BxPC-3 Pancreatic Cancer Cells Research Articles

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.

HLA-B influences integrin beta-1 expression and pancreatic cancer cell migration

Human leukocyte antigen (HLA) class I molecules present antigenic peptides to cytotoxic T cells, causing lysis of malignant cells. Transplantation biology studies have implicated HLA class I molecules in cell migration, but there has been little evidence presented that they influence cancer cell migration, a contributing factor in metastasis. In this study, we examined the effect of HLA-B on pancreatic cancer cell migration. HLA-B siRNA transfection increased the migration of the S2-013 pancreatic cancer cells but, in contrast, reduced migration of the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. Integrin molecules have previously been implicated in the upregulation of pancreatic cancer cell migration, and knockdown of HLA-B in S2-013 cells heightened the expression of integrin beta 1 (ITGB1), but in the PANC-1 and MIA PaCa-2 cells HLA-B knockdown diminished ITGB1 expression. A transmembrane sequence in an S2-013 HLA-B heavy chain matches a corresponding sequence in HLA-B in the BxPC-3 pancreatic cancer cell line, and knockdown of BxPC-3 HLA-B mimics the effect of S2-013 HLA-B knockdown on migration. In total, our findings indicate that HLA-B influences the expression of ITGB1 in pancreatic cancer cells, with concurrent distinctions in transmembrane sequences and effects on the migration of the cells.

NOSH-aspirin (NBS-1120) inhibits pancreatic cancer cell growth in a xenograft mouse model: Modulation of FoxM1, p53, NF-κB, iNOS, caspase-3 and ROS

Pancreatic cancer has poor survival rates and largely ineffective therapies. Aspirin is the prototypical anti-cancer agent but its long-term use is associated with significant side effects. NOSH-aspirin belongs to a new class of anti-inflammatory agents that were designed to be safer alternatives by releasing nitric oxide and hydrogen sulfide. In this study we evaluated the effects of NOSH-aspirin against pancreatic cancer using cell lines and a xenograft mouse model. NOSH-aspirin inhibited growth of MIA PaCa-2 and BxPC-3 pancreatic cancer cells with IC50s of 47 ± 5, and 57 ± 4 nM, respectively, while it did not inhibit growth of a normal pancreatic epithelial cell line at these concentrations. NOSH-aspirin inhibited cell proliferation, caused G0/G1 phase cycle arrest, leading to increased apoptosis. Treated cells displayed increases in reactive oxygen species (ROS) and caspase-3 activity. In MIA PaCa-2 cell xenografts, NOSH-aspirin significantly reduced tumor growth and tumor mass. Growth inhibition was due to reduced proliferation (decreased PCNA expression) and induction of apoptosis (increased TUNEL positive cells). Expressions of ROS, iNOS, and mutated p53 were increased; while that of NF-κB and FoxM1 that were high in vehicle-treated xenografts were significantly inhibited by NOSH-aspirin. Taken together, these molecular events and signaling pathways contribute to NOSH-aspirin mediated growth inhibition and apoptotic death of pancreatic cancer cells in vitro and in vivo.

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.

MicroRNA-608 promotes apoptosis via BRD4 downregulation in pancreatic ductal adenocarcinoma.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) remains poor even among patients with the same Tumor-Node-Metastasis stage. Thus, it is necessary to identify biomarkers that can accurately predict outcomes. There is accumulating evidence suggesting that microRNA (miR) expression influences overall survival (OS) time in patients with PDAC, via the regulation of tumor suppressor genes and oncogene expression. Specifically, miR-608 expression is hypothesized to regulate PDAC progression via the downregulation of bromodomain-containing protein 4 (BRD4) expression and the promotion of cell apoptosis. The present study aimed to investigate this theory. Thus, whole genome expression microarray analysis was performed on three patient samples with OS time >30 months, and compared with three samples with <12 months, in order to identify differentially expressed miRNAs (DEMs), via EdgeR analysis. A total of 591 DEMs were identified that exhibited a fold change >1, including 390 upregulated and 201 downregulated genes. Subsequently, 10 DEMs were identified using quantitative PCR in a different population of 68 tissues, collected from patients with PDAC. Notably, a high level of miRNA-608 expression was associated with longer OS times (P<0.05). Bioinformatics analysis was then performed to predict the molecular mechanism underlying the regulation of cell apoptosis by miRNA-608, and a dual-luciferase assay determined that overexpression of mimics in the Panc-1 and Bxpc-3 pancreatic cancer cell lines increased levels of apoptosis compared with the control. Additionally, high miRNA-608 expression decreased the protein level of BRD4. A luciferase reporting assay was used to elucidate whether miRNA-608 may directly inhibit the expression of BRD4 by binding to the 3′-untranslated region of its mRNA in the same cell lines. A subsequent rescue experiment indicated that the upregulation of BRD4 may reverse the apoptosis-promoting effect induced by miRNA-608. In summary, the present study revealed that miRNA-608 promotes apoptosis in PDAC via the negative regulation of BRD4. The results of the present study provide a theoretical basis that may improve the prediction of prognosis in patients with PDAC, and also indicate an opportunity to develop individualized treatment and investigate novel therapeutics that target these mechanisms.

Abstract B095: Novel anti-CD147 antibodies inducing activation of SMAD signaling and tumor shrinkage in intractable cancer models

Abstract CD147, a type I membrane protein, is highly expressed in various cancers and is involved in the growth, metastasis, and activation of inflammatory signals in cancer cells via interaction with various molecules, such as integrins, CD44, and monocarboxylate transporters. Additionally, the expression of CD147 in tumors positively correlates with poor prognosis in various cancers. Therefore, CD147 is an attractive target for cancer therapy. However, there have been no successful drugs targeting CD147 so far, possibly because it is a multifunctional protein interacting with multiple molecules, making it challenging to discover a relevant CD147-targeting drug candidate by general in vitro screening methodologies. In this study, we developed novel anti-CD147 antibodies, which showed significant antitumor efficacy in immune-deficient mice bearing human pancreatic cancer cell lines, PANC-1 or MIA PaCa-2. Six anti-CD147 antibodies showing in vivo anti-tumor efficacy were obtained; one mouse IgG3, two rat IgG2b, two rat IgG1, and one rat IgG2a antibodies. Analysis of antibody dependent cellular toxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity of chimeric and humanized antibodies with human IgG2 or IgG4P derived from the obtained 6 anti-CD147 antibodies, #147A/B/C/D/E/F, suggested no significant immune-effector function for these antibodies. Despite lacking effector function, these antibodies exhibited significant in vivo antitumor activity, suggesting that the mechanism underlying their anti-tumor efficacy did not involve their effector function. We also found that the anti-tumor efficacy of antibodies #147A and #147B positively correlated with SMAD4 protein expression rather than CD147 protein expression in 10 pancreatic cancer xenograft models (correlation coefficient of #147B: anti-tumor efficacy vs. CD147, 0.245; vs. SMAD4, 0.9328). Moreover, the administration of anti-CD147 antibodies upregulated rhoB expression in an anti-CD147 antibody-sensitive SMAD4-positive MIA PaCa-2 graft mouse model. The stable expression of SMAD4 increased the sensitivity to the anti-CD147 antibody, ch4#147A (chimeric IgG4P derived from #147A) in xenograft mice inoculated with the BxPC-3 pancreatic cancer cell line, which is originally negative for SMAD4 and has low sensitivity to anti-CD147 antibodies. These data suggest that the expression of SMAD4 and activation of SMAD signaling are functionally important for susceptibility to anti-CD147 antibody treatment. The humanized anti-CD147 IgG4P antibody, h4#147D, derived from #147D with cross reactivity to monkey CD147, showed superior anti-tumor efficacy with complete tumor reduction, compared to standard anticancer drugs, including gemcitabine, imatinib, and sorafenib, in xenograft mice bearing corresponding cancer cell lines, MIA PaCa-2, KU812 chronic myeloid leukemia, and HepG2 liver cancer cell lines, respectively. No toxicity of h4#147D was observed in cynomolgus monkeys at a single dose of 100 mg/kg. These data suggest that h4#147D might be a novel anti-CD147 antibody, which could induce SMAD activation and tumor shrinkage in multiple xenograft models. Therefore, it has potential as a promising antitumor therapeutic antibody with superior anti-tumor efficacy to existing therapy. Citation Format: Keisuke Fukuchi, Kayoko Nanai, Shoji Yamamoto, Jun Tsukada, Yusuke Totoki, Koichiro Inaki, Masato Ishigami, Naoyuki Makita, Yoko Nakano, Chigusa Yoshimura, Kozo Yoneda, Masato Amano, Kensuke Nakamura, Yoshiyuki Kanari, Yoko Oda, Haruyuki Nishigohri, Rika Nakano, Atsuko Nishida, Kenji Murakami, Yumi Matsui, Naomi Kasanuki, Shoji Midori, Satoko Funo, Sayako Takahashi, Hironobu Komori, Toshiaki Ohtsuka, Toshinori Agatsuma. Novel anti-CD147 antibodies inducing activation of SMAD signaling and tumor shrinkage in intractable cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B095. doi:10.1158/1535-7163.TARG-19-B095

Therapeutic Potential of LNP-Mediated Delivery of miR-634 for Cancer Therapy

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that negatively regulate gene expression by interfering with the translation or stability of target transcripts. Some tumor-suppressive miRNAs can concurrently target multiple cancer-promoting genes and may be useful as therapeutic anticancer agents. However, the development of drug delivery systems is critical for the implementation of miRNA-based therapeutics. We have previously demonstrated that the enforced expression of miR-634 effectively induces apoptosis by concurrently and directly targeting genes associated with mitochondrial homeostasis, antiapoptosis signaling, antioxidant ability, and autophagy in cancer cells. In the current study, we validated the therapeutic potential of lipid nanoparticle (LNP)-mediated delivery of miR-634 for cancer therapy. We confirmed the ability of enforced expression of miR-634 to induce apoptosis in various cancer cell lines, including pancreatic cancer cells. Intravenous administration of LNPs harboring miR-634 significantly reduced the xenograft tumor growth of BxPC-3 pancreatic cancer cells in mice. These findings suggest that LNP-mediated delivery of miR-634 can potentially be used for cancer therapy.

Verteporfin- and sodium porfimer-mediated photodynamic therapy enhances pancreatic cancer cell death without activating stromal cells in the microenvironment.

.The goal of our study was to determine the susceptibility of different pancreatic cell lines to clinically applicable photodynamic therapy (PDT). The efficacy of PDT of two different commercially available photosensitizers, verteporfin and sodium porfimer, was compared using a panel of four different pancreatic cancer cell lines, PANC-1, BxPC-3, CAPAN-2, and MIA PaCa-2, and an immortalized non-neoplastic pancreatic ductal epithelium cell line, HPNE. The minimum effective concentrations and dose-dependent curves of verteporfin and sodium porfimer on PANC-1 were determined. Since pancreatic cancer is known to have significant stromal components, the effect of PDT on stromal cells was also assessed. To mimic tumor–stroma interaction, a co-culture of primary human fibroblasts or human pancreatic stellate cell (HPSCs) line with PANC-1 was used to test verteporfin-PDT-mediated cell death of PANC-1. Two cytokines (TNF- and IL-) were used for stimulation of primary fibroblasts (derived from human esophageal biopsies) or HPSCs. The increased expression of smooth muscle actin (-SMA) confirmed the activation of fibroblasts or HPSC upon treatment with TNF- and IL-. Cell death assays showed that both sodium porfimer- and verteporfin-mediated PDT-induced cell death in a dose-dependent manner. However, verteporfin-PDT treatment had a greater efficiency with lower concentration than sodium porfimer-PDT in the PANC-1 incubated with stimulated fibroblasts or HPSC. Moreover, activation of stromal cells did not affect the treatment of the pancreatic cancer cell lines, suggesting that the effects of PDT are independent of the inflammatory microenvironment found in this two-dimensional culture model of cancers.

Carboxypeptidase E-∆N Promotes Proliferation and Invasion of Pancreatic Cancer Cells via Upregulation of CXCR2 Gene Expression.

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2.

Robo1-specific CAR-NK Immunotherapy Enhances Efficacy of 125I Seed Brachytherapy in an Orthotopic Mouse Model of Human Pancreatic Carcinoma.

The aim of the current study was to investigate the synergistic efficacy of Robo1 bichimeric antigen receptor-natural killer cell (BiCAR-NK) immunotherapy and 125I seed brachytherapy in an orthotopic pancreatic cancer mouse model. The orthotopic pancreatic tumor model was established with human pancreatic cancer BxPC-3 cells expressing red fluorescent protein. The mice were treated with 125I seed implantation alone or the combination of 125I seeds with Robo1-specific CAR-NK cells. To assess tumor inhibition, in vivo fluorescence imaging was conducted. 7 Tesla magnetic resonance (7T-MR) scanning was applied to measure the changes in the metabolic profiles of tumor tissues. Tumor size was significantly reduced in the 125I and 125I +CAR-NK treated group compared to the untreated group (p<0.05). The 125I seed +CAR-NK treated group showed significantly higher tumor reduction than 125I seed treatment alone (p<0.05). T1 diffusion weighted imaging (T1DWI) sequence showed that the tumors of the 125I +BiCAR-NK treated group had a significantly higher grey scale value than the tumors from the untreated control and the group treated with 125I seed alone (p<0.05). Robo1 specific CAR-NK immunotherapy enhances efficacy of 125I seed brachytherapy in an orthotopic pancreatic cancer mouse model.

The RNA m6A methyltransferase METTL3 promotes pancreatic cancer cell proliferation and invasion

Epigenetic modifications are involved in carcinogenesis and METTL3 is involved in RNA methylation. This study aimed to explore the role of the RNA m6A methyltransferase METTL3 in pancreatic cancer cells. The m6A modification was analyzed in human pancreatic cancer and paracancerous specimens, as well as in the normal HPDE6-C7 pancreatic cell line and the MIA-PaCa-2 and BxPC-3 pancreatic cancer cell lines. Immunohistochemistry (IHC), western blotting, and RT-qPCR were used to detect the expression of METTL3. Cell lines were transfected with siRNAs against METLL3. Proliferation, invasion, and migration were examined. The functions of METTL3 were predicted by bioinformatics analysis. In the 40 patients included, high METTL3 expression was associated with high pathological stage (P = 0.02) and high N stage (P = 0.02). Survival was better in patients with low METTL3 expression compared with those with high MTTL3 expression (P < 0.01). METTL3 and CIITA expression levels were inversely correlated (r = −0.71, P < 0.01). RNA m6A content in tumor specimens was significantly higher than that in paracancerous specimens. METTL3 protein and mRNA levels were significantly higher in tumor specimens compared with paracancerous specimens, as well as in cancerous cell lines vs. normal cells. METTL3 knockdown in MIA PaCa-2 and BxPC-3 cells decreased RNA m6A modifications. Cell proliferation, invasion, and migration were decreased by METTL3 knockdown in cancerous cell lines. A total of 673 differentially expressed genes were identified by bioinformatics: 659 were upregulated and 14 were downregulated. In conclusion, METTL3 is probably involved in pancreatic carcinogenesis. It could eventually be a prognostic marker or a treatment target.

Extracellular vesicles derived from pancreatic cancer cells BXPC3 or breast cancer cells MCF7 induce a permanent procoagulant shift to endothelial cells

The endothelium could be a potential target of cancer cell derived extracellular vesicles (CaCe-dEV). We investigated in vitro the effect of CaCe-dEV on the hemostatic balance of endothelial cells. Extracellular vesicles released from pancreas adenocarcinoma cells (BXPC3) or human breast cancer cells (MCF7) were isolated by differential centrifugation. Human umbilical vein endothelial cells (HUVEC) were cultured for 72 h in the presence or absence of CaCe-dEV. Subsequently, they were washed and re-cultivated over three cycles to get daughter cell generations (DG) which were not exposed to CaCe-dEV. Thrombin generation of normal platelet poor plasma (PPP) added in wells carrying HUVEC was assessed by the Calibrated Automated Thrombogram®. Tissue factor activity (TFa) and procoagulant phospholipid clotting time were assessed.Some traces of TFa were displayed by non-exposed HUVEC (0.18 ± 0.03 pM) and their EVs (1.2 ± 1.0 pM). Non-exposed HUVEC did not induce any detectable thrombin generation. BXPC3-dEV displayed significantly higher TFa as compared to MCF7-dEV (45 ± 5 pM versus 4.6 ± 2.3pM respectively; p < 0.05). HUVEC exposed to CaCe-dEV enhanced thrombin generation. BXPC3-dEV induced significantly higher thrombin generation as compared to those exposed to MCF7-dEV. The procoagulant properties of HUVEC, acquired upon exposure to CaCe-dEV were transferred to DG.In conclusion, CaCe-dEV lead to a procoagulant shift of endothelial cells which, upon exposure, display TFa and enhance thrombin generation which is transferred to DG of HUVEC. The potency of CaCe-dEV to induce procoagulant shift of HUVEC depends on the histological type of the cancer cells. The procoagulant shift of endothelial cells which is transferable to DG could be an additional mechanism - together with cancer-induced blood hypercoagulability - in the pathogenesis of cancer associated thrombosis.

Dauricine suppresses the growth of pancreatic cancer in vivo by modulating the Hedgehog signaling pathway.

Pancreatic cancer is a highly malignant cancer associated with high expression levels of sonic hedgehog signaling molecule (Shh), patched 1 (Ptch1), smoothened frizzled class receptor (Smo) and glioma-associated oncogene family zinc finger 1 (Gli1) in the hedgehog (Hh) signaling pathway. Inhibition of the Hh signaling pathway is a potential therapeutic target for pancreatic cancer. The aim of the present study was to investigate the effects of dauricine in a pancreatic cancer BxPC-3 ×enograft animal model and examine the underlying molecular mechanisms through Hh signaling pathway. High-and low-dose dauricine treatment significantly suppressed tumor growth with no concomitant effect on the spleen index. In addition, dauricine induced apoptosis and cell cycle arrest in pancreatic cancer BxPC-3 cells. The inhibitory effects of dauricine on pancreatic cancer may be mediated by the suppression of the Hh signaling pathway, as indicated by the decreases in the gene and protein expression levels of Shh, Ptch1, Smo and Gli1. The effects of dauricine were similar to those of 5-fluorouracil. Dauricine, a naturally occurring alkaloid, may be a potential anticancer agent for the treatment of pancreatic cancer.

The long coding RNA GSTM3TV2 acts an oncogene to promote chemoresistance in pancreatic cancer

Objectives: To examine the expression of the long coding RNA GSTM3TV2 in pancreatic cancer tissues and to examine its role and mechanism in chemoresistance of pancreatic cancer cells. Methods: The expression of lncRNA GSTM3TV2 in 15 pancreatic cancer specimens and corresponding adjacent to cancer tissue samples diagnosed by Department of Pathology, Peking Union Medical College Hospital was detected by real-time PCR.And the expressions of GSTM3TV2 in pancreatic cancer cell AsPC-1, BxPC-3, MIAPaCa-2, PanC-1, SU86.86, T3M4, and chemoresistant cells AsPC-1/GR and MIAPaCa-2/GR, and human pancreatic nestin-expressing cells hTERT-HPNE were detected. Pancreatic cancer cell lines were transfected with GSTM3TV2-pcDNA3.1(+)in order to get cells with GSTM3TV2 overexpression.GSTM3TV2-siRNA was transfected into pancreatic cancer cells to knock down GSTM3TV2. The cell chemoresistance was measured by CCK-8 and flow cytometry assay when incubated with nab-paclitaxel. At the same time, subcutaneous xenograft tumor models were established in nude mice to observe the effect of GSTM3TV2 on chemoresistance of tumor growth in nude mice.Western blot assay was also performed to detect the molecular mechanism of chemoresistance of GSTM3TV2. Results: Comparing toadjacent tissues(0.084±0.019), GSTM3TV2 expression was significantly upregulated in the pancreatic cancer tissues(0.493±0.084) (t=5.146, P<0.05). GSTM3TV2 expression were higher in the chemotherapy resistance pancreatic cancer cells AsPC-1/GR(210.799±19.788) and MIAPaCa-2/GR(122.408±23.419) than that in the AsPC-1(3.793±0.615) and the MIAPaCa-2(5.179±1.095)(t=21.800,P<0.05;t=-18.490,P<0.05). The results of in vivo experiments showed that the volume of subcutaneously transplanted tumors in the overexpressing GSTM3TV2 group ((1 059.609±102.498)mm(3)) was significantly larger than that in the control group((566.414±81.087) mm(3)) by treated with nab-paclitaxel(t=4.230,P<0.05).Meanwhile, GSTM3TV2 could promote the expression of Cyclin D1, CDK6, Cyclin E1, Vimentin, N-cadherin, ZEB1, Snail and Slug; but decrease cleaved caspase-3, cleaved PARP in pancreatic cancer cells. Conclusions: The expression level of GSTM3TV2 in pancreatic canceris higher than that in paired adjacent tissues. GSTM3TV2 may act as an oncogene to promote chemoresistance in pancreatic cancer through regulation of cell proliferation, apoptosis, and epithelial-mesenchymal transition.

Silencing NEDD9 by lentivirus-delivered shRNA inhibits the growth of BxPC-3 cells in vitro and in vivo.

BackgroundNeural precursor cell-expressed, developmentally downregulated protein 9 (NEDD9) is an invasion and metastasis-related gene. It has been proven to be highly expressed and closely associated with tumor proliferation and invasion in several types of human cancers including pancreatic adenocarcinoma. The present study was aimed to investigate and characterize the efficacy of silencing NEDD9 by lentivirus-delivered shRNA in pancreatic cancer (PC) BxPC-3 cells in vivo and in vitro.MethodsFive kinds of PC cell lines were used to determine the cell line which expressed NEDD9 the most with qRT-PCR and western blotting. Then, we transduced the lentivirus-delivered NEDD9 shRNA into the human PC BxPC-3 cells to obtain a stable cell line expressing shRNA targeting NEDD9. NEDD9 mRNA and protein expression were measured by qRT-PCR and western blotting, respectively. Cell proliferation, migration, and invasion were assessed by cell colony formation, scratch wound healing, and Transwell assays, respectively. Mouse tumor xenografts were established by injecting tumor cells into the right flank of BALB/c nude mice. The effects of silencing NEDD9 on the growth of BxPC-3 cells in vivo were also examined.ResultsAmong 5 kinds of PC cell lines, BxPC-3 cells were selected as the most suitab to carry out the following experiment. Transduction of lentivirus-delivered NEDD9 shRNA efficiently reduced NEDD9 expression in pancreatic adenocarcinoma BxPC-3 cells. Silencing NEDD9 by RNAi inhibited proliferation, migration, and invasion of BxPC-3 cells in cell culture. Importantly, it significantly reduced the growth of BxPC-3 cells in mouse xenografts.ConclusionsSilencing NEDD9 by lentivirus-delivered shRNA efficiently inhibited the growth of PC BxPC-3 cells both in vitro and in vivo, and may prove to be a potential new therapeutic agent for human PC.

Irisin Enhances Doxorubicin-Induced Cell Apoptosis in Pancreatic Cancer by Inhibiting the PI3K/AKT/NF-κB Pathway.

BackgroundIrisin, a myokine released from skeletal muscle following exercise, has been shown to affect the proliferation of some cancer cells and chemosensitivity of anticancer drugs like doxorubicin (DOX). However, the effects of irisin on chemosensitivity in pancreatic cancer (PC) cells have not been studied.Material/MethodsIn this study, the effects of irisin co-treatment with DOX or gemcitabine (GEM) on MIA PaCa-2, BxPC-3 PC cells, and H9c2 cardiomyocytes were investigated. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, and TUNEL (TdT-mediated dUTP nick-end labeling) assays were conducted to evaluate cytotoxicity induced by DOX or GEM. Fluorescence microscopy and flow cytometry experiments were performed to assess the intracellular accumulation of DOX. Cellular levels of apoptosis-related protein expression and protein phosphorylation were determined by Western blot analyses.ResultsThe results showed that irisin can increase the chemosensitivity of PC cells to DOX or GEM. The analyses of apoptosis indicated that irisin enhances DOX-induced cellular apoptosis by increasing the expression of cleaved PARP (poly ADP-ribose polymerase) and cleaved caspase-3, and reducing the expression of B cell lymphoma/lewkmia-2 (BCL-2) and B cell lymphoma-extra large (BCL-xL) in PC cells but not in H9c2 cells. Irisin attenuated serine/threonine kinase AKT (protein kinase B/PKB) phosphorylation and inhibited the activation of nuclear factor κB (NF-κB) signaling in PC cells.ConclusionsIrisin can potentiate the cytotoxicity of doxorubicin in PC cells without increasing cardiotoxicity, possibly through inactivating the PI3K/AKT/NF-κB signaling pathway.

Preparation of Dual-Template Epitope Imprinted Polymers for Targeted Fluorescence Imaging and Targeted Drug Delivery to Pancreatic Cancer BxPC-3 Cells.

Molecularly imprinted polymers were commonly used for drug delivery. However, single-template molecularly imprinted polymers often fail to achieve both drug delivery and precise targeting. To address this issue, a dual-template molecularly imprinted polymer nanoparticle used for targeted diagnosis and drug delivery for pancreatic cancer BxPC-3 cells (FH-MIPNPs) was prepared. In the FH-MIPNPs, the 71-80 peptide of human fibroblast growth-factor-inducible 14 modified with glucose (Glu-FH) and bleomycin (BLM) were used as templates simultaneously, so that the FH-MIPNPs could load BLM and bind to the BxPC-3 cells, which overexpress human fibroblast growth-factor-inducible 14 (FN14). Targeted imaging experiments in vitro show that the FH-MIPNPs could specifically target BxPC-3 cells and that there is no targeting effect on cells without expression of FN14. In vivo antitumor experiment results demonstrated that the FH-MIPNP-loaded BLM (FH-MIPNPs/BLM) could inhibit the growth of xenografts tumor of BxPC-3 (tumor volume increased to 1.05×), which shows that FH-MIPNPs/BLM had obvious targeted therapeutic effect compared to the other three control groups of BLM, FH-NIPNPs/BLM, and physiological saline (tumor volume increased to 1.5×, 1.6×, and 2.4×, respectively). What is more, FH-MIPNPs have low biotoxicity through toxicity experiments in vitro and in vivo, which is favorable toward making molecularly imprinted polymers an effective platform for tumor-targeted imaging and therapy.

MiR-203 regulates JAK-STAT pathway in affecting pancreatic cancer cells proliferation and apoptosis by targeting SOCS3.

Janus kinase (JAK)- signal transducer and transcriptional activator (STAT) pathway overactivation is closely related to tumorigenesis. Cytokine signal transduction inhibitor 3 (SOCS3) is a negative regulator of JAK-STAT. It is shown that miR-203 is significantly elevated in the pancreatic cancer tissues. The bioinformatics analysis revealed a targeted binding site between miR-203 and the 3'-UTR of SOCS3 mRNA. This study investigated the role of miR-203 in regulating SOCS3 expression and the proliferation and apoptosis of the pancreatic cancer cells. Quantitative Real Time-PCR (qRT-PCR) was used to detect the expressions of miR-203 and SCOS3 mRNA in tumor tissues and paracancerous tissues. The Dual-Luciferase reporter gene assay was adopted to validate the target interaction between miR-203 and SOCS3. The PANC-1 cells were cultured in vitro and divided into miR-NC group and miR-203 inhibitor group followed by an analysis of the expressions of SOCS3, p-JAK2, and p-STAT3, cell apoptosis by flow cytometry, and cell proliferation by EdU staining. Compared with the adjacent tissues, miR-203 expression was significantly increased, while SOCS3 mRNA level was significantly declined in the tumor tissues of pancreatic cancer patients. There was a targeted regulatory relationship between miR-203 and SOCS3 mRNA. Compared with those in HPDE6-C7 cells, miR-203 level was upregulated, whereas SOCS3 mRNA and the protein expressions were reduced in pancreatic cancer PANC-1 and BXPC3 cells. The transfection of miR-203 inhibitor significantly increased SOCS3 mRNA and the protein levels, decreased p-JAK2 and p-STAT3 protein expressions, enhanced cell apoptosis, and inhibited cell proliferation in the pancreatic cancer PANC-1 cells. Increased miR-203 expression and reduced SOCS3 level are associated with the pathogenesis of pancreatic cancer. MiR-203 can regulate the proliferation and apoptosis of the pancreatic cancer cells by targeting the inhibited SOCS3 expression and regulating the JAK-STAT pathway activity.

Anti-tumor effect and mechanistic study of elemene on pancreatic carcinoma

BackgroundElemene is an effective anticancer component extracted from Zingiberaceae plants. This work was aimed to evaluate the anti-tumor effect and mechanism actions of elemene on pancreatic carcinoma in vitro and in vivo.MethodsThe anti-proliferation experiment was measured by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) method in the time of 24, 48 and 72 h in three different dosages. The cell cycle was detected by flow cytometer after 12 h treatment. Forty-eight nude mice were subcutaneously xenograft with BxPC-3 pancreatic cancer cells and divided into four groups: Control group and high, medium, low dosage of elemene (20, 40 and 60 mg/kg) treatment groups. Immunoblot and immunohistochemical methods were applied to detect the protein expression of P53 and Bcl-2 in the tumor of pancreatic cancer xenografts. H & E staining was used to detect the histopathological changes in each group.ResultsA significant inhibition effect was observed in the anti-proliferation of BxPC-3 and Panc-1 cells in vitro in the time course of 24, 48 and 72 h with a dose dependent manner. The cell cycle results showed that elemene could arrest pancreatic cancer cells in the S phase after 12 h treatment in BxPC-3 and Panc-1 cell line. The in vivo BxPC-3 xenografts study exhibited that elemene was significantly decreased the tumor size in the high dosage group, compared to control group. And there is no any significant change in body weight of all animals. H&E pathology section result showed that treatment with elemene significantly decreased the inflammation cells and reduced the histopathological changes with a dose-dependent manner. Meanwhile, treatment with elemene significantly up-regulates the protein expression of P53, while down-regulate the protein expression of Bcl-2 in the tumor tissues, respectively. Furthermore, the western blot result showed that treatment with elemene increased the expression of P53 and decreased the expression of Bcl-2, compared with the control group, which is similar to the results of immunohistochemical staining.ConclusionsThis study suggests that elemene has a potential anti pancreatic cancer effect, down-regulation the protein expression of Bcl-2 and up-regulation the protein expression of P53 in a dose dependent manner may be is the anti-tumor mechanism.

Inhibition of EP2/EP4 prostanoid receptor-mediated signaling suppresses IGF-1-induced proliferation of pancreatic cancer BxPC-3 cells via upregulating γ-glutamyl cyclotransferase expression

Inhibition of prostaglandin E2 signaling via EP2/EP4 prostanoid receptors suppresses Insulin-like growth factor (IGF)-1-induced proliferation of pancreatic cancer BxPC-3 cells. To better understand the mechanism of EP2/EP4 signaling for controlling cell proliferation, we performed metabolome analyses in BxPC-3 cells treated with IGF-1 alone or IGF-1 plus EP2/EP4 inhibitors. These analyses revealed increased g-aminobutyric acid and 5-oxoproline production following the addition of EP2/EP4 inhibitors to IGF-1-treated cells. The expression of a 5-oxoproline-catalyzing enzyme, γ-glutamylcyclotransferase (GGCT), was also upregulated by IGF-1 treatment and further enhanced by the addition of EP2/EP4 inhibitors. Knockdown of GGCT expression resulted in the loss of suppressive effects of EP2/EP4 inhibitors on IGF-1-induced BxPC-3 cell proliferation, whereas GGCT overexpression repressed the basal proliferation of BxPC-3 cells but did not affect the suppressive effects of EP2/EP4 inhibitors. To summarize, we propose a role for EP2/EP4 signaling in regulating IGF-1-induced cell proliferation, in which EP2/EP4 signaling represses IGF-1-induced GGCT expression, which mediates and whose amount controls a branch of IGF-1 signaling to promote cell proliferation via extracellular signal-regulated kinase phosphorylation.