Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

Effective anticancer agents based-on two Pillar[5]arene derivatives for pancreas cancer cell lines: synthesis, apoptotic effect, caspase pathway.

This study aimed to evaluate the possible anticancer effects of two different pillar[5]arene derivatives (5Q-[P5] and 10Q-P[5]) on two different pancreatic cancer cell lines in vitro. For this purpose, changes in the expression of major genes that play a role in apoptosis and caspase pathways were investigated. Panc-1 and BxPC-3 cell lines were used in the study and the cytotoxic dose of pillar[5]arenes was determined by the MTT method. Changes in gene expression after pillar[5]arenes treatment were evaluated by real-time polymerase chain reaction (qPCR). Apoptosis was studied by flow cytometry. As a result of analysis, it was determined that proapoptotic genes and genes involved in major caspase activation were upregulated and antiapoptotic genes were down-regulated in Panc-1 cell line treated with pillar[5]arenes. Flow cytometric apoptosis analysis also showed an increased apoptosis rate in this cell line. On the contrary, although MTT analysis showed cytotoxic effect in BxPC-3 cell line treated with two pillar[5]arene derivatives, the apoptosis pathway was not active. This suggested that it may activate different death pathways for BxPC-3 cell line. Thus, it was first determined that the pillar[5]arene derivatives reduced cancer cell proliferation on pancreatic cancer cells.

Elaiophylin Elicits Robust Anti-Tumor Responses via Apoptosis Induction and Attenuation of Proliferation, Migration, Invasion, and Angiogenesis in Pancreatic Cancer Cells.

Pancreatic cancer remains a formidable challenge in oncology due to its aggressive nature and limited treatment options. In this study, we investigate the potential therapeutic efficacy of elaiophylin, a novel compound, in targeting BxPC-3 and PANC-1 pancreatic cancer cells. We comprehensively explore elaiophylin's impact on apoptosis induction, proliferation inhibition, migration suppression, invasion attenuation, and angiogenesis inhibition, key processes contributing to cancer progression and metastasis. The results demonstrate that elaiophylin exerts potent pro-apoptotic effects, inducing a substantial increase in apoptotic cells. Additionally, elaiophylin significantly inhibits proliferation, migration, and invasion of BxPC-3 and PANC-1 cells. Furthermore, elaiophylin exhibits remarkable anti-angiogenic activity, effectively disrupting tube formation in HUVECs. Moreover, elaiophylin significantly inhibits the Wnt/ß-Catenin signaling pathway. Our findings collectively demonstrate the multifaceted potential of elaiophylin as a promising therapeutic agent against pancreatic cancer via inhibition of the Wnt/ß-Catenin signaling pathway. By targeting diverse cellular processes crucial for cancer progression, elaiophylin emerges as a prospective candidate for future targeted therapies. Further investigation of the in vivo efficacy of elaiophylin is warranted, potentially paving the way for novel and effective treatment approaches in pancreatic cancer management.

Gold Nanoparticles Synthesized by an Aqueous Extract of Codium tomentosum as Potential Antitumoral Enhancers of Gemcitabine.

Cancer still poses a global threat, since a lot of tumors remain untreatable despite all the available chemotherapeutic drugs, whose side effects, it must also be noted, still raise concerns. The antitumoral properties of marine seaweeds make them a potential source of new, less toxic, and more active antitumoral agents. Furthermore, these natural extracts can be combined with nanotechnology to increase their efficacy and improve targeting. In this work, a Codium tomentosum (CT) aqueous extract was employed for the green synthesis of gold nanoparticles (Au@CT). The complete characterization of Au@CT was performed by UV-Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, Zeta potential, electron microscopy, X-ray powder diffraction (XRD), high-performance steric exclusion chromatography (HPSEC), and by the determination of their antioxidant capacity. The antiproliferative activity of Au@CT was then tested in hepatic (HEPG-2) and pancreatic (BxPC-3) cell lines. Their potential capacity as enhancers of gemcitabine, a drug frequently used to treat both types of tumors, was also tested. The activity of Au@CT was compared to the activity of the CT extract alone. A synergistic effect with gemcitabine was proven for HEPG-2. Our results showed that gold nanoparticles synthesized from seaweed extracts with antitumoral activity could be a good gemcitabine enhancer.

Salidroside ameliorated hypoxia-induced tumorigenesis of BxPC-3 cells via downregulating hypoxia-inducible factor (HIF)-1α and LOXL2.

Herein, we found that salidroside suppressed hypoxia-inducible factor 1 alpha (HIF-1α) and lysyl oxidase-like protein 2 (LOXL2) within human pancreatic cancer BxPC-3 cells cultured both under normoxia and hypoxia condition. To investigate the effect of salidroside on tumorigenesis of BxPC-3 cells and whether HIF-1α and LXCL2 were involved in this process, cells transfected with or without LOXL2 overexpression vector, were treated with 50 µg/mL of salidroside or 50 µM of KC7F2 (a HIF-1α inhibitor) under hypoxia. Cell viability and invasion were assessed using CCK-8 and Transwell chamber assay, respectively. Expression of E-cadherin and matrix metalloproteinase 2/9 (MMP 2/9) was determined, by Western blot analysis, to assess cell mobility at molecular levels. We confirmed that hypoxia increased LOXL2 and induced tumorigenesis of BxPC-3 cells, as evidenced by promoted cell proliferation and invasion, enhanced MMP2/9 while reduced E-cadherin. Interestingly, hypoxia-induced carcinogenesis was significantly retarded by both salidroside and KC7F2, however, enhanced with LOXL2 overexpression. Besides, salidroside and KC7F2 reduced LOXL2, and reversed the tumorigenesis of BxPC-3 cells induced by LOXL2 overexpression. Given the inhibitory effect of salidroside on HIF-1α expression, our data suggested that: (1) LOXL2 was the mechanism, whereby salidroside and KC7F2 showed inhibitory effect on cancer progression of BxPC-3 cells; (2) salidroside exerted its anticancer effect, most likely, by a HIF-1α/LOXL2 pathway. In conclusion, salidroside was a novel therapeutic drug in pancreatic cancer, and downregulation of HIF-1α and LXCL2 was the underlying mechanism.

BAF45D knockdown decreases cell viability, inhibits colony formation, induces cell apoptosis and S-phase arrest in human pancreatic cancer cells.

Pancreatic cancer, an extremely aggressive malignancy, is resistant to chemo- or radiotherapy. The rapid progression of pancreatic cancer without distinctive clinical sign makes early diagnosing and/or treating very difficult. BAF45D, a member of the d4 domain family, is involved in oncogenic processes. However, the role of BAF45D in pancreatic tumorigenesis is largely unclear. Our goal is to examine BAF45D protein expression after lentivirus-mediated Baf45d RNAi and explore the effects of BAF45D knockdown on cell proliferation, cell apoptosis, and cell cycle of human pancreatic cancer cells. Here our results showed that Baf45d RNAi downregulated BAF45D protein levels and decreased cell viability, increased cell apoptosis, and decreased colony formation in BxPC-3 cells. Moreover, BAF45D knockdown induced S-phase arrest in BxPC-3 cells. Our results here suggest that BAF45D may play a crucial role in tumorigenic properties of human pancreatic cancer cells.

Anti-Cancer Potential of Oxialis obtriangulata in Pancreatic Cancer Cell through Regulation of the ERK/Src/STAT3-Mediated Pathway.

As a plant medicine, Oxalidaceae has been used to treat various diseases in Korea. However, there is little data on the anti-cancer efficacy of Oxalidaceae, particularly O. obtriangulata. This study aimed to investigate the anti-cancer effect of O. obtriangulata methanol extract (OOE) and its regulatory actions on pancreatic carcinoma. OOE showed anti-proliferative effects and induced cell death in the colony formation and cell viability assays, respectively. The Fluorescence-activated cell sorting (FACS) data confirmed that OOE significantly induced cell cycle accumulation at the G2/M phase and apoptotic effects. Additionally, OOE inhibited the activated ERK (extracellular-signal-regulated kinase)/Src (Proto-oncogene tyrosine-protein kinase Src)/STAT3 (signal transducers and activators of transcription 3) pathways including nuclear translocation of STAT3. Furthermore, suppression of Ki67, PARP(Poly ADP-ribose polymerase), caspase-3, P27(Cyclin-dependent kinase inhibitor 1B), and c-Myc as well as the STAT3 target genes CDK(cyclin-dependent kinase)1, CDK2, Cyclin B1, VEGF-1(vascular endothelial growth factor-1), MMP-9(Matrix metallopeptidase 9), and Survivin by OOE was observed in BxPC3. We speculate that these molecular actions might support an anti-cancer effect of OOE. In this study, we demonstrated that OOE may be a promising anti-cancer material and may serve as a natural therapy and alternative remedy for pancreatic cancer treatment.

RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo.

OBJECTIVES: This study was undertaken to investigate the induction of transition of pancreatic cancer to epithelial mesenchyme by RGC-32. METHODS: Primary human pancreatic cancer cell line BXPC-3 was transfected with lentivirus overexpressing the response gene to complement-32 gene (RGC-32) and used to induce tumor in mice. The tumor sizes were measured and the expression of cytokeratin, e-cadherin and vimentin at mRNA using real time PCR and at protein levels by Western blot. RESULTS: Compared with the control, mice inoculated with the cells transfected with empty vector had similar tumor size while those inoculated with the cells transfected with RGC-32 expressing virus had significantly greater tumor size. HE staining showed that tumors were formed in all treatments. Molecular analyses showed that there was no difference in the expression of the cytokeratin, e-cadherin and vimentin genes at mRNA and protein levels between control and empty vector groups. However, mice derived from cells transfected with RGC-32 expressing virus had reduced cytokeratin and e-cadherin expression and increased vimentin expression. CONCLUSIONS: These data suggest that RGC-32 promotes the proliferation of pancreatic cancer and induces the epithelial-mesenchymal transition (EMT). It would be a future direction of research to investigate the regulatory mechanism of signal molecules downstream RGC-32 on EMT-related transcription factors and deliberate the role of RGC-32 in tumorigenicity. As a result, RGC-32 may become a new therapeutic target for cancers.

Anacardic acid inhibits pancreatic cancer cell growth, and potentiates chemotherapeutic effect by Chmp1A - ATM - p53 signaling pathway.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer related death and its incidence has risen steadily. Although anticancer drugs have been developed based on the new molecular findings, the drugs have produced unsatisfactory results due to toxicity and resistance. Thus, a complementary therapeutic intervention is urgently needed for pancreatic cancer patients. METHODS: The aim of this study was to assess the potential therapeutic effect of Anacardic acid on pancreatic cancer in vitro and elucidate its underlying mechanisms. Human pancreatic cancer cells were treated with Anacardic acid and assessed for the cytotoxic effect using MTT and spheroid formation assays. Using the same methods, the synergy between Anacardic acid and 5-Fluorouracil or Gemcitabine was determined. To elucidate the underlying molecular mechanisms, Western blot analysis and immunocytochemistry were performed on cancer cells treated with Anacardic acid alone or in combination with 5-Fluorouracil or Gemcitabine. Chromatin Modifying Protein 1A (Chmp1A), Ataxia Telangiectasia Mutated (ATM), and p53 were the primary signaling molecules examined. In addition, Chmp1A was silenced with shRNA to examine the necessity of Chmp1A for the anticancer effect of Anacardic acid, 5-Fluorouracil, or Gemcitabine. RESULTS: Anacardic acid induced an anticancer effect in pancreatic cancer cell lines in a dose dependent manner, and increased the cytotoxicity of 5-Fluorouracil or Gemcitabine in MTT cell viability assays. In spheroid formation assays, spheroids formed were smaller in size and in number upon Anacardic acid treatment compared to control. Mechanistically, Anacardic acid exerted its anticancer activity via the activation of Chmp1A, ATM, and p53. Interestingly, 5-Fluorouracil and Gemcitabine also induced an increase in Chmp1A protein level, suggesting that Chmp1A might mediate the cytotoxic action of chemotherapeutics. Silencing experiments indicate that Chmp1A is required for the action of Anacardic acid, but not for 5-Fluorouracil or Gemcitabine. CONCLUSIONS: Our data suggests that Anacardic Acid might be a promising complementary supplement to slow the initiation or progression of pancreatic cancer.

Silencing pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer cells to gemcitabine.

Pancreatic adenocarcinoma upregulated factor (PAUF) is a new oncogene that activates signaling pathways that play a critical role in resistance to gemcitabine. We thus speculated that PAUF also plays a role in resistance to gemcitabine of pancreatic cancer cells. We established BxPC-3 cell lines with stable PAUF knockdown (BxPC-3_shPAUF) and controls (BxPC-3_shCtrl) and evaluated sensitivity to gemcitabine in vitro by MTT and flow cytometry. We established a xenograft model of human pancreatic cancer to examine PAUF function in gemcitabine resistance in vivo. Gene chip microarrays were performed to identify differentially expressed genes in BxPC-3_shPAUF and BxPC-3_shCtrl cells. Silencing PAUF increased the sensitivity of BxPC-3 cells to gemcitabine in vitro and in vivo. PAUF-knockdown BxPC-3 cell lines treated with gemcitabine showed increased proliferation inhibition and apoptosis compared with controls. Gemcitabine exhibited a more pronounced effect on reduction of BxPC-3_shPAUF tumors than BxPC-3_shCtrl tumors. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assays confirmed a significantly higher apoptotic rate of BXPC-3_shPAUF tumors compared with BXPC-3_shCtrl tumors. Gene array showed that PAUF function in gemcitabine sensitivity might involve MRP2, MRP3, MDR1, PIK3R1, and NFkB2 genes. PAUF could be considered as a key molecular target for sensitizing pancreatic cancer cells to gemcitabine.

Identification of initial leads directed at the calmodulin-binding region on the Src-SH2 domain that exhibit anti-proliferation activity against pancreatic cancer.

Cellular calmodulin binds to the SH2 domain of Src kinase, and upon Fas activation it recruits Src into the death-inducing signaling complex. This results in Src-ERK activation of cell survival pathway through which pancreatic cancer cells survive and proliferate. We had proposed that the inhibition of the interaction of calmodulin with Src-SH2 domain is an attractive strategy to inhibit the proliferation of pancreatic cancer. Thus we have performed screening of compound libraries by a combination of methods and identified some compounds (initial leads) that target the calmodulin-binding region on the SH2 domain and inhibit the proliferation of pancreatic cancer cells in in vitro assays. Most of these compounds also exhibited varying degrees of cytotoxicity when tested against immortalized breast epithelial cell line (MCF10A). These initial leads are likely candidates for development in targeted delivery of compounds to cancer cells without affecting normal cells.

Oridonin inhibits BxPC-3 cell growth through cell apoptosis.

Oridonin, an ent-kaurene diterpenoid extracted from the traditional Chinese herb Rabdosia rubescens, has multiple biological and pharmaceutical functions and has been used clinically for many years. While the antitumor function of oridonin has been corroborated by numerous lines of evidence, its anticancer mechanism has not been well documented. In this study, the pancreatic cancer cell line BxPC-3 was used as a model to investigate a possible anticancer mechanism of oridonin through examining its effects on cell viability. The results showed that oridonin affected cell viability in a time- and dose-dependent manner. After exposure to different oridonin concentrations, growth rates and cell cycle arrest of BxPC-3 cells were significantly reduced compared with untreated cells, suggesting its effects on proliferation inhibition. Detailed signaling pathway analysis by western blot analysis revealed that low-dose oridonin treatment inhibited BxPC-3 cell proliferation by up-regulating p53 and down-regulating cyclin-dependent kinase 1 (CDK1), which led to cell cycle arrest in the G2/M phase. A high-dose oridonin not only arrested BxPC-3 cells in the G2/M phase but also induced cell accumulation in the S phase, presumably through γH2AX up-regulation and DNA damage. In addition, our results showed that a cell subpopulation was stained with propidium iodide after oridonin treatment. Protein quantification showed that cleaved poly(ADP-ribose) polymerase (PARP) expression was increased after a high-dose oridonin treatment, especially after long-term exposure. Accompanied by the increased level of deactivated PARP in BxPC-3 cells, the apoptosis initiators caspase-3 and caspase-7 expressions were also significantly increased, suggesting that caspase-mediated apoptosis contributed to cell death.

Intracellular distributing and interferon-γ secretion of human interleukin-18 in BxPC-3 cells.

OBJECTIVE: To investigate the characteristics of interleukin-18 (IL-18) in vitro, explore IL-18, interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretive activity in BxPC-3 line cells with interleukin-18 mutants. METHODS: Human IL-18 full-length gene (hIL-18-F) and the hIL-18 presumed mature protein gene (hIL-18-M) were inserted into the expression vector pEGFP-N1, to construct recombinant plasmids as Mu0, Mu1, Mu2, Mu3, and Mu4, and the recombinant plasmids were then transferred into BxPC-3 line cells. There are significant differences between Mu1, Mu2 and the pEGFP-C1 control group (P<0.05) by 3-(4,5-dimethiazol- 2-yl)- 2,5-diphenyltetrazolium bromide (MTT) for a proliferation assay, and the fluorescence of the Mu1 and Mu 2 appeared targeted to the membranous region in the BxPC-3 cells after transfected 24h by confocal laser scanning microscope (OLSM).To characterize the intracellular distribution of hIL-18, recombinant IL-18 were each fused to the enhanced green fluorescent protein gene, and expressed in BxPC-3 cells. RESULTS: Results showed that the Mu1 tended to the membranous region in BxPC-3 cells, this indicates that the N-terminal former amino acid peptide helped ChIL-18 target to BxPC-3 cellS membranes. ELISA results demonstrated that IFN-γ and IL-18 secreted levels of BxPC-3 cells transfecting with recombinant plasmid showed an significant difference (P<0.01); refers to IL-2 expression, the two BxPC-3 cells groups transfecting with recombinant plasmid have no significant function (P>0.05). CONCLUSIONS: The results showed that hIL-18 and hIL-18 presumed mature protein can induce the secretion of IFN-γ in BxPC-3 cells, and increase the expression of IL-18, but they have no effects on IL-2.

Preparation and Characterization of PLGA-based Magnetic Polymer Nanoparticles for Targeting Pancreatic Adenocarcinoma.

AIMS: This study aims to develop a novel tumor-targeted molecular probe for pancreatic cancer imaging. The objective of this is to prepare a CKAAKN peptide-conjugated poly (lactic-co-glycolic acid)-poly (ethylene glycol) amphiphilic polymer (CKAAKN-PEG-PLGA) for the tumor-targeted delivery of magnetic resonance imaging (MRI) contrast agent ultrasmall superparamagnetic iron oxide (USPIO). BACKGROUND: The early diagnosis of pancreatic cancer is crucial for improving its prognosis, but the clinical application of many diagnostic methods is limited owing to a lack of specificity and sensitivity. METHODS: CKAAKN-PEG-PLGA was synthesized by the amidation reaction. USPIO-loaded polymeric magnetic nanoparticles (USPIO@CKAAKN-PEG-PLGA) were prepared by the emulsion solvent evaporation method. The in vitro tumor targeting and bio-safety of nanoparticles were evaluated by targeted cellular uptake, MR imaging and MTT assay. RESULTS: USPIO@CKAAKN-PEG-PLGA nanoparticles showed excellent biosafety with an average diameter of 104.5 ± 4.1 nm. Modification of CKAAKN peptide could improve USPIO binding ability to internalize into CKAAKN-positive BxPC-3 cells compared with non-targeting nanoparticles and the control group. The relative fluorescence intensity in BxPC-3 and HPDE6-C7 cells was 23.77 ± 4.18 and 6.44 ± 2.10 (p < 0.01), and respectively became 16.13 ± 0.83 and 11.74 ± 1.74 after the addition of free CKAAKN peptide. In vitro MR imaging studies showed that an obvious decrease in the signal intensity was observed in the targeted nanoparticles group incubated with BxPC-3 and HPDE6-C7 cells (p < 0.05). CONCLUSION: USPIO@CKAAKN-PEG-PLGA nanoparticles could significantly enhance the tumor specificity of USPIO in CKAAKN-positive pancreatic cancer cell BxPC-3, which is expected as a promising candidate of MRI contrast enhancement for the early diagnosis of pancreatic cancer.

Multifaceted Effects of Kinase Inhibitors on Pancreatic Cancer Cells Reveals Pivotal Entities with Therapeutic Implications.

Pancreatic cancer is one of the most aggressive forms of cancer and is the seventh leading cause of cancer deaths worldwide. Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of pancreatic cancers. Most pancreatic cancers are recalcitrant to radiation, chemotherapy, and immunotherapy, highlighting the urgent need for novel treatment options for this deadly disease. To this end, we screened a library of kinase inhibitors in the PDAC cell lines PANC-1 and BxPC-3 and identified two highly potent molecules: Aurora kinase inhibitor AT 9283 (AT) and EGFR kinase inhibitor WZ 3146 (WZ). Both AT and WZ exhibited a dose-dependent inhibition of viability in both cell lines. Thus, we conducted an in-depth multilevel (cellular, molecular, and proteomic) analysis with AT and WZ in PANC-1 cells, which harbor KRAS mutation and exhibit quasimesenchymal properties representing pancreatic cancer cells as having intrinsic chemoresistance and the potential for differential response to therapy. Elucidation of the molecular mechanism of action of AT and WZ revealed an impact on the programmed cell death pathway with an increase in apoptotic, multicaspase, and caspase 3/7 positive cells. Additionally, the key survival molecule Bcl-2 was impacted. Moreover, cell cycle arrest was observed with both kinase inhibitors. Additionally, an increase in superoxide radicals was observed in the AT-treated group. Importantly, proteomic profiling revealed differentially regulated key entities with multifaceted effects, which could have a deleterious impact on PDAC. These findings suggest potential targets for efficacious treatment, including a possible increase in the efficacy of immunotherapy using PD-L1 antibody due to the upregulation of lactoferrin and radixin. Furthermore, combination therapy outcomes with gemcitabine/platinum drugs may also be more effective due to an increase in the NADH dehydrogenase complex. Notably, protein-protein interaction analysis (STRING) revealed possible enrichment of reactome pathway entities. Additionally, novel therapy options, such as vimentin-antibody--drug conjugates, could be explored. Therefore, future studies with the two kinases as monotherapy/combination therapy are warranted.

Development of visualization and analysis methods for evaluating intratumoral nanoparticle kinetics for tumor-targeted drug delivery using Förster resonance energy transfer in vivo live imaging and tissue clearing techniques.

In this study, the imaging methods for evaluating the kinetics of nanoparticles as drug delivery systems in tumor tissues were improved in BxPC3 tumor-bearing mice. First, Förster resonance energy transfer (FRET) live imaging was selected to quantitatively evaluate nanoparticle kinetics in the tumor tissue of mice. Briefly, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide (as an acceptor)-and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (as a donor)-coloaded nanoparticles were administered intravenously to the mice, and imaging was performed using a fluorescence in vivo imager. The fluorescence intensities of images were acquired in the FRET, donor, and acceptor channels, and the nanoparticle kinetics in the tumor region was quantified by compensating for bleed-through. Second, in the cleared tumor tissue of mice, the difference in evaluation properties between the two- and three-dimensional visualization of the nanoparticles was examined. In brief, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-loaded nanoparticles were intravenously administered to the mice after fluorescently labeled tomato lectin treatment to visualize tumor vessels. Excised tumor tissue was cleared and observed using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. The three-dimensional minimum distances traveled by DiI from the tumor vessels were calculated using information about the two-dimensional distance and the slicing position using the Pythagoras theorem. These imaging techniques should facilitate the development of drug delivery systems for cancer.

Anticancer Activity of Aqueous Fenugreek Seed Extract Against Pancreatic Cancer, Histological Evidence.

OBJECTIVES: Pancreatic cancer is one of the malignant tumors in the digestive system all over the world. It has a very low survival rate. Although there is a great advancement in its therapy either through radiotherapy, chemotherapy, or other therapies, the overall survival is still less than 24 months. Another problem for pancreatic cancer is the resistance to its conventional chemotherapy (e.g., gemcitabine). From this point of view, there is an urgent need to find an effective drug treatment METHODS: The anticancer activity of the germinated fenugreek seed extract is examined in vitro and in vivo against BXPC-3 pancreatic cancer cell lines. Two groups of albino mice were injected with BXPC-3 cells: the first group remained non-treated and the second group was injected with IC50 of the fenugreek extract. The body weight and the survival rate were observed in the two groups and histological examination of the pancreatic tissue was observed RESULTS: Germinated fenugreek seed extract can be efficiently maximized the survival rate in pancreatic cancer mice and protect the pancreatic tissue from lesions related to cancer. CONCLUSION: Germinated fenugreek seed extract can be used to fight pancreatic cancer.

Design, Synthesis and Biological Evaluation of Coumarin Derivatives as NEDD8 Activating Enzyme Inhibitors in Pancreatic Cancer Cells.

BACKGROUND: NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is one of the ubiquitin-like proteins which is activated by the NEDD8 activating enzyme (NAE). The overexpressed NAE can cause a variety of diseases such as numerous cancer types and inflammatory diseases. The selective inhibition of NAE could mediate the rate of ubiquitination and the subsequent degradation of proteins associated with cancer so as to achieve the purpose of treatment. OBJECTIVE: In this article, we decided to study the synthesis and screening of coumarin scaffold derivatives against cancer cell lines, specifically the human pancreatic cancer cell line BxPC-3. METHODS: Twenty-four targeted compounds were synthesized, and their anti-proliferative activity against three cancer cell lines, cytotoxicity against three normal cell lines through CCK-8 and MTT assay were evaluated to screen out the candidate compound. Then the target was further confirmed by both enzyme and cell-based experiments, as well as cell apoptosis research. RESULTS: Several new 4-position substituted coumarin derivatives (12a~x) were synthesized and most of them exhibit antiproliferative activity in three cancer cell lines. A series of experiments were performed to identify the best candidate compound 12v. This compound displayed the highest potency against BxPC-3 with an IC50 value of 0.28 µM. It can also inhibit NAE activity in enzyme and cellbased assay, and induce CRLs-mediated accumulation of the substrate and apoptosis in BxPC-3 cells. Meanwhile, it exhibited relatively low toxicity in three normal cells. CONCLUSION: Based on these results, we found that compound 12v inhibited NAE activity in enzyme and cell-based systems and induced apoptosis in BxPC-3 cells. Additionally, it also had a low toxicity. These results suggested that 12v may be promising lead compounds for the development of new anticancer drugs.

Expression of CD98hc in Pancreatic Cancer and Its Role in Cancer Cell Behavior.

Background: Cluster of differentiation 98 heavy chain (CD98hc) is a transmembrane protein, which functions both as a coreceptor of ß-integrins, enhancing intracellular integrin-dependent downstream signaling, and as a transporter of branched-chain and aromatic amino acids. As such, it is pivotal in cell cycle regulation and protection of oxidative, nutritional and DNA replication stress. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor clinical prognosis. The role of CD98hc in pancreatic cancer remains to be elucidated. The aim of this study was to determine the expression of CD98hc in pancreatic ductal adenocarcinoma and to define its potential functional role in cancer cell biology. Methods: Immunohistochemical staining for CD98hc was performed on 222 tissue samples of patients with pancreatic ductal adenocarcinoma. The pancreatic cancer cell lines PANC-1 and BxPC-3 were used to determine the effect of CD98hc expression on cancer cell behavior using cell adhesion, cell trans-migration and cell spreading assays. Flow cytometry was performed to study the rate of apoptosis after detachment or serum starvation. shRNA-lentiviral constructs were used to knock down or reconstitute full length or mutated CD98hc. Results: Up to 20% of pancreatic ductal adenocarcinomas express CD98hc in the acinar cells (13%) and islet cells (20%) embedded in tumor tissue. Although expression of CD98hc in tumor tissue was not associated with a particular tumor stage or grade, our data show a trend towards longer overall survival of pancreatic cancer patients without CD98hc expression as compared to those with immunohistochemical positivity. In vitro downregulation of CD98hc in the pancreatic cancer cell lines PANC-1 and BxPC-3 significantly inhibits cell proliferation (p<0.05), self-renewal (p<0.05) and anchorage-independent growth (p<0.05). Conclusion: CD98hc is expressed in a remarkable percentage of pancreatic ductal adenocarcinomas. Due to its important role in cell behavior and malignant cell transformation, it may be a promising molecular target for potential new therapeutic approaches in pancreatic cancer in the future.

Targeting Tumor Cells Overexpressing the Human Epidermal Growth Factor Receptor 3 with Potent Drug Conjugates Based on Affibody Molecules.

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.