Human Pancreatic Cancer Cell BxPC-3 Research Articles

Abstract 7134: Treatment of pancreatic cancer cells with tumor treating fields (TTFields) and PARP inhibitors

Abstract Background: Therapeutic options for advanced stage pancreatic cancer are limited. Patients harboring germline BRCA mutations benefit from PARP inhibitors (PARPi); unfortunately, such mutations are rare in pancreatic cancer. Tumor Treating Fields (TTFields), electric fields that disrupt cellular processes crucial for cancer cell viability, have shown effectiveness against pancreatic cancer cells, and are currently under clinical investigation for the treatment of patients with pancreatic cancer. The mechanism of action of TTFields in various cancer types has suggested involvement of BRCA downregulation. The current study examined the potential sensitization of BRCA wild-type pancreatic cancer cell lines to PARPi with TTFields application. Methods: BxPC3 and AsPC1 human pancreatic BRCA wild-type cancer cells were treated for 72 h with TTFields (frequency of 150 kHz; intensity of 1 and 0.5 V/cm RMS, respectively), using the inovitro device. The PARPi niraparib and olaparib were each administered to the cells at various concentrations, with or without co-application of TTFields. At treatment cessation, cell count, colony formation, and apoptosis were measured. Results: Applying PARPi to pancreatic cell lines yielded a dose-dependent cytotoxic and anti-clonogenic effects, and induced apoptosis. TTFields application also induced such effects when applied alone, while delivery of TTFields together with PARPi amplified the effects demonstrated by PARPi alone. Conclusions: Concomitant application of TTFields and PARPi in BRCA wild type pancreatic cancer cells exhibits a potential improvement relative to the mono-therapies. This could be rationalized based on the previously demonstrated ability of TTFields to induce a state of BRCAness in cancer cells, and suggests benefit for TTFields and PARPi co-treatment in the absence of background BRCA mutations. Citation Format: Hila Ene, Kerem Ben Meir, Antonia Martinez-Conde, Roni Frechtel-Gerzi, Hila Gabay, Daria Gerasimova, Rotem Engelman, Eyal Dor-On, Adi Haber, Moshe Giladi, Yoram Palti. Treatment of pancreatic cancer cells with tumor treating fields (TTFields) and PARP inhibitors [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 7134.

Evaluation of the cytotoxic and immunomodulatory effects of sonodynamic therapy in human pancreatic cancer spheroids

Sonodynamic therapy (SDT) exploits the energy generated by ultrasound (US) to activate sound-sensitive drugs (sonosensitizers), leading to the generation of reactive oxygen species (ROS) and cancer cell death. Two-dimensional (2D) and three-dimensional (3D) cultures of human pancreatic cancer BxPC-3 cells were chosen as the models with which to investigate the therapeutic effects of the US-activated sonosensitizer IR-780 as pancreatic cancer is still one of the most lethal types of cancer. The effects of SDT, including ROS production, cancer cell death and immunogenic cell death (ICD), were extensively investigated. When subjected to US, IR-780 triggered significant ROS production and caused cancer cell death after 24 h (p ≤ 0.01). Additionally, the activation of dendritic cells (DCs) led to an effective immune response against the cancer cells undergoing SDT-induced death. BxPC-3 spheroids were developed and studied extensively to validate the findings observed in 2D BxPC-3 cell cultures. An analysis of the pancreatic cancer spheroid section revealed significant SDT-induced cancer cell death after 48 h after the treatment (p ≤ 0.01), with this being accompanied by the presence of SDT-induced damage-associated molecular patterns (DAMPs), such as calreticulin (CRT) and high mobility group box 1 (HMGB1). In conclusion, the data obtained demonstrates the anticancer efficacy of SDT and its immunomodulatory potential via action as an ICD-inducer.

Investigation of the Effects of Juglone-Selenium Treatments on Epithelial-Mesenchimal Transition and Migration in BxPC-3 and PANC-1 Human Pancreatic Cancer Cells

Investigation of the Effects of Juglone-Selenium Treatments on Epithelial-Mesenchimal Transition and Migration in BxPC-3 and PANC-1 Human Pancreatic Cancer Cells

Maslinic acid solid lipid nanoparticles as hydrophobic anticancer drug carriers: Formulation, in vitro activity and in vivo biodistribution

Maslinic acid (MA) is a natural pentacyclic triterpenoid with inherent antitumor activity which has a very low solubility in water. MA solid lipid nanoparticles (SLNs) were prepared using Poloxamer 407 and Dicarboxylic acid-Poloxamer 407 as surfactants. Both MA SLNs are monodisperse, with sizes around 130 nm, and stable. Curcumin has been encapsulated in both types of nanoparticles without altering their colloidal properties. Moreover, SLNs greatly improve the solubility of MA and Curcumin. The cytotoxicity of MA and SLNs has been evaluated in BxPC3 human pancreatic cancer cells, MCF7 human breast cancer cells, and in a human fibroblast primary cell line. MA shows higher cytotoxic effect in BxPC3 and MCF7 cancer cells than in human primary fibroblasts. Nile Red loaded MA SLNs are quickly uptaken by BxPC3 and MCF7 cells, and show different cytoplasmic distributions depending on the cellular line. The oral or intravenous administration of MA SLNs in mice does not report any toxic effect, and the intravenous administration of fluorescent MA SLNs shows a homogeneous distribution in mice, without site-specific accumulation. Results suggest the great potential of MA SLNs as nanocarriers of anticancer drugs and as promising targeted theranostic nanodevices.

Modulatory Effects of Metformin on Farnesoid X receptor and Specificity Protein 1 in Human Pancreatic Cancer BxPC-3 Cells

Background: Pancreatic cancer is the fourth major cause of death, accounting for 7% of cancer-related deaths. Metformin (antihyperglycemic agent) users had a 62% lower risk of developing pancreatic cancer when compared to metformin non-users. Objective: This research aims to study the role of the Farnesoid X receptor (FXR) receptor in human pancreatic cancer cell line BxPC-3 and metformin’s modulatory effects on this receptor. Material and methods: Cell viability was assessed using MTT assay for INT747 (FXR agonist) and metformin, FXR and Specificity Protein 1 (Sp1) mRNA and protein levels were measured by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot, respectively. Results: INT747 increased the growth and viability of BxPc-3 cells in addition to a significant increase in FXR and SP1 mRNA and protein levels. In contrast, BxPC-3 cells viability was significantly reduced by metformin in a dose- and time-dependent manner with downregulation of FXR and SP1 at mRNA and protein levels Conclusion: FXR may act as an oncogenic factor in pancreatic cancer BxPC-3 cells by increasing FXR and SP1 expression, while metformin significantly reduced FXR mRNA and protein levels in BxPC-3 cells, which may be due to a reduction in SP1 expression.

Mychonastes sp. 246 Suppresses Human Pancreatic Cancer Cell Growth via IGFBP3-PI3K-mTOR Signaling.

Previously, we confirmed that Mychonastes sp. 246 methanolic extract (ME) markedly reduced the viability of BxPC-3 human pancreatic cancer cells. However, the underlying mechanism ME remained unclear. Hence, we attempted to elucidate the anticancer effect of ME on BxPC-3 human pancreatic cancer cells. First, we investigated the components of ME and their cytotoxicity in normal cells. Then, we confirmed the G1 phase arrest mediated growth inhibitory effect of ME using a cell counting assay and cell cycle analysis. Moreover, we found that the migration-inhibitory effect of ME using a Transwell migration assay. Through RNA sequencing, Gene Ontology-based network analysis, and western blotting, we explored the intracellular mechanisms of ME in BxPC-3 cells. ME modulated the intracellular energy metabolism-related pathway by altering the mRNA levels of IGFBP3 and PPARGC1A in BxPC-3 cells and reduced PI3K and mTOR phosphorylation by upregulating IGFBP3 and 4E-BP1 expression. Finally, we verified that ME reduced the growth of three-dimensional (3D) pancreatic cancer spheroids. Our study demonstrates that ME suppresses pancreatic cancer proliferation through the IGFBP3-PI3K-mTOR signaling pathway. This is the first study on the anticancer effect of the ME against pancreatic cancer, suggesting therapeutic possibilities and the underlying mechanism of ME action.

MS-5, a Naphthalene Derivative, Induces Apoptosis in Human Pancreatic Cancer BxPC-3 Cells by Modulating Reactive Oxygen Species.

Pancreatic cancer is one of the most fatal cancers with a poor prognosis. Standard chemotherapies have proven largely ineffective because of their toxicity and the development of resistance. Therefore, there is an urgent need to develop novel therapies. In this study, we investigated the antitumor activity of MS-5, a naphthalene derivative, on BxPC-3, a human pancreatic cancer cell line. We observed that MS-5 was cytotoxic to BxPC-3 cells, as well as inhibited the growth of cells in a concentration- and time- dependent manner. Flow cytometry analysis revealed that the percentage of annexin V-positive cells increased after MS-5 treatment. We also observed cleavage of caspases and poly (ADP-ribose) polymerase, and downregulation of Bcl-xL protein. Flow cytometry analysis of intracellular levels of reactive oxygen species (ROS) and mitochondrial superoxide suggested that MS-5 induced the generation of mitochondrial superoxide while lowering the overall intracellular ROS levels. Thus, MS-5 may be potential candidate for pancreatic cancer treatment.

Preclinical Evaluation of 68Ga- and 177Lu-Labeled Integrin αvβ6-Targeting Radiotheranostic Peptides.

The integrin αvβ6, an epithelium-specific cell surface receptor, is overexpressed on numerous malignancies, including the highly lethal pancreatic ductal adenocarcinomas. Here, we developed and tested a novel αvβ6-targeting peptide, DOTA-5G (1) radiolabeled with 68Ga, for PET/CT imaging and 177Lu for treatment. With the goal to develop a radiotheranostic, further modifications were made for increased circulation time, renal recycling, and tumor uptake, yielding DOTA-albumin-binding moiety-5G (2). Methods: Peptides 1 and 2 were synthesized on solid phase, and their affinity for αvβ6 was assessed by enzyme-linked immunosorbent assay. The peptides were radiolabeled with 68Ga and 177Lu. In vitro cell binding, internalization, and efflux of 68Ga-1 and 177Lu-2 were evaluated in αvβ6-positive BxPC-3 human pancreatic cancer cells. PET/CT imaging of 68Ga-1 and 68Ga-2 was performed on female nu/nu mice bearing subcutaneous BxPC-3 tumors. Biodistribution was performed for 68Ga-1 (1 and 2 h after injection), 68Ga-2 (2 and 4 h after injection), and 177Lu-1 and 177Lu-2 (1, 24, 48, and 72 h after injection). The 177Lu-2 biodistribution data were extrapolated for human dosimetry data estimates using OLINDA/EXM 1.1. Therapeutic efficacy of 177Lu-2 was evaluated in mice bearing BxPC-3 tumors. Results: Peptides 1 and 2 demonstrated high affinity (<55 nM) for αvβ6 by enzyme-linked immunosorbent assay. 68Ga-1, 68Ga-2, 177Lu-1, and 177Lu-2 were synthesized in high radiochemical purity. Rapid invitro binding and internalization of 68Ga-1 and 177Lu-2 were observed in BxPC-3 cells. PET/CT imaging and biodistribution studies demonstrated uptake in BxPC-3 tumors. Introduction of the albumin-binding moiety in 177Lu-2 resulted in a 5-fold increase in tumor uptake and retention over time. Based on the extended dosimetry data, the dose-limiting organ for 177Lu-2 is the kidney. Treatment with 177Lu-2 prolonged median survival by 1.5- to 2-fold versus controls. Conclusion: 68Ga-1 and 177Lu-2 demonstrated high affinity for the integrin αvβ6 both invitro and invivo, were rapidly internalized into BxPC-3 cells, and were stable in mouse and human serum. Both radiotracers showed favorable pharmacokinetics in preclinical studies, with predominantly renal excretion and good tumor-to-normal-tissue ratios. Favorable human dosimetry data suggest the potential of 177Lu-2 as a treatment for pancreatic ductal adenocarcinoma.

Hydroxychloroquine Promotes Bcl-xL Inhibition-induced Apoptosis in BxPC-3 Human Pancreatic Cancer Cells.

Anti-apoptotic proteins, including Bcl-2 and Bcl-xL, hinder cancer treatment, and several drugs targeting these molecules have been developed. One is ABT-263 (navitoclax), which targets Bcl-2, Bcl-xL, and Bcl-w. On the other hand, hydroxychloroquine (HCQ) has been used as a drug for malaria infection and autoimmune disease. HCQ can exert a similar effect as chloroquine with fewer adverse events. In addition, HCQ exerts antitumor activity. In the present study, the effects of HCQ on ABT-263-induced antitumor activities were examined using three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3). In vitro effects of HCQ and ABT-263 were examined by cell viability, colony-forming assays, and flow cytometry. Protein expression was determined by immunoblotting. In vivo effects of HCQ and ABT-263 were examined by a xenograft mice model. Combined treatment with HCQ and ABT-263 synergistically decreased the viability of only BxPC-3 cells. This synergistic effect was not observed when HCQ was combined with ABT-199, an inhibitor specific to Bcl-2. The combination of HCQ and ABT-263 induced caspase-dependent apoptosis. Protein expression of Bcl-xL was more highly expressed in BxPC-3 cells than in the other two cell lines, and the combination of HCQ with a Bcl-xL inhibitor or siRNA-mediated knockdown of Bcl-xL induced apoptosis in BxPC-3 cells. Combination therapy with HCQ and ABT 737, an ABT-263 analogue, suppressed the in vivo growth of BxPC-3 with transient body-weight loss. HCQ effectively promotes Bcl-xL inhibition-induced apoptosis in BxPC-3 human pancreatic cancer cells.

Organic cation transporter 3 mediates the non-norepinephrine transporter driven uptake of meta-[211At]astato-benzylguanidine

IntroductionMeta-[211At]astato-benzylguanidine ([211At]MABG) accumulates in pheochromocytoma via norepinephrine transporter (NET) and leads to a strong antitumor effect, but it also distributed in normal tissues non-specifically. Meta-[131I]iodo-benzylguanidine ([131I]MIBG), an iodine-labeled analog of [211At]MABG, is known to be transported by not only NET but also organic cation transporter (OCT). The involvement of OCT in [211At]MABG uptake is still largely unknown. We investigated the involvement of OCT in the non-NET-driven uptake of [211At]MABG both in vitro and in vivo. Methods[123I]MIBG and [211At]MABG uptake was investigated in PC-12 (rat pheochromocytoma cell line), NIH/3T3 (mouse fibroblasts cell line), ACHN (human renal cancer cell line), and BxPC-3 (human pancreatic cancer cell line). Herein, we used desipramine and dl-norepinephrine to inhibit NET, and we used steroids (hydrocortisone and prednisolone) to inhibit OCT3. The [211At]MABG uptake in OCT3-knockdown cells established with OCT3-selective siRNA was also investigated. We investigated the biodistribution of [211At]MABG in PC-12 tumor-bearing mice after a preloading of phosphate-buffered saline (PBS) or hydrocortisone solution. ResultsThe uptake of both [123I]MIBG and [211At]MABG was significantly inhibited by desipramine in PC-12 cells but not the other cell lines. The expression of OCT3 was relatively higher than those of the other OCT subtypes in ACHN and BxPC-3 cells. The expression of OCTs was not observed in NIH/3T3 cells. The uptake of both [123I]MIBG and [211At]MABG in ACHN and BxPC-3 cells was significantly inhibited by the steroid treatments. The [211At]MABG uptake was also reduced in OCT3-knockdown cells (p < 0.001). The radioactivity of [211At]MABG was significantly reduced in normal tissues by the preloading of hydrocortisone. In contrast, there was an increasing trend of [211At]MABG uptake in the PC-12 tumors. The tumor-to-normal tissue ratio was significantly increased by the preloading of hydrocortisone compared to that of PBS. ConclusionOur results suggest that OCT3 is involved in non-NET-driven [211At]MABG uptake. The preloading of hydrocortisone selectively reduced [211At]MABG accumulation in normal organs in vivo. OCT3 inhibition may therefore be beneficial for a reduction of the radiation risk in healthy organs in the treatment of malignant pheochromocytomas.

Abstract 6285: MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and an inherently chemoresistant tumor. Dense fibrotic stroma is a major obstacle for drug delivery to the tumor cells in PDAC. Pancreatic stellate cells (PSCs) are the major stromal cells responsible for fibrotic microenvironment. MicroRNA-217 (miR-217) is known mainly as a tumor suppressor. However, the detailed roles of miR-217 on pathophysiology of PDAC remain unclarified. Methods: Expressions levels of miR-217 were measured in surgically resected human PDAC samples. Using Lipofectamine RNAiMAX, miR-217 or negative control miR (NC) were transfected in human pancreatic cancer cells, Panc-1, MIApaca-2, BxPC-3 and murine PAN02 cells, and chemosensitivities to gemcitabine and paclitaxel were investigated by MTS assay. Human hPSC-1 (obtained from Riken cell bank, Japan) was cultured in 20%FBS+DMEM and the changes of phenotypes and expression levels of miR-217 after the stimulation with TGF-β1 was measured by fluorescein microscope and digital PCR, respectively. Results: The expression level of miR-217 was markedly downregulated in PDAC tissues than in noncancerous pancreatic tissues (P&amp;lt;0.001, n=9). Overexpression of miR-217 significantly inhibited the proliferation of Panc-1, MIApaca-2 and BxPC-3 (P&amp;lt;0.001) and migration of Panc-1, MIApaca-2 and PAN02 (P&amp;lt;0.05). Lipofection of miR-217 further reduced the number of PAN02 exposed to 1-5nM gemcitabine (P&amp;lt;0.0001) and 0.01-0.1uM paclitaxel (P=0.0085) which resulted in the reduced the IC-50 as compared with NC. Human PSC-1 stimulated with TGF-β1 (10ng/ml) significantly reduced miR-217 expression compared to nascent cells. The stimulation with TGF-β1 upregulated the expression of cancer associated fibroblast (CAF) markers, α-smooth muscle actin (SMA) and vimentin. However, the expression levels of these markers were significantly reduced by the kipofection of miR-217. Conclusion: MiR-217 affects both cancer and stromal cells in PDAC which may regulate chemosensitivity in PDAC. Restoring miR-217 levels in tumor cells and/or PSCs could be a future therapeutic strategy. Citation Format: Yuki Kaneko, HIdeyuki Ohzawa, Yuki Kimura, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer [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 6285.

Investigation of Copper-64-Based Host-Guest Chemistry Pretargeted Positron Emission Tomography.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled in vivo at the target site using a radiolabeled small molecule (radioligand) that interacts with the macromolecule with high specificity. We report an investigation of host-guest chemistry-driven pretargeting using copper-64 radiolabeled ferrocene (Fc; guest) compounds and a cucurbit[7]uril (CB7; host) molecule functionalized carcinoembryonic antigen targeting hT84.66-M5A monoclonal antibody (CB7-M5A). Two novel ferrocene-based radioligands ([64Cu]Cu-NOTA-PEG3-Fc and [64Cu]Cu-NOTA-PEG7-Fc) were prepared, and their in vitro stability, pharmacokinetic in vivo profile in healthy mice, and pretargeting performance in a subcutaneous BxPC3 human pancreatic cancer cell xenograft mouse model were compared. The antibody dosing was optimized using a zirconium-89 radiolabeled M5A antibody ([89Zr]Zr-DFO-M5A) in a BxPC3 xenograft model, and the dosimetry of [89Zr]Zr-DFO-M5A and the pretargeting approach were compared. Finally, the effects of varying lag times up to 9 days between CB7-M5A and radioligand injection were investigated. In vivo pretargeting studies with both ferrocene radioligands resulted in specific tumor uptake (p = 0.0006 and p = 0.003) and also showed that the host-guest-based pretargeting approach excels with extended lag times up to 9 days with good tumor localization, suggesting that host-guest pretargeting may be suitable for use without clearing agents which have complicated clinical application of this technique. To our knowledge, the reported lag time of 9 days is the longest investigated lag time in any reported pretargeting studies.

Efficacy of MDX-124, a novel anti-annexin-A1 antibody, in preclinical models of pancreatic cancer.

590 Background: Pancreatic cancer is a highly fatal disease with poor survival and response to both chemotherapy and immunotherapy. Novel approaches to treat this disease are urgently required. Annexin-A1 (ANXA1) is secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic processes. High ANXA1 expression in pancreatic cancer patients is associated with poor overall survival, and influences cancer progression, drug sensitivity, migration and invasion. MDX-124 is a novel humanized antibody targeting ANXA1 and we have previously presented data demonstrating its significant antiproliferative activity. Here we present further data showing the efficacy of MDX-124 in several preclinical models of pancreatic cancer. Methods: In-vitro models utilized MIA PaCa-2, PANC-1 or BxPC-3 human pancreatic cancer cell lines. Cell cycle progression was evaluated by measuring changes in DNA content via flow cytometry. Pancreatic cancer cell viability following incubation with MDX-124 (0-10 µM) and 5FU (IC50) was assessed via MTT assay. A transwell migration assay was used to evaluate the effect of MDX-124 (0-50 µM) on pancreatic cancer cell migration. In-vivo efficacy was evaluated using an orthotopic mouse model of metastatic pancreatic cancer (FC1242luc/zsGreen; KPC-derived cell line) with bioluminescent imaging used to quantify the incidence and burden of lung metastases. Results: When compared to untreated MIA PaCa-2 pancreatic cancer cells, MDX-124 treatment decreased the proportion of cells in S-phase by 29% and G2 phase by 9.1%, with a concomitant increase in G1 of 38.1%. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. MDX-124 significantly reduced the viability of MIA PaCa-2 and PANC-1 cell lines versus an IgG control in a dose-dependent manner. Additionally in these two cell lines, combination of MDX-124 with 5FU (IC50) had a significant synergistic impact reducing cancer cell viability by 99.8% and 91.2% respectively. Furthermore, MDX-124 significantly reduced the migratory ability of MIA PaCa-2 and BxPC-3 pancreatic cancer cells. In the orthotopic model of metastatic pancreatic cancer, the murine analog of MDX-124 (MDX-001), markedly reduced both the incidence and size of lung metastases. Conclusions: MDX-124 demonstrated significant anti-tumor efficacy in several preclinical models of pancreatic cancer as a single agent, with increased potency observed when used in combination with 5FU. Medannex will initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including pancreatic cancer.

LncRNA LINC01857 drives pancreatic adenocarcinoma progression via modulating miR-19a-3p/SMOC2

ObjectivesEmerging evidence has demonstrated that LINC01857 exerts a pivotal function in many cancers. However, its function in Pancreatic Ductal Adenocarcinoma (PDAC) still remains unclear. This study was designed to investigate the regulatory character of LINC01857 in PDAC. MethodsBioinformatic tools and databases were used to seek potential miRNAs and mRNAs. Gene expression was evaluated by Reverse Transcription quantitative real-time Polymerase Chain Reaction (RT-qPCR), and western blot was used for protein level detection. A subcellular fraction assay was done to ascertain the location of LINC01857 in PANC-1 and BxPC-3 human pancreatic cancer cells. CCK-8, EdU, wound healing and Transwell assays were performed to inquire into the influence of LINC01857, and SPARC -related Modular Calcium-binding protein-2 (SMOC2) on cell viability, proliferation, migration, and invasion, respectively. The interaction between LINC01857 and its downstream genes was explored by RNA immunoprecipitation and luciferase reporter assays. ResultsLINC01857 levels were significantly elevated in PDAC. Knockdown of LINC01857 significantly restrained the proliferation, migration, invasion, and Epithelial-Mesenchymal Transition (EMT) process of PDAC cells. MiR-19a-3p was a downstream target of LINC01857, and miR-19a-3p levels were significantly decreased in PDAC cells. In addition, SMOC2 expression had a negative correlation with that of miR-19a-3p, and SMOC2 was a downstream target of miR-19a-3p. Furthermore, SMOC2 upregulation partially abolished the inhibitive influence of LINC01857 downregulation on cell proliferation, migration, invasion, and the EMT process. ConclusionLINC01857 promotes malignant phenotypes of PDAC cells via upregulation of SMOC2 by interacting with miR-19a-3p.

Abstract 1755: E. coli genomic DNA fragments induce cGAS-STING-mediated DUOX2 expression in human pancreatic cancer cells that is associated with H2O2-related DNA damage

Abstract There is a growing appreciation that pancreatic cancers harbor higher numbers of bacteria &amp; fungi compared to normal pancreatic tissue in both humans &amp; mice. These microbes have been suggested to contribute to pancreatic carcinogenesis, &amp; impact therapeutic sensitivity &amp; resistance. Dual oxidase 2 (DUOX2), one of the seven NADPH oxidases, is expressed at the pancreatic cancer plasma membrane &amp; generates extracellular hydrogen peroxide (H2O2). We previously demonstrated that DUOX protein was significantly upregulated in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), &amp; some early stage pancreatic ductal adenocarcinomas (PDACs). Moreover, DUOX expression in these tissues was associated with H2O2-mediated DNA double strand breaks &amp; upregulation of hypoxia markers VEGF &amp; HIF-1α. Incidentally, we also discovered that mycoplasma-contaminated BxPC-3 human pancreatic cancer cells upregulated known oncogenes such as EGFR, MET, &amp; VEGF-A, in concert with ~20-fold upregulation of DUOX2 mRNA. Recently, the cytosolic DNA-sensing cGAS-STING pathway has been implicated as a critical mediator of tumor-targeting immune responses. Using a panel of human colon &amp; pancreatic cancer cell lines coupled with Western blot analysis &amp; qPCR methods, we have demonstrated that the introduction of purified E. coli genomic DNA fragments, as well as several kinds of plasmid DNA, into the pancreatic cancer cell cytosol significantly enhanced DUOX2 RNA &amp; protein expression, leading to an enhanced DNA damage response. For BxPC-3, CFPAC-1, &amp; HTB134 human pancreatic cancer cells, enhanced DUOX2 expression following introduction of exogenous DNA was associated with the activation of the cGAS-STING pathway, as indicated by enhanced IRF-3 and TBK1 phosphorylation. Treatment of these cell lines with exogenous cGAMP for 24h also activated the cGAS-STING pathway and significantly increased DUOX2 expression. Correspondingly, in BxPC-3 cells treated with human cGAS-specific small interfering RNAs, we observed the significant suppression of cGAS RNA and protein expression &amp; related downstream signaling, along with the significant attenuation of DNA-induced DUOX2 RNA &amp; protein. In contrast, exogenous DNA did not upregulate DUOX2 in colon cancer cell lines. In summary, our data suggest that, in pancreatic cancers, a bacteria-containing microenvironment is likely to promote DUOX2-related H2O2 production as a consequence of the bacterial DNA-induced activation of the cGAS-STING innate immunity pathway, which, in turn, could enhance the oxidative milieu, augment leukocyte recruitment, &amp; increase genetic instability, thus contributing to the molecular evolution of pancreatic tumors. Citation Format: Yongzhong Wu, Stephen L. Wang, Smitha Antony, Jennifer L. Meitzler, Jiamo Lu, Guojian Jiang, Becky Diebold, Mallick David, Mariam M. Konate, Roy Krishnendu, James H. Doroshow. E. coli genomic DNA fragments induce cGAS-STING-mediated DUOX2 expression in human pancreatic cancer cells that is associated with H2O2-related DNA damage [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 1755.

ARHGAP25 Inhibits Pancreatic Adenocarcinoma Growth by Suppressing Glycolysis via AKT/mTOR Pathway.

Increasing evidence reveals that the Rho GTPase-activating protein is a crucial negative regulator of Rho family GTPase involved in tumorigenesis. The Rho GTPase-activating protein 25 (ARHGAP25) has been shown to specifically inactivate the Rho family GTPase Rac1, which plays an important role in pancreatic adenocarcinoma (PAAD) progression. Therefore, here we aimed to clarify the expression and functional role of ARHGAP25 in PAAD. The ARHGAP25 expression was lower in PAAD tissues than that in normal pancreatic tissues based on bioinformatics analysis and immunohistochemistry staining. Overexpression of ARHGAP25 inhibited cell growth of AsPC-1 human pancreatic cancer cells in vitro, while opposite results were observed in BxPC-3 human pancreatic cancer cells with ARHGAP25 knockdown. Consistently, in vivo tumorigenicity assays also confirmed that ARHGAP25 overexpression suppressed tumor growth. Mechanically, overexpression of ARHGAP25 inactivated AKT/mTOR signaling pathway by regulating Rac1/PAK1 signaling, which was in line with the results from the Gene set enrichment analysis on The Cancer Genome Atlas dataset. Furthermore, we found that ARHGAP25 reduced HIF-1α-mediated glycolysis in PAAD cells. Treatment with PF-04691502, a dual PI3K/mTOR inhibitor, hampered the increased cell growth and glycolysis due to ARHGAP25 knockdown in PAAD cells. Altogether, these results conclude that ARHGAP25 acts as a tumor suppressor by inhibiting the AKT/mTOR signaling pathway, which might provide a therapeutic target for PAAD.

Chelidonine Induces Apoptosis via GADD45a-p53 Regulation in Human Pancreatic Cancer Cells.

Chelidonium majus has been used as a traditional medicine in China and western countries for various diseases, including inflammation and cancer. However, the anti-cancer effect of chelidonine, a major compound of C. majus extracts, on pancreatic cancer remains poorly understood. In this study, we found that treatment with chelidonine inhibited proliferation of BxPC-3 and MIA PaCa-2 human pancreatic cancer cells. Annexin-V/propidium iodide staining assay showed that this growth inhibitory effect of chelidonine was induced through apoptosis. We found that chelidonine treatment upregulated mRNA levels and transcription factor activity in both cell lines. Increases in protein expression levels of p53, GADD45A, p21 and cleaved caspase-3 were also observed, with more distinct changes in MIA PaCa-2 cells compared to the BxPC-3 cells. These results suggest that chelidonine induces pancreatic cancer apoptosis through the p53 and GADD45A pathways. Our findings provide new insights into the use of chelidonine for the treatment of pancreatic cancer.

Saikosaponin D Inhibits Proliferation and Promotes Apoptosis Through Activation of MKK4-JNK Signaling Pathway in Pancreatic Cancer Cells.

IntroductionPancreatic cancer remains one of the most lethal malignancies and has few treatment options. Saikosaponin D (SSD), a major bioactive triterpene saponin isolated from Bupleurum chinense, has been reported to exert cytotoxicity properties toward many cancer cells. However, the effects of SSD on pancreatic cancer have been little scrutinized.MethodsHere, we investigated the effect of SSD on the proliferation and apoptosis of human pancreatic cancer BxPC3 and PANC1 cells and the mouse pancreatic cancer cell line Pan02. Cell viability was determined by MTT assays and cell apoptosis analyzed by DAPI staining and flow cytometry. Expression levels of apoptosis-regulating markers and activity of the MKK4–JNK signaling pathway were determined by Western blotting. The inhibitor SP600125 was applied to confirm the role of the JNK pathway in SSD efficiency.ResultsSSD significantly inhibited the proliferation of BxPC3, PANC1, and Pan02 cells in a concentration- and time-dependent manner. Flow-cytometry analysis indicated obvious apoptosis induction after SSD exposure. Furthermore, SSD significantly triggered cleavage of caspase 3 and caspase 9 proteins and increased the expression of FoxO3a. In addition, activity of the MKK4–JNK pathway was dramatically increased after treatment with SSD in BxPC3 cells. SSD obviously stimulated phosphorylation of JNK, cJun, and SEK1/MKK4 proteins within 30 minutes. The addition of SP600125 blocked the activation of SSD on the MKK4–JNK regulatory pathway and reversed the effects of SSD on proliferation inhibition and apoptosis induction in BxPC3 cells.ConclusionThese results revealed that SSD was capable of suppressing tumor growth and promoting apoptosis of pancreatic cancer cells via targeting the MKK4–JNK signaling pathway, indicating the possibility of further developing SSD as a potential therapeutic candidate for pancreatic cancer.

Tumor-specific near-infrared nanobody probe rapidly labels tumors in an orthotopic mouse model of pancreatic cancer

BackgroundNanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen (CEA) nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer. MethodsAnti-CEA or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC-3 (high-CEA expressing) or MiaPACA-2 (low-CEA expressing) human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-CEA or control nanobody-IRDye800CW were injected intravenously. Mice were imaged at various time points hours post-injection. ResultsAnti-CEA nanobodies clearly labeled BxPC3 orthotopic pancreatic tumors 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low CEA-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for CEA-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems. ConclusionThis anti-CEA, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-CEA nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.

A novel derivative of valepotriate inhibits the PI3K/AKT pathway and causes Noxa-dependent apoptosis in human pancreatic cancer cells.

Natural compound valepotriate exhibits inhibitory activity against a number of cancers, but the effect of valepotriate against pancreatic cancer is unclear, and the structure–activity relationship of valepotriate has not been characterized. In this study, we performed a structure-based similarity search and found 16 hit compounds. Among the 16 hits, (1S,6S,7R)-6-(acetyloxy)-1-[(3-methylbutanoyl)oxy]-4a,5,6,7a-tetrahydro-1H-spiro[cyclopenta[c]pyran-7,2’-oxiran]-4-ylmethyl 3-methylbutanoate (denoted as Amcp) exhibited superior anticancer activity against human pancreatic cancer BxPC-3 and SW1990 cells. The anti-proliferation activity of Amcp was validated in human pancreatic cancer BxPC-3 and SW1990 cells in vitro. Amcp more effectively induced apoptosis in BxPC-3 and SW1990 cells than gemcitabine. At a concentration of 15 μM, Amcp significantly suppressed the PI3K/AKT pathway and disrupted the mitochondrial membrane equilibrium through modulation of Noxa and Mcl-1 balance in both cell lines. Meanwhile, knockdown of Noxa substantially attenuated Amcp-induced reduction of cell viability and anti-apoptotic protein Mcl-1 level in BxPC-3 cells. In addition, Amcp showed synergistic anticancer effects when combined with gemcitabine in BxPC-3 cells. To conclude, this work not only suggests that Amcp possesses a dual-inhibitory activity towards PI3K/AKT pathway and Mcl-1, but also enlightens further development of bioactive valepotriate derivatives.