Pancreatic Cancer Cell Lines CFPAC-1 Research Articles

IMN041 is an immunotherapeutic and an effective treatment in mouse xenograft models of pancreatic cancer, renal cancer and triple negative breast cancer

BackgroundiMN013 (5-aza-2',2'-difluorodeoxycytidine), a DNA methyl transferase inhibitor and ribonucleotide reductase inhibitor, and its prodrug iMN041 (3',5'-di-trimethylsilyl-2',2'-difluro-5- azadeoxycytidine), have been shown to be active in mouse xenograft models of hematogenous and solid tumors. In a xenograft of non-small cell lung cancer (NCI-H460), iMN041 treated mice demonstrated a marked inflammatory response upon analysis of tumor histology, which was hypothesized to be mediated by upregulation of natural killer (NK) cells. This study aimed to characterize the antitumor immune responses generated by iMN041 and test the efficacy iMN041 in solid tumors with poor prognosis.MethodsIn the Renca syngeneic mouse model, tumors were harvested following two doses of iMN041 or vehicle control, and analyzed by fluorescent-activated cell sorting for an antitumor immune response. iMN041 was also tested for tumor growth inhibition and animal survival for up to 42 days in xenograft models of pancreatic, renal, and triple negative breast cancer.ResultsTumors from mice implanted with the Renca cell line and treated with iMN041 demonstrated an increase in granzyme B in NK (p = 0.024) and NKT cells (p = 0.004), an increase in the ratios of CD8-T to regulatory T cells (Treg) (p = 0.0026) and CD4-T to Treg cells (p = 0.022) and a decrease in myeloid-derived suppressor cells (p = 0.040), compared to vehicle controls. A significant decrease in MAGE-A positive tumor cells in treated mice, concordant with a proportional decrease in all live tumor cells, suggests that these cells are one of the main targets of the activated immune system. Xenograft models of the triple negative breast cancer cell line DU4475, renal cancer cell lines 786-O and Caki-1, and pancreatic cancer cell lines CFPAC-1 and SW1990, demonstrated significantly lower tumor volumes, and, where there were a sufficient number of events, significantly improved survival in treated mice compared to vehicle control mice.ConclusionsIn mouse cancer models, iMN041 is an effective treatment for solid tumors mediated in part through a unique antitumor immune response.

Identification of the O-Glycan Epitope Targeted by the Anti-Human Carcinoma Monoclonal Antibody (mAb) NEO-201.

Simple SummaryGlycosylation is an important post-translational modification made on mammalian proteins and lipids. In cancer cells, the disruption of several glycosylation patterns, such as the O-glycosylation, has been observed. The expression of incomplete/truncated O-glycans in cancer cells occurs in both solid and liquid tumors and is correlated with poor prognosis and tumor progression. The employment of monoclonal antibodies (mAbs) targeting truncated O-glycans in cancer cells could serve as an effective strategy to counteract tumor growth. In previous studies, we reported that the IgG1-humanized mAb NEO-201 binds specifically to tumor-associated variants of CEACAM5 and CEACAM6 expressed by colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers but is not reactive against most normal tissues. Since CEACAMs are highly glycosylated proteins, in this article, we evaluated whether the epitope recognized by NEO-201 is an O-glycan. This study demonstrated that NEO-201 binds to core 1 O-glycans and targets and kills cancer cells expressing core 1 and extended core 1 O-glycans. Usually, GalNAc residue can be added on to threonine and serine to form O-glycans, suggesting that NEO-201 binds to core 1 and extended core 1 O-glycans attached to any protein carrying amino acid regions containing serine and threonineTruncated O-glycans expressed in cancer cells support tumor progression, and they may serve as potential targets to improve the monitoring and treatment of cancers. Previously, we reported that NEO-201 binds to several tumors expressing tumor-associated CEACAM5 and CEACAM6 variants but does not bind to those expressed in healthy tissues. This specific binding may be associated with the presence of truncated O-glycans attached on the protein sequence of these variants. To evaluate the glycosylation pattern targeted by NEO-201 we performed an O-glycan array consisting of 94 O-glycans. O-glycan profiles were elucidated from the human pancreatic cancer cell line CFPAC-1, human hematological neoplastic cells (HL-60, U937, K562) and human neutrophils. The O-glycan array analysis showed that NEO-201 interacts with core 1-4 O-glycans and that the binding to a specific core 1 O-glycan was the strongest. The O-glycan profiling of the NEO-201-reactive cells CFPAC-1, HL-60, U937 and human neutrophils showed that cells recognized by NEO-201 express mostly core 1 and/or extended core 1 O-glycans. In addition, NEO-201 mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells expressing core 1 or extended core 1 O-glycan profiles. These results demonstrated that NEO-201 binds to core 1 and extended core 1 O-glycans expressed in its target cells. Since GalNAc residue can be added onto threonine and serine to form O-glycans, it is very likely that NEO-201 recognizes these O-glycans attached to any protein with amino acid regions containing serine and threonine.

LncRNA LINP1 promotes malignant progression of pancreatic cancer by adsorbing microRNA-491-3p.

The purpose of this study was to explore the mechanism by which long noncoding RNA (lncRNA) LINP1 promoted the development of pancreatic cancer (PCa). Meanwhile, the regulatory relationship between lncRNA LINP1 and microRNA-491-3p was further investigated to provide an effective theoretical basis for the treatment of this cancer. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was carried out to examine lncRNA LINP1 and microRNA-491-3p expression in tumor tissue specimens collected from 56 PCa patients, and the interplay between lncRNA LINP1 expression and some clinical indicators, as well as prognosis of patients with PCa was also analyzed. Meanwhile, in vitro, qRT-PCR further verified lncRNA LINP1 level in PCa cell lines. In addition, lncRNA LINP1 knockdown model was constructed using lentivirus in PCa cell lines CFPAC-1 and BxPC-3, and Cell Counting Kit-8 (CCK-8), transwell, and cell wound healing assays were carried out to evaluate the impact of lncRNA LINP1 on the function of PCa cells. Finally, Dual-Luciferase reporting assay and cell reverse experiments were applied to uncover the potential mechanism. QRT-PCR revealed that lncRNA LINP1 showed a significantly higher expression in pancreatic tumor tissue samples than in adjacent normal ones. Compared with patients with low expression of lncRNA LINP1, patients with highly expressed lncRNA LINP1 showed a higher incidence of distant metastasis, but a lower overall survival rate. In addition, compared to the sh-NC group, the proliferation, invasion, and migration ability of PCa cells decreased remarkably in LINP1 knockdown group. The results of Luciferase reporting assay demonstrated that lncRNA LINP1 could be targeted by microRNA-491-3p through a specific binding site, and qRT-PCR results uncovered a negative correlation between microRNA-491-3p and lncRNA LINP1 expression in PCa tissues. Finally, the recovery experiment revealed a mutual regulation between LINP1 and microRNA-491-3p, which may jointly regulate the malignant progression of PCa. LncRNA LINP1 is able to enhance the proliferation and metastasis of PCa cells by modulating microRNA-491-3p, thus affecting the incidence of lymph node or distant metastasis and prognosis of patients with PCa.

Effects of sinomenine hydrochloride on proliferation, migration and invasion of human pancreatic cancer cell lines CFPAC-1 and PANC-1

Objective To investigate the effects and mechanisms of sinomenine hydrochloride (SH) on prolife ration, migration and invasion of pancreatic cancer cells. Methods Pancreatic cancer CFPAC-1 and PANC-1 cell lines were divided into three groups. The cells not treated with SH were used as a control group, and the cells treated with 0.50 mmol/L and 0.25 mmol/L of SH were used as experimental groups. The effect of cell counting kit-8 (CCK-8) and colony formation assay on cell proliferation ability was examined. The effects of wound healing and Transwell assays on cell migration and invasion. Western blotting was used to detect epithelial-mesenchymal transition (EMT)-associated protein epithelial cadherin (E-cad), neuro-cadherin (N-cad) and Vimentin (Vim). Expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 proteins in the MMPs family. Results In CCK-8 assay, SH inhibited the proliferation of cells in the 0.50 mmol/L group and 0.25 mmol/L group of CFPAC-1 and PANC-1 cells at 24 h and 48 h, and the inhibition rate were (23.97±3.31)% vs. (11.35±3.06)%, t=2.796, P<0.05; (31.62±3.30)% vs. (15.71±3.12)%, t=3.496, P<0.05; (36.09±3.64)% vs. (20.96±3.58)%, t=2.949, P<0.05; (44.80±3.90)% vs. (20.65 ± 3.53)%, t=4.586, P<0.05. The number of colonies in the CFPPAC-1 and PANC-1 0.50 mmol/L groups and the 0.25 mmol/L group were lower than those in the blank control group (13.67±0.88 vs. 49.33±2.33, t=14.300, P<0.01, 23.33±4.91). The ratio was 96.67±8.82, t=7.291, P<0.01, 28.00±1.15 to 49.33±2.33, t=8.194, P<0.01, 50.67±5.21 to 96.67±8.82, t=4.427, P<0.05. The wound healing rate of CFPPAC-1 and PANC-1 in 0.50 mmol/L group and 0.25 mmol/L group was lower than that in the blank control group [(25.33±1.63)% vs. (60.08±1.65)%, t=14.930, P<0.01, (37.55±2.69)% vs. (79.01±2.26)%, t=11.813, P<0.01, (42.49±2.05)% vs. (60.08±1.65)%, t=6.679, P<0.01, (51.67±2.36)% vs. (79.01±2.26)%, t=8.354, P<0.01]. Transwell test CFPPAC-1 and PANC-1 0.50 mmol/L , 0.25 mmol/L group were lower than the blank control group [(44.33±4.48) cells vs. (122.30±5.04) cells, t=11.561, P<0.01, (61.67±7.26) cells vs. (172.70±3.71) cells, t=13.610, P<0.01, (80.33±3.93) cells vs. (122.30±5.04) cells, t=6.568, P<0.01, (96.67±8.81) cells vs. (172.70±3.71) cells, t=7.943, P<0.01]. The expression levels of N-cad, Vim, MMP-2 and MMP-9 proteins in Western blotting assays were significantly decreased, while the protein expression levels of E-cad were increased. These changes showed a concentration-dependent relationship with SH. Conclusion SH can inhibit the proliferation, migration and invasion of pancreatic cancer CFPAC-1 and PANC-1 cells by inhibiting EMT. Key words: Pancreatic cancer; Sinomenine hydrochloride; Proliferation; Migration; Invasion; Epithelial-mesenchymal transition

Quercetin-3-O-glucoside suppresses pancreatic cancer cell migration induced by tumor-deteriorated growth factors in vitro.

Analysis using Universal exPress Codes (UPCs) with the public microarray database GEO indicates significantly higher mRNA expressions of VEGF-A, bFGF, and bFGFR2 in pancreatic cancers than those in normal pancreatic tissues. Human pancreatic cancer cell line CFPAC-1 and SNU-213 had relatively differential sensitivity to exogenous VEGF-A, bFGF, and TGF-β1 in migration property. Treatment of quercetin-3-O-glucoside suppressed the migratory activity induced by TGF-β and VEGF-A even at relatively low dosages in CFPAC-1, but not in bFGF-activated SNU-213 cells. However, high dosages of quercetin-3-O-glucoside sufficiently suppressed the migratory activity induced by bFGF in SNU-213 cells. Furthermore, co-treatment with low dose of gemcitabine plus quercetin-3-O-glucoside showed synergistic inhibition effects on the infiltrate activity induced by bFGF in CFPAC-1 and SNU-213 cells. These results collectively suggested that quercetin-3-O glucoside could act as an inhibitor of local metastasis induced by various growth factors in pancreatic cancers and be an effective adjuvant to boost chemotherapeutic efficacy of gemcitabine, currently used in pancreatic cancers.

SKLB316, a novel small-molecule inhibitor of cell-cycle progression, induces G2/M phase arrest and apoptosis in vitro and inhibits tumor growth in vivo

Benzothiazole derivatives have received considerable attentions for their potencies in cancer therapy. In the present study, we reported that SKLB316, a novel synthesized benzothiazole derivative, exhibits activities to inhibit colorectal and pancreatic cancer in vitro and in vivo by inducing G2/M cell cycle arrest and apoptosis. In vitro, it exhibited significant anti-proliferative activities against human cancer cells derived from different histotypes including the colorectal cancer cell line HCT116 and pancreatic cancer cell line CFPAC-1. We chose these cell lines to study the possible anti-tumor mechanism because they are sensitive to SKLB316 treatment. Flow cytometry assays showed that SKLB316 could induce G2/M cell cycle arrest. Mechanistically, SKLB316 could decrease the activities of cdc2/cyclin B1 complex, including decreasing the synthesis of cyclin B1, cdc2 and cdc25c, while accumulating the levels of phosphorylated cdc2 (Tyr15) and checkpoint kinase 2. SKLB316 could also decrease the level of cyclin E and A2. Moreover, SKLB316 could induce cancer cell apoptosis, which was associated with activation of caspase 9, downregulation of Bcl-2 and upregulation of Bax. SKLB316 could also decrease the mitochondrial membrane potential and induce the generation of reactive oxygen species in cells. The results implied that SKLB316 may induce apoptosis via the mitochondria-mediated apoptotic pathway. Moreover, SKLB316 could suppress the growth of established colorectal and pancreatic cancer tumors in nude mice without causing obvious side effects. TUNEL assays confirmed that SKLB316 could also induce tumor cell apoptosis in vivo. Taken together, these findings demonstrate the potential value of SKLB316 as a novel anti-tumor drug candidate.

Inhibition of cell proliferation by a selective inhibitor of the Ca2+-activated Cl− channel, Ano1

BackgroundIon channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca2+-activated Cl− channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16Ainh-A01 as a specific inhibitor of Ano1. AimTo investigate the effect of the T16Ainh-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. MethodsInhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16Ainh-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. ResultsT16Ainh-A01 inhibited Ca2+-activated Cl− currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16Ainh-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16Ainh-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. ConclusionsThe selective Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl− currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.

Gallic Acid as a Cancer-Selective Agent Induces Apoptosis in Pancreatic Cancer Cells

Background: Gallic acid (GA) is a plant phenol isolated from water caltrop which is reported to have anti-inflammatory and anti-cancer effects. In this study, the antiproliferative effect of GA on human pancreatic cancer cell lines CFPAC-1 and MiaPaCa-2 as well as hepatocytes HL-7702 as normal cells was examined. Particularly, the mechanism of GA-induced apoptosis in MiaPaCa-2 cells in vitro was further studied. Methods: Cell viability was measured using SRB assay, and apoptosis was detected by Hoechst staining and annexin V-PI staining assays. Mitochondrial membrane potential was detected by rhodamine-123 staining. Flow cytometry analysis was employed to detect the apoptosis-related events. Results: GA inhibited the proliferation of CFPAC-1 and MiaPaCa-2 cells in a time- and dose-dependent manner, with IC₅₀S of 102.3 ± 2.4 and 135.2 ± 0.6 µM at 48 h, respectively. GA treatment led to the increased proportion of cell apoptosis from 12.5 ± 0.72 to 78.3 ± 2.48% at the concentrations of 6.25 and 25.0 µg/ml, which was evidenced again by chromatins staining assay. Also, GA activated caspase-3, caspase-9, and reactive oxygen species, elevated Bax expression and [Ca²⁺]_i and reduced mitochondrial membrane potential (ΔΨm) in MiaPaCa-2 cells. Remarkably, when compared with human normal cells HL-7702 (IC₅₀ >100 µg/ml), GA showed selective toxicity for cancer cells. Conclusions: GA can function as a cancer-selective agent by inducing apoptosis in MiaPaCa-2 cells via the mitochondria-mediated pathways. To the best of our knowledge, GA should open up new opportunities for the therapy of pancreatic cancer.

Galectin-1 Secreted by Activated Stellate Cells in Pancreatic Ductal Adenocarcinoma Stroma Promotes Proliferation and Invasion of Pancreatic Cancer Cells

This study aimed to clarify that the activated pancreatic stellate cells (PaSCs) are the origin of the highly expressed galectin-1 in the stroma of pancreatic ductal adenocarcinoma (PDAC) tissue and to evaluate the effect of the secreted galectin-1 on proliferation and invasion ability of pancreatic cancer cell line CFPAC-1 in vitro. Different kinds of PaSCs were isolated from the normal or cancerous pancreatic tissues and cultured. Immunohistochemistry study, quantitative polymerase chain reaction, and Western blot were carried out to check the cellular origin of galectin-1 in PDAC tissue. By using modified Boyden chambers, in vitro coculture system of PaSCs was established with the pancreatic cancer cell line CFPAC-1 and based on which we assessed the proliferation and invasion ability of CFPAC-1 with or without galectin-1 antagonists. We identified PaSCs as the primary source of the highly expressed galectin-1 in PDAC stroma. Galectin-1 secreted by PaSCs increased CFPAC-1 proliferative rate in the proliferation assay and facilitated CFPAC-1 infiltration in the invasion assay. Under malignant circumstances, PaSCs express and secret galectin-1, which could further promote the proliferation and invasion of cancer cells.

Abstract 3943: Affymetrix whole genome microarray analysis of 51 human tumor cell lines representative of 16 different tissues of origin: Unsupervised hierarchical clustering

Abstract The total mRNA for each sample of 51 human tumor cell lines representative of 16 different tissues of origin was split into 3 replicates, and analyzed against the entire human genome using standard Affx WT procedures. Approximately 50% of the samples (25 out of the 51 cell lines tested) exhibited low-level clustering related to their tissue of origin. The remaining 50% (26 out of the 51 cell lines tested) did not cluster with other samples of the same tissue of origin. These data reveal the importance of testing potential anticancer agents in multiple models representative of several different tumors of origin, as there is a 50% chance that the model chosen is not actually representative of the intended tissue of origin. This analysis also showed that the pancreatic cancer cell line CFPAC-1 did not cluster with any other cell line tested, revealing the unique genetic profile of this cell line. Interestingly, the reported lung cancer cell lines NCI-H69 and NCI-H82 clustered more closely with leukemic lines than with lung or any other solid tumor. This is particularly interesting as these lines are known to grow/behave more like a suspension culture than a monolayer. The NCI recently published its genetic analysis of their 60-panel, and they revealed that the MDA-MB-435 cell line, traditionally thought to be a breast cancer cell line, more closely resembled a melanoma line; hence, it was re-classified as a melanoma (likely a metastasized melanoma that was taken from the breast site). Our analysis reveals that another traditional breast cancer cell line, UISO-BCA-1, also clusters more closely with the melanomas (including the MDA-MB-435 cell line), suggesting that this cell line also may have been misclassified. Based on these data, we suggest that any potential anticancer agent showing activity in a particular cell line should be tested in other cell lines that cluster with the active line, and not merely in other lines supposedly representative of the same tissue of origin. Furthermore, in early stage testing, it would be more prudent to test several cell lines from different clusters, rather than several cell lines from different tissues of origin. It follows that by testing orphan drugs against several cell lines from each cluster, it would be possible to significantly narrow (and possibly identify), the likely drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3943. doi:10.1158/1538-7445.AM2011-3943

Hypoxiz-Inducible Factor-1α (HIF-1α) Mediates Chemokine Receptor CXCR4 Expression Under Hypoxic Condition in Human Pancreatic Cancer Line CFPAC-1

Background: Hypoxia is a common characteristic of many aggressive tumors including pancreatic cancer. Under hypoxia, cancer has been observed to be more aggressive. The chemokine receptor CXCR4 has been linked to the invasion and metastasis of many types of cancers. How CXCR4 expression is regulated under the hypoxic condition in pancreatic cancer is still not clear. We hypothesize that in human pancreatic cancer cell line CFPAC-1, CXCR4 is overexpressed under hypoxia and this overexpression is mediated by the hypoxia-inducible factor-1α (HIF-1α).