Pancreatic Cancer Cell Lines AsPC-1 Research Articles

To Study theAnti-cancer Effects of Shigella Flexneri in AspC-1 Pancreatic Cancer Cell Line in Approach to Bax and bcl-2 Genes.

Cancer is the uncontrolled division of cells and can be caused by genetic or environmental factors. Pancreatic cancer is one of the deadliest among all cancers. The role of bacteria as an anticancer agent dates back to almost 100years ago. The microbiome has recently become a focus of research in carcinogenesis and even pancreatic cancer. Shigella flexneri is a gram-negative bacterium, which causes shigellosis with symptoms such as diarrhea, fever, and stomach cramps in human. Shigella flexneri may play a very important role in the internal pathways of apoptosis and may induce apoptosis in some of the cancerous cells. In this experiment bacteria were cultured on Salmonella-Shigella agar, then inoculated into BHI Broth medium. After sonication, the protein concentration of the bacterium was measured by using the ZellBio Sensitive Protein Bradford Assay kit. MTT assay was performed to obtain IC50 for the said bacterial protein. Later by cDNA kit synthesized the cDNA based on the RNA template. In the end, the results were analyzed using real-time PCR and the expression of Bax and Bcl-2 genes was measured before and after treatment. The results showed that Shigella flexneri has the potential anti-proliferative effect in pancreatic cancer. The inhibitory concentration, pro-apoptotic amount to upregulate Bax, and meanwhile also to downregulate the bcl-2 found to be 10μl. In general, due to defects in the apoptotic pathway in cancer cells and the existence of drug-resistant cells, the detection of new apoptotic inducers such as Shigella flexneri cell extract can be used for further studies on cancer therapy.

Inhibition of Migration, Invasion and Drug Resistance of Pancreatic Adenocarcinoma Cells - Role of Snail, Slug and Twist and Small Molecule Inhibitors.

PurposeThe main purpose of this study is to demonstrate the effects of epithelial to mesenchymal transition activating transcription factor silencing (EMT-ATF silencing) on migration, invasion, drug resistance and tumor-forming abilities of various pancreatic cancer cell lines. Additionally, the contribution of small molecule inhibitors of EMT (SD-208 and CX4945) to the effects of gene silencing was evaluated.MethodsEMT activating transcription factors “Snail, Slug and Twist” were silenced by short hairpins on Panc-1, MIA PaCa-2, BxPC-3, and AsPC-1 pancreatic cancer cell lines. The changes in migration, invasion, laminin attachment, cancer stem-like cell properties and tumor-forming abilities were investigated. Chemosensitivity assays and small molecule inhibitors of EMT were applied to the metastatic pancreatic cancer cell line AsPC-1.ResultsEMT-ATF silencing reduced EMT and stem cell-like characteristics of pancreatic cancer cell lines. Following EMT-ATF silencing amongst the four PC cell lines, AsPC-1 showed the best response and was chosen for further chemoresistance and combinational therapy applications. EMT downregulated AsPC-1 cells showed less resistance to select chemotherapeutics compared to the control group. Both small molecule inhibitors enhanced the outcomes of EMT-ATF silencing.ConclusionOverall it was found that EMT-ATF silencing, either by EMT-ATF silencing or with the enhancement by small molecules, is a good candidate to treat pancreatic cancer since it simultaneously minimizes metastasis, stem cell properties, and drug resistance.

Cytotoxic activity of proteins extracted from fennel (Foeniculum vulgare) seeds against human cancer cell lines

Fennel (Foeniculum vulgare) is an aromatic flowering medicinal plant classified into the family Umbelliferae (Apiaceae). It is native to southern Europe and the Mediterranean region with a long history of use by humans as a spice, fresh vegetable, and in traditional folk medicine. Despite its popularity for its medicinal value, the majority of data in scientific literature is based on the crude extracts and essential oils such as trans‐anethole and estragole, fenchone, limonene, and 1‐octen‐3‐ol. Besides, other phytochemicals found from seeds are polyphenols, included rosmarinic acid and luteolin, etc. In present study, we investigated the anticancer potential of proteins purified from fennel seeds against human breast cancer MCF 7 and pancreatic cancer AsPC‐1 cell lines. The crude fennel extract was partially purified using anion‐exchange and size exclusion chromatography techniques to have a comparative analysis for activity guided fractionation. For anion‐exchange chromatography (HiTrap‐Q FF 5ml column in 20mM Tris/HCl, pH 8.00 buffer) and for gel filtration (HiPrep Sephacryl S‐200 HR16/20 column in 20mM, pH 8.00 buffer ) were used. The crude extract and fractions collected were judged for their purity by 10% Tris‐tricine SDS‐PAGE gel electrophoresis. The MTT assay results exhibited that both anion‐exchange and gel filtration fractions demonstrated significant cytotoxic activity against MCF‐7 breast cancer cell line with cell inhibition in a range of 65–80% at 100μg/ml. In contrast, evaluation againist AsPC‐1pancreatic cancer cell line revealed non‐significant inhibition by fractions obtained from both methods. Further work is in progress to identify and fully characterized the primary structure of cytotoxic proteins by mass spectrometry and Edman protein sequencing.

One-pot synthesis of substituted pyrrole-imidazole derivatives with anticancer activity.

A facile and efficient method to synthesize pyrrole-imidazole was developed via a post-Ugi cascade reaction followed by one purification procedure. Synthesized pyrrole-imidazole was collected by performing a mild reaction and a simple procedure, which could be applicable to a broad scope of functionalized anilines. The screening results demonstrated that compound 7e exhibited a high potency of anticancer activity in human pancreatic cancer cell lines PANC and ASPC-1. Our work shed light on the potential of post-Ugi cascade reaction in combinatorial and medicinal chemistry.

The expression of long non-coding RNA-LINC01410 in pancreatic cancer and its effect on proliferation and migration of pancreatic cancer cells

Objective: To explore the expression of long non-coding RNA-LINC01410 in the pancreatic cancer tissues, corresponding paracancerous tissues, human pancreatic cancer cell lines and normal pancreatic ductal epithelial cell line, and analyze the effect of LINC01410 on pancreatic cancer cell proliferation and migration. Methods: Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of LINC01410 in 16 cases of pancreatic cancer tissue and its adjacent tissues. RT-qPCR was performed to analyze LINC01410 expression in the pancreatic cancer cell lines AsPC-1, CAPAN-1, SW1990, BxPC-3 and CFPAC-1, and human normal pancreatic ductal epithelial cell line HPDE6-C7. Transfection of interference plasmid (shRNA) in the pancreatic cancer cell line with the highest expression level of LINC01410 were used to knock-down the expression of LINC01410. CCK-8 assay, colony formation assay, and transwell chamber assay were performed to detect the proliferation and migration of pancreatic cancer cells. The complementary paired miRNAs and downstream genes of LINC01410 were predicted by bioinformatics. The expression of miRNA and downstream genes was detected by RT-qPCR, and the protein expression of downstream genes was determined by Western blot. Results: The expression of LINC01410 in the pancreatic cancer tissues was significantly higher than that in the adjacent tissues [(3.46±0.32) vs (0.65±0.08), P<0.01]. The expression levels of LINC01410 in the pancreatic cancer cell lines were significantly higher than that in the normal human pancreatic ductal epithelial cells (P<0.05). The expression of LINC01410 was highest in BxPC-3 cells (P<0.01). After knock-down of the LINC01410 expression in the pancreatic cancer cell line BxPC-3, the cell proliferation was significantly inhibited (P<0.05), and the cell migration ability was decreased (P<0.05). LINC01410 complementarily paired with miR-497-5p, and miR-497-5p complementarily paired with IFITM3. After inhibiting the expression of LINC01410, the expression of miR-497-5p was increased [(1.04±0.17) vs (5.79±0.43), P<0.01], the mRNA expression of IFITM3 was decreased [(0.39±0.05) vs (1.00±0.03), P<0.01], and the protein expression of IFITM3, CDK6, Cyclin D2, PCNA, Vimentin, and N-cadherin was decreased. Conclusions: The expression of LINC01410 was increased in pancreatic cancer tissues and cell lines. Down-regulation of LINC01410 expression inhibits the proliferation and migration of pancreatic cancer BxPC-3 cells, and its mechanism may be closely related to regulating the miR-497-5p and IFITM3 gene expression.

Low molecular weight heparin and direct oral anticoagulants influence tumour formation, growth, invasion and vascularisation by separate mechanisms

The bidirectional association between coagulation and cancer has been established. However, anticoagulant therapies have been reported to have beneficial outcomes by influencing the vascularisation of the tumours. In this study the influence of a set of anticoagulants on tumour formation, invasion and vascularisation was examined. WM-266-4 melanoma and AsPC-1 pancreatic cancer cell lines were treated with LMWH (Tinzaparin and Dalteparin), and DOAC (Apixaban and Rivaroxaban) and the rate of tumour formation, growth and invasion were measured in vitro. In addition, the influence of these anticoagulants on vascularisation was examined using the chorioallantoic membrane assay (CAM) model and compared to the outcome of treatment with Bevacizumab. Using this model the influence of pharmacological concentrations of the anticoagulant on the growth, invasion and vascularisation of tumours derived from WM-266-4 and AsPC-1 cells was also measured in vivo. Tinzaparin and Daltepain reduced tumour formation and invasion by the cell lines in vitro, but with dissimilar potencies. In addition, treatment of CAM with LMWH reduced the local vascular density beyond that achievable with Bevacizumab, particularly suppressing the formation of larger-diameter blood vessels. In contrast, treatment with DOAC was largely ineffective. Treatment of CAM-implanted tumours with LMWH also reduced tumour vascularisation, while treatment of tumours with Apixaban reduced tumour growth in vivo. In conclusion, LMWH and DOAC appear to have anti-cancer properties that are exerted through different mechanisms.

The effects of microRNA-181b on proliferation and invasion of pancreatic cancer cells

Objective To investigate the effects of microRNA-181b (miR-181b) on cell proliferation and invasion of pancreatic cancer cells. Methods MiR-181b expression levels in 3 pancreatic cancer cell lines PANC-1, BXPC-3, ASPC-1 and normal pancreatic HTERT-HPNE cells were observed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). PANC-1 cells were transfected with 50 nmol/L miR-181b inhibitor or negative control using Lipofectamine 2000. After transfection, the expression of miR-181b was measured by RT-qPCR. Cell proliferation was tested by methylthiazol tetrazolium (MTT) assay. Cell invasion was detected by transwell. Bioinformatics prediction and luciferase activity tests were to verify phosphate and tension homology deleted on chromsome ten (PTEN) was the targeted gene of miR-181b. The expression levels of PTEN, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were measured by RT-qPCR. Results Compared with the HTERT-HPNE cells (1.00±0.06), the expression level of miR-181b in PANC-1 (2.84±0.16), BXPC-3 (1.74±0.18), ASPC-1 (2.21±0.20) was significantly increased. After transfection with 50 nmol/L miR-181b inhibitor, there was no obvious change in PANC-1 cell proliferation (P>0.05), and cell invasive ability was inhibited (P<0.01). Bioinformatics predicted PTEN might be the targeted gene of miR-181b. Luciferase experiments showed that luciferase activity of wild-type PTEN group fell by 39% after transfection of miR-181b mimics, but there was no statistical difference in the mutant group. After transfection with miR-181b inhibitor, PTEN was significantly increased (1.85±0.06 vs. 1.01±0.08, P<0.05), and MMP-2 (0.59±0.02 vs. 1.01±0.04) and MMP-9 (0.66±0.02 vs. 0.99±0.07) were significantly decreased (P<0.05). Conclusion These results demonstrate that miR-181b promotes proliferation and invasion in pancreatic cancer cells, which may be partly related to effecting MMP-2 and MMP-9 expression by PTEN. Therefore, it may be a new target for the biologic therapy for pancreatic cancer. Key words: Pancreatic cancer; MicroRNA-181b; Proliferation; Invasion

Organotin Polymers for the Control of Pancreatic Cancer

Pancreatic cancer is the fourth leading cause of death in the USA. Treatment is seldom successful. A wide variety of organotin polymers exhibit good inhibition of human pancreatic cancer cell lines AsPC-1 and PANC-1. The AsPC-1 is an adenocarcinoma pancreatic cell line and PANC-1 which is an epithelioid carcinoma pancreatic cell line. Synthesis is rapid employing commercially available reactants and the interfacial polymerization that is employed in the commercial synthesis of aramids and polycarbonates.

Rhein sensitizes human pancreatic cancer cells to EGFR inhibitors by inhibiting STAT3 pathway

BackgroundRhein is a lipophilic anthraquinone extensively found in medicinal herbs. Emerging evidence suggests that rhein has significant antitumor effects, supporting its potential use as an antitumor agent. The IL6/STAT3 signaling pathway has been suggested as an attractive target for the discovery of novel cancer therapeutics.MethodsThe human pancreatic cancer cell lines AsPC-1, Patu8988T, BxPC-3 and PANC-1, and immunodeficient mice were chosen as models to study the effects of rhein. The potent antiproliferative and proapoptotic effects of rhein were examined by cell viability, cellular morphology, apoptosis and colony formation assays. The STAT3 luciferase report assay, immunostaining analysis and Western blot analysis revealed the inhibition of the IL6/STAT3 signaling axis.ResultsApoptosis was induced by adjunctive use of rhein with epidermal growth factor receptor (EGFR) inhibitors in pancreatic cancer cells as verified by cell apoptosis analysis and changes in the expression level of apoptotic/anti-apoptotic proteins BCL-2, BAX, Caspase 3 and Cl-PARP. Suppression of the phosphorylation of STAT3 and EGFR were also observed as a result of the treatment with a combination of rhein and EGFR inhibitors. Most interestingly, it was found that rhein considerably sensitized cells to erlotinib, thus suppressing tumor growth in PANC-1 and BxPC-3 xenograft models. The in vivo anti-tumor effect was associated with increased apoptosis and combined inhibition of the STAT3 and EGFR pathways in tumor remnants.ConclusionsRhein sensitizes human pancreatic cancer cells to EGFR inhibitors through inhibition of STAT3. Taken together, the results indicate that rhein offers a novel blueprint for pancreatic cancer therapy, particularly when combined with EGFR inhibitors.

The anti-mesothelin monoclonal antibody amatuximab enhances the anti-tumor effect of gemcitabine against mesothelin-high expressing pancreatic cancer cells in a peritoneal metastasis mouse model.

Pancreatic cancer often has a very poor prognosis, even after complete resection. The recurrence of hepatic and peritoneal metastases is an important prognostic factor; therefore, the development of improved adjuvant therapy is urgently required. Mesothelin is a cell surface glycoprotein whose expression is restricted to a variety of cancer types, including pancreatic cancer. This expression pattern makes mesothelin an attractive target for cancer therapy, and several agents targeting mesothelin are currently in clinical trials. Here, we used the chimerized high-affinity anti-mesothelin monoclonal antibody amatuximab to investigate its effect on peritoneal metastasis. We used the AsPC-1 pancreatic cancer cell line engineered to express Gaussia luciferase (Gluc), (AsPC-1-Gluc) for in vivo experiments. Results showed that while amatuximab was not directly cytotoxic on an AsPC-1-Gluc tumor cells in a peritoneal metastasis model, it prevented the formation of tumor growth. In combination therapy with gemcitabine, amatuximab exhibited synergistic killing. Our results suggest that blockade of mesothelin by amatuximab may be a useful strategy for preventing the peritoneal dissemination of pancreatic cancer under an adjuvant setting.

Phosphoglycerate Mutase 1 (PGAM1) Promotes Pancreatic Ductal Adenocarcinoma (PDAC) Metastasis by Acting as a Novel Downstream Target of the PI3K/Akt/mTOR Pathway.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors known, with an overall 5-year survival rate of less than 6% due to early local invasion and distant metastasis. Exploring suitable therapeutic targets associated with invasion and metastasis is required for improving the prognosis of PDAC. In this study, we investigated the role of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) in PDAC. PGAM1 expression was examined in tissue samples of 54 PDAC patients using immunohistochemistry, and the correlation between clinicopathological expression and PGAM1 expression was determined. A survival curve was generated using the Kaplan–Meier method. After silencing PGAM1 by siRNA in pancreatic cancer cell lines Aspc-1 and Panc-1, the changes in proliferation, migration, and invasion, and signal pathways were determined. In this study, the expression of PGAM1 was found positively related to poor differentiation, metastasis, advanced clinical stage, and poor survival rate. Silencing PGAM1 decreased the proliferation of Aspc-1 and Panc-1 cells with an S phase arrest, but without influencing cell apoptosis. Migration and invasion also decreased significantly, independent of proliferation. PGAM1 was also found to promote EMT of PDAC cell lines by regulating the Wnt/β-catenin pathway. PGAM1 itself was modulated by the PI3K/Akt/mTOR pathway as a novel downstream target and has a positive mutual regulation with HIF-1α. This study indicates that PGAM1 is closely associated with clinical metastasis and poor prognosis of PDAC. PGAM1 is considered as a potential therapeutic target in PDAC metastasis.

Differential expression profiles of microRNAs in highly and weakly invasive/metastatic pancreatic cancer cells.

Pancreatic cancer is the eighth-leading cause of cancer-associated mortality worldwide. To date, the cellular and molecular mechanisms associated with the invasion and metastasis of pancreatic cancer remain unclear. To examine these mechanisms, a microRNA (miRNA/miR) microarray with 1,965 genes was hybridized with labeled miRNA probes from invasive PC-1.0 and non-invasive PC-1 cells for molecular profiling analysis. In addition, reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was utilized to validate the microarray results. Online miRNA target prediction algorithms online were used to predict the target genes of the differentially expressed miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) term enrichment analysis were performed for the potential targets of the differentially expressed miRNAs. The results demonstrated that 54 miRNAs were differentially expressed, of which 33 were upregulated and 21 were downregulated in the PC-1.0 cell line compared with the PC-1 cell line. A total of 6 upregulated miRNAs (miR-31, -34a, -181a, -181b, -193a-3p, and -193b) and 4 downregulated miRNAs (miR-221, -222, -484, and -502-3p) were selected from these 54 miRNAs and validated by RT-qPCR. The differentially expressed miRNAs were further validated by RT-qPCR in the human pancreatic cancer cell lines AsPC-1 (highly invasive) and CAPAN-2 (less invasive). The results revealed that 2 upregulated miRNAs (miR-34a and -193a-3p) and 4 downregulated miRNAs (miR-221, -222, -484, and -502-3p) exhibited a consistent expression pattern between the PC-1.0/PC-1 and AsPC-1/CAPAN-2 pancreatic cancer cells. The GO and KEGG enrichment analysis indicated that the mRNAs potentially targeted by miRNAs were involved in a range of biological functions. These results suggest that different miRNA expression profiles occur between highly and weakly invasive and metastatic pancreatic cancer cell lines, and may affect a variety of biological functions in pancreatic cancer.

Recombination of a dual-CAR-modified T lymphocyte to accurately eliminate pancreatic malignancy

BackgroundThe therapeutic application of T cells endowing with chimeric antigen receptors (CARs) is faced with “on-target, off-tumor” toxicity against solid tumors, particularly in the treatment of the pancreatic cancer. To our best knowledge, the pancreatic cancer cell line AsPC-1 often highly expressed some distinct tumor-associated antigens, such as carcino-embryonic antigen (CEA) and mesothelin (MSLN). Therefore, in this research, we have characterized dual-receptor CAR-modified T cells (dCAR-T) that exert effective and safe cytotoxicity against AsPC-1 cells.MethodsBased on the dual signaling pathway of wild T cells, we designed a novel dCAR diagram specific for CEA and MSLN, which achieved comparable activity relative to that of conventional CAR-T cells (CEA-CAR T or MSLN-CAR T). In this dCAR, a tandem construct containing two physically separate structures, CEA-CD3ζ and MSLN-4/1BB signaling domains were effectively controlled with tumor antigens CEA and MSLN, respectively. Finally, the activity of dCAR-T cells has been verified via in vitro and in vivo experiments.ResultsIn the presence of cognate tumor cells (AsPC-1) expressing both CEA and MSLN, dCAR-T cells exerted high anti-tumor activity relative to that of other single-receptor CAR-T cells bearing only one signaling pathway (e.g., Cζ-CAR and MBB-CAR). In a xenograft model, dCAR-T cells significantly inhibited the growth of AsPC-1 cells yet no effect on the growth of non-cognate tumor cells. Furthermore, the released cytokines and T cell persistence in mice were comparable with that of conventional CAR-T cells, obtaining specific and controllable cytotoxicity.ConclusionsA novel type of CAR-T cells, termed dCAR-T, was designed with specific activities, that is, significant cytotoxicity for two antigen-positive tumor cells yet no cytotoxicity for single antigen-positive tumor cells. Dual-targeted CAR-T cells can be precisely localized at the tumor site and can exert high cytotoxicity against tumor cells, alleviating “on-target, off-tumor” toxicity and enabling accurate application of CAR-T cell therapy.

Abstract LB-174: Novel AC electrokinetic platform for rapid isolation and characterization of extracellular vesicles from NSCLC patients

Abstract We have developed a novel Alternating Current Electrokinetics (ACE) platform to isolate macromolecules such as circulating cell-free DNA (ccfDNA) and nanoparticles, including cell-derived extracellular vesicles (EVs). ccfDNA and EV isolation, and antibody staining using the platform required less than two hours to complete. EVs isolated by ultracentrifugation from the pancreatic cancer cell line ASPC-1 were used to validate the ACE platform's performance. EVs from pancreatic cancer ASPC-1 cells were detected using an anti-CD63 antibody and an antibody to the pancreatic cancer specific membrane protein Glypican-1, confirming that the ACE platform could isolate EVs and identify membrane-bound proteins. Recovered EVs were isolated and EV-associated RNA was amplified by RT-PCR. EVs from isolated from NSCLC patient plasma on the ACE platform contained higher levels of PD-L1 compared to healthy individuals (p&amp;lt;0.001). In parallel on the ACE platform, ccfDNA was isolated and quantified from the same samples, with increased levels measured in cancer patients. Improved differentiation between the NSCLC patients and the healthy normals was achieved when both markers were included in the analysis. Therefore, we believe the ACE platform has the potential to become a multimodal analytics tool for cancer diagnostics and treatment monitoring. Citation Format: Rajaram Krishnan, Juan P. Hinestrosa, David Searson, Robert Turner, Robert Kovelman, Heath Balcer, James Madsen, David Bodkin. Novel AC electrokinetic platform for rapid isolation and characterization of extracellular vesicles from NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-174.

PO-420 Mechanisms of action of anti-proliferative lichen compound protolichesterinic acid

Introduction Protolichesterinic acid (PA), an aliphatic α-methylene γ-lactone isolated from the lichen Iceland moss ( Cetraria islandica ) has an anti-proliferative effect against a variety human cancer cells. In multiple myeloma cells PA induces apoptosis. The anti-proliferative effect of PA is not mediated by the known inhibitory action of PA on 5- and 12-lipoxygenases and effects on growth factor signalling are secondary. PA inhibits DNA replication and earlier studies indicated direct effects of PA on HIV reverse transcriptase (RT) and DNA polymerase. RT inhibitors are known to affect mitochondria. Material and methods Molecular modelling software (Glide, Schrodinger, LLC) was used to test for the ability of PA to bind onto DNA polymerases α, β, γ and θ. The crystal structures of polymerases were acquired from Protein Data Bank (PDB ID: 4QCL, 5TB8, 4ZTZ and 5A9F, respectively) and PA was docked into each structure. The effects of PA on mitochondria were assessed morphologically by electron microscopy and metabolically by measuring extracellular glucose and lactate concentrations in cell medium after 6 and 24 hour (ABL90 FLEX blood gas analyzer) in the pancreatic cancer cell line Aspc-1, the breast cancer cell line T47D, normal skin fibroblast and MCF-10 non-tumorigenic breast epithelial cell line. Results and discussions PA docked to all four types of DNA polymerases, creating multiple hydrogen bonds directly with the enzymes/polymerases and with the metal ion (Mg 2+ ) cofactor. Hydrogen bonds to the metal ions bridging the interaction to the enzyme appeared important and might be necessary/important for the activity of protolichesterinic acid and explain its mechanism of action. Morphological changes in mitochondria were observed following exposure to PA in both cancer cell lines, but these were difficult to interpret. Aspc-1 pancreatic cancer showed a significant increase in lactate production after 24 hour exposure to PA. This was not observed in the breast cell lines, T47D and MCF-10. Aspc-1 is less sensitive to the anti-proliferative effects of PA than the breast cell lines. Further metabolomics studies are in progress. Conclusion Docking study of crystal structures of DNA polymerases indicates that PA could affect activity of several types of DNA polymerase, but this remains to be tested experimentally. PA may affect mitochondrial structure but increased lactate production is associated with relative resistance to its anti-proliferative effects.

Traditional Chinese medicine Astragalus polysaccharide enhanced antitumor effects of the angiogenesis inhibitor apatinib in pancreatic cancer cells on proliferation, invasiveness, and apoptosis.

BackgroundTraditional chemotherapy and molecular targeted therapy have shown modest effects on the survival of patients with pancreatic cancer. The current study aimed to investigate the antitumor effects of apatinib, Astragalus polysaccharide (APS), and the combination of both the drugs in pancreatic cancer cells and further explore the molecular mechanisms in vitro.Materials and methodsExpression of vascular endothelial growth factor receptor-2 (VEGFR-2) in human pancreatic cancer cell lines ASPC-1, PANC-1, and SW1990 was detected by Western blotting. Cell proliferation was measured by MTS, and migration and invasion were detected by wound-healing and Transwell assays, respectively. Cell apoptosis rate was determined by flow cytometry and cellular autophagy level affected by apatinib, and APS was analyzed by Western blotting.ResultsHuman pancreatic cancer cell lines ASPC-1 and PANC-1 expressed VEGFR-2, but VEGFR-2 was not detected in SW1990. Either apatinib or APS inhibited cell proliferation in a dose-dependent manner in ASPC-1 and PANC-1. APS in combination with apatinib showed enhanced inhibitory effects on cell migration and invasion compared with apatinib monotherapy in ASPC-1 and PANC-1. Meanwhile, APS combined with apatinib strongly increased cell apoptosis percentage. Western blotting showed that the combination of APS and apatinib significantly enhanced the downregulation of phosphorylated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) (p-AKT and p-ERK) as well as matrix metalloproteinases-9 (MMP-9) expression. In addition, both apatinib and APS induced cellular autophagy. However, the expression of autophagy-related proteins was not further elevated in the combination group.ConclusionThe study first demonstrated that apatinib showed potentially inhibitory effects in pancreatic cancer cells and that APS enhanced the antitumor effects of apatinib through further downregulating the expression of phosphorylation of AKT and ERK as well as MMP-9.

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells.

Hepatic arterial infusion (HAI) chemotherapy is expected to be a more effective and safer method to treat the hepatic metastasis of pancreatic cancer than intravenous (iv) administration because of higher tumor exposure and lower systemic exposure. To clarify the uptake mechanism of nucleoside anticancer drugs, including gemcitabine (GEM), in pancreatic cancer, we investigated the uptakes of radiolabeled uridine (a general substrate of nucleoside transporters) and GEM in pancreatic cancer cell lines MIA-PaCa2 and As-PC1. Uridine uptake was inhibited by non-labeled GEM and also by S-(4-nitrobenzyl)-6-thioinosine (NBMPR; an inhibitor of equilibrative nucleoside transporters, ENTs) in a concentration-dependent manner, suggesting that ENTs contribute to uridine uptake in pancreatic cancer cells. As for GEM, saturable uptake was mediated by high- and low-affinity components with Km values of micromolar and millimolar orders, respectively. Uptake was inhibited in a concentration-dependent manner by NBMPR and was sodium ion-independent. Moreover, the concentration dependence of uptake in the presence of 0.1μM NBMPR showed a single low-affinity site. These results indicated that the high- and low-affinity sites correspond to hENT1 and hENT2, respectively. The results indicated that at clinically relevant hepatic concentrations of GEM in GEM-HAI therapy, the metastatic tumor exposure of GEM is predominantly determined by hENT2 under unsaturated conditions, suggesting that hENT2 expression in metastatic tumor would be a candidate biomarker for indicating anticancer therapy with GEM-HAI.

Syntheses and anti-pancreatic cancer activities of rakicidin A analogues

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor and resistant to most therapies. Pancreatic cancer stem cells (PCSCs) had critical role in regulating PDAC progression, metastasis, and drug resistance. Therefore, targeting PCSCs is considered to be a promising strategy for treatment of PDAC. However, there is no effective drug that can selectively ablate PCSCs. A series of twenty rakicidin A analogues were synthesized via a combinatorial strategy and evaluated for their anti-PDAC activities, and the structure-activity relationship was also discussed. Compound 32g was prepared in 14 linear steps with 5.05% overall yield, which is much more efficient than our previously reported total synthesis of rakicidin A (19 linear steps with 0.19% yield). In a highly metastatic pancreatic cancer cell line ASPC-1, compound 32g showed about 4 times higher potency (IC50 = 0.022 μM) than rakicidin A (IC50 = 0.082 μM) at hypoxia condition, and 12 folds of hypoxia selectivity (IC50 = 0.27 μM at nomoxia condition). In contrast, the activity of adriamycin in the same hypoxic condition decreased. The percentage of PCSCs (with CD24+CD44+ESA+ biomarker), activity of ALDH, and the number of tumorspheres in PANC-1 cells were greatly reduced after treatment of 32g. More importantly, the tumor-initiating frequency was reduced by 19 folds after the treatment of 32g, which is better than that of rakicidin A (reduction of 4.7 folds).

Differential Organ-targeting and Cellular Characteristics of Metastatic Human Pancreatic Cancer Cell Lines in Mouse Models.

The lethal characteristic of pancreatic cancer is metastasis which is recalcitrant to currently-used chemotherapy. Our aim was to understand metastasis at the cellular level. We previously reported that multi-nucleate cells or spindle cells were more prominent in pancreatic cancer metastasis than in the primary tumor. In the present report, we investigated four representative human pancreatic cell lines for differences in cell morphology between the primary tumor and various metastatic organ targets for each cell line. Human pancreatic cancer cell lines AsPC-1, Panc-1, KP2 and KP3 were used. Pancreatic cancer cells were injected into spleen of nude mice resulting in experimental metastasis to various organs which were observed at the cellular level when the organs were placed into culture. AsPC-1 and KP2 pancreatic cells formed many experimental liver metastases, in contrast to Panc-1 and KP3. Lung metastasis was only observed for AsPC-1. In the cultures established from the primary and metastatic tumors, multi-nucleate cells were found to be more prominent in the metastasis of the pancreatic cell lines with frequent metastasis, AsPC-1 and KP2. Spindle-like cells were observed prominently in AsPC-1 lung metastasis. Human pancreatic cancer cell lines have differential metastatic characteristics with regard to target organs and cell-morphology changes. Multi-nucleate and spindle cells may play an important role in pancreatic cancer metastasis to the liver and lung, respectively.

MEKK3 Sustains EMT and Stemness in Pancreatic Cancer by Regulating YAP and TAZ Transcriptional Activity.

Pancreatic cancer is one of the most threatening and poorly understood human malignancies. MEKK3 (MAP3K3) is a serine/threonine kinase activated by different signaling pathways. YAP and TAZ are critical oncogenic effectors in pancreatic cancer. We hypothesized that MEKK3 could sustain pancreatic cancer by inducing YAP/TAZ oncogenic activities. In Panc1 and AsPC1 pancreatic cancer cell lines MEKK3 was knocked-out (KO) by the CRISPR/Cas9 method. These cells were used to evaluate MEKK3 contribution to the expression of YAP/TAZ and their target genes, cell migration, stemness, and in vivo tumor growth. MEKK3 KO reduced both EMT and cell migration, the size of 3D colonies and the percentage of CD44+/CD24+/EpCAM+ CSC, promoter recruitment of YAP/TAZ and the expression of their target genes. It reduced tumor growth and prolonged mice overall survival. Silencing of MEKK3 represents a valid approach to revert in vivo the aggressiveness of pancreatic cancer by modulating YAP/TAZ transcriptional activities.