Human Pancreatic Tumor Cell Lines Research Articles

Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

Reflection interference contrast microscopy (RICM) allows the visualization of the cell's adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell's spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.

Abstract 3952: Cytochrome P450-derived eicosanoids regulate pancreatic cancer and metastasis.

Abstract Background: 20-hydroxeicosatetraenoic acid (20-HETE) and epoxyeicosatrenoic acids (EETs) are lipid autacoids biosynthesized by cytochrome P450 ω-hydroxylases (e.g. CYP4F2) and epoxygenases, respectively. These eicosanoids regulate inflammation, vascular tone and stimulate angiogenesis which is required for tumor growth and metastasis. However, despite their pleiotropic effects on cells, the role of these cytochrome P450-derived metabolites in pancreatic cancer has not been studied. We hypothesize that 20-HETE and EETs in the tumor microenvironment stimulate pancreatic cancer and metastasis. Methods and Results: Using genetic and pharmacological manipulation of endogenous 20-HETE and EET levels, we demonstrate that 20-HETE and EETs are critical for primary pancreatic tumor growth and metastasis. Native CYP4F2 protein was expressed in a series of human pancreatic tumor cell lines. Transgenic mice with endothelial-specific (tie2-promoter) overexpression of CYP4F2 were used to achieve increased endothelial levels of 20-HETE and stimulated pancreatic tumor growth. Systemic administration of 20-HETE and/or 14,15-EET by osmotic minipump stimulated primary human pancreatic cancer (Panc1) and murine pancreatic (PancOH7) tumor growth in both subcutaneous and orthotopic models. Inhibitors of soluble epoxide hydrolase, the enzyme that metabolizes EETs, elevated EET levels and promoted pancreatic tumor growth. Conversely, systemic administration of a 20-HETE antagonist (HET0016) and/or EET antagonist (14,15-EEZE) suppressed PancOH7 pancreatic tumor growth without toxic effects. Tie2-CYP4F2 transgenic mice exhibited spontaneous liver metastasis after resection of primary pancreatic (PancOH7) subcutaneous tumors. This is of interest because liver metastasis from the injection of pancreatic tumor cells is commonly only achieved by direct injection of tumor cells into the spleen or pancreas. Systemic administration of 20-HETE in WT mice stimulated a 4-fold increase in the number of surface lung metastases compared with control mice and led to an induction of liver and mediastinal lymph node metastasis 26-30 days after resection of the primary PancOH7 tumor. In contrast, the 20-HETE antagonist and/or EET antagonist prolonged survival in the PancOH7 resection metastasis model. Moreover, the 20-HETE antagonist and/or EET antagonist inhibited pancreatic tumor cell proliferation and viability induced by 20-HETE and 14,15-EET, respectively. Furthermore, the 20-HETE antagonist and/or EET antagonist suppressed tumor angiogenesis including a decrease in CD31-positive vessels and VEGF levels in PancOH7 pancreatic tumors. Conclusion: 20-HETE and EET production in the tumor microenvironment may be a critical regulator of pancreatic tumor growth and metastasis, offering a mechanistic rationale for using 20-HETE and EET antagonists as novel anti-cancer therapeutics for pancreatic cancer. Citation Format: Jessica A. Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. Cytochrome P450-derived eicosanoids regulate pancreatic cancer and metastasis. [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 3952. doi:10.1158/1538-7445.AM2013-3952

Abstract 2659: Imaging human pancreatic tumor xenografts with 89Zr-labeled anti-mesothelin antibody.

Abstract Background: Mesothelin (MSLN) is a tumor differentiation antigen that is highly expressed by cells of many epithelial tumors, with limited expression in normal human tissues. Our understanding of therapeutic antibodies targeting MSLN might benefit from immunoPET imaging of antibody uptake. We developed and preclinically validated an 89Zr labeled anti-MSLN antibody (“89Zr-AMA”) for this noninvasive imaging of tumor and normal organ uptake. Methods: 89Zr was attached to an anti-MSLN humanized IgG1 monoclonal antibody derivatized with the bifunctional chelator reagent N-succinyldesferrioxamine-B-tetrafluorphenol. The 89Zr-AMA was characterized in terms of conjugation ratio, aggregation, radiochemical purity, stability, and immunoreactivity. Two human MSLN-expressing pancreatic tumor cell lines, HPAC and CAPAN-2, were used for xenograft studies in mice. Tumor uptake and organ distribution of 89Zr-AMA were studied in the HPAC line at three protein doses (10, 25 and 100 μg) labeled with 1 MBq 89Zr and results were compared with nonspecific 111In-IgG. After dose-finding, CAPAN-2 and HPAC tumor xenograft-bearing mice were scanned with μPET at 1, 3, and 6 days after tracer injection of the optimal AMA dose labeled with 5 MBq 89Zr, followed by ex vivo biodistribution at day 6. Tracer uptake was quantified and expressed as mean standardized uptake values (SUVmean). Results: 89Zr-AMA formed with high specific activity (> 500 MBq/mg), high yield (> 90% without further purification), and high purity (> 95% determined by SE-HPLC analysis). In vitro validation of 89Zr-AMA showed a fully preserved immunoreactivity with a long (> 1 week) stability in 0.9% NaCl. Biodistribution analyses of the dose-finding groups revealed a dose-dependent 89Zr-AMA tumor uptake, with the highest fractional tumor uptake in the 10 μg dose group, 14.2 %ID/g on day 6. Tumor uptake of the non-specific control antibody, 111In-IgG, was lower than that of the 89Zr-AMA (P < 0.05, paired t test). Day 6 89Zr-AMA biodistribution data from the animals that underwent μPET showed ex vivo tumor uptake of 12.0 %ID/g in HPAC and 11.8 %ID/g in CAPAN-2 tumors and 4.6 and 4.4 %ID/g in blood. Uptake of the nonspecific control 111In-IgG was 5.7 %ID/g for HPAC and 3.6 %ID/g for CAPAN-2 tumors, and 10.0 and 7.5 %ID/g for their respective blood pools. MicroPET imaging was consistent with the biodistribution data. 89Zr-AMA showed a progressive increase in tumor uptake over time, whereas the activity in the blood pool decreased; in liver, spleen and kidney it remained stable. Conclusion: 89Zr-AMA tumor uptake is antigen-specific in MSLN-expressing tumors. This tracer can be translated to the clinic for serial non-invasive PET imaging. Citation Format: Eva J. ter Weele, Marjolijn N. Lub-de Hooge, Daniel Maslyar, Anton G.T. Terwisscha van Scheltinga, Jos G.W. Kosterink, Elisabeth G.E. de Vries, Simon P. Williams. Imaging human pancreatic tumor xenografts with 89Zr-labeled anti-mesothelin antibody. [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 2659. doi:10.1158/1538-7445.AM2013-2659

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell-cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif-deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43's endocytosis and assembly into gap junctions.

Acetogenins from Annona muricata

Silica gel chromatography of the dichloromethane extract of the seeds of Annona muricata afforded annoreticuin-9-one (1), while the flesh of the fruit yielded cis-annoreticuin (2) and sabadelin (3). The structures of 1-3 were elucidated by extensive 1 D and 2D NMR spectroscopy and confirmed by mass spectrometry. Acetogenins 1 and 2 were first isolated from A. reticulata and A. montana, respectively. Acetogenin 1 was reported to exhibit cytotoxic activities against the human pancreatic tumor cell line (PACA-2), human prostate adenocarcinoma (PC-3) and human lung carcinoma (A-549), while 2 was reported to exhibit cytotoxicity against human hepatoma carcinoma cell line (Hep G2).

EGF Receptor Signaling Is Essential for K-Ras Oncogene-Driven Pancreatic Ductal Adenocarcinoma

Clinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with thepresence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, development of pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53-defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic.

Ready, Set, Go: The EGF Receptor at the Pancreatic Cancer Starting Line

Acinar-to-ductal metaplasia (ADM) results from pancreatic injury or KRAS activation, and is an early step in pancreatic cancer progression. In this issue of Cancer Cell, Ardito and colleagues and Navas and colleagues demonstrate that ADM- and KRAS-driven pancreatic cancer require EGFR signaling, revealing a mechanism for developmental reprogramming that primes tumorigenesis.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

AbstractCancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells

Background:The combination of the reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib with gemcitabine obtained FDA approval for treating patients with pancreatic cancer. However, duration of response is often limited and there is currently no reliable predictive marker.Methods:We determined the sensitivity of a panel of human pancreatic tumour cell lines to treatment with afatinib, erlotinib, monoclonal antibody (mAb) ICR62, and gemcitabine, using the Sulforhodamine B colorimetric assay. The effect of these agents on cell signalling and cell-cycle distribution was determined by western blot and flow cytometry, respectively.Results:At 200 nM, ICR62 had no effect on growth of these tumour cells with the exception of BxPC-3 cells. BxPC-3 cells were also sensitive to treatment with afatinib and erlotinib with respective IC50 values of 11 and 1200 nM. Compared with erlotinib, afatinib was also more effective in inhibiting the growth of the other human pancreatic tumour cell lines and in blocking the EGF-induced phosphorylation of tyrosine, EGFR, MAPK, and AKT. When tested in BxPC-3 xenografts, afatinib induced significant delay in tumour growth.Conclusion:The superiority of afatinib in this study encourages further investigation on the therapeutic potential of afatinib as a single agent or in combination with gemcitabine in pancreatic cancer.

In vitro cytotoxic activity of abietane diterpenes from Peltodon longipes as well as Salvia miltiorrhiza and Salvia sahendica

Phytochemical investigations of the n-hexane extract from the roots of Peltodon longipes (Lamiaceae) resulted in the isolation of 12 known abietane diterpenes (1–12). Structures were established on the basis of one and two dimensional nuclear magnetic resonance spectroscopic data (1H and 13C, COSY, HSQC and HMBC), electron ionization mass spectrometric analysis (EIMS) as well as comparison with data from literature. These compounds, as well as eight known diterpenes (13–19) from Salvia miltiorrhiza, and two from Salvia sahendica (20 and 21) were evaluated for their cytotoxic effects in human pancreatic (MIAPaCa-2) and melanoma (MV-3) tumor cell lines using the MTT assay. Tanshinone IIa (13), 7α-acetoxyroyleanone (1), 1,2-dihydrotanshinone (16) and cryptotanshinone (14) had the highest cytotoxic effects in MIAPaCa-2, displaying IC50 of 1.9, 4.7, 5.6, and 5.8μM, respectively. Structure–activity relationships of abietane diterpenoid quinones are discussed.

Development of a new stool biomarker ELISA for the early detection of colorectal cancer.

e14128 Background: An ELISA was developed using NPC-1C, a novel antibody that reacts with an antigen expressed specifically by human colorectal and pancreatic tumor tissues and cell lines. The target antigen that NPC-1C recognizes was shown to be related to MUC5AC, a member of the mucin family of glycoproteins. NPC-1C reacts with neither normal tissues from healthy donors (rare, weak binding to normal colon and esophagus), nor cell lines derived from other tumor types, thereby providing the basis for a proof of concept comparative evaluation with stool from normal healthy donors. Methods: An ELISA that uses NPC-1C antibody as both capture and detection reagent was developed. Samples of stool collected during colonoscopy from colorectal cancer patients (n=4), stool from people with small polyps (n=4), stool from people with multiple polyps (n=2), stool from people with large polyps (n=3), and stool from healthy adults (n=13) were applied to the ELISA. A soluble extract of stool was prepared by detergent lysis and centrifugation. The level of NPC-1C-specific MUC5AC antigen measured in this ELISA was compared among all groups. Results: Preliminary results demonstrated that healthy people did not express NPC-1C antigen in their stool. The signal in the assay was similar to background levels (average 723 units). In contrast, people with small polyps had higher levels (average 3,819 units); people with multiple polyps expressed higher levels (average 7,369 units); people with large polyps had even higher levels (average 10,189 units); and colon cancer patients had the highest levels (average 175,983 units), more than 240 times the level of MUC5AC-related antigen compared with healthy people. Conclusions: The proof of concept has been established to correlate the level of NPC-1C reactive antigen, measured by a novel stool-based ELISA, with colon cancer disease progression. The level of NPC-1C-specific MUC5AC detected increased concomitantly with the number and size of polyps observed during colonoscopy, and reached the highest levels in patients with colon cancer. Our goal is to apply this ELISA test for early non-invasive diagnostic screening for colorectal cancer, an area of still largely unmet need.

Abstract 2972: In vitro quantification and modeling of DNA damage by histone H2A after exposure to gemcitabine and a Chk1 inhibitor

Abstract Background: Gemcitabine is a nucleoside analog used as an anti-cancer chemotherapeutic drug. To enhance its disruption of DNA replication and repair, Chk1 inhibitors are being developed for use in combination with Gemcitabine and other cytotoxic agents. Evaluation of these therapies with mechanistic biomarkers can be an important part of the drug development process. One such biomarker is the phosphorylation of histone H2A (γH2AX), which is a DNA damage response biomarker. An ELISA based assay has been developed to quantify γH2AX. This quantitative assay provides data that is incorporated into a mathematical model relating drug exposure to DNA damage and cell viability. Experiment Design: MiaPaCa-2 cells, a human pancreatic tumor cell line, were subjected to Gemcitabine alone (0.1 to 5uM for 2 hours), a Chk1 inhibitor alone (0.1-5uM for 2 – 48 hours), or the Chk1 inhibitor in combination with 0.5 and 5uM Gemcitabine. Cellular concentrations of Gemcitabine and the Chk1 inhibitor were detected with LC/MS. DNA damage was assessed by the γH2AX ELISA at 2, 6, 24 and 48 hours after the start of incubation with drug. In addition to γH2AX, cell viability was measured by cell number at 48 hours. A mathematical model was developed to quantify the modulation of γH2AX when exposed to each drug separately and in combination. Result: Transient exposure of the cells to Gemcitabine resulted in an increase in γH2AX at later times (24 and 48 hours). The Chk1 inhibitor resulted in a dose dependent increase in γH2AX, with a greater effect being observed at later times of exposure. The combination therapy showed a greater response than Gemcitabine or Chk1 alone. However it was observed that higher doses of Gemcitabine and Chk1 in combination did not always yield the largest γH2AX response or the greatest effect on cell viability. This may be due to cellular resistance and is still being investigated. Conclusion: The ELISA based assay for γH2AX provides a quantitative assessment of the level of DNA damage. This information can then be utilized in developing mathematical models of drug action alone and in combination with other drugs. 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 2972. doi:10.1158/1538-7445.AM2011-2972

Abstract 630: Responses of human pancreatic cancer cells to treatment with insulin-like growth factor receptor (IGF-IR) tyrosine kinase inhibitor NVP-AEW541 alone and in combination with anti-EGFR mAb ICR62 or cytotoxic drugs

Abstract Aberrant expression and activation of growth factor receptor signalling pathways including epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-IR) have been reported in a wide range of epithelial cancers and have been associated with increased cell proliferation, reduced apoptosis, invasion and metastasis. Of the growth factor receptor inhibitors, the EGFR inhibitor erlotinib has been approved for the treatment of pancreatic cancer. However, its overall therapeutic efficacy can be of short duration. In some studies, signalling with the IGF-IR has been associated with resistance to therapy with the EGFR inhibitors. In this study, using the Sulforhodamine B colorimetric assay, we investigated the sensitivity of a panel of human pancreatic cancer cell lines (PT-45, AsPC1, PANC1, MiaPaca2, BxPC3, Capan1 and FA6) to treatment with IGF-IR tyrosine kinase inhibitor NVP-AEW541 alone or in combination with anti-EGFR monoclonal antibody (mAb) ICR62 and cytotoxic agents (i.e. 5-FU, doxycycline and gemcitabine). We also investigated the association between the expression levels of IGF-IR and EGFR in these tumour cells, determined by flow cytometry, and their responses to treatment with NVP-AEW541 and/or ICR62. At concentrations above 5μM, NVP-AEW541 inhibited completely the growth of most of the pancreatic cancer cell lines with IC50 values ranging from 342nM (FA6) to 2.73μM (PT45). At maximum concentration of 150nM used in this study, ICR62 did not have any effect on the growth of human pancreatic tumour cell lines. In addition, treatment with a combination of ICR62 and NVP-AEW541 did not enhance the inhibitory effect of the single agent in pancreatic cancer cells. Interestingly, treatment with a combination of NVP-AEW541 and gemcitabine was found to be synergistic for AsPC1 and PANC1 cells, additive for BxPC3, MiaPaca2 and PT-45 cells, but antagonistic for FA6 and CAPAN1 cells. We also examined the cell cycle distribution of BxPC3 cells following treatment with NVP-AEW541 and found an increase in the population of cells in sub-G1 and G0/G1 phases. In contrast, gemcitabine treatment of BxPC3 cells was accompanied by an increase in the populations of cells in both the sub-G1 and S phases of the cell cycle. Taken together, our results suggest that dual targeting of the EGFR and IGF-IR in pancreatic cancer cells by a combination of ICR62 and NVP-AEW541 is not superior to treatment with the single agent in vitro. Further investigations in vivo on the anti-tumour activity of the NVP-AEW541 in combination with ICR62 and cytotoxic drugs in pancreatic cancer are warranted. 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 630. doi:10.1158/1538-7445.AM2011-630

Abstract 4584: Pre-clinical studies of a novel antibody to treat pancreatic and colorectal cancers

Abstract Introduction: NPC-1C is a chimeric monoclonal antibody being developed as a novel biological treatment for pancreatic and colorectal cancers. NPC-1C appears to recognize a variant form of MUC5AC expressed specifically by human pancreatic and colorectal tumor tissues and cell lines. Here we show pre-clinical studies in preparation for testing this therapeutic for a Phase I clinical trial. Methods: The NPC-1C antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the GMP-grade antibody. Results: Here we show that NPC-1C reacts with colorectal and pancreatic tumor tissues, but does not cross-react with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. The NPC-1C antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against control tumor cell lines. In vivo, the anti-tumor efficacy of NPC-1C was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. The pre-clinical toxicity profile of NPC-1C in normal BALB/c mice demonstrated little, if any, toxicity following single or multiple intravenous injections. The bio-distribution of radiolabeled NPC-1C in mice with pre-established human tumors revealed specific accumulation of NPC-1C at the tumor site, with little or no binding or accumulation in several major organ systems. Finally, the cytokine and antibody responses to NPC-1C were studied in the pre-clinical mouse model. Although anti-NPC-1C antibodies were detected (as expected) in a time-dependent fashion, there were no changes detect in Type I and Type II cytokines either 3 days or 14 days after intravenous administration. Conclusions: The pre-clinical development of NPC-1C indicates that it is safe and efficacious, and suggests that it may have clinical activity in patients whose tumor expresses the variant MUC5AC epitope. The potential clinical application for this antibody was bolstered by showing approximately 50-70% of human pancreatic and colon tumor tissues stained positively with NPC-1C. The therapeutic antibody is now being tested in a Phase I clinical trial in patients with advanced pancreatic and colorectal cancer. 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 4584. doi:10.1158/1538-7445.AM2011-4584

Development of a new stool biomarker ELISA for the early detection of colorectal cancer.

390 Background: An ELISA was developed using NPC-1C, a novel antibody that reacts with an antigen expressed specifically by human colorectal and pancreatic tumor tissues and cell lines. The target antigen that NPC-1C recognizes was shown to be related to MUC5AC, a member of the mucin family of glycoproteins. NPC-1C reacts with neither normal tissues from healthy donors (rare, weak binding to normal colon and esophagus), nor cell lines derived from other tumor types, thereby providing the basis for a proof of concept comparative evaluation with stool from normal healthy donors. Methods: An ELISA that uses NPC-1C antibody as both capture and detection reagent was developed. Samples of stool collected during colonoscopy from colorectal cancer patients (n = 4), stool from people with small polyps (n = 4), stool from people with multiple polyps (n = 2), stool from people with large polyps (n = 3), and stool from healthy adults (n = 13) were applied to the ELISA. A soluble extract of stool was prepared by detergent lysis and centrifugation. The level of NPC-1C-specific MUC5AC antigen measured in this ELISA was compared among all groups. Results: Preliminary results demonstrated that healthy people did not express NPC-1C antigen in their stool. The signal in the assay was similar to background levels (average 723 units). In contrast, people with small polyps had higher levels (average 3,819 units); people with multiple polyps expressed higher levels (average 7,369 units); people with large polyps had even higher levels (average 10,189 units); and colon cancer patients had the highest levels (average 175,983 units), more than 240 times the level of MUC5AC-related antigen compared with healthy people. Conclusions: The proof of concept has been established to correlate the level of NPC-1C reactive antigen, measured by a novel stool-based ELISA, with colon cancer disease progression. The level of NPC-1C-specific MUC5AC detected increased concomitantly with the number and size of polyps observed during colonoscopy, and reached the highest levels in patients with colon cancer. Our goal is to apply this ELISA test for early noninvasive diagnostic screening for colorectal cancer, an area of still largely unmet need. [Table: see text]

NPC-1C: A novel, therapeutic antibody to treat pancreas and colorectal cancers.

235 Background: NPC-1C (ensituximab) is a chimeric monoclonal antibody being developed as a novel biologic treatment for pancreatic and colorectal cancers. This antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. The NPC-1C epitope appears to be expressed specifically by human colon and pancreatic tumor tissues and cell lines. Methods: Antitumor activity was established in vitro by measuring ADCC with a standard 4-hour 111-Indium release assay on pancreatic and colorectal cancer cell lines. In vivo antitumor efficacy of NPC-1C was tested using pre-established subcutaneous human pancreatic tumor xenograft models. Results: In vitro, the NPC-1C antibody exhibits ADCC activity specifically against human colon and pancreatic tumor cells, but not against control tumor cell lines. The in vivo data showed significant, and reproducible, antitumor action, including some complete tumor regressions. The clinical application for this antibody was bolstered by several examples of human tumor tissues stained with biotin-conjugated NPC-1C that showed a strong correlation of NPC-1C staining against pancreatic and colon tumors. Approximately 45% of tumors stained strongly positive. The staining pattern was typical of elaborated mucin expression, but also showed cytoplasmic and cell membrane staining. Conclusions: A phase I open label, multicenter dose escalation clinical trial with NPC-1C is currently accruing patients with advanced pancreatic and colorectal cancer who are refractory to standard therapy. The primary objectives of the phase I clinical trial are to determine the safety and tolerability of escalating doses of NPC-1C monoclonal antibody therapy and to assess pharmacokinetics and select immune responses to the antibody at each dose level. Secondary objectives are to evaluate evidence of clinical benefit and to explore the immunologic correlates associated with administration of NPC-1C. Results from this trial will determine the minimum standard dosage levels to be used in further trials. [Table: see text]

Comazaphilones A−F, Azaphilone Derivatives from the Marine Sediment-Derived Fungus Penicillium commune QSD-17

Chemical investigation of Penicillium commune QSD-17, a fungus isolated from a marine sediment sample collected in the southern China Sea, yielded six new azaphilone derivatives, namely, comazaphilones A-F (1-6). The structures of these compounds were established on the basis of spectroscopic analysis. Attempts to define the absolute configuration of these azaphilones through investigation of Mosher's esters failed, possibly due to steric crowding at C-6 and C-7 and due to the degradation of these azaphilone derivatives under the reaction conditions. The inhibitory activities of the six azaphilones against four bacteria, one pathogenic fungus, and seven tumor cell lines were evaluated. Compounds 3-5 displayed potent inhibitory activity against several of these bacteria, while compounds 4-6 showed cytotoxic activity against human pancreatic tumor cell line SW1990. The preliminary SAR results indicated that the double bond at C-10 and the location of the orsellinic acid unit at C-6 in these azaphilones are important for the antibacterial activity and cytotoxicity, respectively. This is the first report of the isolation of azaphilone derivatives from a marine sediment-derived fungus.

Enhancement of gemcitabine sensitivity in pancreatic cancer by co-regulation of dCK and p8 expression

The purpose of this study was to improve the gemcitabine sensitivity in pancreatic cancer by adenovirus-mediated co-regulation of dCK and p8 expression. Firstly, we analyzed the sensitivity of three human pancreatic tumor cell lines (Capan-2, Panc-1 and BxPc-3) to gemcitabine using MTT assays, and found Panc-1 to be relatively resistant to gemcitabine. Further, we investigated the expression of dCK and p8 in different pancreatic cancer cell lines using real-time PCR and Western blot analysis, and found that the expression levels of these two genes were related to the gemcitabine sensitivity of pancreatic cancer cells. We constructed recombinant adenovirus vectors, Ad-dCK and Ad-p8-siRNA, that overexpressed dCK and knocked down p8, respectively. Using MTT assays, we observed that combined infection using Ad-dCK and Ad-p8-siRNA in vitro led to a significant decrease in the gemcitabine IC50 with an increase in apoptosis and caspase-3 activity in Panc-1 cells, which are relatively resistant to gemcitabine. Furthermore, in established subcutaneous pancreatic cancer models in nude mice, the tumor inhibition was markedly enhanced accompanied by elevation of the apoptosis index after intratumoral injection of Ad-dCK and Ad-p8-siRNA on the basis of intraperitoneal gemcitabine chemotherapy. Taken together, the present findings suggest that, dCK and p8 may be the important factors in the regulation of gemcitabine sensitivity in pancreatic cancer cells. Moreover, co-regulation of the two factors achieved better effects than regulation of either one alone.

Influence of Cell Cycle Checkpoints and p53 Function on the Toxicity of Temozolomide in Human Pancreatic Cancer Cells

Background: Though an increased efficacy of carmustine and temozolomide (TMZ) has been demonstrated by inactivation of O6-methylguanine-DNAmethyltransferase(MGMT) with O6-benzyl-guanine (BG) in human pancreatic tumors refractive to alkylating agents, the regulatory mechanisms have not been explored. Methods: The effects of TMZ and BG on apoptosis, cell growth, the mitotic index, cell cycle distribution, and protein expression were studied byTUNEL, cell counting, flow cytometry, and Western blot analysis, respectively. Results: The wt-p53 human pancreatic tumor cell line Capan-2 and p53-efficient mouse embryonic fibroblasts (MEFs) were more responsive to treatment with TMZ + BG than mutant p53 Capan-1 and p53-null MEFs. S phase delay with a subsequent G2/M arrest was observed in Capans in response to BG + TMZ. The G1-to-S transition delay in Capan-2 was associated with p53-dependent apoptosis and was distinctly different from the presumed mismatch repair (MMR) killing operative during the G2/M arrest. The effect of p53on BG +TMZ toxicity was supported by a marked change in apoptosis when p53 function was restored/inactivated. There was an early induction of MMR proteins in p53-efficient lines. Conclusion: p53 provokes a classic proapoptotic response by delaying G1-to-S progression, but it may also facilitate cell killing by enhancing MMR-related cell cycle arrest and cell death.

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.