventional DNA demethylating agent, 5-aza-cytidine (Aza-CR), to a human embryonic kidney cell line 293T and analyzed gene expression profiles by microarray; 138 and 202 genes that are upregulated 5-fold or more were identified by MeTA and Aza-CR, respectively. ChIP-Seq analysis indicated that MeTA-inducing protein containing MBD binds to more than 80% of genes selected by MeTA-array. We found associations between expressional restorations by MeTA, methylation status, and NFκB (AD)-MBD fusion protein bindings in CpG islands (CGIs) around the transcription start site of the genes. Importantly, MeTA can upregulate genes meeting the stringent criteria of CGIs defined by Takai and Jones (Proc Natl Acad Sci USA 99: 3740-3745, 2002) at the promoter region at higher frequency; 109 of 138 (79.0%) genes in MeTA vs. 121 of 202 (59.9%) genes in Aza-CR. We further applied MeTA-array to 12 pancreatic cancer cell lines along with HPDE (normal pancreatic ductal epithelial cell line) and identified 31 candidate tumor-specific hypermethylated genes; 26 of them have never been reported previously using the conventional DNA demethylating agents. Seven genes, IRX4, LHX6, NEFH, NEFL, NEFM, NPTX2 and TMEM204 were further examined their methylation statuses by MSP, and we found that 100% (21/21) of IRX4, 62% (13/21) of LHX6, 100% (21/21) of NEFH, 100% (21/21) of NEFL, 100% (21/21) of NEFM, 100% (21/21) of NPTX2 and 95% (20/21) of TMEM204 were methylated in our series of pancreatic cancer cell lines. Furthermore, 68% (15/22) of IRX4, 55% (12/22) of LHX6, 55% (12/22) of NEFH, 59% (23/22) of NEFL, 82% (18/22) of NEFM and 82% (18/22) of NPTX2 were also hypermethylated in primary pancreatic cancer specimens in a tumor-specific manner. Our results suggest that MeTA-array is a highly efficient method to identify methylation-mediated transcriptionally silenced genes in human cancer. Citation Format: Shinichi Fukushige, Akira Horii. MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4233. doi:10.1158/1538-7445.AM2013-4233

Abstract 1025: Optimized DZNep exposure presensitizes pancreatic cancer cells to anticancer nucleoside analogues: potential clinical implications.

Abstract We evaluated the potential of a histone methylation reversal agent 3-deazaneplanocin A (DZNep) in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents produced a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs and emphasize the promising clinical utilities of histone methylation reversal agents in future pancreatic cancer combination therapies. Citation Format: Sau Wai Hung, Hardik Mody, Sean Marrache, Yangzom D. Bhutia, Franklin Davis, Jong Hyun Cho, Shanta Dhar, Chung K. Chu, Rajgopal Govindarajan. Optimized DZNep exposure presensitizes pancreatic cancer cells to anticancer nucleoside analogues: potential clinical implications. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1025. doi:10.1158/1538-7445.AM2013-1025

Decreased expression of DAB2IP in pancreatic cancer with wild-type KRAS

KRAS mutation plays an important role in the pathogenesis of pancreatic cancer. However, the role of wild-type KRAS in the progression of pancreatic cancer remains unknown. The present study was to investigate the expression of the Ras GTPase activating protein (DAB2IP) in pancreatic cancer and its clinical significance. The expression of DAB2IP in pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was analyzed by Western blotting and real-time quantitative reverse transcription-PCR (qRT-PCR). The KRAS mutational types of pancreatic cancer tissues obtained from pancreatic cancer patients (n=20) were also analyzed. Subsequently, DAB2IP expression was detected in pancreatic cancer tissues, adjacent and normal pancreatic tissues (n=2) by immunohistochemistry, and the relationship between DAB2IP expression and the clinical characteristics of patients was evaluated. Western blotting and qRT-PCR results showed that DAB2IP expression in pancreatic cancer cells with wild-type KRAS was lower than that in those with mutation-type KRAS and normal human pancreatic ductal epithelial cells (P<0.05). Immunohistochemistry showed that DAB2IP expression was lower in pancreatic cancer tissues than that in adjacent and normal pancreatic tissues (Z=-4.000, P=0.000). DAB2IP expression was lower in pancreatic cancer patients with the wild-type KRAS gene than that in those with KRAS mutations (WilcoxonW=35.000, P=0.042). Furthermore, DAB2IP expression in patients with perineurial invasion was lower than that in those without invasion (WilcoxonW=71.500, P=0.028). DAB2IP expression was lower in patients with more advanced stage than that in those with early clinical stage (WilcoxonW=54.000, P=0.002). DAB2IP expression was reduced in patients with pancreatic cancer compared with those with no cancer. DAB2IP expression was correlated with the KRAS gene, perineurial invasion and clinical stage of the disease. Our data indicated that DAP2IP expression can be used as a potential prognostic indicator and a promising molecular target for therapeutic intervention in patients with pancreatic cancer.

Cl− and K+ channels in human pancreatic ductal adenocarcinoma (PDAC) cells

The Cl‐ channel anoctamin‐1 (ANO1 or TMEM16A) is overexpressed in oral, head, prostate and colon cancer and predicted as a pharmaceutical target or biomarker for distant metastasis. The K2p K+ channel family (e.g. TREKs, TWIKs, and TASKs) is also associated with the pathogenesis of cancer. ANO1 and the K2p channels are involved in regulation of cell migration, cell volume, and apoptosis and might contribute to cancer development through modulating these processes. Here, we aim at determining the roles of ANO1 and K2p channels in pancreatic ductal adenocarcinoma (PDAC). Channel expression levels in 4 human PDAC lines (AsPC‐1, BxPC‐3, PANC‐1, MIAPaCa‐2) were compared to the normal pancreatic ductal epithelial cell line (HPDE*) by qPCR analysis. We found ANO1, TWIK‐1, TREK‐1 and TASK‐3 to be over‐expressed in PDAC cells. Whole‐cell patch‐clamp measurements of ANO1 in AsPC‐1 cells revealed a time‐, voltage‐and temperature dependent Cl‐ current with the instantaneous ICl/V relationship exhibiting GHK‐rectification. ANO1‐like currents were in our experiments not recorded in HPDE cells. We conclude that ANO1 upregulation may be a marker in pancreatic cancer cell lines, however, its function is yet unknown and ongoing studies evaluate the potential roles of ANO1 in PDAC growth and chemotherapy resistance. Financed by FP7 Marie Curie Initial Training Network “IonTraC”.

Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial-mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells.

Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.

Class I and Class II Histone Deacetylases Are Potential Therapeutic Targets for Treating Pancreatic Cancer

BackgroundPancreatic cancer is a highly malignant disease with an extremely poor prognosis. Histone deacetylase inhibitors (HDACIs) have shown promising antitumor activities against preclinical models of pancreatic cancer, either alone or in combination with chemotherapeutic agents. In this study, we sought to identify clinically relevant histone deacetylases (HDACs) to guide the selection of HDAC inhibitors (HDACIs) tailored to the treatment of pancreatic cancer.MethodologyHDAC expression in seven pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was determined by Western blotting. Antitumor interactions between class I- and class II-selective HDACIs were determined by MTT assays and standard isobologram/CompuSyn software analyses. The effects of HDACIs on cell death, apoptosis and cell cycle progression, and histone H4, alpha-tubulin, p21, and γH2AX levels were determined by colony formation assays, flow cytometry analysis, and Western blotting, respectively.ResultsThe majority of classes I and II HDACs were detected in the pancreatic cancer cell lines, albeit at variable levels. Treatments with MGCD0103 (a class I-selective HDACI) resulted in dose-dependent growth arrest, cell death/apoptosis, and cell cycle arrest in G2/M phase, accompanied by induction of p21 and DNA double-strand breaks (DSBs). In contrast, MC1568 (a class IIa-selective HDACI) or Tubastatin A (a HDAC6-selective inhibitor) showed minimal effects. When combined simultaneously, MC1568 significantly enhanced MGCD0103-induced growth arrest, cell death/apoptosis, and G2/M cell cycle arrest, while Tubastatin A only synergistically enhanced MGCD0103-induced growth arrest. Although MC1568 or Tubastatin A alone had no obvious effects on DNA DSBs and p21 expression, their combination with MGCD0103 resulted in cooperative induction of p21 in the cells.ConclusionOur results suggest that classes I and II HDACs are potential therapeutic targets for treating pancreatic cancer. Accordingly, treating pancreatic cancer with pan-HDACIs may be more beneficial than class- or isoform-selective inhibitors.

Role of the protein kinase C signaling pathway in the regulation of claudin-4 in normal human pancreatic duct epithelial cells and cancer cells

Role of the protein kinase C signaling pathway in the regulation of claudin-4 in normal human pancreatic duct epithelial cells and cancer cells

A Potential Window onto Early Pancreatic Cancer Development: Evidence of Cancer Stem Cell Growth after Exposure to Cadmium Chloride in Vitro

Cancer stem cells are a small subset of tumor cells that are postulated to underlie tumor initiation, growth, and metastasis. Research suggests that these cells may have begun as normal stem cells that underwent mutation or other changes that derail cellular programming and alter growth control pathways. Investigating a previously established link between cadmium exposure and pancreatic cancer, a new study finds that normal human pancreatic cells chronically exposed in vitro to a low level of cadmium chloride acquired cancer cell characteristics and generated what appeared to be cancer stem cells [EHP 120(9):1265–1271; Qu et al.]. Normal human pancreatic ductal epithelial cells were cultured with or without 1.0 µM of cadmium chloride for 29 weeks. The treated cells were sampled periodically and assessed for cancer cell characteristics, including increased secretion of matrix metalloproteinase-9 (MMP-9), increased invasiveness, increased anchorage-independent growth (i.e., ability to grow while floating freely in medium), and altered colony formation. Various subpopulations of pancreatic cancer stem cells have been shown to overexpress the genes CD44, CXCR4, OCT4, and S100P, which are involved in various aspects of cancer growth and spread. Samples therefore were also analyzed for expression of these genes and their associated proteins. The results showed that, compared with control cells, those cells chronically exposed to cadmium (which the authors dubbed CCE cells) demonstrated characteristics typical of cancer cells. Spheres derived from CCE cells (bottom) rapidly outpaced those derived from control cells (top). Stem cells and cancer stem cells, when studied in vitro, often form free-floating spheres of cells for reasons not completely understood. The investigators isolated and further cultured spheres generated by control and CCE cells, then submitted them to the same analyses as earlier samples. Spheres derived from CCE cell cultures exhibited cancer cell characteristics and were more numerous and larger than control spheres. They also exhibited increased transcription and expression of OCT4, CD44, and CXCR4. In gel cultures CCE spheres grew aggressively and formed more intricate structures than control spheres; microscopically, these structures appeared malignant, with poorly differentiated cells of many shapes and sizes. The study shows that in vitro exposure of normal human pancreatic cells to a nontoxic level of cadmium chloride can promote the development of cancer characteristics, thus strengthening the evidence for cadmium as a potential cause of pancreatic cancer in humans. Additionally, the study may help investigators define the very early stages of pancreatic cancer, a major advantage for a disease that typically is diagnosed only after it has metastasized.

Regulation of tight junctions in human normal pancreatic duct epithelial cells and cancer cells

To investigate the regulation of tight junction molecules in normal human pancreatic duct epithelial (HPDE) cells and pancreatic cancer cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture and compared them to pancreatic cancer cell lines. The hTERT-transfected HPDE cells were positive for PDE markers and expressed claudin-1, claudin-4, claudin-7, and claudin-18, occludin, tricellulin, marvelD3, JAM-A, zonula occludens (ZO)-1, and ZO-2. The tight junction molecules, including claudin-4 and claudin-18 of normal HPDE cells, were in part regulated via a protein kinase C signal pathway by transcriptional control. In addition, claudin-18 in normal HPDE cells and pancreatic cancer cells was markedly induced by a PKC activator, and claudin-18 in pancreatic cancer cells was also modified by DNA methylation. In the marvel family of normal HPDE cells and pancreatic cancer cells, tricellulin was upregulated via a c-Jun N-terminal kinase pathway, and marvelD3 was downregulated during Snail-induced epithelial-mesenchymal transition.

Overexpression and oncogenic function of aldo-keto reductase family 1B10 (AKR1B10) in pancreatic carcinoma

AbstractAldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), and metabolizing these lipid substrates has a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohistochemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 overexpressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared with a normal pancreatic ductal epithelial cell (HPDE6E7), all of the six cultured pancreatic adenocarcinoma cell lines had an overexpression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in (1) increased cell apoptosis, (2) increased non-farnesyled HDJ2 protein and (3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation.

Abstract LB-150: Binding selectivity of RNAs by eukaryotic initiation factor 3f in pancreatic cells

Abstract Deregulation of translation is necessary for an increase in protein production that benefits cancer cells to increase proliferation and tumor growth. Eukaryotic initiation factor 3f (eIF3f), a subunit in the eIF3 protein complex, has been shown to be a regulator for translation initiation. Our group was the first to describe eIF3f as a tumor suppressor, where we show its expression is lost in melanoma and pancreatic tumor tissues. In this study, we show that eIF3f binds to various RNAs for genes involved in GTP-binding, RNA binding, RNA capping and other cellular processes, through the use of RNA-Immunoprecipitation assay followed by a microarray analysis, coined RIP-Chip in normal human pancreatic ductal epithelial (HPDE) cells. We confirmed the binding of eIF3f to these RNAs through RNA-Immunoprecipitation (RIP) assay using an antibody against eIF3f to pulldown of our sample RNA and visualized by RT-PCR analysis. To determine the role of this selective binding of eIF3f to these particular RNAs, we observed their expression in the HPDE cells, were eIF3f was stably knocked-down with lentivirus containing eIF3f-siRNA. Preliminary results demonstrate some correlation between the absence of eIF3f and the protein levels of the genes encoding for the eIF3f pulled-down RNA. Our results demonstrate that eIF3f may be involved in directly targeting specific oncogenic RNA sequences for degradation. Further investigations will be needed to elucidate the role of these genes and eIF3f expression in the transformation of pancreatic tissues to the aggressive phenotype of pancreatic cancer. 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 LB-150. doi:1538-7445.AM2012-LB-150