Pancreatic Cancer Cell Lines PANC-1 Research Articles

Leukotriene B4 induces EMT and vimentin expression in PANC-1 pancreatic cancer cells: Involvement of BLT2 via ERK2 activation

Leukotriene B4 (LTB4) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB4 is known to be correlated with cancer progression. LTB4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown.We examined whether LTB4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB4-induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB4-or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pCBLT2) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells.Taken together, these results suggest that BLT2 is involved in LTB4-induced vimentin expression through ERK2 in PANC-1 cells.

271 - Catalase as a Potential Biomarker of Pharmacological Ascorbate Cancer Therapy

Pharmacological ascorbate (P-AscH–) has been shown to induce toxicity in a various cancer cell lines both in vitro and in vivo. P-AscH– can readily oxidize and deliver a high flux of H2O2, which can easily cross cell membrane and convert to more reactive species inside the cell. We hypothesize differential sensitivity of cells to P-AscH– is due to their ability to remove H2O2. The principal enzymes responsible for the elimination of H2O2 are catalase, glutathione peroxidase (GPx) and peroxiredoxins (Prx). Among these, catalase is the principal enzyme that involved in the detoxification of H2O2 at high concentration. In this study, we compared catalase levels and peroxide removal capacity in several pancreatic cancer cell lines MIA PaCa-2, AsPC-1, 403, 339 and Panc-1. Preliminary results indicated that MIA PaCa-2 cells had the lowest level of catalase and were most sensitive to P-AscH–, while Panc-1 had the highest level of catalase and were the least sensitive. In mice with pre-established xenograft pancreatic tumor, P-AscH– (4g/kg, i.p. twice daily) showed a greater inhibition of tumor growth for MIA PaCa-2 xenografts in comparison to Panc-1 xenografts. Furthermore, immunofluorescent staining of catalase in xenograft tumors also indicated that MIA PaCa-2 had lower level of catalase than Panc-1 in vivo. Taken together, catalase activity in tumors may predict which cancers will respond to P-AscH– therapy. Supported by VA Merit grant and NIH grants CA184051, CA137230, and CA169046.

RETRACTED ARTICLE: Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis

In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation.

BackgroundPancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition.MethodsAn immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay.ResultsInitial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities.ConclusionThis study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as anti-EMT and anti-CSC drug leads.

128P - Modulation of specificity protein 1 (SP1) is a novel therapeutic strategy for pancreatic cancer

Pancreatic cancer is one of the most aggressive and lethal malignancies. Specificity Protein 1 (SP1) is a transcription factor regulates and promotes tumor progression. Some studies have reported SP1 promotes epithelial-mesenchymal transition (EMT) and is associated with aggressive and poor patient prognosis. However, there is a paucity of clinical evidence regarding the role of SP1 in pancreatic cancer. In this study, we aimed to confirm the function of SP1 in invasiveness of pancreatic cancer cells and to evaluate the clinical impact in patients with pancreatic cancer. Between June 2002 and December 2012, 81 patients underwent radical curative resection for pancreatic cancer at Gangnam Severance Hospital, Seoul, Korea. Pancreatic cancer cell lines MIA PaCa-2, PANC-1, AsPC-1, and BxPC-3 were used for in vitro study. To evaluate the endogenous expression level of SP1, we purified the whole RNA and protein to perform the qPCR, RT-PCR and Western blot. si-SP1 was used for specifically inhibit the function of SP1. The invasive potential of pancreatic cancer cells were assessed in matrigel coated chambers. Among the 81 patients, 32 (39.5%) were positive for SP1. On univariate and multivariate analyses, poor differentiation tumor and SP1-positive status were identified as independent prognostic factors for DFS. High expression of SP1 was observed and correlated with the expression of mesenchymal markers (Snail, L1CAM, Vimentin) unlike epithelial markers (CDH1). Importantly, silencing of SP1 showed markedly decrease in motility and the invasiveness of cancer cells (p < 0.001) as determined from transwell invasion and transendothelial migration assays, respectively. Our results demonstrated that SP1 is an independent marker for metastatic disease and death in patients with pancreatic cancer. Additionally, our in vitro study demonstrated that SP1 expression promotes EMT and the invasiveness of pancreatic cancer cell lines, a finding compatible with the results of previous in vitro studies. These findings suggest that SP1 can potentially be a valuable target for the improvement of survival rates in patients with pancreatic cancer.

Interplay between cell cycle and autophagy induced by boswellic acid analog.

In this study, we investigated the role of autophagy induced by boswellic acid analog BA145 on cell cycle progression in pancreatic cancer cells. BA145 induced robust autophagy in pancreatic cancer cell line PANC-1 and exhibited cell proliferation inhibition by inducing cells to undergo G2/M arrest. Inhibition of G2/M progression was associated with decreased expression of cyclin A, cyclin B, cyclin E, cdc2, cdc25c and CDK-1. Pre-treatment of cells with autophagy inhibitors or silencing the expression of key autophagy genes abrogated BA145 induced G2/M arrest and downregulation of cell cycle regulatory proteins. It was further observed that BA145 induced autophagy by targeting mTOR kinase (IC50 1 μM), leading to reduced expression of p-mTOR, p-p70S6K (T389), p-4EBP (T37/46) and p-S6 (S240/244). Notably, inhibition of mTOR signalling by BA145 was followed by attendant activation of AKT and its membrane translocation. Inhibition of Akt through pharmacological inhibitors or siRNAs enhanced BA145 mediated autophagy, G2/M arrest and reduced expression of G2/M regulators. Further studies revealed that BA145 arbitrated inhibition of mTOR led to the activation of Akt through IGFR/PI3k/Akt feedback loop. Intervention in IGFR/PI3k/Akt loop further depreciated Akt phosphorylation and its membrane translocation that culminates in augmented autophagy with concomitant G2/M arrest and cell death.

Numb/Notch signaling pathway modulation enhances human pancreatic cancer cell radiosensitivity

The present study aims to evaluate whether repression of the Numb/Notch signaling pathway affects the radiosensitivity of human pancreatic cancer cell lines. Different doses of X-rays (0, 2, 3, 4, and 5Gy) were applied to the PANC-1, SW1990, and MIA PaCa-2 human pancreatic cancer cell lines, and the Numb/Notch pathway inhibitor DAPT was added at different doses (0, 1, 3, and 5μmol/l). MTT assay, colony formation assay, flow cytometry, scratch assay, and Transwell experiments were performed, and qRT-PCR and Western blot were conducted for the detection of Numb expression. Tumorigenicity assay in nude mice was carried out to verify the influence of blocker of the Numb/Notch signaling pathway on the radiosensitivity of xenograft tumors. The MTT assay, colony formation assay and flow cytometry experiments revealed that proliferation decreased as radiation dose increased. The viability of PANC-1 cells at 5Gy, SW 1990 cells at 4Gy and 5Gy, and MIA PaCa-2 cells at 2-5Gy was significantly lower than that of non-irradiated cells (all P<0.05). The migration and invasion assays indicated that the PANC-1 cell line was least radiosensitive, while the MIA PaCa-2 cell line was the most radiosensitive. Numb expression significantly increased with increasing radiation dose, whereas the expression of Hes1, Notch1, and Hes5 significantly decreased compared to non-irradiated cells (P<0.05). Compared to untreated control cells, DAPT dose dependently increased Numb expression and inhibited Notch1, Hes1, and Hes5 expressions at 2Gy (P<0.05). Subcutaneous tumorigenicity assay in nude mice demonstrated that DAPT increased the radiosensitivity of PANC-1, SW 1990, and MIA PaCa-2 cells. These findings suggest that Numb/Notch signaling in pancreatic cancer cells is associated with X-ray radiation and that inhibition of the Numb/Notch signaling pathway can enhance radiosensitivity, suggesting that inhibition of the Numb/Notch signaling pathway may serve as a potential target for clinical improvement of the radiosensitivity of pancreatic cancer.

MicroRNA miR-374, a potential radiosensitizer for carbon ion beam radiotherapy.

In this study, we compared the microRNA(miRNA) profiles of a control and X-ray- and carbon ion beam-resistant cells to identify miRNAs that can be used as radiosensitizers and biomarkers. Mouse squamous cell carcinoma line NR-S1, its X-ray-resistant derivative X60, and its carbon ion beam‑resistant derivative C30 were subjected to miRNA microarray analysis. Expression of miRNAs shown to be upregulated or downregulated in the microarray analysis was confirmed by qRT-PCR. Downregulated miRNAs were overexpressed in human pancreatic cancer cell lines PANC1 and MIA PaCa-2, and the resulting cells were tested for radiosensitivity using colony-forming and sphere-forming assays. Of 1,265miRNAs analyzed, 4were downregulated and11 were upregulated in X-ray-resistant and carbon ion beam-resistant cells. Two of the downregulated miRNAs, miR-196 and miR-374, were selected for overexpression in PANC1 and MIA PaCa-2 cells. Overexpression of miR-374 sensitized PANC-1 and MIA PaCa-2 cells toward carbon ion beam radiation. miRNA miR-374 has the potential to be a new radiosensitizer for carbon ion beam radiotherapy and a new biomarker to determine the optimal treatment for cancer.

A Size-Selective Intracellular Delivery Platform.

Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 μm wide constriction is effective for delivery of cargo to primary human T-cells that have an average diameter of 6.7 μm. In contrast, a 6 or 7 μm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 μm) and PANC-1 (12.3 μm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T-cells. The application of this approach is demonstrated by selectively delivering dextran-conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.

Cell-cycle-controlled radiation therapy was effective for treating a murine malignant melanoma cell line in vitro and in vivo.

Radiotherapy is a commonly used regimen for treating various types of intractable cancers, although the effects depend on the cell cycle of the targeted cancer cell lines, and for irradiation purposes it is therefore critical to establish a protocol for controlling the cell cycle. Here, we showed that a common murine melanoma cell line B16BL6 was more vulnerable to irradiation during the early S phase, and that synchronisation of the cell cycle greatly increased the therapeutic effects of radiotherapy. Cell-sorting experiments, according to cell-cycle phase, using B16BL6 cells demonstrated that cells in the early S phase were the most susceptible to radiotherapy. Gemcitabine, a clinically utilised anti-cancer drug, induced cell-cycle arrest during the early S phase in B16BL6 cells, and thus a synergistic therapeutic effect was observed when irradiation was administered at the right time. Human pancreatic cancer cell line PANC-1 exhibited similar properties to B16BL6 in terms of its radiosensitivity during the S/G2/M phase and also demonstrated a synergistic effect of cell cycle synchronisation. These results show the importance of cell-cycle control in the application of irradiation and suggest a suitable time interval between chemotherapy and radiotherapy, as well as providing useful information for treating intractable cancer.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Interleukin-32α inactivates JAK2/STAT3 signaling and reverses interleukin-6-induced epithelial-mesenchymal transition, invasion, and metastasis in pancreatic cancer cells.

Interleukin (IL)-32 is a newly discovered cytokine that has multifaceted roles in inflammatory bowel disease, cancer, and autoimmune diseases and participates in cell apoptosis, cancer cell growth inhibition, accentuation of inflammation, and angiogenesis. Here, we investigated the potential effects of IL-32α on epithelial–mesenchymal transition, metastasis, and invasion, and the JAK2/STAT3 signaling pathway in pancreatic cancer cells. The human pancreatic cancer cell lines PANC-1 and SW1990 were used. Epithelial–mesenchymal transition-related markers, including E-cadherin, N-cadherin, Vimentin, Snail, and Zeb1, as well as extracellular matrix metalloproteinases (MMPs), including MMP2, MMP7, and MMP9, were detected by immunofluorescence, Western blotting, and real-time polymerase chain reaction. The activation of JAK2/STAT3 signaling proteins was detected by Western blotting. Wound healing assays, real-time polymerase chain reaction, and Western blotting were performed to assess cell migration and invasion. The effects of IL-32α on the IL-6-induced activation of JAK2/STAT3 were also evaluated. In vitro, we found that IL-32α inhibits the expressions of the related markers N-cadherin, Vimentin, Snail, and Zeb1, as well as JAK2/STAT3 proteins, in a dose-dependent manner in pancreatic cancer cell lines. Furthermore, E-cadherin expression was increased significantly after IL-32α treatment. IL-32α downregulated the expression of MMPs, including MMP2, MMP7, and MMP9, and decreased wound healing in pancreatic cancer cells. These consistent changes were also found in IL-6-induced pancreatic cancer cells following IL-32α treatment. This study showed that reversion of epithelial–mesenchymal transition, inhibition of invasiveness and metastasis, and activation of the JAK2/STAT3 signaling pathway could be achieved through the application of exogenous IL-32α.

The hepatitis B virus X protein promotes pancreatic cancer through modulation of the PI3K/AKT signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer, with poor outcomes. Infection with the hepatitis B virus (HBV) may be associated as a worse prognosis for PDAC patients; however, the mechanisms involved in this process are unclear. We evaluated whether HBV infection leads to PDAC with a more aggressive phenotype, and attempted to elucidate the mechanisms involved. Clinicopathological data and outcomes from 64 patients with PDAC were collected and compared between serum HBsAg+ and HBsAg− patients. Furthermore, we examined the effects of the HBV X protein (HBx) on proliferation and migration of the pancreatic cancer cell lines PANC-1 and SW1990. We investigated expression changes of over 500 proteins by protein array analysis and identified several HBV- and PDAC-related candidates, which were further validated by immunoblotting and enzyme-linked immunosorbent assay. No differences in clinicopathological features were observed between HBsAg+ and HBsAg− patients; however, HBsAg+ patients had a shorter median survival time (8 vs. 13 months), although the differences were not significant. HBV DNA was detected in clinical specimens, even in PDAC patients considered “HBV-free”, potentially due to occult infection. HBx expression significantly enhanced cellular proliferation and migration and induced an epithelial-mesenchymal transition phenotype. Expression of ErbB4 and TGF-α was increased in parallel with HBx expression, and several downstream pathways including PI3K/AKT, MAPK, and ERK were upregulated. Inhibition of the PI3K/AKT pathway reversed the effects of HBx in PDAC cell lines. HBx may, therefore, contribute to the progression of PDAC through modulation of these pathways.

A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells

Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties – e.g. low sample dilution, low contamination – that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.

Investigation of juglone effects on metastasis and angiogenesis in pancreatic cancer cells

Juglone, a natural component, is shown to have cytotoxic and apoptotic effects in several cancer cell lines. However, little is known about its effects on invasion and metastasis. In this study, we aimed to determine the antimetastatic effect of juglone in the BxPC-3 and PANC-1 pancreatic cancer cell lines. Cytotoxic effect of juglone was evaluated by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test. The cells were treated with juglone at <IC50 doses (5, 10, 15 and 20μM ) for 24h. After the cell adhesion and invasion analysis, expression profiles of the MMP-2, MMP-9 and Phactr-1 genes were determined by qPCR. The IC50 dose of juglone was found to be 21.05μM in the BxPC-3 cell line and 21.25μM in the PANC-1 cell line for 24h. According to the cell adhesion and invasion analysis, treatment of juglone for 24h reduced the adhesion and invasion features of pancreatic cancer cells. A significant reduction of MMP-2, MMP-9 and Phactr-1 expressions was observed in pancreatic cancer cells after the treatment of juglone at <IC50 doses. By this study, it has been shown for the first time that juglone inhibits cell invasion and metastasis in pancreatic cancer line and can be evaluated as an effective anticancer agent in pancreatic cancer.

Study on effect and mechanism of ellagic acid on the proliferation and apoptosis of pancreatic cancer cell line PANC-1

Objective To investigate the effect of ellagic acid on the proliferation and apoptosis of pancreatic cell line PANC-1 in vitro and explore possible mechanism of ellagic acid's effect on PANC-1 cells. Methods PANC-1 cells were treated with ellagic acid at concentrations of 0, 2, 4, 6, 8 μg/ml for 24, 48 and 72 h respectively. Inverted Microscope showed cells' morphological changes. The cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay. When the cells were treated with ellagic acid at concentrations of 0, 2, 4, 6 μg/ml for 48 h, the morphological changes of apoptotic cells were observed by cell staining, apoptotic rate and cell cycle were detected by flow cytometry, the protein expression of cyclo-oxgenase 2 (COX-2) and nuclear factor-κB (NF-κB) were measured by Western blotting. Results Ellagic acid could significantly inhibited the growth of PANC-1 cells in a concentration-dependent and time-dependent manner. The difference among these groups were statistically significant(P<0.05). After treatment with ellagic acid, apoptosis of PANC-1 cells was seen. Apoptosis rates in the different concentration groups were respectively (11.18±0.99)%, (20.70±2.32)%, (39.68±0.69)%, the apoptosis rate of control group [(0.62±0.23)%] compared with statistically significant difference among the different concentration groups (P<0.05). The percentages of G0/G1 phase cells were respectively(48.98±2.36)%, (60.80±1.40)%, (72.82±0.59)%, the percentage of control group [(42.78±0.74)%] compared with statistically significant difference among the different concentration groups(P<0.05). The protein expression of COX-2 and NF-κB measured by Western blotting were significantly down-regulated dose-dependently. Conclusion Ellagic acid can inhibit the proliferation of pancreatic cancer cell line PANC-1 and induce apoptosis. The mechanism may depend on arresting cell cycle and decreasing protein expression of COX-2 and NF-κB. Key words: Ellagic acid; Pancreatic neoplasm; Proliferation; Apoptosis; Cyclo-oxgenase 2; Nuclear factor-κB

Synthesis of new tricyclic thiolactams as potent antitumor agent for pancreatic cancer

We synthesized the novel tricyclic thiolactams 2a–d, 3d–k, having a benzyl or substituted benzyl substituent on the nitrogen of indole subunit, and their preferential cytotoxicity under both nutrient-deprived medium (NDM) and Dulbecco’s modified Eagle’s medium (DMEM) was evaluated against a human pancreatic cancer cell line PANC-1. Among the tested compounds, the 4′-hydroxy derivative 3d showed the most potent cytotoxicity in NDM (PC50 1.68μM) although the moderate preferential cytotoxicity (PC50 1.68μM in NDM vs PC50 20μM in DMEM). The 3′-hydroxy derivative 3e exhibited the most preferential cytotoxicity (PC50 1.96μM in NDM vs less than 50% inhibition at 30μM in DMEM). The benzyl 2a and halogenated benzyl derivatives 2b,c showed no cytotoxicity in NDM. In addition, the indole (10, PC50 173.7μM), lactone (11, PC50 131.7μM), and lactam (12, PC50 44.8μM) derivatives showed week or moderate cytotoxicity in NDM. These results indicated that the hydroxy group on the benzyl substituent and tricyclic thiolactam ring were essential for the cytotoxicity in NDM against PANC-1 cell line. Moreover, 3′-hydroxy derivative 3e compound exhibited antitumor activity against the pancreatic ductal adenocarcinoma (PDAC) xenograft model in vivo.

Curcumin inhibits hypoxia-induced epithelial‑mesenchymal transition in pancreatic cancer cells via suppression of the hedgehog signaling pathway.

Hypoxic microenvironment, a common feature of pancreatic cancer, is associated with tumor proliferation, metastasis and epithelial-mesenchymal transition (EMT) changes. In recent years, many natural agents, including curcumin, have been proven to possess the ability to inhibit the progression of pancreatic cancer. However, whether curcumin is able to suppress hypoxia-induced pancreatic cancer progression and the underlying mechanisms are still not fully elucidated. The aim of the present study was to evaluate whether curcumin affects hypoxia-induced EMT and the activation of Hh signaling pathway in pancreatic cancer. The human pancreatic cancer cell line Panc-1, was treated with hypoxic condition and curcumin. Cell proliferation was assessed by the MTT assay. Wound healing assay and transwell invasion assay were used to detect the migratory and invasive activity of cancer cells. The EMT-related factors, E-cadherin, N-cadherin, vimentin were detected by QT-PCR, western blot analysis and immunofluorescence staining. The Hh signaling-related factors, SHH, SMO and GLI1 were detected by western blot analysis. The results of present study showed that curcumin could not only inhibit the hypoxia-induced cell proliferation, migration and invasion in pancreatic cancer, but also mediate the expression of EMT-related factors. In addition, curcumin remarkably inhibited hypoxia-mediated activation of Hh signaling pathway. Taken together, these data indicate that curcumin plays an important role in suppressing hypoxia-induced pancreatic cancer metastasis by inhibiting the Hh signaling pathway. Curcumin might be a potential candidate for chemoprevention of this severe disease.

Investigating the role of mucin in the delivery of nanoparticles to cellular models of human cancer disease: an in vitro study

Mucin, a glycosylated protein, is aberrantly overexpressed in a variety of tumor cells. The glycoprotein mesh decreases the rate of intracellular drug uptake and effectiveness. We investigated the influence of the mucin mesh on the cellular uptake of anti-MUC1 antibody and nanoparticles by fluorescence spectroscopy and microscopy. A glycosylation inhibitor (benzyl-α-GalNAc) was employed to regulate mucin glycosylation events. In our panel of pancreatic cell lines, only PANC-1 cells exhibited a significant increase in the uptake of liposomes following glycosylation inhibition, resulting in improved cytotoxicity of gemcitabine-loaded liposomes. Interestingly, areas devoid of liposome uptake were observed for pancreatic cancer cell lines PANC-1, Capan-1, and Capan-2; however, these restricted regions could be diminished for PANC1 cells only. In conclusion, investigating the reason(s) for differential cellular uptake of nanoparticles, in association with the production of mucin glycosylation mesh, should provide valuable leads to the future development of nanomedicine for cancer treatment.

Cdc7 is a potent anti-cancer target in pancreatic cancer due to abrogation of the DNA origin activation checkpoint.

PurposeCdc7 is a serine/threonine kinase which is responsible for the ‘firing’ of replication origins leading to initiation of DNA replication. Inhibition or depletion of Cdc7 in normal cells triggers a DNA origin activation checkpoint causing a reversible G1 arrest. Here we investigate Cdc7 as a novel therapeutic target in pancreatic cancer.Experimental designCdc7 target validation was performed by immunoexpression profiling in a cohort of 73 patients with pancreatic adenocarcinoma including 24 controls. Secondly Cdc7 kinase was targeted in Capan-1 and PANC-1 pancreatic cancer cell line models using either an siRNA against Cdc7 or alternatively a small molecule inhibitor (SMI) of Cdc7 (PHA-767491).ResultsCdc7 was significantly overexpressed in pancreatic adenocarcinoma compared to benign pancreatic tissue (median LI 34.3% vs. 1.3%; P<0.0001). Cdc7 knockdown using siRNA in Capan-1 and PANC-1 cells resulted in marked apoptotic cell death when compared with control cells. A prominent sub-G1 peak was seen on flow cytometry (sub-G1 51% vs. 3% and 45% vs. 0.7% in Capan-1 and PANC-1 cells, respectively). Annexin V labelling confirmed apoptosis in 64% vs. 11% and 75% vs. 8%, respectively. Western blotting showed cleavage of PARP-1 and caspase-3 and presence of γH2A.X. TUNEL assay showed strong staining in treated cells. These results were mirrored following Cdc7 kinase inhibition with PHA-767491.ConclusionsOur findings show that Cdc7 is a potent anti-cancer target in pancreatic adenocarcinoma and that Cdc7 immunoexpression levels might be used as a companion diagnostic to predict response to therapeutic siRNAs or SMIs directed against this kinase.