BxPC-3 Cells Research Articles

Abstract 1318: Interleukin-1 promotes enhanced functional expression of dual oxidase 2 in human pancreatic cancer cells

Abstract Pancreatic inflammation contributes to increased risk of pancreatic adenocarcinoma (PDAC) development through the release of pro-inflammatory cytokines and generation of reactive oxygen species (ROS). Interleukin-1 (IL-1) is a major upstream pro-inflammatory cytokine secreted by malignant or microenvironmental cells that supports pancreatic tumorigenesis, invasiveness, and tumor heterogeneity. Previously, our laboratory has established that dual oxidase 2 (DUOX2), one of seven NADPH oxidase (NOX) family members, plays a critical role in H2O2-mediated host defense and chronic inflammation in the gastrointestinal tract. DUOX2 is upregulated and expressed along with its maturation factor, DUOXA2, in several human pancreatic cancer cell lines following exposure to various pro-inflammatory cytokines, including IFN-γ, IL-4, and IL-17A. Using a panel of PDAC cells lines, we report the induction of DUOX2/DUOXA2 mRNA and protein expression by both IL-1α and IL-1ß, two major agonists of IL-1. In BxPC-3, CFPAC-1 and Hs 766T (HTB-134) cells, prolonged treatment with IL-1α or IL-1ß results in transient activation of STAT1 and NF-kB signaling followed by prolonged induction of DUOX2/DUOXA2 mRNA and protein expression. Stimulation by IL-1α or IL-1ß results in a specific increase in the expression of DUOX2/DUOXA2; increased expression of other NOX family members was not observed. Activation of canonical IL-1 signaling in our PDAC cell lines was supported by the observation of IL-1-related upregulation of IL-8 (CXCL8) mRNA, a chemokine essential for neutrophil recruitment and a potent promoter of angiogenesis, in IL-1-treated PDAC cells. Moreover, when the anti-inflammatory compound anakinra (an IL-1R antagonist) was co-administered in vitro with IL-1, cytokine-induced DUOX2/DUOXA2 and CXCL8 induction was significantly suppressed. Finally, xenografts established with BxPC-3 cells demonstrated slower growth and significant downregulation of IL-1ß and DUOX2 when the immunosuppressive corticosteroid dexamethasone was administered. Given our previous findings that DUOX2 is highly expressed in the pancreas during episodes of chronic pancreatitis or pancreatic cancer, and contributes significantly to extracellular ROS formation, these studies suggest that IL-1 may play a role in promoting inflammation and tumorigenesis in pancreatic malignancies through upregulation of DUOX2. Citation Format: David J. Mallick, Yongzhong Wu, Mariam M. Konaté, Becky A. Diebold, Smitha Antony, Jennifer L. Meitzler, Guojian Jiang, Jiamo Lu, Krishnendu Roy, James H. Doroshow. Interleukin-1 promotes enhanced functional expression of dual oxidase 2 in human pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1318.

Abstract 3463: Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy due to the lack of early diagnosis and limited response to all treatment modalities. Early metastatic dissemination, rich desmoplastic stroma and immunosuppressive infiltrate are some of the features contributing to the poor prognosis of PDAC patients (5-year survival rate of less than 10%). Despite scientific progress in the development of new therapeutic regimes, PDAC patients in low- and middle-income countries have restricted access to high-cost innovative drugs. Recently, attention has been given to the concept of drug repurposing that could meet the need for novel but affordable and widely accessible therapies. Imidazoline receptor 1 Nischarin (NISCH, IR1, IRAS) is an integrin α5-interacting protein that acts as a tumor suppressor in breast cancer through modulation of cancer cell motility and survival. Importantly, NISCH has several clinically approved agonists that are used for the treatment of hypertension and are shown to modulate autophagy and reduce inflammation and fibrosis, which makes NISCH a potentially good therapeutic target for defying PDAC progression. Based on our GSEA analysis of NISCH expression in PDAC patient samples, NISCH was predicted to have an effect on cancer cell focal adhesion assembly and cytoskeletal organization. Therefore, we examined the effects of NISCH agonists on PDAC cells fitness in vitro. A panel of PDAC cell lines was tested in MTT assay to determine the effects of NISCH agonists rilmenidine, clonidine and moxonidine on cell viability. Flow cytometry, western blot and immunofluorescence were used to examine the effects of NISCH agonists on PDAC cell metabolism, migration and cytoskeletal organization. Tg(fli1:EGFP) zebrafish model was used to examine the effects of NISCH agonists on PDAC cell growth and invasion. We found that NISCH was expressed in PDAC cell lines, and that out of the three tested agonists, rilmenidine most potently inhibited cancer cell viability. Treatment with rilmenidine significantly reduced PANC-1, MIA PaCa-2 and BxPC-3 cell attachment to extracellular matrix (collagen type I and fibronectin) and migratory potential in wound healing and transwell assays. Furthermore, treatment with rilmenidine altered the organization of actin cytoskeleton and focal adhesion assembly, potentially through the reduction of intracellular reactive oxygen species (ROS) levels. Importantly, treatment with rilmenidine inhibited invasion of PANC-1 cells in Tg(fli1:EGFP) zebrafish model. With these antimigratory and potentially antimetastatic effects of rilmenidine, our study lays the ground for a more extensive examination of the biological role of NISCH in PDAC progression and implies that its agonists may be good candidates for drug repurposing in this type of cancer. Citation Format: Marijana Pavlovic, Marija Ostojic, Kristina Zivic, Aleksandar Pavic, Tatjana Srdic-Rajic, Jelena Grahovac. Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3463.

Investigation of the potential role of TGR5 in pancreatic cancer by a comprehensive molecular experiments and the liquid chromatography mass spectrometry (LC–MS) based metabolomics

BackgroundTakeda G protein receptor 5 (TGR5) is widely recognized as a potential drug target for the treatment of metabolic diseases. TGR5 is not only a metabolic regulator, but also has a potential role that participating in developing and progressing of gastrointestinal cancer. We aimed to investigate the potential role of TGR5 in pancreatic cancer by utilizing molecular experiments and the liquid chromatography mass spectrometry (LC–MS) based metabolomics.MethodsHerein, we assessed pancreatic cancer proliferation, migration and invasion in response to TGR5 antagonist SBI-115 in vitro experiments. Cell death was examined by using TUNEL assay on agarose-embedded sections. Then we investigated the effects of TGR5 on PANC-1 and BXPC3 cells via transmission electron microscopy (TEM). Moreover, LC–MS-based metabolomics was performed to explore the potential underlying mechanisms of TGR5 in pancreatic cancer. The correlations between TGR5 and the metabolism-related genes were further analysed by GEPIA 2 database.ResultsWe found the proliferation capacities were decreased significantly in PANC-1 and BXPC3 cells after the treatment of SBI-115 for 48 h. The results of TUNEL assay showed that antagonism of TGR5 by SBI-115 had a remarkable effect on inducing cell death. Analysis of TEM demonstrated that SBI-115 treatment could impair the morphology of mitochondria in most PANC-1 and BXPC3 cells. The LC–MS-based analyses revealed that antagonism of TGR5 could alter the metabolic profiles of PANC-1 cells in vitro. Moreover, TGR5 was associated with some metabolism-related genes in pancreatic cancer.ConclusionOur data suggests that antagonism of TGR5 may suppress cell proliferation and induce apoptosis in pancreatic cancer cells. TGR5 may affect the metabolism of pancreatic cancer, and TGR5 would be an attractive target for pancreatic cancer treatment.

Pancreatic cancer cells spectral library by DIA-MS and the phenotype analysis of gemcitabine sensitivity

Data-independent acquisition (DIA)-mass spectrometry (MS)-based proteome strategies are increasingly used for detecting and validating protein biomarkers and therapeutic targets. Here, based on an in-depth proteome analysis of seven pancreatic cancer cell lines, we built a pancreas-specific mass spectrum library containing 10633 protein groups and 184551 peptides. The proteome difference among the seven pancreatic cancer cells was significant, especially for the divergent expression of proteins related to epithelial-mesenchymal transition (EMT). The spectra library was applied to explore the proteome difference of PANC-1 and BxPC-3 cells upon gemcitabine (GEM) treatment, and potential GEM targets were identified. The cytotoxicity test and GEM target analysis found that HPAC, CFPAC-1, and BxPC-3 were sensitive to GEM treatment, whereas PANC-1 and AsPC-1 were resistant. Finally, we found EMT was significant for CFPAC-1, AsPC-1, and PANC-1 cells, whereas BxPC-3 and HPAC cells showed more typical epithelial features. This library provides a valuable resource for in-depth proteomic analysis on pancreatic cancer cell lines, meeting the urgent demands for cell line-dependent protein differences and targeted drug analysis.

Zebrafish Xenograft Model for Studying Pancreatic Cancer-Instructed Innate Immune Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has up to half the tumor mass of tumor-associated myeloid cells. Myeloid innate immune cells play important roles in regulating cancer cell recognition and tumor growth. PDAC cells often mold myeloid cells into pro-tumoral state to fuel cancer growth and induce immune suppression. However, how tumor cells educate the innate immune responses remains largely unknown. In this study, we used four different human PDAC cell lines (PANC1, BxPC3, AsPC1, and CFPAC1) to establish the zebrafish xenograft model and investigated the interaction between pancreatic cancer and innate immune cells. The primary tumor-derived cancer cells PANC1 and BxPC3 activated innate immune anti-tumoral responses efficiently, while cancer cells from metastatic tissues AsPC1 and CFPAC1 induced an innate immune suppression and educated innate immune cells towards pro-tumoral state. Chemical conversion of innate immune cells to anti-tumoral state inhibited tumor growth for AsPC1 and CFPAC1. Moreover, genetic and pharmacological inhibition of macrophages also significantly reduced tumor growth, supporting the important roles of macrophages in innate immune suppression. REG4 expression is high in AsPC1 and CFPAC1. Knockdown of REG4 induced innate immune activation and reduced tumor growth in the xenografts, indicating that REG4 is a beneficial target for PDAC therapy. Our study provides a fast in-vivo model to study PDAC-innate immune interaction and their plasticity that could be used to study the related mechanism as well as identify new drugs to enhance immunotherapy.

Human amniotic fluid mesenchymal stem cells attenuate pancreatic cancer cell proliferation and tumor growth in an orthotopic xenograft mouse model

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a malignant cancer and chemotherapy ineffectively treats PDAC, leading to the requirement for alternative tumor-targeted treatment. Human amniotic fluid mesenchymal stem cells (hAFMSCs) have been revealed to suppress tumor growth in various cancers and they are a strong candidate for treating PDAC.MethodsTo evaluate the effects of hAFMSCs on human pancreatic carcinoma cells (PANC1, AsPC1 and BxPC3 cell lines) and the possible mechanism involved, an in vitro cell coculture system was used. A PANC1 orthotopic xenograft mouse model was established and hAFMSCs were injected intravenously at 4 weeks post-xenograft.ResultsAn in vitro coculture assay showed that hAFMSCs inhibited PANC1 cell proliferation by inducing S phase cell cycle arrest and increased cell apoptosis in a time-dependent manner. In PANC1 cells, hAFMSCs caused the downregulation of Cyclin A and Cyclin B1 as well as the upregulation of p21 (CDKN1A) at 24 h post coculture. The upregulation of pro-apoptotic factors Caspase-3/-8 and Bax at 24 h post coculture reduced the migration and invasion ability of PANC1 cells through inhibiting the epithelial-mesenchymal transition (EMT) process. In a PANC1 orthotopic xenograft mouse model, a single injection of hAFMSCs showed significant tumor growth inhibition with evidence of the modulation of cell cycle and pro-apoptotic regulatory genes and various genes involved in matrix metallopeptidase 7 (MMP7) signaling-triggered EMT process. Histopathological staining showed lower Ki67 levels in tumors from hAFMSCs-treated mice.ConclusionsOur data demonstrated that hAFMSCs strongly inhibit PDAC cell proliferation, tumor growth and invasion, possibly by altering cell cycle arrest and MMP7 signaling-triggered EMT.

Targeting Tumor Cells Overexpressing the Human Epidermal Growth Factor Receptor 3 with Potent Drug Conjugates Based on Affibody Molecules.

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

Isoprenylated Coumarin Exhibits Anti-proliferative Effects in Pancreatic Cancer Cells Under Nutrient Starvation by Inhibiting Autophagy.

Due to the lack of early detection methods and effective treatments, pancreatic cancer has one of the lowest five-year survival rates among all cancers. We have previously identified novel isoprenylated coumarin compounds that exhibit preferential cytotoxicity against pancreatic adenocarcinoma cell line PANC-1 exclusively under glucose deprivation conditions. Using cell cytotoxicity assays, we investigated the anti-proliferative mechanism of our most potent isoprenylated coumarin compound of the series, DCM-MJ-I-21, with respect to time, against two other pancreatic cancer cell lines, BxPC-3 and Capan-2. We used western blotting to quantify the autophagic flux influenced by our compound, autophagy inducers (starvation and Rapamycin), and autophagy inhibitors (chloroquine and wortmannin). We observed a clear dependence on glucose in DCM-MJ-I-21 in BxPC-3 and Capan-2 pancreatic cancer cell lines, suggesting that our compound targets a pathway shared by these cancer cell lines when glycolysis is not an option for survival. Our lead compound increased the conversion of LC3-I to LC3-II in PANC-1, similar to the effect of chloroquine, an autophagy inhibitor. In addition, Spautin-1, another autophagy inhibitor, showed almost the same anti-proliferative activities at the same concentration under nutrient-deprived conditions as our lead compound in both 2D and 3D cell cultures. Our lead isoprenylated coumarin compound induces selective pancreatic cancer cell death under nutrient-deprived conditions through inhibition of autophagy, potentially providing insights into new therapeutic options.

Gossypol Induces Apoptosis of Human Pancreatic Cancer Cells via CHOP/Endoplasmic Reticulum Stress Signaling Pathway.

Gossypol, a natural phenolic aldehyde present in cotton plants, was originally used as a means of contraception, but is currently being studied for its anti-proliferative and anti-metastatic effects on various cancers. However, the intracellular mechanism of action regarding the effects of gossypol on pancreatic cancer cells remains unclear. Here, we investigated the anti-cancer effects of gossypol on human pancreatic cancer cells (BxPC-3 and MIA PaCa-2). Cell counting kit-8 assays, annexin V/propidium iodide staining assays, and transmission electron microscopy showed that gossypol induced apoptotic cell death and apoptotic body formation in both cell lines. RNA sequencing analysis also showed that gossypol increased the mRNA levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and activating transcription factor 3 (ATF3) in pancreatic cancer cell lines. In addition, gossypol facilitated the cleavage of caspase-3 via protein kinase RNA-like ER kinase (PERK), CHOP, and Bax/Bcl-2 upregulation in both cells, whereas the upregulation of ATF was limited to BxPC-3 cells. Finally, a three-dimensional culture experiment confirmed the successful suppression of cancer cell spheroids via gossypol treatment. Taken together, our data suggest that gossypol may trigger apoptosis in pancreatic cancer cells via the PERK-CHOP signaling pathway. These findings propose a promising therapeutic approach to pancreatic cancer treatment using gossypol.

Biogenic silver/silver chloride nanoparticles inhibit human cancer cells proliferation in vitro and Ehrlich ascites carcinoma cells growth in vivo

Silver/silver chloride nanoparticles (Ag/AgCl-NPs) were synthesized for the first time from the herbal Geodorum densiflorum rhizome extracts and characterized by different techniques. The surface plasmon resonance peak at 455 nm was observed in the UV–Visible spectrum, the average particle size of 25 nm was determined by SEM, XRD reflection peaks (28.00°, 32.42°, 38.28°, 46.38°, 54.94°, 57.60°, 64.64°, and 67.48°) indicated the presence of Ag-NPs and AgCl-NPs, heat stability was confirmed by TGA and FTIR analysis indicated the presence of alcohol/phenol, alkanes, primary amines, nitro compounds, alkyl chloride functional groups. The synthesized Ag/AgCl-NPs, previously synthesized Kaempferia rotunda and Zizyphus mauritiana mediated Ag/AgCl-NPs separately inhibited the proliferation of BxPC-3 cells with the IC50 values of 7.8, 17.1, and 20.1 µg/ml, respectively. In the case of MCF-7 cells, the IC50 values of G. densiflorum- Ag/AgCl-NPs and K. rotunda-Ag/AgCl-NPs were 21.5 and 23.5 µg/ml, respectively. Whereas the IC50 of G. densiflorum-Ag/AgCl-NPs was 28.0 µg/ml against glioblastoma stem cells (GSCs). Induction of apoptosis in GSCs, BxPC-3 and MCF-7 cells was noted followed by NPs treatment. In GSCs, the expression level of NFκB, TNFα, p21, and TLR9 genes were upregulated after treatment with G. densiflorum-Ag/AgCl-NPs while in the MCF-7 cells, the expression of p53, FAS, Caspase-8 and -9, NFκB, MAPK, JNK and p21 genes were increased. G. densiflorum-Ag/AgCl-NPs inhibited 60% and 95% of EAC cells growth at the doses of 2 and 4 mg/Kg/day after intraperitoneal treatment with five consequent days, respectively. A remarkable improvement of hematological parameters with the decreased average tumor weight and increase of 75% life span of G. densiflorum-Ag/AgCl-NPs treated mice were observed. Altogether, this study reported for the first time in vitro anticancer activity of biogenic G. densiflorum-Ag/AgCl-NPs against GSC cells along with MCF-7 and BxPC-3 cells and in vivo anticancer properties against EAC cells.

Generation of a poly-functionalized indolizine scaffold and its anticancer activity in pancreatic cancer cells

A highly efficient domino [4 + 2] annulation process was employed to construct a novel indolizine chemical scaffold. Biological investigation led us to identify 6w as a potent anticancer agent. 6w significantly inhibited cell viability in BxPC3 pancreatic cancer, MCF7 breast cancer, and PC3 prostate cancer cell lines with IC50 values of 0.47 ± 0.04, 1.82 ± 0.08 and 2.68 ± 0.08 µM, respectively. Remarkably, 6w showed a weak effect on cell viability of nontumorigenic human keratinocyte cell line HaCaT compared to the above three types of cancer cells. 6w most potently inhibited cell viability of BxPC3 cells, and 6w also potently reduced cell migration and induced apoptosis in BxPC3 cells through activation of caspase-3 and cleavage of PARP in a dose-dependent manner. These results suggest that 6w can be used for the development of potential anticancer drugs for the treatment of pancreatic cancer.

Design and Development of a New Type of Hybrid PLGA/Lipid Nanoparticle as an Ursolic Acid Delivery System against Pancreatic Ductal Adenocarcinoma Cells.

Despite many attempts, trials, and treatment procedures, pancreatic ductal adenocarcinoma (PDAC) still ranks among the most deadly and treatment-resistant types of cancer. Hence, there is still an urgent need to develop new molecules, drugs, and therapeutic methods against PDAC. Naturally derived compounds, such as pentacyclic terpenoids, have gained attention because of their high cytotoxic activity toward pancreatic cancer cells. Ursolic acid (UA), as an example, possesses a wide anticancer activity spectrum and can potentially be a good candidate for anti-PDAC therapy. However, due to its minimal water solubility, it is necessary to prepare an optimal nano-sized vehicle to overcome the low bioavailability issue. Poly(lactic-co-glycolic acid) (PLGA) polymeric nanocarriers seem to be an essential tool for ursolic acid delivery and can overcome the lack of biological activity observed after being incorporated within liposomes. PLGA modification, with the addition of PEGylated phospholipids forming the lipid shell around the polymeric core, can provide additional beneficial properties to the designed nanocarrier. We prepared UA-loaded hybrid PLGA/lipid nanoparticles using a nanoprecipitation method and subsequently performed an MTT cytotoxicity assay for AsPC-1 and BxPC-3 cells and determined the hemolytic effect on human erythrocytes with transmission electron microscopic (TEM) visualization of the nanoparticles and their cellular uptake. Hybrid UA-loaded lipid nanoparticles were also examined in terms of their stability, coating dynamics, and ursolic acid loading. We established innovative and repeatable preparation procedures for novel hybrid nanoparticles and obtained biologically active nanocarriers for ursolic acid with an IC50 below 20 µM, with an appropriate size for intravenous dosage (around 150 nm), high homogeneity of the sample (below 0.2), satisfactory encapsulation efficiency (up to 70%) and excellent stability. The new type of hybrid UA-PLGA nanoparticles represents a further step in the development of potentially effective PDAC therapies based on novel, biologically active, and promising triterpenoids.

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.

FGFR3 Nuclear Translocation Contributes to Proliferative Potential and Poor Prognosis in Pancreatic Ductal Adenocarcinoma.

Fibroblast growth factor receptor 3 (FGFR3) was revealed to have divergent, even opposite roles in different neoplasms. In pancreatic ductal adenocarcinoma (PDAC), its impact on biological behavior and prognosis was not well elucidated. Fibroblast growth factor receptor 3 was downregulated by RNA interference to explore its impact on cell proliferative proclivity in PDAC cells. Furthermore, tissue microarray-based immunohistochemistry for FGFR3 was performed in 326 patients with PDAC who underwent radical resection, and its clinicopathologic and prognostic implications were then evaluated. First, successful FGFR3 knockdown remarkably decreased its expression, cell proliferation, and S-phase ratio in the cell cycle in 2 PDAC cell lines, BxPC-3 and AsPC-1. Meanwhile, alterations in p-Akt, cyclin D1, cyclin B1, and p21 were also observed. Subsequently, high nuclear FGFR3 expression, but not cytoplasmic, was significantly common in tumor tissues and positively associated with N stage and dismal overall survival in the entire cohort. In addition, nuclear FGFR3 expression was also prognostic in 10 of 14 subsets. Univariate and multivariate Cox regression analyses identified nuclear expression of FGFR3 as an independent prognosticator in the entire cohort. Our data showed that FGFR3 nuclear translocation contributes to cell proliferative potential and predicts poor long-term prognosis in PDAC after surgical resection.

Hyperthermia Enhances Efficacy of Chemotherapeutic Agents in Pancreatic Cancer Cell Lines.

Chemotherapy (CT) is the standard care for advanced pancreatic ductal adenocarcinoma (PDAC); however, with limited efficacy. Hyperthermia (HT) treatment has been suggested as a sensitizer to improve outcomes. However, the direct effect of the HT and CT combination is not fully understood. Therefore, we aim to assess the direct cytotoxic effect of HT in PDAC cells as monotherapy or in combination with chemotherapeutics. Different temperatures (37-, 40.5-, 41-, and 41.5 °C) and durations (6-, 12-, and 24 h) were tested in PDAC cell lines (BxPC-3, Capan-1, Capan-2, PANC-1, and MIA-PaCa-2). Different concentrations of gemcitabine, 5-fluorouracil, and cisplatin were also tested in these conditions. The impact on cell metabolic activity was determined by an MTS assay. Enhancement of chemosensitivity was assessed by a reduction in half-maximal inhibitory concentration (). HT and chemotherapeutics interactions were classified as antagonistic, additive, or synergistic using the combination index. HT inhibited cell proliferation in a cell type, temperature, and duration-dependent manner. The induction of apoptosis was seen after 6 h of HT treatment, eventually followed by secondary necrosis. The HT and CT combination led to an reduction of the tested CT. At 12 h of HT, this effect was between 25 to 90% and reached a 95% reduction at 24 h. The additive or synergistic effect was demonstrated in all cell lines and chemotherapeutics, although, again, this depended on cell type, duration, and temperature. HT is cytotoxic and enhances the therapeutic effectiveness of gemcitabine, 5-fluorouracil, and cisplatin on PDAC cells. This result was further confirmed by the decrease in the expression of RRM2, TS, and ERCC1 in BxPC-3 and Capan-2 cells. These observations warrant further study in specific subsets of PDAC patients to improve their clinical outcomes.

The recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis shows potent efficacy against pancreatic cancer

KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF2-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF2-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF2-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF2-HSA internalized via macropinocytosis. DF2-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF2-HSA induced apoptosis in cancer cells. Notably, DF2-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF2-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF2-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF2-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF2-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.

Timosaponin A3 Inhibits Palmitate and Stearate through Suppression of SREBP-1 in Pancreatic Cancer

Timosaponin A3 (TA3) was demonstrated as a potent anticancer chemical by several studies. Although the effects of inhibiting growth, metastasis, and angiogenesis in various cancer cells were demonstrated through multiple mechanisms, the pharmacological mechanism of TA3 shown in pancreatic cancer (PC) is insufficient compared to other cancers. In this study, we aimed to explore the key molecular mechanisms underlying the growth inhibitory effects of TA3 using PC cells and a xenograft model. First, from the microarray results, we found that TA3 regulated INSIG-1 and HMGCR in BxPC-3 cells. Furthermore, we showed that inhibition of sterol regulatory element-binding protein-1 (SREBP-1) by TA3 reduced the fatty acid synthases FASN and ACC, thereby controlling the growth of BxPC-3 cells. We also tried to find mechanisms involved with SREBP-1, such as Akt, Gsk3β, mTOR, and AMPK, but these were not related to SREBP-1 inhibition by TA3. In the BxPC-3 xenograft model, the TA3 group had more reduced tumor formation and lower toxicity than the gemcitabine group. Interestingly, the level of the fatty acid metabolites palmitate and stearate were significantly reduced in the tumor tissue in the TA3 group. Overall, our study demonstrated that SREBP-1 was a key transcription factor involved in pancreatic cancer growth and it remained a precursor form due to TA3, reducing the adipogenesis and growth in BxPC-3 cells. Our results improve our understanding of novel mechanisms of TA3 for the regulation of lipogenesis and provide a new approach to the prevention and treatment of PC.

Oncogenic Kras-Mediated Cytokine CCL15 Regulates Pancreatic Cancer Cell Migration and Invasion through ROS.

Simple SummaryOncogenic KrasG12D and tumor inflammation are critical components of the development and dissemination of pancreatic ductal adenocarcinoma (PDAC). The aim of this study is to investigate a lesser-known cytokine, CCL15, that functions as a new downstream target of KrasG12D with the purpose of regulating PDAC cell migration and invasion. We showed increased levels of CCL15 as well as the presence of its receptors, including CCR1 and CCR3, in PDAC tissues and cell lines. The knockdown of CCL15 diminished metastatic Panc-1 cell migration, whereas the treatment of CCL15 in non-metastatic BxPC-3 cells promoted BxPC-3 cell motility. Similar results were verified using murine metastatic PDAC KP-2 cells. Furthermore, we demonstrated that CCL15-modulated PDAC cell migration through the upregulation of cellular reactive oxygen species (ROS) levels and the knockdown of KrasG12D resulted in a decrease in CCL15. Altogether, our data unveiled a new mechanism of oncogenic KrasG12D in modulating PDAC inflammation and spreading.Pancreatic ductal adenocarcinoma (PDAC) is well known for its high death rate due to prompt cancer metastasis caused by cancer cell migration and invasion within the early stages of its development. Here, we reveal a new function of cytokine CCL15, namely the upregulation of PDAC cell migration and invasion. We showed increased levels of CCL15 transcripts and protein expressions in human PDAC tissue samples, as well as in cultured cell lines. Furthermore, PDAC cells also expressed CCL15 receptors, including CCR1 and CCR3. Murine PDAC cell lines and tissues strengthened this finding. The manipulation of CCL15 in metastatic Panc-1 cells through CCL15 knockdown or CCL15 neutralization decreased Panc-1 cell motility and invasiveness. In addition, treating non-metastatic BxPC-3 cells with recombinant CCL15 accelerated the cell migration of BxPC-3. A reduction in the levels of reactive oxygen species (ROS) by either N-Acetyl-L-Cysteine treatment or p22phox knockdown led to a decrease in Panc-1 cell migration and a reversed effect on recombinant CCL15-promoted BxPC-3 cell movement. Importantly, the knockdown of oncogenic Kras in Panc-1 cells abolished CCL15 protein expression and impeded cell migration without affecting PDAC cell growth. Altogether, our work elucidates an additional molecular pathway of oncogenic Kras to promote PDAC metastasis through the upregulation of cell migration and invasion by the Kras downstream CCL15, a lesser-known cytokine within the cancer research field.

Mechanical Cues, E-Cadherin Expression and Cell "Sociality" Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior.

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior.

Aquaporin-3 and Aquaporin-5 Facilitate Migration and Cell–Cell Adhesion in Pancreatic Cancer by Modulating Cell Biomechanical Properties

Background: Aquaporins are membrane channels responsible for the bidirectional transfer of water and small non-charged solutes across cell membranes. AQP3 and AQP5 are overexpressed in pancreatic ductal adenocarcinoma, playing key roles in cell migration, proliferation, and invasion. Here, we evaluated AQP3 and AQP5 involvement in cell biomechanical properties, cell–cell adhesion, and cell migration, following a loss-of-function strategy on BxPC-3 cells. Results: Silencing of AQP3 and AQP5 was functionally validated by reduced membrane permeability and had implications on cell migration, slowing wound recovery. Moreover, silenced AQP5 and AQP3/5 cells showed higher membrane fluidity. Biomechanical and morphological changes were assessed by atomic force microscopy (AFM), revealing AQP5 and AQP3/5 silenced cells with a lower stiffness than their control. Through cell–cell adhesion measurements, the work (energy) necessary to detach two cells was found to be lower for AQP-silenced cells than control, showing that these AQPs have implications on cell–cell adhesion. Conclusion: These findings highlight AQP3 and AQP5 involvement in the biophysical properties of cell membranes, whole cell biomechanical properties, and cell–cell adhesion, thus having potential implication in the settings of tumor development.