Pancreatic Cancer Cell Lines AsPC-1 Research Articles

Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

BackgroundThe membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3.ResultsChromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer.ConclusionsOur studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways might inhibit the expression of MUC4 and prevent the proliferation and invasion of pancreatic cancer cells.

Parecoxib inhibits cell proliferation and induces apoptosis in human pancreatic cancer cell lines BxPC-3 and AsPC-1

Parecoxib inhibits cell proliferation and induces apoptosis in human pancreatic cancer cell lines BxPC-3 and AsPC-1

Multiple Kinase Pathways Involved in the Different De Novo Sensitivity of Pancreatic Cancer Cell Lines to 17-AAG

17-Allylamino-17-demethoxygeldanamycin (17-AAG) specifically targets heat shock protein (HSP)90 and inhibits its chaperoning functions for multiple kinases involved in cancer cell growth and survival. To select responsive patients, the molecular mechanisms underlying the sensitivity of cancer cells to 17-AAG must be elucidated. We used cytotoxicity assays and Western blotting to explore the effects of 17-AAG and sorafenib on cell survival and expression of multiple kinases in the pancreatic cancer cell lines AsPC-1 and Panc-1. Gene cloning and transfection, siRNA silencing, and immunohistochemistry were used to evaluate the effects of mutant p53 protein on 17-AAG sensitivity. AsPC-1 and Panc-1 responded differently to 17-AAG, with half maximal inhibitory concentration (IC(50)) values of 0.12 and 3.18 μM, respectively. Comparable expression of HSP90, HSP70, and HSP27 was induced by 17-AAG in AsPC-1 and Panc-1 cells. P-glycoprotein and mutant p53 did not affect 17-AAG sensitivity in these cell lines. Multiple kinases are more sensitive to HSP90 inhibition in AsPC-1 than in Panc-1 cells. After 17-AAG treatment, p-Bad (S112) decreased in AsPC-1 cells and increased in Panc-1 cells. Sorafenib markedly increased p-Akt, p-ERK1/2, p-GSK-3β, and p-S6 in both cell lines. Accordingly, 17-AAG and sorafenib acted antagonistically in AsPC-1 and Panc-1 cells, except at high concentrations in AsPC-1 cells. Differential inhibition of multiple kinases is responsible for the different de novo sensitivity of AsPC-1 and Panc-1 cells to HSP90 inhibition. P-glycoprotein and mutant p53 protein did not play a role in the sensitivity of pancreatic cancer cells to 17-AAG.

Abstract B63: Synergistic combination between resveratrol and tanshinone I for effective prevention of pancreatic cancer

Abstract Pancreatic cancer is the fourth most common cause of cancer-related mortality in the United States. Pancreatic cancer is often dubbed the silent killer because it seldom causes symptoms until advanced. As a result, few pancreatic cancers are operable at diagnosis. Furthermore, pancreatic cancer is often resistant to conventional radiotherapy or chemotherapy, and the morbidity rate almost equals the mortality rate. Therefore, search for effective regimens with moderate side effects for pancreatic cancer therapy and prevention is an urgent necessity. Chinese herbal medicines have been used for centuries with demonstrated safety profiles, thus their bioactive ingredients may be effective candidates for the prevention and/or treatment of pancreatic cancer. Danshen (Salvia miltiorrhiza) has been widely adopted in the traditional Chinese medicinal preparations for treatment of cardiovascular disease with minimal side effects. In the present study, we evaluated the effect of several major Danshen components, tanshinone IIA (T2A), tanshinone I (T1) and cryptotanshinone (CT) on pancreatic cancer cells. We found that CT, T2A and T1 all showed inhibitory effects on the growth of human pancreatic cancer cell lines MIA PaCa-2 and ASPC-1 with IC50 around 30μM, 3–8μM and 3–5μM, respectively. The drug-resistant human pancreatic cancer cell line PANC-1 showed less sensitivity to CT and T2A treatment but was very sensitive to T1 treatment with IC50 around 3.5μM. The potent T1 activity against pancreatic cancer cells was associated with downregulation of several protein markers related to cell cycle (cyclin D and cyclin B) and anti-apoptosis (Bcl2 and survivin), c-myc and Aurora A and B kinases in both MIA PaCa-2 and PANC-1 cells. To further identify more potent synergistic combination effect of natural bioactive compounds against pancreatic cancer, we determined the anti-growth activity of the combination between T1 and resveratrol, a natural compound found in the skin of red grape and other fruits. T1 and resveratrol combinations consistently showed synergistic effect on inhibiting the growth of MIA PaCa-2, ASPC-1 and PANC-1 cells. Protein assays showed that the synergistic combination effect of T1 and resveratrol was associated with combined downregulation of c-myc and Aurora A protein levels, suggesting that T1 and resveratrol may inhibit the growth of pancreatic cancer cells in part via combined inhibition on the functions of c-myc and Aurora A. Aurora A is the kinase that plays a critical role in controlling mitosis and cell growth. We further confirmed if Aurora A was a functional target for the synergistic combination effect of T1 and resveratrol. Knockdown of Aurora A dramatically inhibited PANC-1 cell growth and decreased the T1 and resveratrol combination activity, suggesting that Aurora A could be one of the targets by the T1 and resveratrol combination effect. Aurora A gene promoter contains the c-myc binding site, suggesting that Aurora A may be regulated by c-myc and the inhibition of c-myc expression may be one of the causes of reduced Aurora A expression following T1 treatment. To confirm this, we treated PANC-1 cells with a c-myc inhibitor and found decreased Aurora A gene expression in a time-dependent manner, indicating the regulation of c-myc on Aurora A gene expression. Our results suggest that the T1 and resveratrol combination may serve as a novel chemopreventive regimen for effective prevention of pancreatic cancer. Supported in part by DOD (PC073988) and NCI/NIH (R21CA127794) Citation Information: Cancer Prev Res 2011;4(10 Suppl):B63.

Modulation of Pancreatic Cancer Chemoresistance by Inhibition of TAK1

TGF-β-activated kinase-1 (TAK1), a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor κB (NF-κB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer. NF-κB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n = 40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. The results of in vitro proliferation were analyzed for statistical significance of differences by nonlinear regression analysis; differences in mouse survival were determined using a log-rank test. All statistical tests were two-sided. AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-κB activity than did their respective control cell lines (relative luciferase activity: AsPc-1, mean = 0.18, 95% confidence interval [CI] = 0.10 to 0.27; control, mean = 3.06, 95% CI = 2.31 to 3.80; MDAPanc-28, mean = 0.30, 95% CI = 0.13 to 0.46; control, mean = 4.53, 95% CI = 3.43 to 5.63; both P < .001). TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC(50) between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells (P < .001). In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden (gemcitabine vs gemcitabine plus LYTAK1, P = .03) and prolonged survival duration (median survival: gemcitabine, 82 days vs gemcitabine plus LYTAK1, 122 days; hazard ratio = 0.334, 95% CI = 0.027 to 0.826, P = .029). The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.

Omeprazole Inhibits Proliferation and Modulates Autophagy in Pancreatic Cancer Cells

BackgroundOmeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines.Methodology/Principal FindingsDose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results.ConclusionsWe hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.

The effect of dual PI3K/mTOR inhibitor NVP-BGT226 on cell cycle and survivin and STAT3 gene expression in human pancreatic cancer cell lines.

e14523 Background: The phosphatidylinositol-3-kinase (PI3K) pathway is one of the most commonly activated signaling pathways in pancreatic cancer. The inhibition of this transducing pathway may open new options for the treatment of pancreatic cancer. NVP-BGT226 is a novel dual class PI3K/mammalian target of rapamycin (mTOR) inhibitor that has entered Phase I/II clinical trials. Methods: We have tested the effects of NVP-BGT226 on cell viability (M'TT assay), apoptosis (AnnexinV/PI-FACS and PARP1 cleavage), cell cycle (FACS PI/RNAse-staining) and expression of Survivin, STAT3 and BCL-xL with Real-time RT-PCR and Western blot in the human pancreatic cancer cell lines Panc-1, BxPc-3, AsPC-1 and MiaPaCa-2. Results: The cell viability decreased within 24-72 hours exposure to about 50% compared to untreated control cells in a concentration and but not time dependent manner. Apoptosis was not detected, but cell cycle analysis revealed that NVP-BGT226 induced predominantly a G0/G1-arrest to about 90-95%. Analysis with Real-time RT-PCR and Western blot showed a remarkable reduction of Survivin and STAT3 expression but an increase of the anti-apoptotic BCL-xL gene expression. Conclusions: The PI3K/mTOR inhibitor NVP-BGT226 induces G0/G1-arrest and and acts via downregulation of Survivin and STAT3.

Combination Paclitaxel and Inhibitor of Nuclear Factor κB Activation Improves Therapeutic Outcome for Model Mice With Peritoneal Dissemination of Pancreatic Cancer

Paclitaxel (PTX) is a useful treatment for peritoneal dissemination of malignant tumors. However, chemoresistance due to PTX-induced nuclear factor κB (NF-κB) activation is an important cause of suboptimal therapeutic effect. We previously reported nafamostat mesilate (FUT175) inhibits NF-κB activation and promotes apoptosis in pancreatic cancer. We hypothesized that addition of FUT175 to PTX may enhance the antitumor effect in peritoneal dissemination of pancreatic cancer. In vitro, we assessed NF-κB activity and apoptosis by the combination of FUT175 and PTX using human pancreatic cancer cell line (AsPc-1). In vivo, we established peritoneal dissemination in nude mice by intraperitoneal injection of AsPc-1 cells. The animals were treated with intraperitoneal injection thrice a week of FUT175, once a week of PTX, or a combination of thrice a week of FUT175 and once a week of PTX (combination group). In the combination groups, PTX-induced NF-κB activation was inhibited, and apoptosis was enhanced in comparison with other groups both in vitro and in vivo. In the combination group, tumor growth, serum tumor marker, and survival rate were significantly better than those in other groups (P < 0.05). Combination chemotherapy using PTX with FUT175 exerts an antitumor effect for peritoneal dissemination of pancreatic cancer.

Abstract 257: Targeted inhibition of mutant k-ras by siRNA in pancreatic cancer cells results in increased sensitivity to anti-EGFR therapy

Abstract Cancer patients are frequently resistant to anti-epidermal growth factor receptor (EGFR) therapy due to secondary mutations in k-ras. This is particularly true for pancreatic cancer patients where the frequency of k-ras mutations is exceptionally high. A variety of therapeutic strategies to target RAS effector pathways have been pursued with limited success. We report here on the use of small interfering RNA (siRNA) to silence oncogenic k-ras in a pancreatic cancer cell line with the goal of sensitizing the cells to anti-EGFR chemotherapies. The AsPC-1 pancreatic cancer cell line expresses EGFR and is homozygous for the mutant k-ras GAT (GGT to GAT in codon 12) allele resulting in the constitutive activation of downstream pathways and reduced apoptotic function. We independently transfected (lipofectamine 2000) AsPC-1 with two siRNAs, one targeted to the mutant (si-k-ras GAT) and one to the wild type (si-k-ras GGT) alleles. We found that levels of k-ras expression (RT-PCR) were significantly reduced after si-k-ras GAT transfection but not after si-k-ras GGT transfection indicating target specificity against the mutant allele. As a preliminary test of the potential clinical utility of si-k-ras GAT mediated inhibition, we measured the relative viability (Tox-8 assay) of AsPC-1 cells transfected with si-k-ras GAT and the EGFR inhibitors Gemcitabine and Erbitux. The results demonstrate a significant increase in sensitivity to both drugs in cells transfected with si-k-ras GAT. We conclude that siRNA mediated inhibition of mutant k-ras may be a promising method by which to sensitize pancreatic cancer cells to anti-EGFR therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 257. doi:10.1158/1538-7445.AM2011-257

Abstract 540: The pro-inflammatory protein S100A9 promotes pancreatic cancer cell growth

Abstract S100A9 is a pro-inflammatory protein originally found in macrophages which is felt to be important in promoting inflammatory responses and cancer cell growth. The protein is found in cancer cells of gastric, colon, breast cervical and thyroid cancer. Generally, S100A9 is a cytoplasmic, cell surface and secreted protein that promotes cancer cell growth via inflammatory mechanisms and migration of immune cells (e.g. PBMCs) to the cancer microenvironment via non-covalent interactions with glycosaminoglycans on endothelial surfaces. Previously, this protein was reported in the stroma of pancreatic cancers but not in pancreatic cancer cells themselves. This report iilustrates S100A9 up-regulation on the surface of p53 mutant pancreatic cancer cell lines BxPC3 and AsPC1 as determined by immunoblot analysis and flow cytometry. In this study, pentamidine a known inhibitor of S100B, inhibited S100A9 dependent cell growth in BxPC3 cell lines (IC50 = 20.1 +/- 10.3 μM). Pentamidine treated BxPC3 cells (20 μM for 96 hrs) exhibited 30.1% apoptotic cell death as measured by Annexin/PI statining vs less than 3% for control treated cells. Given that S100A9 was found in other cancer models to promote recruitment of pro-infammatory cells (e.g. PBMCs) and subsequent support of cancer cell growth, we studied the effect of pentamidine on PBMC migration. Pentamidine also blocked the migration of PBMC from a metastatic pancreatic cancer patient (67%) to a similar degree as an Anti-s100A9 (68%) Ab or an anti-s100A8/A9 (65%) antibody. S100A8/A9 antibody is known to bind to helix 1 of S100A8 and helix 4 of S100A9, areas of the protein considered important for target interactions. It is likely that pentamidine indiscriminately binds to both the S100A8/A9 heterodimer as well as the homodimer in addition to other S100 proteins. S100A9 levels in BxPC3 cells as detected via Western analysis also increase with 20 μM pentamidine treatment, but not 10 fold as reported in the literature for S100B. As p53 was also shown previously to bind to the S100A9 promoter and S100A9 is not as robustly expressed in mutant p53 cell lines, p53 independent pathways are clearly important for cancer cell survival. This study suggests that S100A9 is up-regulated in pancreatic cancer and that inhibition causes pancreatic cancer cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2011-540

A novel antiangiogenic approach for adjuvant therapy of pancreatic carcinoma

Surgical therapy remains the only curative option for pancreatic ductal adenocarcinoma. But even after complete resection, almost all patients suffer from local tumor recurrence. Current standard adjuvant therapy with gemcitabine does not impressively affect the recurrence rate. The aim of this study was to evaluate a novel anti-angiogenic adjuvant treatment strategy by targeting the vascular endothelial growth factor receptor (VEGFR). We assayed the effects of a novel VEGFR inhibitor (ZK261991) on pancreatic carcinoma. ZK261991 is a highly selective and potent VEGFR-kinase inhibitor, which is orally available. We used a previously established nude mouse orthotopic pancreatic cancer resection model. Subcutaneous donor tumor fragments (1mm(3)) derived from human pancreatic cancer cell lines HPAF-2 and AsPC-1 were implanted in the pancreatic tail of 48 nude mice. Fourteen days afterwards, all mice underwent a histologically confirmed curative tumor resection followed by daily adjuvant oral therapy with ZK261991 (50mg/kg; n = 24) vs. placebo (n = 24). The mice were sacrificed after 12weeks of therapy or in case of defined endpoints. All sacrificed mice underwent autopsy. A dissemination score (local and systemic tumor spread), size of recurrent tumor mass, survival, and weight loss/gain were surveyed. Kaplan-Meier analysis of survival showed a significant benefit for mice treated with ZK261991 after HPAF-2 tumor resection: 83.8days (95% CI 73.9-93.6) vs. 60.9days (95% CI 48.9-73.0), p = 0.006. Adjuvant treatment with ZK261991 of AsPC-1-derived tumors showed a tendency towards a benefit compared to control but no significant difference: 75.8days (95% CI 59.7-91.9) vs. 65.7days (95% CI 51.6-79.7). There were no significant differences in dissemination score and size of recurrent tumor mass between the treatment groups. Adjuvant anti-angiogenic therapy with the novel VEGFR-inhibitor ZK261991 resulted in a significant survival benefit after curative tumor resection in a clinically relevant orthotopic animal model of pancreatic cancer. Combination of anti-angiogenic treatment with cytotoxic agents may further improve the results of adjuvant therapy.

Construction of MKK4 adenovirus and its effects on the proliferation and invasion of pancreatic cancer cells

Objective To explore the effects of MKK4 gene on the proliferation and invasion of pancreatic cancer cells. Methods Western blotting was used to assay the expression of MKK4 in pancreatic cancer cell lines AsPC1, BxPC3, Hs766t, MiaPaCa2, and Pancl. MKK4 adenovirus was constructed and transfected into MKK4 negative pancreatic cancer cells. Thymidine incorporation assays were used to measure the DNA synthesis rate and Matrigel assays to measure the invasive ability of pancreatic cells. Results In pancreatic cancer cells, MKK4 lost expression in AsPC1 and BxPC3 cells. MKK4 adenovirus was constructed and transfected into MKK4 negative pancreatic cancer cell lines AsPC1 and BxPC3. The exogenous expression of MKK4 in AsPC1 and BxPC3 increased the proliferation ability by 53% and 61%( P ＜ 0. 05 ), increased the invasion ability by ( 8. 2 ± 2. 7) fold and ( 15.7 ± 1.6) fold ( P ＜ 0. 01 ), respectively. Conclusion The exogenous expression of MKK4 increases the proliferation and invasion of pancreatic cancer cells, which hints that MKK4 gene is a potential oncogene in pancreatic cancer. Key words: Pancreatic carcinoma; MKK4

Abstract 4430: A novel mitotic kinesin Eg5 inhibitor exerts the growth-inhibitory effect in cancer cells in a manner independent of induction of monoastral formation but dependent on KRAS mutation status

Abstract Mitotic kinesin Eg5 plays an important role in mitosis, as it is critical for proper bipolar spindle assembly. After the discovery of the first Eg5 inhibitor monastrol, a number of Eg5 inhibitors have been developed as anticancer drugs. We have previously synthesized a series of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors and demonstrated potent inhibitory effect on Eg5 ATPase activity and induction of mitotic arrest with characteristic monoastral spindles in HeLa cells. In this study, we evaluated the growth-inhibitory acitivities of novel STLC derivatives, Compound1, Compound2, and Compound3 by MTT assay in a wide range of human cancer cell lines derived from gastric (MKN1, MKN45, MKN74, NUGC3, NUGC4, NCI-N87), colon (C170, DLD1, HCT15, COLO205), pancreatic (AsPC1, BxPC3, SUIT2) and lung (PC-9, PC-14, H1975, H441, A549) cancers and investigated potential predictive biomarkers. Among the derivatives, Compound3 showed the most potent growth-inhibitory effect and sensitivity between cell lines showed marked differences, with IC50 values ranging from 0.134 to 7.14 μM. Pancreatic cancer cell line AsPC1 and non-small cell lung cancer (NSCLC) cell lines H441 and A549 which harbor KRAS mutation showed relatively high IC50 values compared to other cell lines without KRAS mutation. Compound3 induced G2/M arrest in all cell lines as early as 8 hours after the treatment by flowcytometry, while the increase of the apoptotic sub-G1 fraction was not observed in these AsPC1, H441 and A549 cells harboring KRAS mutation. These results suggested that differential sensitivity to Compound3 and attenuated induction of apoptosis by Compound3 were attributed to KRAS mutation. Induction of monoastral spindles formation by Compound3 was observed as early as 8 hours after the treatment not only in sensitive cell lines but in refractory cell lines such as AsPC1 and H441 cells by immunocytochemistry, suggesting the induction of monoastral spindles formation does not work as a predictive biomarker for clinical development. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level of each cell line by western blot analysis. No significant difference was observed in the induction of a spindle checkpoint kinase BubR1 phosphorylation among the cells, a potential predictive biomarker in response to the Eg5 inhibitor treatment by western blot analysis. Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development and KRAS mutation status was suggested to be a candidate for a predictive biomarker of poor response to Eg5 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4430.

Abstract 5416: Delta-tocotrienol potentiates antitumor activity of gemcitabine in pancreatic cancer through inhibition of nuclear factor-κB

Abstract Background: Gemcitabine is currently the standard therapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance. Chemoresistance to gemcitabine seems to be due to constitutive activation of nuclear factor-kappaB (NF-κB) and induction of its regulated gene products. We have already shown that δ-tocotrienol is the most active natural vitamin E in depleting the NF-κB activity in pancreatic cancer. Therefore in this study we used δ-tocotrienol to enhance the antitumor activity of gemcitabine in pancreatic cancer in vitro and in vivo. Methods: The antiproliferative (MTT assay) and proapoptotic (Cell Death ELISA assay) activities of gemcitabine and δ-tocotrienol alone and in combination were evaluated in human pancreatic cancer cell lines MiaPaCa-2 and AsPc-1. Additive/synergistic effects of the combination was determined using combination index and isobologram plot. The effect of the two drugs and combination on malignant transformation in MiaPaca-2 cells was determined by colony formation assay. The in vivo activity of the two drugs and combination was evaluated in SCID nude mice xenografted with AsPc-1 cells. Because the NF-κB has a central role in gemcitabine chemoresistance, we investigated the effect of the two drugs alone and in combination on the NF-κB activity and its regulated gene products in vitro and in vivo. Results: The combination of gemcitabine and δ-Tocotrienol inhibited the cell proliferation of pancreatic cancer cells (AsPc-1 and Mia PaCa-2) more than either drug alone. Gemcitabine and δ-tocotrienol combination at 1:2 ratio resulted in additive to synergistic effects on inhibition of cell proliferation of Mia PaCa-2 cells. The inhibition of malignant transformation and apoptosis induction (histone associated DNA fragments, caspase-3 activity, PARP-1 cleavage, and BAX expression) in pancreatic cancer cells was greater with δ-tocotrienol compared with gemcitabine. The combination of the two drugs resulted in additive/synergistic effects on apoptosis induction. Gemcitabine and δ-tocotrienol combination was more effective in inhibiting AsPc-1 tumor growth and apoptosis induction in mice than either drug alone. Gemcitabine was less effective in inhibiting the nuclear DNA binding of NF-κB/p65 activity, p65/p50 subunits, and antiapototic proteins Bcl-XL, XIAP and survivin expression compared to δ-tocotrienol. However the combination of gemcitabine and δ-Tocotrienol showed additive/synergistic effects on depletion of NF-κB activity and its regulated gene products in vitro and in vivo. Conclusion: This combination chemotherapy could be a potential novel strategy for advanced pancreatic cancer. This study supports the selection of δ-tocotrienol as a potential therapeutic adjuvant to potentiate gemcitabine antitumor activity in human pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5416.

Abstract C108: A novel mitotic kinesin Eg5 inhibitor exerts the growth-inhibitory effect in cancer cells in a manner independent of induction of monoastral formation but dependent of KRAS mutation status

Abstract Mitotic kinesin Eg5 plays an important role in mitosis, as it is critical for proper bipolar spindle assembly. After the discovery of the first Eg5 inhibitor monastrol, a number of Eg5 inhibitors have been developed as anticancer drugs. We have previously synthesized a series of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors and demonstrated potent inhibitory effect on Eg5 ATPase activity and induction of mitotic arrest with characteristic monoastral spindles in HeLa cells. In this study, we evaluated the growth-inhibitory acitivities of novel STLC derivatives, Compound1, Compound2, and Compound3 by MTT assay in a wide range of human cancer cell lines derived from gastric (MKN1, MKN45, MKN74, NUGC3, NUGC4, NCI-N87), colon (C170, DLD1, HCT15, COLO205), pancreatic (AsPC1, BxPC3, SUIT2) and lung (PC-9, PC-14, H1975, H441, A549) cancers and investigated possible predictive biomarkers. Among the derivatives, Compound3 showed the most potent growth-inhibitory effect and sensitivity between cell lines showed marked differences, with IC50 values ranging from 0.134 to 7.14 M. Pancreatic cancer cell line AsPC1 and non-small cell lung cancer (NSCLC) cell lines H441 and A549 which harbor KRAS mutation showed relatively high IC50 values compared to other cell lines without KRAS mutation. Compound3 induced G2/M arrest in all cell lines as early as 8 hours after the treatment by flowcytometry, while the increase of the apoptotic sub-G1 fraction was not observed in these AsPC1, H441 and A549 cells harboring KRAS mutation. These results suggested that differential sensitivity to Compound3 and attenuated induction of apoptosis by Compound3 were attributed to KRAS mutation. Induction of monoastral spindles formation by Compound3 was observed as early as 8 hours after the treatment not only in sensitive cell lines but in refractory cell lines such as AsPC1 and H441 cells by immunocytochemistry, suggesting the induction of monoastral spindles formation does not work as a predictive biomarker for clinical development. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level of each cell line by western blot analysis. No significant difference was observed in the induction of a spindle checkpoint kinase BubR1 phosphorylation among the cells, a possible predictive biomarker in response to the Eg5 inhibitor treatment by western blot analysis. Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development and KRAS mutation status was suggested to be a candidate for a predictive biomarker of poor response to Eg5 inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C108.

Abstract A67: Molecular evidence of increased antitumor activity of gemcitabine in combination with a Cdk inhibitor, P276 in pancreatic cancer

Abstract Purpose: Gemcitabine is an inhibitor of ribonucleotide reductase and DNA synthesis and is a standard of care for the treatment of pancreatic cancer. The present study investigates whether the cdk inhibitor P276 would be synergistic with gemcitabine in pancreatic cancers. Experimental design: Various pancreatic cancer cell lines were treated with gemcitabine and P276 and the cytotoxicity was assessed using the combination index. The effect of gemcitabine and P276 on cell cycle and apoptosis was analyzed by flow cytometry. Several genes that are known to inhibit apoptosis and contribute to chemoresistance were analyzed using western blot analysis and RT-PCR. Finally, in vivo efficacy of the combination was studied in Panc-1 xenograft model in SCID mice. Results: The combination of gemcitabine followed by P276 was found to be synergistic in various pancreatic cancer cell lines Panc-1, Capan-1, BxPc-3, AsPc-1 and MiaPaca-2 as assessed by the combination index using the Compusyn software. Mechanistic studies indicated enhancement of apoptosis in Panc-1 cells as seen by flow cytometry and increase in the antiapoptotic Bcl-2 protein by western blot analysis. Several genes that are known to inhibit apoptosis and contribute to chemoresistance such as p8, cox-2 and survivin were assessed to investigate the basis for the observed chemosensitizing effects of P276. P276 potentiated the gemcitabine induced cytotoxicity by downregulation of p8, cox-2 and survivin. In contrast, cox-2 was found to be significantly up regulated when the cells were exposed to gemcitabine alone, while there was no effect on p8 or survivin as compared to control, suggesting the potential mechanism of acquired chemoresistance to gemcitabine. BNIP3 the tumor suppressor is also downregulated in most pancreatic cancer cell lines and is associated with chemoresistance to gemcitabine. Interestingly BNIP3 was significantly up regulated in the combination and P276 alone but not by gemcitabine alone. In addition to in vitro results, we show here that gemcitabine in combination with P276 is much more effective as an antitumor agent compared with either agent alone in our Panc-1 xenograft tumor model in SCID mice. Conclusion: These data demonstrate that the gemcitabine and a Cdk inhibitor, P276 combination displays synergistic cytotoxic activity, induces apoptosis and sensitizes the pancreatic cells to gemcitabine by modulating the pro- and anti apoptotic proteins. In vivo evidence of the synergistic combination is also demonstrated in Panc-1 xenograft model. The chemosensitzation of pancreatic tumors to gemcitabine would likely be an important and novel strategy for treatment of pancreatic cancer and enable the use of lower and safer concentrations, to pave the way for a more effective treatment in this devastating disease. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A67.

Down-regulation of ZIP4 by RNA Interference Inhibits Pancreatic Cancer Growth and Increases the Survival of Nude Mice with Pancreatic Cancer Xenografts

Zinc levels have been correlated with cancer risk, although the role of zinc and zinc transporters in cancer progression is largely unknown. We recently found that a zinc transporter, ZIP4, is overexpressed in pancreatic cancer. In this study, we further deciphered the role that ZIP4 plays in a pancreatic cancer mouse model by silencing ZIP4. ZIP4 stable silencing was established in pancreatic cancer cell lines ASPC-1 (ASPC-shZIP4) and BxPC-3 (BxPC-shZIP4) by short hairpin RNA using retrovirus vectors. The stable cells were characterized in vitro and in vivo using a nude mouse xenograft model. Silencing of ZIP4 was associated with decreased cell proliferation, migration, and invasion. Both ASPC-shZIP4 and BxPC-shZIP4 cells showed a significant reduction in tumor volume and weight in the s.c. model, and decreased primary tumor weight in the orthotopic model compared with the vector control cells (ASPC-shV and BxPC-shV). Silencing of ZIP4 also caused reduced incidence of tumor metastasis in the mice and downsized the tumor grade. More importantly, silencing of ZIP4 significantly increased the survival rate of nude mice with orthotopic xenografts (P = 0.005). All ASPC-shZIP4-injected mice (100%) remained alive up to 32 days after tumor implantation, whereas only 30% of the ASPC-shV mice were alive at the same time point. CyclinD1 expression was decreased in the ASPC-shZIP4 xenografts. These results identify a previously uncharacterized role of ZIP4 in pancreatic cancer progression, and indicate that knocking down ZIP4 by short hairpin RNA might be a novel treatment strategy for pancreatic cancers with ZIP4 overexpression.

Selective cytotoxic activity of the marine-derived batzelline compounds against pancreatic cancer cell lines

Pancreatic cancer is the fourth leading cause of cancer death in the United States. The prognosis of the disease is very negative, because the cancer will be usually metastasized by the time a patient manifests symptoms. Although combination therapy shows some promise, new drugs to treat the disease are needed. Given our interest in finding new therapies for pancreatic cancer, we sought to determine whether the known cytotoxic activity of the batzellines extended to pancreatic cancer cell lines. The batzellines are pyrroloiminoquinones alkaloids obtained from the deep-water Caribbean sponge Batzella sp (family Esperiopsidae, order Poecilosclerida). We show here that batzellines exhibit selective cytotoxicity towards the pancreatic cancer cell lines AsPC-1, Panc-1, BxPC-3, and MIA PaCa2 compared with the normal African green monkey kidney epithelial cell line Vero. The batzellines cause cytotoxicity by inducing cell cycle arrest that is mediated by their ability to intercalate into DNA and/or inhibit topoisomerase II activity. The cytotoxic abilities of isobatzellines A and C against pancreatic cancer cell lines, their low toxicity against normal cells, and their reported ability to be synthesized makes them interesting compounds with potential chemotherapeutic effects that may merit further research.

Insulin‐like growth factor stimulation increases radiosensitivity of a pancreatic cancer cell line through endoplasmic reticulum stress under hypoxic conditions

Tumor hypoxia is an obstacle to radiotherapy. Radiosensitivity under hypoxic conditions is determined by molecular oxygen levels, as well as by various biological cellular responses. The insulin-like growth factor (IGF) signaling pathway is a widely recognized survival signal that confers radioresistance. However, under hypoxic conditions the role of IGF signaling in radiosensitivity is still poorly understood. Here, we demonstrate that IGF-II stimulation decreases clonogenic survival under hypoxic conditions in the pancreatic cancer cell lines AsPC-1 and Panc-1, and in the human breast cancer cell line MCF-7. IGF treatment under hypoxic conditions suppressed increased radiation sensitivity in these cell lines by pharmacologically inhibiting the phosphoinositide 3-kinase-mammalian target of rapamycin pathway, a major IGF signal-transduction pathway. Meanwhile, IGF-II induced the endoplasmic reticulum stress response under hypoxia, including increased protein levels of CHOP and ATF4, mRNA levels of CHOP, GADD34, and BiP, as well as splicing levels of XBP-1. The response was suppressed by inhibiting phosphoinositide 3-kinase and mammalian target of rapamycin activity. Overexpression of CHOP in AsPC-1 cells increased radiation sensitivity by IGF-II simulation under hypoxic conditions, whereas suppression of CHOP expression levels with small hairpin RNA or a dominant negative form of a proline-rich extensin-like receptor protein kinase in hypoxia decreased IGF-induced radiosensitivity. IGF-induced endoplasmic reticulum stress contributed to radiosensitization independent of cell cycle status. Taken together, IGF stimulation increased radiosensitivity through the endoplasmic reticulum stress response under hypoxic conditions.

Metformin induces apoptosis of pancreatic cancer cells

To assess the role and mechanism of metformin in inducing apoptosis of pancreatic cancer cells. The human pancreatic cancer cell lines ASPC-1, BxPc-3, PANC-1 and SW1990 were exposed to metformin. The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of metformin was tested. In each pancreatic cancer cell line tested, metformin inhibited cell proliferation in a dose dependent manner in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays). Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction. Enzymelinked immunosorbent assay (ELISA) showed that metformin induced apoptosis in all pancreatic cancer cell lines. In Western blot studies, metformin induced poly-ADP-ribose polymerase (PARP) cleavage (an indicator of caspase activation) in all pancreatic cancer cell lines. The general caspase inhibitor (VAD-fmk) completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1, the caspase-8 specific inhibitor (IETD-fmk) and the caspase-9 specific inhibitor (LEHD-fmk) only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells. We also observed that metformin treatment dramatically reduced epidermal growth factor receptor (EGFR) and phosphorylated mitogen activated protein kinase (P-MAPK) in both a time- and dose-dependent manner in all cell lines tested. Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines. And the metformin-induced apoptosis is associated with PARP cleavage, activation of caspase-3, -8, and -9 in a time- and dose-dependent manner. Hence, both caspase-8 and -9-initiated apoptotic signaling pathways contribute to metformin-induced apoptosis in pancreatic cell lines.