Pancreatic Cancer Cell Lines Capan-1 Research Articles

CD74 Promotes a Pro-Inflammatory Tumor Microenvironment by Inducing S100A8 and S100A9 Secretion in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of pancreatic cancer with a poor prognosis and low survival rates. The prognostic and predictive biomarkers of PDAC are still largely unknown. The receptor CD74 was recently identified as a regulator of oncogenic properties in various cancers. However, the precise molecular mechanism of CD74 action in PDAC remains little understood. We investigated the role of CD74 by silencing CD74 in the pancreatic cancer cell line Capan-1. CD74 knockdown led to reductions in cell proliferation, migration, and invasion and increased apoptosis. Moreover, silencing CD74 resulted in the decreased expression and secretion of S100A8 and S100A9. An indirect co-culture of fibroblasts and tumor cells revealed that fibroblasts exposed to conditioned media from CD74 knockdown cells exhibited a reduced expression of inflammatory cytokines, suggesting a role of CD74 in influencing cytokine secretion in the tumor microenvironment. Overall, our study provides valuable insights into the critical role of CD74 in regulating the oncogenic properties of pancreatic cancer cells and its influence on the expression and secretion of S100A8 and S100A9. Taken together, these findings indicate CD74 as a potential diagnostic biomarker and therapeutic target for pancreatic cancer.

Pancreatic Stromal Cell-derived Oncostatin M Confers Drug Resistance to a Multi-tyrosine Kinase Inhibitor in Pancreatic Cancer Cells.

Pancreatic cancer is known to have one of the worst prognoses of all cancers, and its tumor cells are highly resistant to chemotherapeutic drugs. Pancreatic cancer cells coexist with stromal cells; however, their involvement in anticancer drug resistance remains poorly understood. Thus, in this study, we analyzed drug sensitivity using an in vitro co-culture system containing pancreatic cancer cells and stromal cells treated with a compound library. We examined the viability of the pancreatic cancer cell lines BxPC-3, Capan-1, and Panc-1 against compounds in an in vitro co-culture model containing pancreatic stromal cells (PSCs) and analyzed the protein expression for drug resistance by western blotting. We found that co-cultured pancreatic cancer cells were resistant to vandetanib, which is an inhibitor of multi-tyrosine kinases. The key factor involved in drug resistance in these pancreatic cancer cells was oncostatin M, which was secreted by stromal cells. The addition of oncostatin M increased the vandetanib resistance of pancreatic cancer cells, while it inhibited the suppression of insulin receptor substrate-1 (IRS1) and the phosphorylation of extracellular signal-regulated kinase (ERK) by vandetanib. Oncostatin M secreted by stromal cells derived from the pancreas activates the IRS1-ERK axis, causing resistance to vandetanib.

A Defucosylated Anti-EpCAM Monoclonal Antibody (EpMab-37-mG2a-f) Exerts Antitumor Activity in Xenograft Model.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (KD): 2.9 × 10-8 M and 1.8 × 10-8 M, respectively] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers.

Abstract 5328: Near-infrared photoimmunotherapy for pancreatic cancer: Targeting cell surface glycans using lectin-photoabsorber conjugate (LPC)

Abstract Introduction: Since protein targets in cancer therapy largely have been explored, glycans would be attractive alternative targets. We had previously discovered a fucosylated glycan epitope highly expressed on pancreatic cancer cells, and a lectin probe called rBC2LCN, which can be used as a specific targeting bullet to this glycan (Shimomura O, Oda T, et al: Mol Cancer Ther 2018). We now then hypothesized that, this lectin-based cancer-targeting strategies should be reasonably applicable to near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT have already developed using a conjugate of a photoabsorber, IRdye700DX (IR700), and a monoclonal antibody which recognizes protein antigens on the target cells. In this study, we firstly developed a lectin-photoabsorber conjugate (BC2-IR700) and investigated a new cancer therapy targeting cancer cell surface glycans. Material and Methods: We developed BC2-IR700 by conjugation of rBC2LCN lectin with IR700 NHS ester. A pancreatic cancer cell line Capan-1 was used as a model which is bound by rBC2LCN. The cytotoxic effect of NIR-PIT with BC2-IR700 was first evaluated in vitro. We then evaluated the anti-cancer effect of BC2-IR700 in vivo using pancreatic cancer xenograft mice including subcutaneous tumor model. Results: The product of BC2-IR700 was confirmed by SDS-PAGE, that showed reasonable 16KDa sharp band with clear fluorescence intensity. After incubation with BC2-IR700, Capan-1 cells showed high fluorescence signal, which was confirmed with flow cytometry and fluorescence microscopy. BC2-IR700 was detected on the cell surface of Capan-1 after incubation for 1h. Immediately after exposure to NIR light, the cell death of Capan-1 was induced and the cytotoxicity significantly increased in a light-dose-dependent manner. After intravenous administration of BC2-IR700 into pancreatic cancer xenograft mice, BC2-IR700 was observed in vivo fluorescence imaging to be accumulated in tumor. NIR-PIT was performed by exposing to NIR light (100 J/cm2) at 6 h following BC2-IR700 intravenous administration (20 μg/mice). Tumor growth was significantly inhibited in the NIR-PIT treatment group compared with the other control groups such as no treatment, BC2-IR700 only, and NIR light exposure only (P < 0.05). Conclusions: The lectin-photoabsorber conjugate, BC2-IR700, was able to be sufficiently applied for NIR-PIT in in vivo mouse model. The novel NIR-PIT targeting cell surface glycans could be a promising therapeutic strategy for the treatment of pancreatic cancer. Citation Format: Yukihito Kuroda, Osamu Shimomura, Hiromitsu Nakahashi, Tomoaki Furuta, Yoshihiro Miyazaki, Takahito Nakajima, Hiroaki Tateno, Tatsuya Oda. Near-infrared photoimmunotherapy for pancreatic cancer: Targeting cell surface glycans using lectin-photoabsorber conjugate (LPC) [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 5328.

Four new compounds with cytotoxic and neuroprotective activity from Notopterygium incisum

Phytochemical research on an extract of Notopterygium incisum yielded twenty-four compounds ( 1 – 24 ), including four new compounds 7 , 10 , 13 and 17 . The structures of new compounds were elucidated by spectroscopic analysis, and the absolute configuration of 5 were assigned via Mosher's method. In addition, compounds 2 , 9 , 19 and 24 were analyzed by single crystal X-ray analysis for the first time. The new compound 7 exhibited remarkable cytotoxic activity against the human pancreatic cancer cell lines PANC-1, CAPAN-2, CFPAC-1, and SW1990 with IC 50 values of 6.25 ± 0.54, 7.74 ± 1.12, 6.68 ± 1.11, and 5.35 ± 0.33 μM, respectively. The primary mechanistic study indicated that 7 caused cell death probably due to induced defects in the mitotic process. Moreover, compounds 2 and 7 - 9 effectively protected against H 2 O 2 -induced neurotoxicity in the SH-SY5Y cells, and 1 , 3 , 6 , 10 , 11 , 16 and 19 exhibited strong DPPH scavenging ability. • Four undescribed and twenty known compounds were reported. • Compound 7 displayed cytotoxicity against four pancreatic cancer cell lines. • Coumarins protected against H 2 O 2 -induced neurotoxicity in the SH-SY5Y cells.

Abstract PO-039: Antiproliferative activity of inhibitors of RAD51, singly and in combination with chemotherapy drugs, against pancreatic cancer cell lines

Abstract Despite the innovations in chemotherapeutic treatment of pancreatic cancer, the generally poor outcome of this disease begs the search for novel targets. RAD51 is a critical component of homologous recombination DNA repair, binding with BRCA2 and forming polymeric filaments essential for its function. RAD51 is often elevated in cancer cells compared to normal cells, particularly in pancreatic cancer: RAD51 is a negative prognostic biomarker and promotes tumor cell proliferation in that disease (Cancer Cell Int 19: 356, 2019; doi: 10.1186/s12935-019-1077-6). Therefore, RAD51 is an attractive target for anticancer treatment in that its elevated level and activity in tumor cells provide a potential level of selectivity for such an agent, increasing the therapeutic index. We demonstrated previously that novel inhibitors of RAD51, IBR2 [2-(Benzylsulfonyl)-1-(1H-indol-3-yl)-1,2-dihydroisoquinoline] and IBR120 (an isoindolinyl derivative of IBR2) (Eur J Med Chem. 96:196-208, 2015; doi: 10.1016/j.ejmech.2015.04.021), not only inhibited proliferation of a range of tumor cell lines at micromolar concentrations but also acted in combination with commonly used cytotoxic chemotherapy and molecularly targeted drugs to synergistically inhibit proliferation (J Pharmacol Expt Ther, 364: 46-54, 2018; doi.org/10.1124/jpet.117.241661). To improve ADME properties, enhance activity, and increase potential to selectively target RAD51, advanced computational tools and rational approaches were employed to predict possible modification to the IBR chemical backbone. This effort resulted in discovery of the compound JKYN-1. As a model system in which to test the activity of JKYN-1 in vitro, human pancreatic cancer cell lines PANC-1, Capan-1 and Capan-2 were used. All lines have mutant K-Ras and p53, and Capan-1 has mutant BRCA2 (https://www.cancer.gov/research/key-initiatives/ras; https://web.expasy.org/cellosaurus). JKYN-1 or its water-soluble methylsulfonate salt (JKYN-1-mesylate) inhibit proliferation of PANC-1 cells approximately 5 times more effectively than IBR120 and inhibit Capan-1 and Capan-2 cells even better. Against PANC-1, JKYN-1-mesylate inhibits proliferation synergistically in combination with afatinib (inhibitor of epidermal growth factor receptor) or with IBR120, and is additive with gemcitabine. Biochemical characterization of the interaction between RAD51 and these inhibitors demonstrated the following: (1) IBR2 and IBR120 promote disassembly of RAD51 multimers in an ex vivo native polyacrylamide gel electrophoresis assay using clonal constructs of RAD51, and (2) JKYN-1 binds to RAD51 with higher affinity than IBR120 and B02, a commercially available RAD51 inhibitor, in a surface plasmon resonance spectroscopy assay. Therefore, JKYN-1 is a potential novel small molecule therapeutic agent for treatment of pancreatic cancer, both as a single agent and in combination with standard chemotherapy drugs. Citation Format: Peter Ferguson, Mark D. Vincent, Yousef Najajreh, Brian Shilton, Stephen Ritter, Rima Al-awar, Richard Marcellus, Mohammed Mohammed, Methvin Isaac, James Koropatnnick. Antiproliferative activity of inhibitors of RAD51, singly and in combination with chemotherapy drugs, against pancreatic cancer cell lines [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-039.

Abstract PO-092: Influence of the IL-13-receptor alpha 1 chain on the malignant phenotype of pancreatic cancer cells

Abstract Introduction: Increasing evidence indicates salient activities of Interleukin (IL)-4, IL-13 and their specific Type-II-IL-4 receptor complex (IL-4Rα/IL-13Rα1) in carcinomas including pancreatic cancer. While IL-4 seems to be tumor-promoting in PDAC, the specific role of the IL-13/IL-13Rα1 axis was yet to be determined. Both cytokines are abundant in PDAC through cells of the tumor microenvironment. In this project, we planned to determine the specific effects of the IL-13Rα1-receptor chain on the malignant phenotype of pancreatic cancer cells. Methods: Expression of IL-13Rα1, IL-4Rα, and the human common gamma chain (γc) in cultured pancreatic cancer cell lines AsPC-1, Capan-1, PANC-1, MIA PaCa-2 and A818-6 as well as downstream signal transduction factors of IL13-Rα1 were detected by Western blot. The role of IL-13Rα1 in pancreatic cancer cell proliferation, mobility and migration was demonstrated based on the successful establishment of stable shRNA based IL-13Rα1-knockdown (KD) clones in Capan-1 and MIA PaCa-2 cell lines. Results: Not only the basal anchorage-dependent growth but also the anchorage-independent growth in soft agar were inhibited in Capan-1 and MIA PaCa-2 cells due to IL-13Rα1-downregulation. Mechanistically, IL-13Rα1-KD was associated with increased apoptosis. Additionally, signal transduction factors STAT3, STAT6, PI3K, Akt and ERK1/2 were activated in Capan-1 exposed to exogenous IL-4 and IL-13. Interestingly, IL-13Rα1-KD reduced the basal expression of STAT3, Akt and ERK1/2. IL-4 and IL-13 induced activation of STAT3, ERK1/2, Akt and STAT6 was significantly suppressed in Capan-1-IL-13Rα1-KD (C-KD) clones. Furthermore, IL-13Rα1-KD resulted in enhanced mobility and migration in Capan-1 cells. Conclusion: It was demonstrated that endogenous IL-13Rα1 is vital for pancreatic cancer cell growth with the involvement of STAT3/6, ERK1/2 and Akt. Surprisingly, IL-13Rα1-KD resulted in enhanced mobility and migration. Overall, IL-13Rα1 plays a critical but heterogenous role in PDAC. Our findings increase the understanding of the different functions and mechanisms involving IL-13Rα1 in pancreatic cancer progression. Citation Format: Jingwei Shi, Marko Kornmann, Benno Traub. Influence of the IL-13-receptor alpha 1 chain on the malignant phenotype of pancreatic cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-092.

Synthetic lethality of LP-184, a next generation acylfulvene, in ex vivo PDX models with homologous recombination defects.

e15064 Background: The clinical success of PARP inhibitors (PARPi) in homologous recombination (HR) deficient (HRD+) solid tumors has broadened the scope of identifying additional agents and vulnerabilities in cancers with DNA repair deficiencies. However, with more than 40% of BRCA1/2-deficient patients failing to respond to PARPi or acquiring resistance with prolonged PARPi administration, newer agents are also needed. LP-184, an acylfulvene, is a prodrug activated by PTGR1. Threshold expression levels of PTGR1 are higher in several tumors, providing a window of specificity for its cytotoxic action. DNA damage inflicted by acylfulvene (AF) agents is reliant upon HR pathway genes including BRCA1 for correction and removal. We hypothesized that tumors with high PTGR1 expression and HR deficiency will therefore be uniquely targeted and demonstrate synthetic lethality when exposed to LP-184. Methods: We evaluated ex vivo antitumor activity of LP-184 in selected PDX models representing lung, pancreatic and prostate cancers with high PTGR1 and known HR defects. Dissociated tumor fragments were treated with LP-184 across a concentration range of 5 nM to 36 uM for 5 days. Cell viability was quantified by CellTiter Glo. LP-184 IC50s were compared with PARPi efficacy. We further confirmed the dependency of PTGR1 in HR deficient tumor cells by comparing LP-184 sensitivity in the BRCA2 deficient cell line CAPAN-1 and the ATM mutant cell line PANC03.27, with and without PTGR1 suppression following an engineered CRISPR knockout of PTGR1. We also analyzed TCGA data to estimate the percentage of tumors with elevated PTGR1 and co-occurring damaging mutations in a panel of 60 HR genes. Results: The mean LP-184 IC50 across 15 HRD+ PDX models tested was 288 nM (range 31 - 2900 nM). LP-184 turned out to be 6 - 340X more potent ex vivo than the PARPi Olaparib in these models. 9 of 15 models were associated with no clinical response to or initial response followed by progression on approved standard of care (SOC) agents. 6 of 15 models showed < 10% tumor growth inhibition in vivo with SOC treatment. Regardless of cancer type, models with high-impact, loss-of-function mutations in ATM, ATR and BRCA1 showed exquisite sensitivity to LP-184 (mean IC50 ̃ 60 nM). CRISPRi-mediated stable suppression of PTGR1 in the pancreatic cancer cell lines CAPAN-1 and PANC03.27 entirely abrogated LP-184 sensitivity relative to isogenic parental cell lines. 17.6% of lung adenocarcinomas (n = 517), 4.5% of pancreatic adenocarcinomas (n = 179) and 9.6% of prostate adenocarcinomas (n = 498) displayed elevated PTGR1 along with damaging HR related mutations, and are likely to be responsive to LP-184 based on analysis of TCGA data. Conclusions: LP-184 is broadly effective in HRD+ tumors that may be less responsive to SOC including PARPi and could be useful clinically in a subset of tumors with high PTGR1 and HR defects.

Novel Pancreatic Cancer Therapy Targeting Cell Surface Glycans by Liposomes Modified with rBC2LCN Lectin

Introduction: Since the outermost layer of cancer cells is covered with various glycans, targeting these groups may serve as an effective strategy in cancer therapy. We previously reported that fucosylated glycans are specifically expressed on pancreatic cancer cells, and that a protein specifically binding to these glycans, namely rBC2LCN lectin, is a potential guiding drug carrier. In the present study, a novel type of glycan-targeting nanoparticle was developed by modifying the surface of doxorubicin-containing liposomes with rBC2LCN lectin. The efficiency and specificity of this formulation, termed Lec-Doxosome, were examined in vitro and in vivo in human pancreatic cancer models. Methods: Lec-Doxosome was prepared by a post-insertion method based on the insertion of rBC2LCN lectin into the liposomal surface via a lipid linker. The in vitro cellular binding, uptake, and cytotoxicity of Lec-Doxosome were compared with the corresponding parameters in the unmodified liposomes by applying to human pancreatic cancer cell line (Capan-1) with affinity for rBC2LCN lectin. For the in vivo assay, Lec-Doxosome was intravenously injected once per week for a total of 3 weeks into mice bearing subcutaneous tumors. Results: The in vitro application of Lec-Doxosome resulted in a 1.2- to 1.6-fold higher intracellular doxorubicin accumulation and a 1.5-fold stronger cytotoxicity compared with the respective rates of accumulation and cytotoxicity in the unmodified liposomes. In vivo, Lec-Doxosome reduced the mean tumor weight (368 mg) compared with that in mice treated with unmodified liposomes (456 mg), without causing any additional adverse events. Conclusion: It was demonstrated from the results obtained herein that rBC2LCN lectin is a potent modifier, as a means for boosting the efficiency of nanoparticles in the targeting of cancer cell surface glycans.

Abstract 6598: Eradication of EpCAM expressing solid tumors by low-affinity CAR T cells

Abstract Background: Adoptive cell transfer therapy with genetically engineered chimeric antigen receptor (CAR)-T cells is rapidly revolutionizing treatment against cancers. However, limited success of CAR T cells against large metastatic solid tumors has been made to date. It is largely due to safety concerns regarding on-target off-tumor toxicity to vital healthy tissues, and poor efficacy after adoptive transfer. Epithelial cell adhesion molecule (EpCAM) was found to be frequently over-expressed in a wide variety of carcinomas, including colon, gastric, pancreas, and breast cancers, making EpCAM an attractive molecule for targeted therapeutics. However, clinical trials with anti-EpCAM antibodies (ING-1, 3622W94) and bispecific T cell engager antibodies (Solitomab) have shown significant dose-limiting toxicities, resulting in very limited clinical responses. To selectively target EpCAM high tumors, we are developing affinity tuned EpCAM CAR T cells with varying affinities to EpCAM. Methods: The human glioblastoma cell line U251, breast cancer cell lines MDA-MB-231 and SK-BR-3, gastric cancer cell lines MKN28 and MKN45, pancreatic cancer cell lines SW1990 and Capan2, and colon cancer cell line HT29 were transduced with Luciferase-F2A-GFP (FLuc/GFP) lentivirus. EpCAM expression levels on tumor cell lines were measured by flow cytometry. Primary human T cells were transduced with EpCAM CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. NSG mice bearing intraperitoneal or subcutaneous MKN28 xenografts, systemic metastatic MKN45 xenografts, and orthotopic Capan2 xenografts were treated with EpCAM CAR T cells to test the in vivo efficacy. PET/CT imaging was adopted to monitor T cell dynamics using 18F-NOTA-octreotide which binds specifically to somatostatin receptor 2 (SSTR2) introduced in CAR T cells. Results: Jurkat cells transduced with C215 and UBS54 CARs displayed 3.5 nM and 264.3 nM binding affinities to soluble EpCAM, respectively. EpCAM CAR expression in primary human T cells was higher than 50%. CD8+ to CD4+ T cell ratios were 1:1. Compared to high-affinity C215 CAR T cells, low-affinity UBS54 CAR showed equivalent cytotoxicity against high-density EpCAM-expressing targets (MKN28, MKN45 and HT29), but slower killing of low-density EpCAM-expressing cell line MDA-MB-231. T cell activation as monitored by CD69 upregulation and cytokine secretion (IL2, IFN-γ, TNF-α, GM-CSF and MIP-1β) of C215 and UBS54 CAR T cells were similar, and correlate well with EpCAM density on tumor cell surfaces. In immunodeficient mouse models, low-affinity UBS54 CAR potently eliminated intraperitoneal and subcutaneous gastric tumors (MKN28), systemic metastatic gastric tumor (MKN45), and orthotopic pancreatic tumor (Capan2). Notably, the level of T cell expansion in the course of tumor killing, observed by PET/CT imaging, was significantly lower for UBS54 CAR than high-affinity C215 CAR. Conclusions: Low-affinity EpCAM targeting CAR T cells demonstrated durable antitumor activity against various tumor models in vivo. To enhance safety and therapeutic efficacy, micromolar affinity anti-EpCAM CAR T cells are currently being evaluated to limit cell killing to EpCAM-positive tumor cells while sparing normal cells with basal levels of EpCAM expression. Citation Format: Yanping Yang, Jaclyn E. McCloskey, Huan Yang, Janusz Puc, Angel A. Gomez Gallegos, Yogindra Vedvyas, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Eradication of EpCAM expressing solid tumors by low-affinity CAR T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6598.

MiR-19b-3p promotes human pancreatic cancer Capan-2 cells proliferation by targeting phosphatase and tension homolog.

Pancreatic cancer is a common cancer with a poor prognosis and an increasing morbidity. miR-19b-3p has been implicated in some cancers, however, its role in pancreatic cancer is unclear. Human pancreatic cancer cell line Capan-2 cells were transfected with miR-19b-3p mimic and inhibitor. Cell proliferation was measured by 5-Ethynyl-2'-deoxyuridine (EdU) staining assays. Cell cycle of Capan-2 cells was examined by flow cytometry. The expression of phosphatase and tension homolog (PTEN) was determined by real-time quantitative polymerase chain reaction (PCR) and western blotting analysis. Functional rescue experiments were performed through PTEN overexpression and miR-19b-3p mimic by using EdU staining assays. miR-19b-3p mimic significantly increased miR-19b-3p while miR-19b-3p inhibitor decreased that. EdU staining showed that miR-19b-3p overexpression promoted Capan-2 cells proliferation while miR-19b-3p inhibition decreased that. Flow cytometry analysis of cell cycle indicated that miR-19b-3p overexpression increased the percentage of Capan-2 cells in S phase while miR-19b-3p inhibition decreased that. PTEN was confirmed to be a target gene of miR-19b-3p and PTEN overexpression eliminated the pro-proliferation effects of miR-19b-3p in Capan-2 cells. Our study demonstrates that miR-19b-3p promotes Capan-2 cells proliferation by targeting PTEN.

Anti-tumor Efficacy Assessment of the Sigma Receptor Pan Modulator RC-106. A Promising Therapeutic Tool for Pancreatic Cancer.

Introduction: Pancreatic cancer (PC) is one of the most lethal tumor worldwide, with no prognosis improvement over the past 20-years. The silent progressive nature of this neoplasia hampers the early diagnosis, and the surgical resection of the tumor, thus chemotherapy remains the only available therapeutic option. Sigma receptors (SRs) are a class of receptors proposed as new cancer therapeutic targets due to their over-expression in tumor cells and their involvement in cancer biology. The main localization of these receptors strongly suggests their potential role in ER unfolded protein response (ER-UPR), a condition frequently occurring in several pathological settings, including cancer. Our group has recently identified RC-106, a novel pan-SR modulator with good in vitro antiproliferative activities toward a panel of different cancer cell lines. In the present study, we investigated the in vitro properties and pharmacological profile of RC-106 in PC cell lines with the aim to identify a potential lead candidate for the treatment of this tumor.Methods: Pancreatic cancer cell lines Panc-1, Capan-1, and Capan-2 have been used in all experiments. S1R and TMEM97/S2R expression in PC cell lines was quantified by Real-Time qRT-PCR and Western Blot experiments. MTS assay was used to assess the antiproliferative effect of RC-106. The apoptotic properties of RC-106 was evaluated by TUNEL and caspase activation assays. GRP78/BiP, ATF4, and CHOP was quantified to evaluate ER-UPR. Proteasome activity was investigated by a specific fluorescent-based assay. Scratch wound healing assay was used to asses RC-106 effect on cell migration. In addition, we delineated the in vivo pharmacokinetic profile and pancreas distribution of RC-106 in male CD-1 mice.Results: Panc-1, Capan-1, and Capan-2 express both SRs. RC-106 exerts an antiproliferative and pro-apoptotic effect in all examined cell lines. Cells exposure to RC-106 induces the increase of the expression of ER-UPR related proteins, and the inhibition of proteasome activity. Moreover, RC-106 is able to decrease PC cell lines motility. The in vivo results show that RC-106 is more concentrated in pancreas than plasma.Conclusion: Overall, our data evidenced that the pan-SR modulator RC-106 is an optimal candidate for in vivo studies in animal models of PC.

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

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

Long non-coding RNA CASC2 upregulates PTEN to suppress pancreatic carcinoma cell metastasis by downregulating miR-21

BackgroundThe mechanism of pancreatic cancer metastasis remains poorly understood. Recently, lncRNA CASC2 has been demonstrated to be a tumor suppressor in various types of cancer. This study aimed to explore the mechanism of CASC2 in the regulation of pancreatic cancer metastasis.MethodsThe expression levels of CASC2 and miR-21 in pancreatic cells were detected by qRT-PCR. Using specific expression vectors, including mimics or shRNA, the expression levels of CASC2, miR-21 and PTEN in pancreatic cells were altered. The association between CASC2, miR-21 and PTEN was detected. Then, cell migration and invasion were assessed using the transwell assay.ResultsCASC2 expression was downregulated in the pancreatic cancer cell lines CAPAN-1, BxPC-3, JF305, PANC-1 and SW1990 compared with levels in normal human pancreatic HPDE6-C7 cells. CACS2 overexpression inhibited the migration and invasion of PANC-1 cells and significantly inhibited the expression of miR-21 and PTEN. MiR-21 was a direct target of CACS2. The overexpression of miR-21 significantly abolished the antimetastatic effects of CASC2 on PANC-1 cells. Moreover, the downregulation of PTEN significantly abolished the antimetastatic effects of CASC2.ConclusionCASC2 functions as a tumor suppressor in pancreatic cancer cells to inhibit tumor cell migration and invasion. Our work revealed a novel regulatory mechanism of the CASC2/miR-21/PTEN axis that may be important in pancreatic cancer.

Anti-CD137 monoclonal antibody enhances trastuzumab-induced, natural killer cell-mediated cytotoxicity against pancreatic cancer cell lines with low human epidermal growth factor-like receptor 2 expression.

Because human epidermal growth factor-like receptor (HER) 2 is expressed on the surface of human pancreatic carcinoma cells to varying degrees, trastuzumab, an anti-HER2 monoclonal antibody (mAb), is expected to exert antibody-dependent, natural killer (NK) cell-mediated cytotoxicity (ADCC) against the cells. However, some reports found that the effect of trastuzumab against human pancreatic carcinoma cells was limited because most express only limited HER2. We examined whether anti-CD137 stimulating mAb could enhance trastuzumab-mediated ADCC against Panc-1, a human pancreatic cancer cell line with low HER2 expression, in vitro. Supplementation of anti-CD137 mAb could improve trastuzumab-mediated ADCC against Panc-1 which was insufficient without this stimulating antibody. The ADCC differed in individual cells, and this was related to the expression of CD137 on the surface of NK cells after trastuzumab stimulation in association with the Fcγ-RIIIA polymorphism. NK cells with Fcγ-RIIIA-VV/VF showed high levels of ADCC against Panc-1, but those with Fcγ-RIIIA-FF did not show optimal ADCC. In addition, trastuzumab-mediated ADCC against the human pancreatic cancer cell line Capan-1 with high HER2 expression was generally high and not affected by the Fcγ-RIIIA polymorphism. These results demonstrated that in Fcγ-RIIIA-VV/VF-carrying healthy individuals, trastuzumab plus αCD137 mAb could induce effective ADCC against HER2-low-expressing pancreatic cancer cell lines, and that such an approach may result in similar findings in patients with pancreatic cancer.

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

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

Identification of PGAM1 as a putative therapeutic target for pancreatic ductal adenocarcinoma metastasis using quantitative proteomics.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive gastrointestinal cancer characterized by an extremely low survival rate because of early metastasis. Identifying satisfactory therapeutic targets associated with metastasis is crucial to improve the treatment effect of PDAC.Materials and methodsIn this research, we used stable isotope labeling by amino acids in cell culture, 1-dodecyl-3-methylimidazolium chloride-assisted sample preparation method preparing protein sample and nano-reversed-phase liquid chromatography-mass spectrometry/mass spectrometry analysis to perform the comparative proteomics of two homologous hamster pancreatic cancer cell lines that are different in metastatic ability: PC-1.0 (highly metastatic) and PC-1 (weakly metastatic). Verifications are through immunohistochemistry on clinical human PDAC pathologic tissues as well as by Western blot of human pancreatic cancer cell lines. siRNA silencing methods were used to study the effect of molecules on invasion and metastasis of pancreatic cancer cell lines.ResultsBioinformatic analysis indicated that a total of 141 differentially expressed proteins (82 upregulated and 59 downregulated in PC-1.0 cells) were identified showing obviously differential expression (>1.5-fold change). These differentially expressed proteins were involved in a number of different biologic functions, metabolic pathways, and pathophysiologic processes. Phosphoglycerate mutase 1 (PGAM1) and HSPE1 are the top two upregulated proteins, and PDIA3 and CALR are the top two downregulated proteins in PC-1.0 cells compared to PC-1 cells. PGAM1 and HSPE1 showed higher expressions in PDAC tissue with clinical metastasis and highly metastatic pancreatic cancer cell lines PC-1.0 and Aspc-1. PDIA3 and CALR showed higher expressions in weakly metastatic pancreatic cancer cell lines PC-1 and Capan-2. The Western blot results were consistent with the MS quantification data. Silencing PGAM1 was found to decrease the migration and invasion of pancreatic cancer cell lines with statistical significance, especially in highly metastatic PC-1.0 and Aspc-1 cell lines.ConclusionThese data indicated that PGAM1 may be a potential therapeutic target for PDAC metastasis.

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice

AimsThe prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths.In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. MethodsComparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. ResultsHere we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. ConclusionsTogether our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.

Ursodeoxycholic acid suppresses epithelial-mesenchymal transition and cancer stem cell formation by reducing the levels of peroxiredoxin II and reactive oxygen species in pancreatic cancer cells.

Reactive oxygen species (ROS) play a key role in cancer development and progression. Ursodeoxycholic acid (UDCA) may possess antioxidant, anti-inflammatory and chemoprophylatic effects. Therefore, we aimed to investigate the effects and mechanisms of UDCA treatment on pancreatic cancer cells. The pancreatic cancer cell lines HPAC and Capan-1 were treated with 0.2mM UDCA. To examine alterations in the levels of intracellular ROS, the DCF-DA stain was used and both stemness and epithelial-mesenchymal transition (EMT)-related genes were quantified using qRT-PCR and western blot analysis. The pancreatic cancer sphere culture was performed following seven days of treatment with 0.2mM UDCA, as an indicator of stemness. Following treatment with UDCA, the level of intracellular ROS was decreased in the pancreatic cancer cells. UDCA decreased both the phosphorylation of STAT3 and the expression of peroxiredoxin II (Prx2). Furthermore, the treatment resulted in the upregulation of E-cadherin and in the downregulation of N-cadherin. In addition, UDCA decreased the expression of sex determining region Y-box 2 (Sox2) and it diminished the number of pancreatic cancer spheres formed. In conclusion UDCA suppressed the levels of intracellular ROS and Prx2 and it decreased EMT and stem cell formation in pancreatic cancer cells. Therefore, UDCA may provide favorable therapeutic benefits, through its antioxidant effects, for patients with pancreatic cancer.

Paeoniflorin suppresses pancreatic cancer cell growth by upregulating HTRA3 expression.

Paeoniflorin (PF) is an active monoterpene glycoside extracted from Paeonia lactiflora Pall. PF has exhibited antitumor effects in various cancer types. However, the effects of PF in pancreatic cancer are largely unexplored. Here, we showed that PF suppressed growth of pancreatic cancer cell lines Capan-1 and MIAPaCa-2 and profoundly sensitized these cells to X-ray irradiation. Through microarray analysis, we identified HTRA3, a tumor-suppressor candidate gene, as the most increased gene upon PF treatment in Capan-1 cells. Ectopic expression of HTRA3 led to reduced cell proliferation and increased expression of apoptotic protein Bax, suggesting a tumor suppressive role of HTRA3 in pancreatic cancer cells. Together, our results provide a set group of genetic proofs and biological proofs that PF inhibited pancreatic cancer growth by upregulating HTRA3.