Human Pancreatic Cancer MIA PaCa-2 Research Articles

Abietane diterpenes from Abies spectabilis and their anti-pancreatic cancer activity against the MIA PaCa-2 cell line

An ethanolic extract of the stem of Abies spectabilis exhibited strong cytotoxicity against MIA PaCa-2 human pancreatic cancer cells preferentially under nutrient-deprived conditions. Therefore, phytochemical investigation of this bioactive extract was carried out, and that led the isolation of ten compounds (1–10) including a new abietane-type diterpene (1). The structure of the new compound (1) was elucidated by combined spectroscopic techniques, including HRFABMS, NMR and quantum ECD calculation. All the isolated compounds were evaluated for their efficacy against MIA PaCa-2 human pancreatic cancer cell line by employing an anti-austerity strategy. Among the tested compounds, dehydroabietinol (5) displayed the most potent activity with a PC50 value of 6.6 μM. Dehydroabietinol (5) was also found to retard the MIA PaCa-2 cell migration under normal nutrient-rich conditions displaying its anti-metastatic potential. Investigation on the mechanism suggested that dehydroabietinol (5) is an inhibitor of the key cancer cell survival Akt/mTOR/autophagy signaling pathway.

4-Acetylantroquinonol B enhances cell death and inhibits autophagy by downregulating the PI3K/Akt/MDR1 pathway in gemcitabine-resistant pancreatic cancer cells.

Gemcitabine (GEM) is a typical chemotherapeutic drug used to treat pancreatic cancer, but GEM resistance develops within weeks after chemotherapy. Hence, the development of a new strategy to overcome drug resistance is urgent. 4-Acetylantroquinonol B (4-AAQB), a ubiquinone derived from Taiwanofungus camphoratus, has hepatoprotective, anti-obesity, and antitumor activities. However, the role of 4-AAQB in enhancing GEM sensitivity is unclear. This study aimed to determine the underlying mechanisms by which 4-AAQB enhances cytotoxicity and GEM sensitivity. Cell viability was dramatically reduced by 4-AAQB (2 and 5 µM) treatment in the MiaPaCa-2 and GEM-resistant MiaPaCa-2 (MiaPaCa-2GEMR) human pancreatic cancer cells. 4-AAQB led to cell cycle arrest, upregulated the levels of reactive oxygen species (ROS), promoted apoptosis, and inhibited autophagy, which subsequently enhanced GEM chemosensitivity by suppressing the receptor for advanced glycation end products (RAGE)/high mobility group box 1 (HMGB1)-initiated PI3K/Akt/multidrug resistance protein 1 (MDR1) signaling pathway in both cell lines. Vascular endothelial growth factor A (VEGFA) expression, cell migration, and invasion were also inhibited by the 4-AAQB incubation. Overall, this combination treatment strategy might represent a novel approach for GEM-resistant pancreatic cancer.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

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

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

Caveolin‐1 increases glycolysis in pancreatic cancer cells and triggers cachectic states

In pancreatic cancer, autocrine insulin-like growth factor-1 (IGF-1) and paracrine insulin stimulate both IGF-1 receptor (IGF1R) and insulin receptor (IR) to increase tumor growth and glycolysis. In pancreatic cancer patients, cancer-induced glycolysis increases hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis and, thereby, causes cancer cachexia. As a protein coexisting with IGF1R and IR, caveolin-1 (cav-1) may be involved in pancreatic cancer-induced cachexia. We undertook the present study to test this hypothesis. Out of wild-type MiaPaCa2 and AsPC1 human pancreatic cancer cell lines, we created their stable sub-lines whose cav-1 expression was diminished with RNA interference or increased with transgene expression. When these cells were studied in vitro, we found that cav-1 regulated IGF1R/IR expression and activation and also regulated cellular glycolysis. We transplanted the different types of MiaPaCa2 cells in growing athymic mice for 8 weeks, using intact athymic mice as tumor-free controls. We found that cav-1 levels in tumor grafts were correlated with expression levels of the enzymes that regulated hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis in the respective tissues. When the tumors had original or increased cav-1, their carriers’ body weight gain was less than the tumor-free reference. When cav-1 was diminished in tumors, the tumor carriers’ body weight gain was not changed significantly, compared to the tumor-free reference. In conclusion, cav-1 in pancreatic cancer cells stimulated IGF1R/IR and glycolysis in the cancer cells and triggered cachectic states in the tumor carrier.

Abstract 3108: Human telomerase reverse transcriptase regulates stemness of pancreatic cancer

Abstract Backgrounds: Human telomerase reverse transcriptase (hTERT) plays a key role on immortalization of cancer cells. In the present study, we clarified the effect of small interfering RNA (siRNA) targeting hTERT in pancreatic cancer cell lines. Methods: Two sequences of siRNA targeting hTERT and negative control siRNA were transfected into PANC-1 and MIA PaCa-2 human pancreatic cancer cell lines. Cell proliferation (WST-8 cell counting assay), stemness (sphere formation) and migration (scratch assay) were analyzed using hTERT-siRNA transfected cells and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin were analyzed by real time PCR. Results: As compared to control-siRNA, hTERT-siRNA suppressed cell proliferation and stemness of pancreatic cancer cell lines. Cell migration did not show any difference between hTERT-siRNA and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin in hTERT-siRNA transfected cells were lower than those in control-siRNA transfected cells. Conclusion: The present study indicates that inhibition of hTERT in pancreatic cancer cells suppressed stemness via alteration of the expression levels of CDK1, SOX9 and nestin. Citation Format: Juanjuan Ye, Keiko Yamakawa, Yuko Narusawa, Yoko Matsuda. Human telomerase reverse transcriptase regulates stemness of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3108.

TGF-β1 Facilitates TAp63α Protein Lysosomal Degradation to Promote Pancreatic Cancer Cell Migration.

Simple SummaryNumerous studies demonstrate that the activation of transforming growth factor-β (TGF-β) signaling is a critical driving force for promoting cancer cell migration and tumor metastasis. Our recent study indicates that TGF-β1 promotes FBXO3-mediated ΔNp63α protein degradation to facilitate cancer metastasis. In this study, we show that TGF-β1 can inhibit TAp63α protein stability in a lysosome-dependent, but canonical Smad pathway-independent manner, which leads to upregulation of p53-R248W expression, and consequently results in increased pancreatic cancer cell migration.TGF-β signaling plays a pivotal role in promoting tumor cell migration and cancer metastasis. ΔNp63α and TAp63α are two major isoforms of p53-related p63 protein. Our recent study has shown that TGF-β1 promotes ΔNp63α protein degradation to facilitate cancer metastasis. However, whether TAp63α is involved in TGF-β1-induced cancer metastasis remains unclear. In this study, we show that, in human pancreatic cancer MIA PaCa-2 cells harboring p53-R248W allele, TGF-β1 can significantly inhibit TAp63α protein stability in a Smad pathway-independent manner. Lysosome inhibitor, chloroquine, but not proteasome inhibitor MG132, can rescue TGF-β1-induced downregulation of TAp63α protein. In addition, we show that either TGF-β1 treatment or silencing of TAp63α can dramatically increase migration of MIA PaCa-2 cells. Importantly, the restored expression of TAp63α can effectively block TGF-β1-induced migration of MIA PaCa-2 cells. Mechanistically, we show that TGF-β1 promotes TAp63α protein degradation, leading to upregulation of p53-R248W protein expression, and consequently resulting in elevated MIA PaCa-2 cell migration. Together, this study indicates that lysosomal degradation is an important way for regulating TAp63α protein fate and highlights that TGF-β1-TAp63α-mutant p53 axis is critically important in pancreatic cancer metastasis.

Gemcitabine-Based Chemoradiotherapy Enhanced by a PARP Inhibitor in Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma is a devastating disease with a 5-year overall survival of 9% for all stages. Gemcitabine-based chemoradiotherapy for locally advanced pancreatic cancer is highly toxic. We conducted an in vitro study to determine whether poly(ADP-ribose) polymerase-1 inhibition radiosensitized gemcitabine-based chemotherapy. Human pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, BxPC-3 and PANC-1 were treated with gemcitabine (10 nM) and/or olaparib (1 µM). Low-LET gamma single dose of 2, 5 and 10 Gy radiations were carried out. Clonogenic assay, PAR immunoblotting, cell cycle distribution, γH2Ax, necrotic and autophagic cell death quantifications were performed. Treatment with olaparib alone was not cytotoxic, but highly radiosensitized cell lines, particularly at high dose per fraction A non-cytotoxic concentration of gemcitabine radiosensitized cells, but less than olaparib. Interestingly, olaparib significantly enhanced gemcitabine-based radiosensitization in PDAC cell lines with synergistic effect in BxPC-3 cell line. All cell lines were radiosensitized by the combination of gemcitabine and olaparib, through an increase of unrepaired double-strand, a G2 phase block and cell death. Radiosensitization was increased with high dose of radiation. The combination of olaparib with gemcitabine-based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.

Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer

PurposeRadioresistance in pancreatic cancer patients remains a critical obstacle to overcome. Understanding the molecular mechanisms underlying radioresistance may achieve better response to radiotherapy and thereby improving the poor treatment outcome. The aim of the present study was to elucidate the mechanisms leading to radioresistance by detailed characterization of isogenic radioresistant and radiosensitive cell lines. MethodsThe human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2 were repeatedly exposed to radiation to generate radioresistant (RR) isogenic cell lines. The surviving cells were expanded, and their radiosensitivity was measured using colony formation assay. Tumor growth delay after irradiation was determined in a mouse pancreatic cancer xenograft model. Gene and protein expression were analyzed using RNA sequencing and Western blot, respectively. Cell cycle distribution and apoptosis (Caspase 3/7) were measured by FACS analysis. Reactive oxygen species generation and DNA damage were analyzed by detection of CM-H2DCFDA and γH2AX staining, respectively. Transwell chamber assays were used to investigate cell migration and invasion. ResultsThe acquired radioresistance of RR cell lines was demonstrated in vitro and validated in vivo. Ingenuity pathway analysis of RNA sequencing data predicted activation of cell viability in both RR cell lines. RR cancer cell lines demonstrated greater DNA repair efficiency and lower basal and radiation-induced reactive oxygen species levels. Migration and invasion were differentially affected in RR cell lines. ConclusionsOur data indicate that repeated exposure to irradiation increases the expression of genes involved in cell viability and thereby leads to radioresistance. Mechanistically, increased DNA repair capacity and reduced oxidative stress might contribute to the radioresistant phenotype.

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

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

Synthesis, in vitro, and in vivo evaluation of novel N-phenylindazolyl diarylureas as potential anti-cancer agents

Novel N-phenylindazole based diarylureas have been designed, synthesized and evaluated as potential anticancer agents. In vitro cell viability studies of these derivatives illustrate good potency with IC50 values in the range of 0.4–50 μM in several cancer cell lines including murine metastatic breast cancer 4T1, murine glioblastoma GL261, human triple negative breast cancer MDA-MB-231, human pancreatic cancer MIAPaCa-2, and human colorectal cancer cell line WiDr. The ester group in the lead compound 8i was modified to incorporate amino-amides to increase solubility and stability while retaining biological activity. Further in vitro studies reveal that lead candidates inhibit tube length in HUVEC cells. In vivo systemic toxicity studies indicate that these candidate compounds are well tolerated in mice without any significant side effects. Anticancer efficacy studies in WiDr tumor xenograft and 4T1 tumor syngraft models demonstrate that the lead candidate 11 exhibits significant antitumor properties as a single agent in these tumor models.

Transcriptome sequencing analysis reveals unique and shared antitumor effects of three statins in pancreatic cancer.

Statins, a class of commonly prescribed cholesterol-lowering medications, have been revealed to influence the risk of multiple types of cancer. However, the antitumor effects of statins on pancreatic cancer and their differential efficacy among a variety of statins are not currently well-defined. The aim of the present study was therefore to identify and compare the genes and related biological pathways that were affected by each individual statin on pancreatic cancer. Two human pancreatic cancer cell lines, MiaPaCa2 and PANC1, were exposed to three statins, lovastatin, fluvastatin and simvastatin. The inhibitory effect of statins on pancreatic cancer cell proliferation was first validated. Next, RNA-seq analysis was used to determine the gene expression alterations in either low (2 µM) or high (20 µM) statin concentration-treated cancer cells. Marked differences in gene transcription profiles of both pancreatic cancer cell lines exposed to high concentration statins were observed. Notably, the high concentration statins significantly suppressed core-gene CCNA2-associated cell cycle and DNA replication pathways and upregulated genes involved in ribosome and autophagy pathways. However, the low concentration statin-induced gene expression alterations were only detected in MiaPaCa2 cells. In conclusion, a marked difference in the intra and inter cell-type performance of pancreatic cancer cells exposed to a variety of statins at low or high concentrations was reported herein, which may provide insights for the potential clinical use of statins in future pancreatic cancer therapeutics.

Machine Learning Approach to Raman Spectrum Analysis of MIA PaCa-2 Pancreatic Cancer Tumor Repopulating Cells for Classification and Feature Analysis.

A machine learning approach is applied to Raman spectra of cells from the MIA PaCa-2 human pancreatic cancer cell line to distinguish between tumor repopulating cells (TRCs) and parental control cells, and to aid in the identification of molecular signatures. Fifty-one Raman spectra from the two types of cells are analyzed to determine the best combination of data type, dimension size, and classification technique to differentiate the cell types. An accuracy of 0.98 is obtained from support vector machine (SVM) and k-nearest neighbor (kNN) classifiers with various dimension reduction and feature selection tools. We also identify some possible biomolecules that cause the spectral peaks that led to the best results.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Mechanism of miRNA-26a on the Proliferation of Pancreatic Tumor Cells by Regulating the Expression of Cycline2

Background: qRT-PCR was used to measure the expression of miRNA-26a in pancreatic epithelial cells (HPDE) and human pancreatic cancer cell lines PANC-1 and MIA PaCa-2. Methods: PANC-1 and MIA PaCa-2 cell lines were infected with lentiviruses to construct PANC-miR-26a and MIA-miR-26a, and RT-PCR was used to detect the infection efficiency. The cell proliferation ability of PANC-miR-26a and MIA-miR-26a were examined by CCK-8 assay, and apoptosis was detected by flow cytometry. Western blotting was used to detect the expressions of CyclinE2 protein and mitochondria-associated apoptotic proteins. Results: miR-26a was expressed in human normal pancreatic epithelial cells (HPDE), and not detected in PANC-1 and MIA PaCa-2; miR-26a was highly expressed in the cell lines PANC-miR-26a and MIA-miR-26a infected by the virus particles. The absorbance values of PANC-miR-26a and MIA-miR-26a were lower than those of NC1 and PANC-1 in control group. The apoptosis rates of PANC-miR-26a and MIA-miR-26a were substantially higher than those of the control group. The overexpression of miR-26a inhibited the expression of the target protein CyclinE2 in PANC-miR-26a and MIA-miR-26a. The expression of the anti-apoptotic protein Bcl-2 was decreased in PANC-miR-26a and MIA-miR-26a, while the expression of the pro-apoptotic protein Bax was increased. Compared with HPDE, miR-26a was down-regulated in PANC-1 and MIA PaCa-2. After overexpression of miR-26a, the proliferation of PANC-1 and MIA PaCa-2 cell lines was weakened. Conclusion: Molecular mechanism is the negative regulation of CyclinE2 by miR-26a as well as the expressions of downstream mitochondrial apoptosis proteins Bcl-2 and Bax.

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.

Modification of radiosensitivity by Curcumin in human pancreatic cancer cell lines

Pancreatic cancer is one of the most aggressive malignancies and is characterized by a low 5-year survival rate, a broad genetic diversity and a high resistance to conventional therapies. As a result, novel therapeutic agents to improve the current situation are needed urgently. Curcumin, a polyphenolic colorant derived from Curcuma longa root, showed pleiotropic influences on cellular pathways in vitro and amongst others anti-cancer properties including sensitization of tumor cells to chemo- and radiation-therapy. In this study, we evaluated the impact of Curcumin on the radiosensitivity of the established human pancreatic cancer cell lines Panc-1 and MiaPaCa-2 in vitro. In contrast to MiaPaCa-2 cells, we found a significant radiosensitization by Curcumin in the more radioresistant Panc-1 cells, possibly caused by cell cycle arrest in the most radiation-sensitive G2/M-phase at the time of irradiation. Furthermore, a significant enhancement of radiation-induced apoptosis, DNA-double-strand breaks and G2/M-arrest after curcumin treatment was observed in both cell lines. These in vitro findings suggest that especially patients with more radioresistant tumors could benefit from a radiation-concomitant, phytotherapeutic therapy with Curcumin.

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

Influence of Drying Method on NMR-Based Metabolic Profiling of Human Cell Lines.

Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40–50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metabolites were only present in SpeedVac dried samples, for example, trimethylamine. This research demonstrates that the choice of drying technique used during the preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome, making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling.

Reactive oxygen species modulates the molecular targets of NBS‐1120 in pancreatic cancer

BackgroundPancreatic 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. NBS‐1120 belongs to a new class of “enhanced NSAIDs” that were designed to be safer alternatives by releasing nitric oxide and hydrogen sulfide. NBS‐1120 is built on an aspirin scaffold and while it retains all the classical pharmacological/therapeutic activities of aspirin, it is devoid of GI side effects. In this study, we evaluated the effects of NBS‐1120 against pancreatic cancer using cell lines and a xenograft mouse model.MethodsCell lines: MIA PaCa2 and BxPC3 human pancreatic cancer, ACBRI 515, human normal pancreatic epithelial. Cell kinetics: growth by MTT; Cell cycle phase distribution and apoptosis: Flow cytometry; Proliferation: PCNA. mRNA: qRT‐PCR. ROS: measured hydrogen peroxide and super oxide by flow cytometry, using DCFDA and DHE dyes. Xenografts: Male athymic nude mice (N=10) implanted s.c. in the right flank with MIA PaCa2 cells, after 10 days the mice were randomly divided into 2 groups (N=5/gp) and gavaged daily with NBS‐1120 (100 mg/kg) or vehicle. Tumor volume and animal wt were recorded every 3 days. After 30 days, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for IHC studies.ResultsNBS‐1120 inhibited growth of MIA PaCa‐2 and BxPC‐3 pancreatic cancer cells with IC50s of 52 ± 4, and 62 ± 5 nM, respectively, while it did not inhibit the growth of a normal pancreatic epithelial cell line at these concentrations. NBS‐1120 effects on cancer cell growth appear to be cyclo‐oxygenase (COX) independent as MIA PaCa2 cells are COX null while BxPC3 cells express both COX‐1 and COX‐2. NBS‐1120 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, NBS‐1120 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). ROS, iNOS, and p53 were increased, while NF‐κB and FoxM1 that were activated in untreated xenografts were significantly inhibited by NBS‐1120. Reduced FoxM1 protein expression was associated with reduced FoxM1 mRNA levels and increased p21 mRNA as measured by qRT‐PCR, likely explaining cell proliferative inhibition.ConclusionsNBS‐1120 preferentially affects cancer cells, targeting parameters important in determining cellular mass, and inflammation, and merits further evaluation.Support or Funding InformationSupported in part by the National Institutes of Health [R24DA018055; R01GM123508].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.