Human Pancreatic Cancer Cell BxPC-3 Research Articles

Shikonin promotes autophagy in BXPC-3 human pancreatic cancer cells through the PI3K/Akt signaling pathway.

The present study aimed to investigate the effect of shikonin on autophagy in BXPC-3 human pancreatic cancer cells and its underlying mechanism. Cell viability was assessed using the Cell Counting Kit-8 assay and the expression of light chain (LC) 3, p62, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-PI3K and p-Akt was analyzed using western blot analysis. Following treatment with 1 μmol/l shikonin for 48 h and 2.5 and 5 μmol/l shikonin for 24 and 48 h, the viability of the BXPC-3 cells was found to be significantly reduced and the protein expression of LC3-II/LC3-I was observed to be increased, while the protein expression of p62, PI3K, Akt, p-PI3K and p-Akt was decreased. These findings suggest that shikonin promotes autophagy in BXPC-3 cells and that the underlying mechanism may be associated with the PI3K/Akt signaling pathway.

Tumour‐educated macrophages display a mixed polarisation and enhance pancreatic cancer cell invasion

At the time of diagnosis, almost 80% of pancreatic cancer patients present with new-onset type 2 diabetes (T2D) or impaired glucose tolerance. T2D and pancreatic cancer are both associated with low-grade inflammation. Tumour-associated macrophages (TAMs) have a key role in cancer-related inflammation, immune escape, matrix remodelling and metastasis. In this study, the interplay between tumour cells and immune cells under the influence of different glucose levels was investigated. Human peripheral blood mononuclear cells were exposed in vitro to conditioned medium from BxPC-3 human pancreatic cancer cells, in normal (5 mM) or high (25 mM) glucose levels. Flow cytometry analyses demonstrated that tumour-derived factors stimulated differentiation of macrophages, with a mixed classical (M1-like) and alternatively activated (M2-like) phenotype polarisation (CD11c(+)CD206(+)). High-glucose conditions further enhanced the tumour-driven macrophage enrichment and associated interleukin (IL)-6 and IL-8 cytokine levels. In addition, hyperglycaemia enhanced the responsiveness of tumour-educated macrophages to lipopolysaccharide, with elevated cytokine secretion compared with normal glucose levels. Tumour-educated macrophages were found to promote pancreatic cancer cell invasion in vitro, which was significantly enhanced at high glucose. The anti-diabetic drug metformin shifted the macrophage phenotype polarisation and reduced the tumour cell invasion at normal, but not high, glucose levels. In conclusion, this study demonstrates that pancreatic cancer cells stimulate differentiation of macrophages with pro-tumour properties that are further enhanced by hyperglycaemia. These findings highlight important crosstalk between tumour cells and TAMs in the local tumour microenvironment that may contribute to disease progression in pancreatic cancer patients with hyperglycaemia and T2D.

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.

Flavonoid apigenin modified gene expression associated with inflammation and cancer and induced apoptosis in human pancreatic cancer cells through inhibition of GSK‐3β/NF‐κB signaling cascade

The objective was to examine the inhibitory effects of citrus fruit bioactive compounds on BxPC-3 and PANC-1 human pancreatic cancer cells, focusing on the antiproliferative mechanism of action of the flavonoid apigenin related to the glycogen synthase kinase-3β/nuclear factor kappa B signaling pathway. Flavonoids, limonoids, phenolic acids, and ascorbic acid were tested for cytotoxic effects on BxPC-3 and PANC-1 cells; apigenin was the most potent (IC50 = 23 and 12 μM for 24 and 48 h for BxPC-3 and IC50 = 71 and 41 μM for 24 and 48 h for PANC-1). Apigenin induced pancreatic cell death through inhibition of the glycogen synthase kinase-3β/nuclear factor kappa B signaling pathway. Apigenin arrested cell cycle at G2 /M phase (36 and 32% at 50 μM for BxPC-3 and PANC-1, respectively) with concomitant decrease in the expression of cyclin B1. Apigenin activated the mitochondrial pathway of apoptosis (44 and 14% at 50 μM for BxPC-3 and PANC-1, respectively) and modified the expression of apoptotic proteins. Apigenin highly upregulated the expression of cytokine genes IL17F (114.2-fold), LTA (33.1-fold), IL17C (23.2-fold), IL17A (11.3-fold), and IFNB1 (8.9-fold) in BxPC-3 cells, which potentially contributed to the anticancer properties. Flavonoids have a protective role in pancreatic cancer tumorigenesis.

Interactions between dietary flavonoids apigenin or luteolin and chemotherapeutic drugs to potentiate anti-proliferative effect on human pancreatic cancer cells, in vitro

The objectives were to assess the potential of dietary flavonoids apigenin (Api) and luteolin (Lut) to enhance the anti-proliferative effects of chemotherapeutic drugs on BxPC-3 human pancreatic cancer cells and to investigate the potential molecular mechanism of action. Simultaneous treatment or pretreatment (0, 6, 24 and 42 h) of flavonoids and chemotherapeutic drugs at various concentrations (0-50 μM) were assessed using the MTS cell proliferation assay. Simultaneous treatment with either flavonoid (13, 25 or 50 μM) and chemotherapeutic drugs 5-fluorouracil (5-FU, 50 μM) or gemcitabine (Gem, 10 μM) for 60 h resulted in mostly less-than-additive effects (p<0.05). Pretreatment for 24h with 13 μM of either Api or Lut, followed by Gem for 36 h was optimal to inhibit cell proliferation. Pretreatment of cells with 11-19 μM of either flavonoid for 24h resulted in 59-73% growth inhibition when followed by Gem (10 μM, 36 h). Lut (15 μM, 24h) pretreatment followed by Gem (10 μM, 36 h), significantly decreased protein expression of nuclear GSK-3β and NF-κB p65 and increased pro-apoptotic cytosolic cytochrome c. Pretreatment of BxPC-3 human pancreatic cancer cells with low concentrations of Api or Lut effectively aid in the anti-proliferative activity of chemotherapeutic drugs.

Localization of the anti‐cancer peptide EGFR‐lytic hybrid peptide in human pancreatic cancer BxPC‐3 cells by immunocytochemistry

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti-cancer epithelial growth factor receptor (EGFR)-lytic hybrid peptide, a 32-amino-acid peptide composed of an EGFR-binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR-lytic hybrid peptide in BxPC-3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR-lytic peptide compared with non-treated cells. In confocal laser scanning microscopy, immunostaining of EGFR-lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR-lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR-lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR-lytic peptide; however, no effect on cell death after EGFR-lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR-lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death.

Inhibitory effect of geraniol in combination with gemcitabine on proliferation of BXPC-3 human pancreatic cancer cells

To investigate the inhibitory effect of geraniol alone, or in combination with gemcitabine, on the proliferation of BXPC-3 pancreatic cancer cells. BXPC-3 cells were treated under different conditions: with geraniol at 10, 20, 40, 80 and 160 µmol/l each for 24 h, 48 h or 72 h; with 20 µmol/l geraniol for 24 h or 0 h before 20 µmol/l gemcitabine for 24 h; with 20 µmol/l geraniol for 24 h, 48 h and 72 h following 20 µmol/l gemcitabine for 24 h; or with 20 µmol/l gemcitabine alone as a control. Cell proliferation was assessed and changes in cell morphology were assessed by light and fluorescence microscopy. Apoptosis was detected using flow cytometry. Geraniol inhibited BXPC-3 cell proliferation in a time- and dosa-dependent manner. Geraniol alone or combined with gemcitabine induced BXPC-3 cell apoptosis. BXPC-3 inhibition rates with combined treatment were 55.24%, 50.69%, 49.83%, 41.85% and 45.27% following treatment with 20 µmol/l geraniol for 24 h or 0 h before 20 µmol/l gemcitabine for 24 h, or 20 µmol/l geraniol for 24 h, 48 h and 72 h, following 20 µmol/l gemcitabine for 24 h, respectively. Geraniol inhibited the proliferation of BXPC-3 cells. Geraniol significantly increased the antiproliferative and apoptosis-inducing effects of gemcitabine on BXPC-3 cells. Maximum inhibition of BXPC-3 cells was achieved with geraniol treatment for 24 h before gemcitabine treatment.

Sann-Joong-Kuey-Jian-Tang decreases the protein expression of Mcl-1 and TCTP and increases that of TNF-α and Bax in BxPC-3 pancreatic carcinoma cells

Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicinal prescription, has been used for the treatment of lymphadenopathy and solid tumors, and has shown therapeutic potential in several human malignant tumor cell lines. However, the efficacy and molecular mechanisms of action of SJKJT in human pancreatic cancer have not yet been elucidated. In the present study, we evaluated the cytotoxic effects of SJKJT on BxPC-3 human pancreatic carcinoma cells by MTT assay. The protein expression levels of myeloid cell leukemia 1 protein (Mcl-1), translationally controlled tumor protein (TCTP), tumor necrosis factor-α (TNF‑α), caspase-8, caspase-3, Bax and Bcl-2 family in the BxPC-3 cells were measured by western blot analysis. The cell cycle was analyzed by flow cytometry. The protein expression of caspase-3 was also detected by immunocytochemistry (ICC). The results revealed that SJKJT inhibited the proliferation of BxPC-3 cells in a time- and dose-dependent manner. The protein expression levels of TNF-α, caspase-8, caspase-3 and Bax increased in the BxPC-3 cells treated with SJKJT; however, the levels of Mcl-1, TCTP and Bcl-xL decreased. The results also demonstrated that SJKJT increased the percentage of BxPC-3 cells in the sub-G1 phase. In addition, ICC staining indicated that the protein expression of caspase-3 was upregulated in the BxPC-3 cells treated with SJKJT. These findings indicate that SJKJT inhibits the proliferation of BxPC-3 cells through the extrinsic and intrinsic pathway, inducing apoptosis in vitro. Our study, using BxPC-3 human pancreatic cancer cells, demonstrates that SJKJT has potential as a chemotherapeutic agent for the treatment of pancreatic cancer. Further sutdies are warranted to fully elucidate its mechanisms of action.

Autophagy in BxPC-3 human pancreatic cancer cells is similar to PANC-1 when treated with anticancer drugs and inhibitors of autophagy

Autophagy in BxPC-3 human pancreatic cancer cells is similar to PANC-1 when treated with anticancer drugs and inhibitors of autophagy

Abstract 722: Evaluation of a chimeric anti CEA-conjugated antibody with AlexaFlour or Dylight fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models.

Abstract In this study, we evaluated multiple wavelengths of different fluorophores of the AlexaFlour (Invitrogen) and Dylight (Thermofisher scientific) series of fluorescent probes for labeling of mouse models of pancreatic cancer. BxPC-3 human pancreatic cancer cells were cultured in-vitro and subsequently injected subcutaneously in nude mice. After the development of subcutaneous tumors, the tumors were harvested and orthotopically implanted into the pancreatic tails of nude mice (1x1 mm fragments). The following fluorescent dyes were evaluated: 488 group (Alexa 488 and Dylight 488); 550 group (Alexa 555 and Dylight 550); 650 group (Alexa 660 and Dylight 650). The dyes were conjugated to chimeric anti-CEA antibody at a ratio of 3-4moles of dye per mole of protein. 50-100 μg of the fluorophore conjugated antibody was injected per mouse intravenously. Mice were then imaged with the OV100 Small Animal Imager (Olympus Corp, Tokyo, Japan). Whole body and intravital imaging were done at various time points (post implantation day 7-14). Mice that had tumor visible on whole body imaging were sacrificed, after which tumor depth, size, weight and fluorescence intensity were recorded. The smallest tumor size was first discernible on whole body imaging with the 650 group of dyes, followed by the 550 group and finally the 488 group with significantly larger tumors. The fluorescence intensity of the Dylight panel of dyes overall was brighter than the AlexaFluor dyes and therefore able to detect smaller and deeper tumors. The 550 and 650 group of dyes had the least amount background fluorescence with the highest amount of tumor to normal tissue contrast. In evaluation of tumor depth the 650 group of dyes were able to detect the deepest tumors; of the group the 488 group had the least tissue penetrance. In this study, the Dylight group of dyes had brighter conjugates with better tissue penetration and tumor delineation when compared to the AlexaFluor dyes. The 650 group of dyes had the most tissue penetration. The 550 group of dyes had similar background autofluorescence to the 650 group that was signficantly less than the 488 group. Both the 550 and 650 group had the highest tumor to background ratio of fluoresence that allows for very high fidelity in the delineation of neoplastic tissue from normal tissue. Citation Format: Ali A. Maawy, Sharmeela Kaushal, Cynthia S. Snyder, George A. Luiken, Mark A. Talamini, Robert M. Hoffman, Michael Bouvet. Evaluation of a chimeric anti CEA-conjugated antibody with AlexaFlour or Dylight fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models. [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 722. doi:10.1158/1538-7445.AM2013-722

Abstract 2951: DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by marked chemoresistance to a broad spectrum of chemotherapeutic drugs, as a consequence of intrinsic cell-autonomous factors, and extrinsic stroma-associated factors. Dual specificity protein phosphatase 1 (DUSP1) has been reported to be overexpressed in pancreatic cancer cells in PDAC. Moreover, DUSP1 was shown to be essential for efficient in vitro colony formation and in vivo tumorigenicity, indicating that DUSP1 may contribute to the biological aggressiveness of PDAC. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human pancreatic cancer cells, we show that gemcitabine activated c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Activation of JNK and p38 MAPK was associated with increased apoptosis, as evidenced by increased cleavage of PARP and Caspase 3. However, gemcitabine also enhanced DUSP1 transcription, as demonstrated by increased DUSP1 mRNA levels, and increased RNA polymerase II loading at both DUSP1 promoter and gene body. Moreover, shRNA-mediated inhibition of DUSP1 enhanced JNK and p38 MAPK activation and sensitized both cell lines to gemcitabine. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the dual advantage of suppressing proliferation while enhancing gemcitabine chemosensitivity. Citation Format: Fang Liu, Murray Korc. DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine. [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 2951. doi:10.1158/1538-7445.AM2013-2951

Effect of Jinlong capsule on proliferation and apoptosis of human pancreatic cancer cells BxPC-3

ObjectiveTo study the possible roles of Jinlong capsule (JLC) on the proliferation and apoptosis of human pancreatic cancer cells BxPC-3. MethodsThe human pancreatic cancer cells BxPC-3 were treated with JLC at the concentration of 0.05-1.00 mg/mL for 24-120 h. The inhibition rate of JLC on human pancreatic cancer cells BxPC-3 was detected by 3-(4,5-dimethiylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometry was employed to measure cell apoptosis using Annexin V-FITC/Propidium iodide (AV-FITC/PI) method. Cell cycles were determined by PI staining. The expression of S100 Calcium binding protein A4 (S100A4) in cell matrix was measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of apoptosis-related protein such as BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), B-cell lymphoma/leukemia-2 (Bcl-2) and Cys-teinylaspartate specific proteinase 3 (Caspase-3) were detected by Western blotting. ResultsJLC significantly inhibited the proliferation of human pancreatic cancer cells BxPC-3 in a dose-dependent and time-dependent manner. JLC promoted cell apoptosis and maintained cell cycle in S and G2/M phase rather than G1/G0 phase. The expression of S100A4 in the cell matrix was reduced. The expression of cell apoptotic protein BNIP3 was increased while Bcl-2 was decreased. ConclusionJLC can inhibit the proliferation of human pancreatic cancer cells BxPC-3 by stimulating cell apoptosis, arresting the cell cycle at S and G2/M phase which blocks the circulation of normal cell cycle and reducing the expression of S100A4 protein. Higher pro-apoptosis protein BNIP3 and lower anti-apoptosis protein Bcl-2 levels were found, which may be related to the apoptotic effects of JLC.

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.

Local drug delivery to a human pancreatic tumor via a newly designed multiple injectable needle

Endoscopic ultrasound-guided fine needle injection (EUS-FNI) has been proposed as a novel technique for local delivery of antitumor agents to refractory tumors, including pancreatic cancer. However, the present outcome of this strategy remains insufficient, and further improvements as well as novel agents and injection devices are required. The aim of the study was to determine the feasibility of a newly designed 'multiple injectable needle' (MIN) for EUS-FNI and the resulting improvements in the drug distribution to the tumor in comparison with straight-type needles. Human pancreatic cancer BxPC-3 cells or orthotopic tumor were transplanted subcutaneously into athymic rats. Ethanol was injected into subcutaneous tumors using a MIN or straight-type needle. Sequential sections of subcutaneous tumors were stained with hematoxylin and eosin, and tumors and injury areas were quantified using ImageJ software. In the orthotopic tumors, injection of the outer needle of the MIN, advancement of the inner needle and ethanol infusion were monitored by EUS. Injury volume of subcutaneous tumors using a MIN was greater than that with the straight-type needles, except for large tumors. Injecting process using MIN was monitored with endoscopic ultrasound (EUS). This study showed improvement in the drug distribution in a tumor, following injection with MIN compared to straight-type needles, suggesting the novel device to be feasible for use in EUS-FNI.

Apigenin Inhibits NNK-Induced Focal Adhesion Kinase Activation in Pancreatic Cancer Cells

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

In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells

Silibinin suppresses the growth of many cancers; however, its efficacy against pancreatic cancer has not been evaluated in established preclinical models. Here, we investigated in vitro and in vivo effects of silibinin against lower and advanced stages of human pancreatic carcinoma cells. Silibinin (25–100μM) treatment for 24–72h caused a dose- and time-dependent cell growth inhibition of 27–77% (P<0.05–0.001) in BxPC-3 cells, and 22–45% (P<0.01–0.001) in PANC-1 cells. Silibinin showed a strong dose-dependent G1 arrest in BxPC-3 cells (upto 72% versus 45% in control; P<0.001), but a moderate response in advanced PANC-1 cells. Cell death observed in cell growth assay, was accompanied by up to 3-fold increase (P<0.001) in apoptosis in BxPC-3 cells, and showed only slight effect on PANC-1 cells. Dietary feeding of silibinin (0.5%, w/w in AIN-93M diet for 7weeks) inhibited BxPC-3 and PANC-1 tumor xenografts growth in nude mice without any apparent change in body weight gain and diet consumption. Tumor volume and weight were decreased by 47% and 34% (P⩽0.001) in BxPC-3 xenograft, respectively. PANC-1 xenograft showed slower growth kinetics and silibinin decreased tumor volume by 34% (P<0.001) by 7weeks. Another 4weeks of silibinin treatment to PANC-1 xenograft showed 28% and 33% decrease in tumor volume and weight, respectively. Silibinin-fed group of BxPC-3 tumors showed decreased cell proliferation and angiogenesis and an increased apoptosis, however, considerable inhibitory effect was observed only for angiogenesis in PANC-1 tumors. Overall, these findings show both in vitro as well as in vivo anticancer efficacy of silibinin against pancreatic cancer that could involve inhibition of cell proliferation, cell cycle arrest, apoptosis induction and/or decrease in tumor angiogenesis.

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells.

1697P - Semuloparin Efficiently Inhibits Thrombin Generation Triggered by Pancreas Adenocarcinoma Cells BXPC3. Distinct Roles of Anti-XA and Anti-IIA Activity

ABSTRACT Background Venous thromboembolism (VTE) is a common complication in patients with cancer receiving chemotherapy. Currently no anticoagulant is approved for VTE prophylaxis in this setting. Semuloparin is an ultra-LMWH generated through a highly selective depolymerization of heparin which protects the antithrombin (AT) binding site in order to improve the benefit/risk ratio compared to existing anitcoagulants. Aims We studied in vitro the mechanism of action of semuloparin on the inhibition of thrombin generation (TG) of human platelet poor plasma (PPP) triggered by human pancreatic cancer cells BXPC3. We compared the antithrombotic efficiency of semuloparin to that of enoxaparin and the specific AT-dependent factor Xa inhibitor fondaparinux. Materials and methods BXPC3 cells were suspended in PPP spiked with clinically relevant concentrations of semuloparin, enoxaparin and fondaparinux. The endogenous thrombin potential (ETP) and the mean rate index (MRI) of the propagation phase of TG were monitored with the CAT assay (Stago France) as described previously (Gerotziafas et al Thromb Res 2011). Anti-Xa and anti-IIa specific activities were assessed assays obtained from Diagnostica Stago. Results Both semuloparin and enoxaparin, at the concentration of 0.4 anti-Xa IU/ml, completely abrogated TG. Total inhibition of TG occured in the presence of 0.002 anti-IIa IU/ml of semuloparin and 0.05 anti-IIa IU/ml of enoxaparin. Fondaparinux, even at concentrations higher than 2 µg/ml, reduced the MRI but did not significantly affect the ETP and did not completely inhibited TG,. The IC50 for the ETP of the anti-IIa activity of enoxaparin was 37-fold higher as compared to that of semuloparin. Conclusion In a cancer cell model of hypercoagulability, semuloparin reduced efficiently thrombin generation. The unique anticoagulant profile of semuloparin based on its high AT-affinity differentiates it from enoxaparin and fondaparinux. The residual anti-IIa activity of semuloparin amplifies its antithrombotic efficacy. This profile is expected to translate into an improved benefit/risk ratio. Disclosure All authors have declared no conflicts of interest.

Development of a unique mouse model for pancreatic cancer lymphatic metastasis

Lymphatic metastasis of pancreatic cancer is a predictor of poor prognosis. However, the molecular mechanisms are largely unknown, thus, making the development of appropriate cell lines and experimental models critically important for future investigations. The purpose of the present study was to establish a 'pancreatic cancer cell and mouse model with high lymphatic metastasis potential' for in-depth study of the underlying mechanisms. The BxPC-3-LN subline, derived from the BxPC-3 human pancreatic cancer cell line, was established through serial passages in nude mice via footpad injections. The subline was able to develop notable lymphatic metastases in 100% of the recipient mice 8 weeks after tumor cell implantation. Compared with the parental BxPC-3 cells, BxPC-3-LN cells were more aggressive, displaying invasive ultrastructure, increased migration and invasion ability, and chemoresistance. Metastasis-related gene alteration including upregulation of MMP14, MMP24, MIF and ADRM1, and downregulation of TGFB2 and ROBO1 were also observed in BxPC-3-LN cells by cDNA microarrays. Thus, the newly selected BxPC-3-LN subline can serve as a unique model for further study of lymphatic metastasis of pancreatic cancer.

The basic helix-loop-helix transcription factor DEC2 inhibits TGF-β-induced tumor progression in human pancreatic cancer BxPC-3 cells

The basic helix loop helix (bHLH) transcription factor DEC2 is associated with the regulation of apoptosis, circadian rhythm and the response to hypoxia. However, the significance of DEC2 in pancreatic cancer remains unknown. Here, we showed for the first time that DEC2 inhibits the progression of human pancreatic cancer. Human pancreatic cancer BxPC-3 cells were treated with or without transforming growth factor-β (TGF-β), siRNA against DEC2, or a combination of TGF-β and DEC2 siRNA or DEC2 overexpression. The cells were analyzed by RT-PCR, real-time PCR, western blotting, immunofluorescent staining and ChIP assay. We also performed immunohistochemical analyses of DEC2 expression in surgically-resected pancreatic cancers. The expression of DEC2 was increased in TGF-β-treated BxPC-3 cells. In the presence of TGF-β, DEC2 overexpression decreased the migration and invasion of BxPC-3 cells. Knockdown of DEC2 by siRNA in the presence of TGF-β significantly increased the expression and nuclear concentration of slug. We also showed that DEC2 siRNA decreased the binding of DEC2 to the E-box of the slug promoter. Immunohistochemically, little DEC2 was detected in pancreatic cancer tissues, whereas significant amounts were detected in the adjacent non-cancerous pancreatic tissues. These results indicate that DEC2 has inhibitory effects against human pancreatic cancer that involve TGF-β and slug.