MiaPaCa-2 Human Pancreatic Cancer Cells Research Articles

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.

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.

Heparan sulfate binds the extracellular Annexin A1 and blocks its effects on pancreatic cancer cells

In pancreatic cancer (PC) progression the protein Annexin A1 (ANXA1) has been described as oncogenic factor. Thus, the need to inhibit its action, mainly the extracellular form, has become an appealing cue for the anti-cancer research. Heparan sulfate (HS) is a glycosaminoglycan of the extracellular matrix known to bind several molecules, as growth factors and cytokines, generating a kind of reservoir in the extracellular environment.Here, we started our study by showing the physical calcium-dependent interaction between HS and ANXA1 as both full-length protein and N-terminal portion, Ac2-26 by biophysical techniques. HS is able to inhibit the migration/invasion process of human PC MIA PaCa-2 cells and partially revert their mesenchymal phenotype as reported through the expression of specific protein markers and the growth in colonies and in 3D-spheroids. Furthermore, both on MIA PaCa-2 and PANC-1 cells, HS blocks the effects of Ac2-26, which enhances the aggressive behavior of PC cells if added alone. These effects appear evident also on endothelial cells whose activation is promoted by Ac2-26 but not in presence of HS. Thus, the interference of the interaction ANXA1-HS on angiogenesis strongly emerges. Moreover, once sequestered by HS, ANXA1 is not more able to bind the formil-peptide receptors (FPRs) preventing the increase of calcium mobilization, peculiar for cell motility.These findings introduce a new important tale in the knowledge about the inhibition of the ANXA1 action in PC development. Further information will be useful to highlight the interaction of HS with the protein, focusing on the characterization of the glycosaminoglycan and on in vivo assays.

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.

Combination of a novel microtubule inhibitor MBRI-001 and gemcitabine synergistically induces cell apoptosis by increasing DNA damage in pancreatic cancer cell lines.

Pancreatic cancer (PC) is a highly malignant cancer with poor prognosis. Although gemcitabine (GEM; 2',2'-difluoro-deoxycytidine) has been used as the first-line chemotherapeutic agent in PC treatment for decades, its limited efficacy remains a significant clinical issue, which may be resolved by GEM combination therapy. In this study, we aimed to investigate the anti-tumor effects of MBRI-001 in combination with GEM in BxPC-3 and MIA PaCa-2 human PC cell lines. In vitro and in vivo results indicate that MBRI-001 showed synergistic activity with GEM. GEM induced apoptosis by increasing DNA damage (phosphorylated core histone protein H2AX (γ-H2AX)), MBRI-001 activated mitochondrial-apoptotic pathway (cleaved poly-ADP ribose polymerase (PARP)). Thus, the combination of the two intensified both apoptosis and DNA damage and showed significantly superior anti-tumor activity compared to each agent alone. The adoption of combination of MBRI-001 with GEM may be beneficial as they act synergistically and thus, can be a potential therapeutic choice for improving the prognosis of PC patients in the future.

Increased dietary fatty acids determine the fatty-acid profiles of human pancreatic cancer cells and their carrier's plasma, pancreas and liver.

Primary contents of dietary fat are three or four types of fatty acids, namely saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), n6-polyunsaturated fatty acid (n6PUFA) and, to less extent, n3-polyunsaturated fatty acid (n3PUFA). Previous studies suggest that increased SFA, MUFA, and n6PUFA in high fat diets (HFDs) stimulate the origination, growth, and liver metastasis of pancreatic cancer cells, whereas increased n3PUFA has the opposite effects. It is unclear whether the fatty acid-induced effects are based on changed fatty-acid composition of involved cells. Here, we investigated whether increased SFA, MUFA, n6PUFA, and n3PUFA in different HFDs determine the FA profiles of pancreatic cancer cells and their carrier's plasma, pancreas, and liver. We transplanted MiaPaCa2 human pancreatic cancer cells in athymic mice and fed them normal diet or four HFDs enriched with SFA, MUFA, n6PUFA, and n3PUFA, respectively. After 7 weeks, fatty acids were profiled in tumor, plasma, pancreas, and liver, using gas chromatography. When tumor carriers were fed four HFDs, the fatty acids that were increased dietarily were also increased in the plasma. When tumor carriers were fed MUFA-, n6PUFA-, and n3PUFA-enriched HFDs, the dietarily increased fatty acids were also increased in tumor, pancreas, and liver. When tumor-carriers were fed the SFA-enriched HFD featuring lauric and myristic acids (C12:0 and C14:0), tumor, pancreas, and liver showed an increase not in the same SFAs but palmitic acid (C16:0) and/or stearic acid (C18:0). In conclusion, predominant fatty acids in HFDs determine the fatty-acid profiles of pancreatic cancer cells and their murine carriers.

In vivo tissue distribution and safety of polyacrylic acid-modified titanium peroxide nanoparticles as novel radiosensitizers

Polyacrylic acid (PAA)-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers. PAA-TiOx NPs were synthesized from commercial TiO2 nanoparticles that were modified with PAA and functionalized by H2O2 treatment. To realize practical clinical uses for PAA-TiOx NPs, their tissue distribution and acute toxicity were evaluated using healthy mice and mice bearing tumors derived from xenografted MIAPaCa-2 human pancreatic cancer cells. Healthy mice were injected with PAA-TiOx NPs at 25mg/kg body weight via the tail vein, and tumor-bearing mice were injected either into the tumor locally or via the tail vein. The concentration of PAA-TiOx NPs in major organs was determined over time using inductively coupled-plasma atomic emission spectrometry. After 1h, 12% of the PAA-TiOx NP dose had accumulated in the tumor, and 2.8% of the dose remained after 1 week. Such high accumulation could be associated with enhanced permeability and retention effects of the tumor, as PAA-TiOx NPs are composed of inorganic particles and polymers, without tumor-targeting molecules. The liver accumulated the largest proportion of the injected nanoparticles, up to 42% in tumor-bearing mice. Blood biochemical parameters were also investigated after intravenous injection of PAA-TiOx NPs in healthy mice. PAA-TiOx NPs invoked a slight change in various liver-related biochemical parameters, but no liver injury was observed over the practical dose range. In the future, PAA-TiOx NPs should be modified to prevent accumulation in the liver and minimize risk to patients.

Antitumor effect of Japanese apricot extract (MK615) on human cancer cells in vitro and in vivo through a reactive oxygen species-dependent mechanism.

MK615 is produced from Japanese apricot and contains several cyclic triterpenes, such as oleanolic and ursolic acids. MK615 was shown to strongly suppress cutaneous in-transit metastasis in a patient with malignant melanoma. The present investigation was undertaken to clarify the antitumor effects of MK615 in vitro and in vivo. Several human cancer cell lines were exposed to MK615 for 7 days to examine its antiproliferative effects. The effect of MK615 on in vivo growth of human pancreatic cancer MIAPaCa-2 cells was also examined. MK615 inhibited the growth of several human cancer cell lines in a concentration-dependent way. Pancreatic cancer MIAPaCa-2 cells were highly sensitive to the growth-inhibiting effects of MK615. Treatment with MK615 preferentially induced cell death in human cancer cells while sparing normal cells such as human umbilical vein endothelial cells (HUVEC) and mouse bone marrow cells. When MIAPaCa-2 cells were incubated with MK615 in the presence of antioxidant, growth-inhibition was significantly reduced, and MK615 induced the accumulation of reactive oxygen species in cancer cells but not in HUVEC. MK615, in both the presence and absence of gemcitabine, significantly inhibited the growth of human pancreatic cancer cells as xenografts without apparent adverse effects. MK615, a supplement produced from Japanese apricot, may have therapeutic value in treating human cancers through a reactive oxygen species-dependent mechanism.

Imaging DNA Repair After UV Irradiation Damage of Cancer Cells in Gelfoam® Histoculture.

DNA damage repair in response to UVC irradiation was imaged in cancer cells growing in Gelfoam® histoculture. UVC-induced DNA damage repair was imaged with green fluorescent protein (GFP)fused to the DNA damage response (DDR)-related binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP nuclear foci to be observed within 1h after UVC irradiation, indicating the onset of DNA damage repair response. Induction of UV-induced 53BP1-GFP focus formation was limited up to a depth of 40 μm in Gelfoam®histoculture of MiaPaCa-2 cells, indicating this was the depth limit of UVC irradiation.

Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells

BackgroundStatin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins.MethodsThese effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 μM). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis.ResultsAll tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway’s intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway.ConclusionsOur data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

Neutral and cationic half-sandwich arene ruthenium, Cp*Rh and Cp*Ir oximato and oxime complexes: Synthesis, structural, DFT and biological studies

The reaction of [(p-cymene)RuCl2]2 and [Cp*MCl2]2 (M = Rh/Ir) with chelating ligand 2-pyridyl cyanoxime {pyC(CN)NOH} leads to the formation of neutral oximato complexes having the general formula [(arene)M{pyC(CN)NO}Cl] {arene = p-cymene, M = Ru, (1); Cp*, M = Rh (2); Cp*, M = Ir (3)}. Whereas the reaction of 2-pyridyl phenyloxime {pyC(Ph)NOH} and 2-thiazolyl methyloxime {tzC(Me)NOH} with precursor compounds afforded the cationic oxime complexes bearing formula [(arene)M{pyC(ph)NOH}Cl]+ and [(arene)M{tzC(Me)NOH}Cl]+ {arene = p-cymene M = Ru, (4), (7); Cp*, M = Rh (5), (8); Cp*, M = Ir (6), (9)}. The cationic complexes were isolated as their hexafluorophosphate salts. All these complexes were fully characterized by analytical, spectroscopic and X-ray diffraction studies. The molecular structures of the complexes revealed typical piano stool geometry around the metal center within which the ligand acts as a NN′ donor chelating ligand. The Chemo-sensitivity activities of the complexes evaluated against HT-29 (human colorectal cancer), and MIAPaCa-2 (human pancreatic cancer) cell line showed that the iridium-based complexes are much more potent than the ruthenium and rhodium analogues. Theoretical studies were carried out to have a deeper understanding about the charge distribution pattern and the various electronic transitions occurring in the complexes.

Abstract 3083: Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) or pancreatic cancer is one of the most lethal malignancies in humans. The survival rate for two years is very limited in most of cases (10%) after diagnosis of this disease. This is because current therapeutic regimens have limited effectiveness. Novel therapeutic targets are needed to treat this disease. GZ17-06.12 contains a nutraceutical for its anti-cancer properties, in several cancers. Crude extracts of the plant Arum palaestinum Boiss used in traditional Israeli and Palestinian medicine, have demonstrated cytostatic and cytotoxic activities in colon, prostrate and lung carcinoma. Therefore, we hypothesize that GZ17-06.12 will inhibit tumor progression and metastasis in PDAC. Experimental Procedure: In this study, we have determined cell proliferation, pancosphere formation and apoptosis following treatment of different doses oGZ17-06.12 for 24-72 hours using MiaPaCa-2 and S2-007 human pancreatic cancer cells. Cell cycle distribution and apoptosis were measured using flow cytometic analysis. Orthotopic pancreatic cancer model in athymic mice was developed and GZ17-06.12 was given orally for 20 days to those mice. Proliferative markers, pEGFR/pAkt and apoptotic markers, Bax/Bcl-2, were monitored following treatment with GZ17-06.12 in both in vivo and in vitro models. Metastatic markers, MMP-2 and MMP-9 were measured in metastatic tissues in orthotopic models. Results: GZ17-06.12 inhibited proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. Cell cycle analysis suggested that cells were arrested in S and G2 phase following treatment. In addition, GZ17-06.12 induced apoptosis in both in vitro and in vivo pancreatic cancer. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, GZ17-06.12 decreased the number and size of the pancospheres in S2-007 cells with concomitant inhibition of pancreatic cancer stem cell markers, DCLK1, Lgr5 and EpCam. The effect of GZ17-06.12 suppressed tumor growth and metastatic potential as indicative of MMP-2 and MMP-9 activity in primary and metastatic tumors. Conclusions: GZ17-06.12 significantly reduces tumorigenesis and metastasis in both in vitro and in vivo pancreatic cancer models, inhibiting proliferation with concomitant increase of apoptosis. Citation Format: Santanu Paul, Lisa Stehno-Bittel, Animesh Dhar. Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3083.

Titanium peroxide nanoparticles enhanced cytotoxic effects of X-ray irradiation against pancreatic cancer model through reactive oxygen species generation in vitro and in vivo.

BackgroundBiological applications of nanoparticles are rapidly increasing, which introduces new possibilities to improve the efficacy of radiotherapy. Here, we synthesized titanium peroxide nanoparticles (TiOxNPs) and investigated their efficacy as novel agents that can potently enhance the effects of radiation in the treatment of pancreatic cancer.MethodsTiOxNPs and polyacrylic acid-modified TiOxNPs (PAA-TiOxNPs) were synthesized from anatase-type titanium dioxide nanoparticles (TiO2NPs). The size and morphology of the PAA-TiOxNPs was evaluated using transmission electron microscopy and dynamic light scattering. The crystalline structures of the TiO2NPs and PAA-TiOxNPs with and without X-ray irradiation were analyzed using X-ray absorption. The ability of TiOxNPs and PAA-TiOxNPs to produce reactive oxygen species in response to X-ray irradiation was evaluated in a cell-free system and confirmed by flow cytometric analysis in vitro. DNA damage after X-ray exposure with or without PAA-TiOxNPs was assessed by immunohistochemical analysis of γ-H2AX foci formation in vitro and in vivo. Cytotoxicity was evaluated by a colony forming assay in vitro. Xenografts were prepared using human pancreatic cancer MIAPaCa-2 cells and used to evaluate the inhibition of tumor growth caused by X-ray exposure, PAA-TiOxNPs, and the combination of the two.ResultsThe core structures of the PAA-TiOxNPs were found to be of the anatase type. The TiOxNPs and PAA-TiOxNPs showed a distinct ability to produce hydroxyl radicals in response to X-ray irradiation in a dose- and concentration-dependent manner, whereas the TiO2NPs did not. At the highest concentration of TiOxNPs, the amount of hydroxyl radicals increased by >8.5-fold following treatment with 30 Gy of radiation. The absorption of PAA-TiOxNPs enhanced DNA damage and resulted in higher cytotoxicity in response to X-ray irradiation in vitro. The combination of the PAA-TiOxNPs and X-ray irradiation induced significantly stronger tumor growth inhibition compared to treatment with either PAA-TiOxNPs or X-ray alone (p < 0.05). No apparent toxicity or weight loss was observed for 43 days after irradiation.ConclusionsTiOxNPs are potential agents for enhancing the effects of radiation on pancreatic cancer and act via hydroxyl radical production; owing to this ability, they can be used for pancreatic cancer therapy in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-016-0666-y) contains supplementary material, which is available to authorized users.

Kaempferol Inhibits Pancreatic Cancer Cell Growth and Migration through the Blockade of EGFR-Related Pathway In Vitro.

Pancreatic cancer is one of the most appalling cancers with a pessimistic prognosis. Despite many therapies, there has been no improvement of survival rates. In this study, we assessed the anti-cancer effects of kaempferol, a well known flavonoid having functional bio-activity against various malignant tumors. Kaempferol had anti-cancer effects on Miapaca-2, Panc-1, and SNU-213 human pancreatic cancer cells. In a dose-dependent manner, kaempferol decreased viability of these pancreatic cancer cells by increasing apoptosis. In particular, kaempferol effectively inhibited the migratory activity of human pancreatic cancer cells at relatively low dosages without any toxicity. The anti-cancer effect of kaempferol was mediated by inhibition of EGFR related Src, ERK1/2, and AKT pathways. These results collectively indicate that kaempferol, a phytochemical ingredient reported to have anti-viability and anti-oxidant properties, can act as a safety anti-migration reagent in human pancreatic cancer cells, which provide the rationale for further investigation of kaempferol as a strong candidate for the potential clinical trial of malignant pancreatic cancers.

Anti-metastatic potential of somatostatin analog SOM230: Indirect pharmacological targeting of pancreatic cancer-associated fibroblasts.

Pancreatic ductal adenocarcinoma (PDA) shows a rich stroma where cancer-associated fibroblasts (CAFs) represent the major cell type. CAFs are master secretors of proteins with pro-tumor features. CAF targeting remains a promising challenge for PDA, a devastating disease where treatments focusing on cancer cells have failed. We previously introduced a novel pharmacological CAF-targeting approach using the somatostatin analog SOM230 (pasireotide) that inhibits protein synthesis in CAFs, and subsequent chemoprotective features of CAF secretome. Using primary cultures of CAF isolated from human PDA resections, we here report that CAF secretome stimulates in vitro cancer cell survival, migration and invasive features, that are abolished when CAFs are treated with SOM230. Mechanistically, SOM230 inhibitory effect on CAFs depends on the somatostatin receptor subtype sst1 expressed in CAFs but not in non-activated pancreatic fibroblasts, and on protein synthesis shutdown through eiF4E-Binding Protein-1 (4E-BP1) expression decrease. We identify interleukin-6 as a SOM230-inhibited CAF-secreted effector, which stimulates cancer cell features through phosphoinositide 3-kinase activation. In vivo, mice orthotopically co-xenografted with the human pancreatic cancer MiaPaCa-2 cells and CAFs develop pancreatic tumors, on which SOM230 treatment does not inhibit growth but abrogates metastasis. Consistently, CAF secretome stimulates epithelial-to-mesenchymal transition in cancer cells, which is reversed upon CAF treatment with SOM230. Our results highlight a novel promising anti-metastatic potential for SOM230 indirectly targeting pancreatic cancer cell invasion through pharmacological inhibition of stromal CAFs.

Targeting the insulin growth factor-1 receptor with fluorescent antibodies enables high resolution imaging of human pancreatic cancer in orthotopic mouse models.

The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of pancreatic cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm or 650 nm fluorophores. Western blotting confirmed the expression of IGF-1R in Panc-1, BxPC3, and MIAPaCa-2 human pancreatic cancer cell lines. Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of the pancreatic cancer cells. Subcutaneous Panc-1, BxPC-3, and MIA PaCa-2 tumors became fluorescent after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted BxPC-3 tumors became fluorescent with the conjugated IGF-1R antibodies, and were easily visible with intravital imaging. Gross and microscopic ex vivo imaging of resected pancreatic tumor and normal pancreas confirmed that fluorescence indeed came from the membrane of cancer cells, and it was stronger from the tumor than the normal tissue. The present study demonstrates that fluorophore-conjugated IGF-1R antibodies can visualize pancreatic cancer and it can be used with various imaging devices such as endoscopy and laparoscopy for diagnosis and fluorescence-guided surgery.

Hyaluronic acid-conjugated polyamidoamine dendrimers for targeted delivery of 3,4-difluorobenzylidene curcumin to CD44 overexpressing pancreatic cancer cells

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM) dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3±1.5nm and −7.02±9.53mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1 human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-formulation resulted in 1.71 fold increase in the IC50 value compared to non-targeted formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF. Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic margin and safety.

Hyaluronic Acid Engineered Nanomicelles Loaded with 3,4-Difluorobenzylidene Curcumin for Targeted Killing of CD44+ Stem-Like Pancreatic Cancer Cells.

Cancer stem-like cells (CSLCs) play a pivotal role in acquiring multidrug resistant (MDR) phenotypes. It has been established that pancreatic cancers overexpressing CD44 receptors (a target of hyaluronic acid; HA) is one of the major contributors for causing MDR. Therefore, targeted killing of CD44 expressing tumor cells using HA based active targeting strategies may be beneficial for eradicating MDR-pancreatic cancers. Here, we report the synthesis of a new HA conjugate of copoly(styrene maleic acid) (HA-SMA) that could be engineered to form nanomicelles with a potent anticancer agent, 3,4-difluorobenzylidene curcumin (CDF). The anticancer activity of CDF loaded nanomicelles against MiaPaCa-2 and AsPC-1 human pancreatic cancer cells revealed dose-dependent cell killing. Results of cellular internalization further confirmed better uptake of HA engineered nanomicelles in triple-marker positive (CD44+/CD133+/EpCAM+) pancreatic CSLCs compared with triple-marker negative (CD44-/CD133-/EpCAM-) counterparts. More importantly, HA-SMA-CDF exhibited superior anticancer response toward CD44+ pancreatic CSLCs. Results further confirmed that triple-marker positive cells treated with HA-SMA-CDF caused significant reduction in CD44 expression and marked inhibition of NF-κB that in-turn can mitigate their proliferative and invasive behavior. Conclusively, these results suggest that the newly developed CD44 targeted nanomicelles may have great implications in treating pancreatic cancers including the more aggressive pancreatic CSLCs.

Abstract 2019: Photoimmunotherapy with an anti-EGFR antibody conjugated to an IRDye700 results in extensive and rapid cell death in vitro and in vivo in human pancreatic cancer cell lines

Abstract Photoimmunotherapy (PIT) of cancer utilizes tumor-specific monoclonal antibodies conjugated to a photosensitizer phthalocyanine dye IR700 which becomes cytotoxic upon irradiation with near infrared light. In this study, we aimed to evaluate the efficacy of PIT on human pancreatic cancer cells in vitro and in vivo in a nude mouse model. An in vitro cytotoxicity assay was used to determine cell death following treatment with PIT for Panc-1, MiaPaCa2, and BxPC-3 human pancreatic cancer cells. For in vivo determination of PIT efficacy, nude mice were implanted with BxPC-3 pancreatic tumors expressing green fluorescent protein (GFP). After tumor engraftment, the mice were divided into two groups: treatment with anti-EGFR-IR700 + 690 nm laser and treatment with 690 nm laser only. Anti-EGFR-IR700 (100 μg) was administered to the treatment group via tail vein injection 24 hours prior to therapy. Tumors were then exposed and treated with phototherapy at an intensity of 150 mW/cm2 for 30 minutes. Whole body imaging was done using an OV-100 small animal imaging system. Anti-EGFR-IR700 antibody bound to the BxPC3 cells to a high degree based on image co-localization. Anti-EGFR-IR700 caused extensive cancer cell killing after light activation compared to control cells in cytotoxicity assays. In the mouse experiments, the treatment group had significantly smaller tumors as measured by GFP than the control after just 96 hours. The present results indicate the high potential of PIT for pancreatic cancer therapy. Citation Format: Nzola de Magalhães, Takashi Murakami, Roger Heim, Lew Makings, Miguel Garcia-Guzman, Hisataka Kobayashi, Robert M. Hoffman, Michael Bouvet. Photoimmunotherapy with an anti-EGFR antibody conjugated to an IRDye700 results in extensive and rapid cell death in vitro and in vivo in human pancreatic cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2019. doi:10.1158/1538-7445.AM2015-2019

Abstract A35: Insulin and hypoxia-inducible factor-1 cooperate to increase the viability of pancreatic cancer cells

Abstract Insulin receptor (IR) and hypoxia-inducible factor-1 (HIF-1) master two different intracellular signaling pathways. Both the pathways regulate growth, glycolysis, migration and angiogenesis in cancer. We hypothesized that the IR and HIF-1 pathways are interrelated to each other in pancreatic cancer cells. To test this hypothesis, we designated 45 athymic mice to six tumor-carrying groups (6-7 mice per group) and an intact control group (n=5). All tumor carriers were subject to one of the following conditions: (i) injection of beta-cell-toxin streptozotosin (SZ) before cancer induction, (ii) daily injection of insulin after cancer induction, and (iii) no treatment other than cancer induction. In a 12-week experiment, wild-type MiaPaCa2 human pancreatic cancer cells and HIF-1-null si-MiaPaCa2 cells were implanted orthotopically. When wild-type cells were used, the resulting groups were mice(wt-sz), mice(wt-in), and mice(wt-un). When si-MiaPaCa2 cells were used, the resulting groups were mice(si-sz), mice(si-in), and mice(si-un). In the end of the experiment, insulin was determined in pancreas and plasma by radioimmunoassay. Tumor viability was assessed by histology and Western blots. We found that pancreatic insulin was decreased in all tumor carriers except mice(wt-in), which suggests that tumor carriage per se impaired pancreatic beta-cells. When plasma insulin was determined, significant decreases were seen in mice(wt-sz) and mice(si-sz). Thus, SZ treatment caused additional damage on pancreatic beta-cells and thereby decreased plasma insulin. When glucose and lactate were determined in the plasma, decreased glucose and increased lactate were seen in four out of six tumor-carrying groups. These changes may be due to an uncontrolled glycolysis in cancer cells (also known as the Warburg effect). When tumors were weighed, the biggest ones came from mice(wt-in). Necrosis was seen in all tumors, but tumors from mice(wt-un) had the least necrosis. It suggests that normal pancreas is the best environment for insulin-HIF-1 cooperation. We performed Western blots to determine IR, hexokinase-II (HK-II, a glycolytic enzyme), and vascular endothelial growth factor (VEGF) in tumor grafts. HK-II and VEGF are known to be regulated by HIF-1. In addition, HK-II and VEGF are also known to be involved in IR-motivated signaling cascades. In the present study, insulin injection in mice(wt-in) and mice(si-in) increased IR expression. However, increased HK-II and VEGF expression were only seen in tumors from mice(wt-in) but not mice(si-in). This suggests that HIF-1 is a prerequisite for insulin to stimulate glycolysis and angiogenesis. Finally, we determined caveolin-1 in tumors. Caveolin-1 is an IR regulator, and caveolin-1 gene is regulated by HIF-1. In this study, we found that wild-type tumors had greater caveolin-1 expression than HIF-1-null tumors. In conclusion, insulin and HIF-1 systems cooperate to give survival advantage to pancreatic cancer cells. HIF-1 expression is a prerequisite for insulin to stimulate pancreatic cancer cells. HIF-1 is also necessary for pancreatic cancer cells to express caveolin-1. Citation Format: Yue Chen, Dapeng Zhang, Lihua Cui, Feng Wang. Insulin and hypoxia-inducible factor-1 cooperate to increase the viability of pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A35.