Pancreatic Cancer Cell Lines MiaPaCa-2 Research Articles

New Analogues of the Nicotinamide Phosphoribosyltransferase Inhibitor FK866 as Potential Anti-Pancreatic Cancer Agents.

During the past two decades, many nicotinamide phosphoribosyltransferase (NAMPT) inhibitors were prepared and tested because this enzyme is overexpressed in pancreatic cancer. Although FK866 is a well-known, strong NAMPT inhibitor, it suffers severe drawbacks. Our work aimed to synthesize efficient NAMPT inhibitors featuring better pharmacokinetic properties than the pyridine-containing FK866. To this aim, the new anticancer agents were based on benzene, pyridazine, or benzothiazole moieties as a cap group instead of the pyridine unit found in FK866 and other NAMPT inhibitors. The new compounds, prepared exploiting standard heterocycle chemistry and coupling reactions (e.g., formation of amides, ureas, and cyanoguanidines, copper-mediated azide-alkyne cycloaddition), have been fully characterized using NMR and HRMS analyses. Their activity has been evaluated using cytotoxicity and intracellular NAD depletion assays in the human pancreatic cancer cell line MiaPaCa-2. Among the 14 products obtained, compound 28, bearing a pyridazine unit as the cap group and a thiophene moiety as the tail group, showed 6.7 nanomolar inhibition activity in the intracellular NAD depletion assay and 43 nanomolar inhibition in the MiaPaCa-2 cells cytotoxicity assay, comparable to that observed for FK866. The positive results observed for some newly synthesized molecules, particularly those carrying a thiophene unit as a tail group, indicate that they could act as in vivo anti-pancreatic cancer agents.

Abstract 4677: Characterizing resistant and metastatic phenotypes to pharmacological ascorbate in pancreatic cancer

Abstract Background and purpose: Pharmacological ascorbate (P-AscH) is a prooxidant that oxidizes to produce high levels of hydrogen peroxide. It is currently being investigated as an anti-cancer neoadjuvant for pancreatic cancer (PC). In phase I clinical trials, P-AscH has generated encouraging results in terms of efficiency and safety. However, 50% of patients do not respond to P-AscH suggesting that resistance occurs in a subset of patients. We have generated PC cell lines resistant to P-AscH to characterize mechanisms of resistance. Methodology: PANC-1 and MIA PaCa-2 PC cell lines were treated with a stepwise progression of increasing concentrations of P-AscH until 10-fold IC50 concentration was fully tolerated (AscR, ascorbate resistant) as determined by clonogenic survival. Once resistance was achieved, various redox enzyme protein expression and activity were measured to determine their role in resistance to P-AscH. Proliferation and cell cycle analysis were conducted on resistant and sensitive cells. An RNA sequencing screen was conducted as an unbiased approach to identify potential candidates crucial for resistance. Finally, phenotypes associated to increased metastatic potential, including collagen invasion and in vivo tumor colonization were explored for characterization. Results: PANC-1 and MIA PaCa-2 AscR cells developed unique mechanisms to tolerate high levels of P-AscH. PANC-1 AscR cells almost exclusively rely on catalase to detoxify H2O2 generated during P-AscH treatments, while MIA PaCa-2 AscR cells do not. It is interesting to note that not every redox enzyme was upregulated in response to P-AscH treatments, including GPx1/4. The AscR cell lines also differed in growth rates. PANC-1 AscR cells developed a faster proliferation rate, whereas MIA PaCa-2 AscR cells significantly decreased their growth. This is consistent with a shortened G1/G0 cell cycle phase and an accumulation in G2/M, respectively. The RNA sequencing screen revealed a metastatic signature in association with resistance to P-AscH, including upregulation of the PI3K/AKT axis. We observed a significant increase in collagen matrix invasion, as well as metastatic colonization in vivo. Additionally, we have observed increased expression of active AKT in both AscR cell lines, indicating a potential mechanism for the increased metastatic phenotype. Conclusion: Our findings demonstrate distinct mechanisms for AcsR cells to tolerate high doses of P-AscH. Although catalase has demonstrated to be a key factor in controlling redox flux, it may not be necessary for all cell types. Not only can P-AscH resistance alter redox enzyme expression, but we have also observed substantial increase in metastatic potential to drive invasion and tumor colonization. In summary, our data indicate a potential route for PC to tolerate high levels of P-AscH and may explain how some patients do not respond to this treatment regimen. Citation Format: Amanda N. Pope, Brianne R. O'Leary, Juan Du, Garry R. Buettner, Michael D. Henry, Joseph J. Cullen. Characterizing resistant and metastatic phenotypes to pharmacological ascorbate in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4677.

Abstract 5904: Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways

Abstract Pancreatic cancer (PaCa) ranks as the fourth leading cause of cancer-related deaths in the United States and is among the top five worldwide. Its hallmark characteristics include early invasion, metastasis, resistance to chemotherapy and radiotherapy, and aggressive tumor progression. Evaluating new generation treatments with significant potential in this ongoing battle against cancer is crucial. Voltage-gated sodium channels (VGSC), long detected in various metastatic cell lines and tissue samples beyond breast, lung, and prostate cancer, are of particular interest. This study aimed to explore the therapeutic effects of inhibiting NaV1.7 and its neonatal variant, nNaV1.7, in pancreatic cancer cells. The study utilized human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2. It investigated the roles and effects of adult and neonatal isoforms of the NaV1.7 channel in pancreatic cancer proliferation, invasion, and metastasis. The findings revealed significantly higher adult and neonatal NaV1.7 mRNA expressions in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells. Specific siRNAs effectively downregulated channel mRNA and protein expressions. Notably, NaV1.7 siRNA treatments significantly inhibited cell proliferation and colony-forming capacity in PANC-1 and MiaPaCa-2 cells, without cytotoxic effects on HaCaT cells. Combining gemcitabine with specific NaV1.7 siRNAs enhanced drug efficacy in the PANC-1 cell line. Furthermore, NaV1.7 siRNAs significantly reduced cell invasion, migration, and wound healing in both cell lines. These siRNAs also induced apoptosis and G1 arrest in the cell cycle. They inhibited protein expressions involved in key signaling pathways, including P-Src/P-Fak/Integrin β1, EF2K, P-Akt, P-mTOR, PARP, Caspase-3/9, Cyclin D1/E1. This study is pioneering, as literature lacks research on the expression and function of NaV1.7 and nNaV1.7 in pancreatic cancer cells or tissue samples. In conclusion, NaV1.7 channel isoforms play a significant role in the development, progression, and metastasis of pancreatic cancer and present as a potential therapeutic target in PaCa. Citation Format: Mumin Alper Erdogan, Ayse Caner. Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5904.

Abstract C175: The potential effect of KRAS inhibitors on tumor microenvironments

Abstract KRAS is one of the most common driver oncogenes among various cancer types. Researches aiming to develop KRAS inhibitors has been surging recently and the first clinical KRAS inhibitor sotorasib was approved for non-small cell lung cancer in the US and Japan. As KRAS activity has been reported to have certain effects on tumor microenvironments, KRAS inhibitor could potentially induce collateral outcomes on tumor microenvironment. Therefore, I have analyzed the potential influences of resistance to KRAS inhibitors on the tumor microenvironment. At first, I have established pancreatic cancer cell line MIA-Paca2 that acquired resistance to sotorasib, MIA/AMG-R, by continuously exposing them to sotorasib (1 µmol/L). An RNA sequencing analysis were performed with MIA/AMG-R and revealed that a gene set related to hypoxia were upregulated. When non-small cell lung cancer cell line NCI-H358 cells were exposed to sotorasib (1 µmol/L) for 4 hrs, the protein levels of HIF-1A decreased while that in MIA Paca-2 cells were unaffected. To evaluate at which steps of protein synthesis from gene transcription to through protein degradation of HIF-1A, NCI-H358 cells were treated with transcription inhibitor actinomycin D, translation inhibitor cycloheximide or proteasome inhibitor MG-132. The treatment of transcription inhibitor actinomycin D on NCI-H358 cells for 6 hrs did not lead to the decrease of HIF-1A protein level however, translation inhibitor cycloheximide induced drastic decrease in HIF-1A protein. Although exposure of NCI-H358 cells to MG-132 increased the expression level of HIF-1A, the combinational treatment of sotorasib and MG-132 still led to slight decrease in HIF-1A protein level. Also, MG-132 failed to recover the protein expression level decreased by cycloheximide treatment. In total, the cellular expression level of HIF-1A is possibly dependent on translation activity and KRAS inhibitor potentially affect the HIF-1A expression through the regulation of its translation. Conclusively, KRAS inhibitors could potentially be used to reprogram the hypoxic tumor microenvironments besides its primary effect against the downstream signaling of KRAS. Citation Format: Noritaka Tanaka, Takeharu Sakamoto. The potential effect of KRAS inhibitors on tumor microenvironments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C175.

Targeting a transcription factor NF-κB by green tea catechins using in silico and in vitro studies in pancreatic cancer.

Pancreatic cancer remains a lethal disease and a major public health problem globally. Nuclear factor-kappa B (NF-κB) has been identified as a therapeutic target in several cancers and plays an important role in inflammatory responses. Many phytochemicals, including catechins, have been reported in the scientific literature with efficient anticancer potential and minimal side effects. This study aims to gain insights into the inhibitory mechanism of catechin derivatives epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) using in silico and in vitro studies especially considering NF-κB targeting. We explored the binding pose, interacting residues and molecular interactions for catechin derivatives with NF-κB. Docking analysis showed that the catechin derivatives acted as covalent inhibitors with the p65 subunit of NF-κB and interacted with other residues through non-bonding interactions and hydrogen bonds. Further, we validated the effect of EGCG on NF-κB activity in pancreatic cancer cell lines MIAPaCa-2 and SU 86.86. Our in vitro data showed EGCG effectively reduced cell growth and proliferation, induced apoptosis, and inhibited NF-κB activity in the studied cell lines. In addition, EGCG repressed the expression of NF-κB target genes including MMP9, MMP2, cMyc, and BCL-2. Thus, targeting NF-κB with EGCG could be a potential therapeutic alternative for pancreatic cancer treatment.

Pomegranate juice anthocyanidins induce cell death in human cancer cells by mobilizing intracellular copper ions and producing reactive oxygen species.

Anthocyanidins are the most abundant polyphenols in pomegranate juice. This class of molecules includes Delphinidin (Del), Cyanidin (Cya), and Pelargonidin (Pel). Using prostate, breast and pancreatic cancer cell lines PC3, MDA-MB-231, BxPC-3 and MiaPaCa-2, we show that anthocyanidins inhibit cell proliferation (measured by MTT assay) and induce apoptosis like cell death (measured by DNA/Histone ELISA). Copper chelator neocuproine and reactive oxygen species scavengers (thiourea for hydroxyl radical and superoxide dismutase for superoxide anion) significantly inhibit this reaction thus demonstrating that intracellular copper reacts with anthocyanidins in cancer cells to cause DNA damage via ROS generation. We further show that copper-supplemented media sensitizes normal breast epithelial cells (MCF-10A) to Del-mediated growth inhibition as determined by decreased cell proliferation. Copper supplementation results in increased expression of copper transporters Ctr1 and ATP7A in MCF-10A cells, which is attenuated by the addition of Del in the medium. We propose that the copper mediated, ROS-induced mechanism of selective cell death of cancer cells may in part explain the anticancer effects of anthocyanidins.

Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS‑activated pancreatic cancer cells.

Pyruvate dehydrogenase kinase 4 (PDK4) is an important regulator of energy metabolism. Previously, knockdown of PDK4 by specific small interfering RNAs (siRNAs) have been shown to suppress the expression of Kirsten rat sarcoma viral oncogene homolog (KRAS) and the growth of lung and colorectal cancer cells, indicating that PDK4 is an attractive target of cancer therapy by altering energy metabolism. The authors previously reported that a novel small molecule, cryptotanshinone (CPT), which inhibits PDK4 activity, suppresses the in vitro three-dimensional (3D)-spheroid formation and in vivo tumorigenesis of KRAS-activated human pancreatic and colorectal cancer cells. The present study investigated the molecular mechanism of CPT-induced tumor suppression via alteration of glutamine and lipid metabolism in human pancreatic and colon cancer cell lines with mutant and wild-type KRAS. The antitumor effect of CPT was more pronounced in the cancer cells containing mutant KRAS compared with those containing wild-type KRAS. CPT treatment decreased glutamine and lipid metabolism, affected redox regulation and increased reactive oxygen species (ROS) production in the pancreatic cancer cell line MIAPaCa-2 containing mutant KRAS. Suppression of activated KRAS by specific siRNAs decreased 3D-spheroid formation, the expression of acetyl-CoA carboxylase 1 and fatty acid synthase (FASN) and lipid synthesis. The suppression also reduced glutathione-SH/glutathione disulfide and increased the production of ROS. Knockdown of FASN suppressed lipid synthesis in MIAPaCa-2 cells, partially promoted ROS production and mildly suppressed 3D-spheroid formation. These results indicated that CPT reduced tumorigenesis by inhibiting lipid metabolism and promoting ROS production in a mutant KRAS-dependent manner. This PDK4 inhibitor could serve as a novel therapeutic drug for KRAS-driven intractable cancers via alteration of cell metabolism.

A Substance P (SP)/Neurokinin-1 Receptor Axis Promotes Perineural Invasion of Pancreatic Cancer and Is Affected by lncRNA LOC389641.

Perineural invasion (PNI) is considered to be a main reason for the poor prognosis of pancreatic cancer. In the present study, we analyzed the roles of substance P (SP)/neurokinin-1 receptor (NK-1R) and lncRNA LOC389641 in pancreatic cancer PNI. Pancreatic cancer cell lines BxPC-3 and MIAPaCa-2 were cocultured with SH-SY5Y cells and then stimulated with SP to simulate the in vivo influence of ganglia on pancreatic cancer. The BxPC-3 and MIAPaCa-2 cells were transfected with a neurokinin-1 receptor (NK-1R) overexpression vector, NK-1R silencing vector, LOC389641 overexpression vector, or LOC389641 silencing vector, respectively. The proliferative abilities of BxPC-3 and MIAPaCa-2 cells were assessed using the cell counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays. Wound-healing and Transwell assays were performed to determine the migration and invasion abilities of the cells. When SP was added to the coculture system, it positively regulated cancer cell proliferation, migration, and PNI and significantly activated the NK-1R/Akt/NF-κB signaling pathway. Incubation with 100 nmol/L SP for 24 h was selected as the optimal condition for treatment. The activated NK-1R positively regulated the proliferation, migration, and invasion of pancreatic cancer cells. However, the levels of lncRNA LOC389641 and tumor necrosis factor receptor SF10A (TNFRSF10A) mRNA in BxPC-3 and MIAPaCa-2 cells were not affected by SP treatment. Overexpression or silencing of LOC389641 changed the effect of SP stimulation on pancreatic cancer PNI. When taken together, these results revealed that SP/NK-1R and LOC389641 promoted the progression of pancreatic cancer PNI. Moreover, we found that pancreatic cancer PNI promoted by the SP/NK-1R axis could be blocked by the TNFRSF10A/NF-κB pathway mediated by LOC389641.

RET‐Dependent Axonal Sprouting from Spinal Afferent Neurons in a Pancreatic Cancer Model

ObjectiveActivation of the RET receptor tyrosine kinase stimulates signaling pathways associated with cell proliferation, growth, differentiation, and survival. Changes in the expression of this receptor and its associated ligands have been linked to tumorigenesis in several tumor types, including pancreatic cancer. It has been established that tumor cells can migrate towards and invade cells of the nervous system, a process called perineural invasion that is associated with metastasis and reduced survival. Given that RET ligands play important roles in spinal afferent neuronal development and morphology, we hypothesized that the release of RET ligands by pancreatic cancer cells has a neurotrophic effect on spinal afferent neurons, drawing their axons towards the tumor and facilitating perineural invasion.MethodsThoraco‐lumbar (T10‐L2) dorsal root ganglion (DRG) neurons from male C57BL/6 mice were collected, dissociated, and cultured for 4 days in F12 media along with a 1:3 dilution of conditioned media from two pancreatic cancer cell lines (PanC1 and MiaPaCa2) or a cell‐free media control (F12 + 1:3 DMEM). In a separate set of experiments, DRG neurons were exposed to the same conditioned media along with the RET inhibitor, Selpercatinib (5 µM, diluted in DMSO). For the RET inhibitor experiments two control groups were used, the 1st contained F12 media + 1:3 DMEM + DMSO (vehicle controls, VC), and the 2nd group of controls consisted of F12 media + 1:3 DMEM + Selpercatinib (VC + Selp). Neurons were stained with calcein‐AM (1 µM) and visualized using a fluorescent microscope. Changes in DRG neuron morphology (neurite length and branching relative to distance from cell soma) were quantified using Sholl analysis.ResultsSholl plots revealed significant changes in DRG neuron morphology following treatment with either PanC1 or MiaPaCa2 conditioned media compared to DRG neurons treated with the RET inhibitor, or the different control groups. Where DRG neurons incubated with PanC1 conditioned media demonstrated an approximate 3 to 4‐fold increase in neurite length compared to VC and VC + Selp (229 ± 32.7 µm vs 74.7 ± 12.2 µm vs 50.2 ± 6.3 µm; respectively). These effects on neurite length were significantly diminished following treatment of the DRG neurons with PanC1 + Selp (116.6 ± 15.5 µm). Furthermore, treatment of DRG neurons with the PanC1 conditioned media led to a 2‐fold increase in the maximum branching of neurites (10.4 ± 2.1 intersections; n=36) compared to VCs (5.75 ± 1.3 intersections; n=35). Again, these effects were significantly diminished following treatment of DRG neurons with PanC1 conditioned media + Selp (3.75 ± 0.6 intersections; n=40) (Kruskal‐Wallis with Dunn’s post‐hoc multiple comparison test, p<0.001). Similar RET‐dependent effects were observed for DRG neurons treated with conditioned media from the MiaPaCa2 cell line.ConclusionThese data suggest that pancreatic cell lines release RET ligands that may attract axons of spinal afferent neurons towards them, which may facilitate perineural invasion.

Proteomics Approach Highlights Early Changes in Human Fibroblasts-Pancreatic Ductal Adenocarcinoma Cells Crosstalk.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality worldwide. Non-specific symptoms, lack of biomarkers in the early stages, and drug resistance due to the presence of a dense fibrous stroma all contribute to the poor outcome of this disease. The extracellular matrix secreted by activated fibroblasts contributes to the desmoplastic tumor microenvironment formation. Given the importance of fibroblast activation in PDAC pathology, it is critical to recognize the mechanisms involved in the transformation of normal fibroblasts in the early stages of tumorigenesis. To this aim, we first identified the proteins released from the pancreatic cancer cell line MIA-PaCa2 by proteomic analysis of their conditioned medium (CM). Second, normal fibroblasts were treated with MIA-PaCa2 CM for 24 h and 48 h and their proteostatic changes were detected by proteomics. Pathway analysis indicated that treated fibroblasts undergo changes compatible with the activation of migration, vasculogenesis, cellular homeostasis and metabolism of amino acids and reduced apoptosis. These biological activities are possibly regulated by ITGB3 and TGFB1/2 followed by SMAD3, STAT3 and BAG3 activation. In conclusion, this study sheds light on the crosstalk between PDAC cells and associated fibroblasts. Data are available via ProteomeXchange with identifier PXD030974.

Retraction Note to: 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.

Abstract 1323: Novel autophagy inhibitor to treat pancreatic cancer

Abstract In this study, we develop a novel autophagy inhibitor targeting one of the most aggressive cell populations present in pancreatic tumors, the pancreatic cancer stem cell (CSC). Targeting this population which has the malignant properties of tumorigenesis/tumor recurrence, the ability to differentiate into a heterogenous tumor cell types and can self-renew is a new strategy to alleviate the current dismal 10% five-year survival rate of pancreatic cancer. Here we characterize an autophagy inhibitor which is a first-in-class nanoparticle comprised of a chloroquine-derived small molecule drug that can self-assemble into a micelle. Due to its favorable pharmacokinetic properties and the CSC reliance on autophagy, our compound is able to selectively target this cell population both in vitro and in vivo. Additionally, the compound has shown synergy with the current standard of care chemotherapeutic, gemcitabine, with an 88.9% reduction in tumor volume in the combination of the two drugs compared to gemcitabine treated alone (p<0.0001) in two separate treatment regimens using a pancreatic PDX mouse model. We further illustrate that the stem cell population is targeted by inhibiting tumorsphere formation in established pancreatic cancer cell lines MIA-PaCa2 and PANC-1 and in pancreatic PDX-derived CSCs in vitro. The drug treatment also promotes a decrease in pluripotency markers, particularly in Sox2 levels. Finally, the drug is well-tolerated with a maximum tolerated dose above 100 mg/kg in mice and is orally bioavailable. Citation Format: Mythili Ramachandran, Zhao Ma, Yuanpei Li. Novel autophagy inhibitor to treat pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1323.

In vitro assessment of a synergistic combination of gemcitabine and zebularine in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with an extremely poor prognosis. Gemcitabine (Gem) is still the mainstay drug for the treatment of PDAC. However, rapid inactivation by cytidine deaminase (CDA) present in pancreatic cancer cells severely limits anticancer efficacy of Gem. In this study, we investigated the effect of a CDA inhibitor - Zebularine (Zeb) on anticancer activity of Gem in pancreatic cancer cell lines MiaPaCa-2, BxPC-3, and Panc-1. Zeb treatment synergistically increased Gem-induced cytotoxicity in all three pancreatic cancer cell lines. The strongest synergistic activity was found at 1:10 M ratio of Gem/Zeb (combination index 0.04–0.4). Additionally, Gem + Zeb treated cells showed marked decreased in the expressions of anti-apoptotic protein including Bcl-2 and survivin while significantly increased the cleaved caspase-3, and loss of mitochondrial membrane potential was observed. Multicellular 3D spheroids of MiaPaCa-2 cells treated with combination showed significant reduction (25–60%) in spheroid size, weight compared to single drug and control group. Live/dead cell imaging showed that Gem + Zeb treated spheroids exhibited a highly distorted surface with significantly higher number of dead cells (red). The results of the present study confirm that this synergistic combination is worthy of future investigations as a potential approach for the treatment of PDAC.

Therapeutic Effects of Paclitaxel Loaded Polyethylene Glycol-Polylactic Acid-Glycolic Acid Copolymer Nanoparticles on Pancreatic Cancer in Rats.

To establish a simple and safe method for the preparation of paclitaxel PEG-PLGA nanoparticles emulsified in tpgs (PTX-pegpllga-np), for high drug loading; and to study its effect on proliferation and apoptosis of human pancreatic cancer cell line MIAPACA-2. PTX-PEG-PLGA-NP was prepared by one-step precipitation, using tpgs as emulsifier. The drug loading and particle size were used as an index to optimize the formulation, and the physical and chemical properties such as in vitro release and stability were characterized. The uptake of fluorescein coumarin 6 (C6) loaded PEG-PLGA-NP by MIAPACA-2 cells was observed by fluorescence microscope, and the growth and apoptosis of MIAPACA-2 cells after PTX-PEG-PLGA-NP were detected by MTT and flow cytometry respectively. The entrapment efficiency of the nanoparticles was 90.26%, the drug loading was 10.13%, the average particle size was 92.3±3.1 nm, and the zeta potential was 10.48±1.54 mV. The cumulative releases of nano preparation and general preparation (Taxol injection) in four hours were 25.9% and 98.5%, respectively; and the former had a strong sustained-release effect. The results of cell uptake experiments showed that the uptake of c6-PEG-PLGA-NP by MIAPACA-2 cells increased gradually with time. MTT results showed that PTX-PEG-PLGA-NP had no significant difference in the inhibition rate of MIAPACA-2 cells compared with PTX group. Flow cytometry showed that PTX-PEG-PLGAnp was superior better than PTX in inducing apoptosis in MIAPACA-2 cells. The tpgs emulsification method is simple and environment-friendly. The paclitaxel loaded nanoparticles prepared through the optimization of the formulation have large drug loading capacity and uniform particle size, which can target the pancreatic cancer MIAPACA-2 cells, and do not weaken its ability to inhibit the growth of MIAPACA-2 cells. The nanoparticles also induce apoptosis in cancer MIAPACA-2 cells, and could be used for further clinical treatment of pancreatic cancer.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Molecular and Functional Analysis of Choline Transporters and Antitumor Effects of Choline Transporter-Like Protein 1 Inhibitors in Human Pancreatic Cancer Cells.

Choline, an organic cation, is one of the biofactors that play an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Choline positron emission tomography-computed tomography (PET/CT) provides useful information for the imaging diagnosis of cancers, and increased choline accumulation has been identified in a variety of tumors. However, the molecular mechanisms of choline uptake and choline transporters in pancreatic cancer have not been elucidated. Here, we examined molecular and functional analyses of choline transporters in human pancreatic-cancer cell line MIA PaCa-2 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and its derivative Amb4269675. CTL1 and CTL2 mRNAs were highly expressed in MIA PaCa-2 cells, and CTL1 and CTL2 proteins were localized in the plasma membrane and the intracellular compartments, respectively. Choline uptake was characterized by Na+-independence, a single-uptake mechanism, and inhibition by choline-uptake inhibitor HC-3, similar to the function of CTL1. These results suggest that the uptake of extracellular choline in MIA PaCa-2 cells is mediated by CTL1. Choline deficiency and HC-3 treatment inhibited cell viability and increased caspase 3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. Ceramide, which is increased by inhibiting choline uptake, also inhibited cell survival and increased caspase-3/7 activity. Lastly, both Amb4269951 and Amb4269675 significantly inhibited tumor growth in a mouse-xenograft model without any adverse effects such as weight loss. CTL1 is a target molecule for the treatment of pancreatic cancer, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.

Modification of radiosensitivity by Curcumin in human pancreatic cancer cell lines

Pancreatic cancer is one of the most aggressive malignancies and is characterized by a low 5-year survival rate, a broad genetic diversity and a high resistance to conventional therapies. As a result, novel therapeutic agents to improve the current situation are needed urgently. Curcumin, a polyphenolic colorant derived from Curcuma longa root, showed pleiotropic influences on cellular pathways in vitro and amongst others anti-cancer properties including sensitization of tumor cells to chemo- and radiation-therapy. In this study, we evaluated the impact of Curcumin on the radiosensitivity of the established human pancreatic cancer cell lines Panc-1 and MiaPaCa-2 in vitro. In contrast to MiaPaCa-2 cells, we found a significant radiosensitization by Curcumin in the more radioresistant Panc-1 cells, possibly caused by cell cycle arrest in the most radiation-sensitive G2/M-phase at the time of irradiation. Furthermore, a significant enhancement of radiation-induced apoptosis, DNA-double-strand breaks and G2/M-arrest after curcumin treatment was observed in both cell lines. These in vitro findings suggest that especially patients with more radioresistant tumors could benefit from a radiation-concomitant, phytotherapeutic therapy with Curcumin.

Activation of GLP-1 receptor enhances the chemosensitivity of pancreatic cancer cells.

This study aimed to determine whether and how the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide affects the chemoresistance and chemosensitivity of pancreatic cancer cells to gemcitabine in vitro and in vivo. The GLP-1R and protein kinase A (PKA) levels were compared between the human pancreatic cancer cell line PANC-1 and the gemcitabine-resistant cell line PANC-GR. The in vitro effects of liraglutide on the cell proliferation and apoptosis as well as the nuclear factor-kappa B NF-κB expression levels of PANC-GR cells were evaluated. In addition, a mouse xenograft model of human pancreatic cancer was established by s.c. injection of PANC-1 cells, and the effects of liraglutide on the chemosensitivity were evaluated in vitro and in vivo. In contrast to PANC-1 cells, PANC-GR cells exhibited lower expression levels of GLP-1R and PKA. Incubation with liraglutide dose dependently inhibited the growth, promoted the apoptosis, and increased the expression of GLP-1R and PKA of PANC-GR cells. Similar effects of liraglutide were observed in another human pancreatic cancer cell line MiaPaCa-2/MiaPaCa-2-GR. Either the GLP-1R antagonist Ex-9, the PKA inhibitor H89, or the NF-κB activator lipopolysaccharide (LPS) could abolish the antiproliferative and proapoptotic activities of liraglutide. Additionally, each of these agents could reverse the expression of NF-κB and ABCG2, which was decreased by liraglutide treatment. Furthermore, liraglutide treatment increased the chemosensitivity of pancreatic cancer cells to gemcitabine, as evidenced by in vitro and in vivo experiments. Thus, GLP-1R agonists are safe and beneficial for patients complicated with pancreatic cancer and diabetes, especially for gemcitabine-resistant pancreatic cancer.

Biodistribution and Pharmacokinetic Study of Gemcitabine Hydrochloride Loaded Biocompatible Iron-Based Metal Organic Framework

This study was designed to improve bioavailability and therapeutic efficacy of Gemcitabine (GEM) with reduced side effects using MOF MIL-100 as cargo. MIL-100 was synthesized, and characterized by microscopic and spectroscopic techniques. Impregnation approach was used for encapsulation of GEM inside the MIL-100 (i.e., MIL100-GEM). In-vitro release studies of MIL100-GEM was carried out in different media (PBS, deionized water and Tris buffer, pH = 7.4, 9.5 mM) to find out the drug release mechanism. Cytotoxicity and apoptosis assays were evaluated using MTT and fluorescence-activated cell sorting (FACS) assay in MiaPaCa-2 pancreatic cancer cell lines. Biocompatibility, pharmacokinetic and biodistribution studies of MIL100-GEM were assessed in Wistar rats. MIL100-GEM exhibited high encapsulation efficiency (78.6 ± 0.5%) and maximum payload (23.6 ± 1%). PXRD confirmed crystallinity of MIL-100, and did not show any effect on its structural integrity after encapsulation of GEM. In-vitro release studies revealed a biphasic release pattern in PBS buffer which followed Higuchi diffusion kinetics. In-vitro cytotoxicity studies showed low IC50 value for MIL100-GEM (3.50 ± 1.33 µg/ml) compared to GEM (6.22 ± 1.55 µg/ml), ensuring adequate cell proliferation after 72 h. Hemolysis study showed that MIL100-GEM (14.54 ± 1.3%) had better biocompatibility than the native GEM (30.52 ± 1.67%). Furthermore, pharmacokinetic and biodistribution studies exhibited ∼17-fold increased bioavailability, ∼20-fold increased distribution half-life and ∼15-folds elimination half-life of GEM with less accumulation of drug in the kidneys. MIL-100 MOF was synthesized and characterized to address the metabolic degradation issue of GEM. Biocompatible, MIL100-GEM demonstrated efficient drug (GEM) loading and enhanced cytotoxic activity in pancreatic cancer cell line with augmented bioavailability, providing MIL-100 a promising drug cargo. MIL-100 was synthesized using a microwave-assisted method. The anticancer drug Gemcitabine Hydrochloride (GEM) was loaded into the MIL-100 using the impregnation method. Encapsulation protected the drug from its metabolic inactivation to enhance bioavailability in target organs and reduce side effects.

High glucose microenvironment accelerates tumor growth via SREBP1-autophagy axis in pancreatic cancer

BackgroundDiabetes is recognized to be a risk factor of pancreatic cancer, but the mechanism has not been fully elucidated. Sterol regulatory element binding protein 1 (SREBP1) is an important transcription factor involved in both lipid metabolism and tumor progression. However, the relationship between high glucose microenvironment, SREBP1 and pancreatic cancer remains to be explored.MethodsClinical data and surgical specimens were collected. Pancreatic cancer cell lines BxPc-3 and MiaPaCa-2 were cultured in specified medium. Immunohistochemistry (IHC) and western blotting were performed to detect the expression of SREBP1. MTT and colony formation assays were applied to investigate cell proliferation. Immunofluorescence, mRFP-GFP adenoviral vector and transmission electron microscopy were performed to evaluate autophagy. We used streptozotocin (STZ) to establish a high glucose mouse model for the in vivo study.ResultsWe found that high blood glucose levels were associated with poor prognosis in pancreatic cancer patients. SREBP1 was overexpressed in both pancreatic cancer tissues and pancreatic cancer cell lines. High glucose microenvironment promoted tumor proliferation, suppressed apoptosis and inhibited autophagy level by enhancing SREBP1 expression. In addition, activation of autophagy accelerated SREBP1 expression and suppressed apoptosis. Moreover, high glucose promotes tumor growth in vivo by enhancing SREBP1 expression.ConclusionOur results indicate that SREBP1-autophagy axis plays a crucial role in tumor progression induced by high glucose microenvironment. SREBP1 may represent a novel target for pancreatic cancer prevention and treatment.