Pancreatic Cancer Cell Lines Research Articles

Synthesis of liposomal nanoparticles to load 4-farnesyloxycoumarin and investigating its anti-cancer and anti-metastatic effects.

The aim of this study was to load 4-farnesyloxycoumarin (4-FLC) in nanoliposomes (4-FLC-LNPs) and evaluate its anti-cancer and anti-metastatic effects. 4-FLC-LNPs were synthesized using a combination of lecithin-cholesterol-polyethylene glycol. The physicochemical properties were evaluated using DLS, FTIR, and microscopy methods. The toxicity against breast cancer (MCF-7), prostate cancer (PS3), pancreatic cancer (PANC), gastric cancer (AGS), and normal cell lines (HUVEC) was evaluated using the MTT assay. Fluorescent staining and flow cytometry were used to assess the occurrence of apoptosis. Molecular analysis methods were used to study the apoptosis and metastasis effects of these nanoliposomes. The antioxidant power of 4-FLC-LNPs was measured using the ABTS and DPPH free radicals methods. 4-FLC-LNPs exhibit a spherical morphology, with an average size of 57.43 nm, a polydispersity index of 0.29, and a zeta potential of -31.4 mV. They demonstrate an encapsulation efficiency of 82.4% for 4-FLC. The IC50 value of 4-FLC-LNPs against the breast cancer cell line was reported as the most sensitive, at approximately 60 μg/mL. ABTS and DPPH results were reported at approximately 30 µg/mL. The inductive effects of nanoliposomes on the apoptosis process were confirmed by an increase in the number of apoptotic cells, as well as the arrest of cells in various phases of cell growth. The increased expression of BAX and decreased expression of Bcl-2, MMP-2, and MMP-9 confirmed the pro-apoptotic and anti-metastatic effects of 4-FLC-LNPs. These finding validate the therapeutic potential of 4-FLC-LNPs, which may be utilized in preclinical studies.

Radioiodinated Nanobody immunoPET probe for in vivo detection of CD147 in pan-cancer.

To develop the extracellular matrix metalloproteinase inducer (CD147)-targeting therapeutic strategies, accurate detection of CD147 expression in tumors is crucial. Owing to their relatively low molecular weights and high affinities, nanobodies (Nbs) may be powerful candidates for cancer diagnosis and therapy. In this study, we developed a novel CD147-targeted nanobody radiotracer, [124I]I-NB147, which provides guidance for the noninvasive detection of CD147-overexpressing cancers. CD147 expression in human cancers was detected via immunohistochemistry (IHC) on tissue microarrays (TMAs). Western blot (WB) and flow cytometry were used to screen CD147-positive malignant melanoma (MM), triple-negative breast cancer (TNBC), and pancreatic cancer (PCA) cell lines. The CD147 nanobody (NB147) was labeled with [124I]INa using Iodogen as the oxidizing agent and was purified by the PD-10 column. The physicochemical properties, affinity, metabolic characteristics, biodistribution, and immunoPET imaging of [124I]I-NB147 were evaluated Moreover, [18F]F-FDG was used as a control. Finally, CD147 expression analysis was performed via multiplex immunofluorescence (MxIF) and autoradiography on human cancer specimens and tumor-bearing mice tissues. TMAs results revealed that CD147 is highly expressed in MM, TNBC, and PCA. A CD147-specific nanobody, NB147, was successfully generated with excellent in vitro binding characteristics. [124I]I-NB147 was obtained with high radiochemical yield and purity, and was stable for at least 4h in vitro. WB and FCM revealed that CD147 was positive in A375, MDA-MB-435 and ASPC1 cells, whereas SK-MEL-28, 4T1 and BXPC3 cells presented low expression levels. The radio-ELISA results indicated that [124I]I-NB147 had a high binding affinity to CD147. The uptake of [124I]I-NB147 was significantly different between CD147 high-expression cells and CD147 low-expression cells (P < 0.001). The biological half-life of the distribution and clearance phases were 0.05h and 1.58h, respectively. In CD147-positive tumor models, the [124I]I-NB147 accumulated in A375, MDA-MB-435, and ASPC1 tumors, and the uptake value was significantly higher than that of [18F]F-FDG. Uptake in SK-MEL-28, BXPC3, and 4T1 tumors was not clearly observed. Finally, through autoradiography and histological studies, the correlation analysis between tumor uptake and CD147 expression level was determined. The high expression of CD147 in MM, TNBC, and PCA tissuesand in tumor cells was verified. The CD147 nanobody, NB147 was produced and radiolabeled to obtain the immunoPET probe, [124I]I-NB147, which showed high affinity to CD147 and precise visualization for accurate diagnosis of CD147-expressing lesions in different cancers. These results provide insight into the imaging and binding properties of nanobody NB147 over extended periods of time, reinforcing its potential in developing radionuclide therapies for CD147-positive cancer patients.

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

Pancreatic cancer-derived exosomal miR-510 promotes macrophage M2 polarization and facilitates cancer cell aggressive phenotypes.

Extensive tumor microenvironment (TME) and tumor-associated macrophages (TAMs) contribute to the initiation and progression of pancreatic cancer (PC). Cancer cell-derived exosomal miRNAs that stimulate macrophage M2 polarization might play an important role in the process. In the current study, we observed significant upregulation of miR-510 in PC cell lines in comparison to normal HPDE cell line, with PANC-1 exhibiting the highest and MIA PaCa-2 the lowest miR-510 levels. Functional assays demonstrated that miR-510 overexpression enhanced, while its inhibition reduced, PC cell viability, migration, invasion, and EMT. In vivo, miR-510 mimics promoted tumor growth and macrophage M2 polarization, whereas miR-510 inhibition had the opposite effect. Exosomes from PANC-1 and MIA PaCa-2 cells, characterized by nanoparticle tracking analysis and TEM, contained significantly higher miR-510 levels than those from HPDE cells. Macrophages incubated with conditioned media from these PC cells showed increased M2 polarization markers, a process inhibited by the exosome inhibitor GW4869. The delivery of miR-510 via PC cell-derived exosomes facilitated macrophage M2 polarization and regulated the STAT signaling pathway, suggesting that exosomal miR-510 plays a crucial role in the tumor microenvironment of PC by modulating macrophage M2 polarization.

Synthesis and Biological Evaluation of N-(1H-Indol-6-ylmethyl)benzenesulfonamide Analogs as Metabolic Inhibitors of Mitochondrial ATP Production in Pancreatic Cancer Cells.

A library of 26 indolyl sulfonamides and 12 amide and ester analogs based upon the 6-indolyl framework has been synthesized in an effort to target pancreatic cancer. The cytotoxicity of the indolyl sulfonamide compounds has been determined using a traditional (48-h compound exposure) assay against 7 pancreatic cancer cell lines and 1 non-cancerous cell line. The potential role of the compounds as metabolic inhibitors of ATP production was evaluated using a rapid screening (2-h compound exposure) assay developed within our laboratories. The IC50 values of the active compounds were determined using the rapid assay and six compounds displayed an IC50 value <5 μM against one or more pancreatic cancer cell lines. The ester analogs also display activity as potential metabolic inhibitors of ATP production with four of the six compounds displaying an IC50 value <5 μM against one or more pancreatic cancer cell lines.

Dendritic cell maturation is induced by p53-armed oncolytic adenovirus via tumor-derived exosomes enhancing systemic antitumor immunity.

Dendritic cells (DCs) are crucial in cancer immunity, because they activate cytotoxic T cells by presenting tumor antigens. Recently, oncolytic virus therapy has been recognized as a systemic immune stimulator. We previously developed a telomerase-specific oncolytic adenovirus (OBP-301) and a p53-armed OBP-301 (OBP-702), demonstrating that these viruses strongly activate systemic antitumor immunity. However, their effects on DCs remained unclear. In the present study, the aim was to elucidate the mechanisms of DC activation by OBP-702, focusing particularly on tumor-derived exosomes. Exosomes (Exo53, Exo301, or Exo702) were isolated from conditioned media of human or murine pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and PAN02) after treatment with Ad-p53, OBP-301, or OBP-702. Exo702 derived from Panc-1 and MiaPaCa-2 cells significantly upregulated CD86, CD80, CD83 (markers of DC maturation), and IFN-γ in DCs in vitro. Similarly, Exo702 derived from PAN02 cells upregulated CD86 and IFN-γ in bone marrow-derived DCs in a bilateral PAN02 subcutaneous tumor model. This DC maturation was inhibited by GW4869, an inhibitor of exosome release, and anti-CD63, an antibody targeting the exosome marker. Intratumoral injection of OBP-702 into PAN02 subcutaneous tumors significantly increased the presence of mature DCs and CD8-positive T cells in draining lymph nodes, leading to long-lasting antitumor effects through the durable activation of systemic antitumor immunity. In conclusion, tumor-derived exosomes play a significant role in DC maturation following OBP-702 treatment and are critical for the systemic activation of antitumor immunity, leading to the abscopal effect.

Discovery of novel TANK-Binding Kinase 1 (TBK1) inhibitor against pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has limited treatment options, underscoring the urgent need for developing new therapies. The upregulation of TBK1 activity plays a crucial role in multiple pancreatic cancer-related signaling pathways, suggesting that inhibiting the kinase activity of TBK1 could be a promising strategy. Herein, we discovered a novel TBK1 inhibitor, LIB3S0280, using a structure-based virtual screening (SBVS) strategy. In the anti-proliferative and viability assays, LIB3S0280 showed significant inhibition against pancreatic cancer cell lines that highly express TBK1 with the GI50 values of 2.24 and 4.71 μM and IC50 values of 6.64 and 10.98 μM at 96 h. For the downstream targets, LIB3S0280 can inhibit TBK1 downstream signaling by decreasing the phosphorylation of IκBα and AKT better than a known TBK1 inhibitor, BX-795. Furthermore, PDAC cells were arrested in G2/M and underwent apoptosis or senescence with the treatment of LIB3S0280. These findings suggest that TBK1 inhibitor LIB3S0280 has great potential as a lead compound in the further development of a novel treatment for PDAC.

Genome-wide microRNA Analysis Identified miR-210-3p Over-expression in Pancreatic Cancer Tissues as a Predictor of their Local Invasiveness.

The severe malignancy of pancreatic ductal adenocarcinoma (PDAC) is mainly due to frequent local invasiveness and distant metastasis. As for local invasiveness, we previously reported that cancer-specific molecular alterations detected on resected PDAC specimen surfaces, so-called molecular surgical margin (MSM) positiveness, were significantly associated with postoperative locoregional recurrence and distant metastasis. However, due to anatomical limitations, achieving adequate surgical margins during pancreatic cancer resection is often challenging. Therefore, predicting local invasiveness based on the primary tumor's gene profile is crucial to avoid positive MSM. Genome-wide miRNA expression profiles were examined and compared between MSM-positive and negative cases. Candidate miRNAs were evaluated in another validation cohort, and their clinicopathological characteristics were examined. Mimic or inhibitor constructs of the candidate miRNA were transfected to PDAC cell lines to evaluate the miRNA function in the pancreatic cancer cell lines and detect the downstream targets. Among some candidates with highly expressed miRNAs in MSM-positive cases by miRNA expression array, recurrence-free survival (RFS) was significantly shorter in the miR-210-3p high expression group (p=0.015). High miR-210-3p was significantly associated with large tumor diameter (p=0.001), anterior invasion positive (p=0.010), and positive lymph node metastasis (p<0.001). miR-210-3p inhibition in PDAC cell lines resulted in decreased proliferation and invasiveness. The iron-sulfur cluster assembly enzyme (ISCU) gene was identified as a target of miR-210-3p. ISCU reduction was significantly observed in PDAC primary tumors with high levels of miR-210-3p, leading to mitochondrial dysfunction in miR-210-3p-overexpressing PDAC cell lines, as demonstrated by glycolysis stress tests. Highly expressed hypoxia-inducible miR-210-3p in primary PDAC tissues induces locally invasive characteristics through mitochondrial dysfunction by suppressing ISCU expression, which may result in poor postoperative RFS outcomes.

Integrated bulk and single-cell profiling characterize sphingolipid metabolism in pancreatic cancer

BackgroundAbnormal sphingolipid metabolism (SM) is closely linked to the incidence of cancers. However, the role of SM in pancreatic cancer (PC) remains unclear. This study aims to explore the significance of SM in the prognosis, immune microenvironment, and treatment of PC.MethodsSingle-cell and bulk transcriptome data of PC were acquired via TCGA and GEO databases. SM-related genes (SMRGs) were obtained via MSigDB database. Consensus clustering was utilized to construct SM-related molecular subtypes. LASSO and Cox regression were utilized to build SM-related prognostic signature. ESTIMATE and CIBERSORT algorithms were employed to assess the tumour immune microenvironment. OncoPredict package was used to predict drug sensitivity. CCK-8, scratch, and transwell experiments were performed to analyze the function of ANKRD22 in PC cell line PANC-1 and BxPC-3.ResultsA total of 153 SMRGs were acquired, of which 48 were linked to PC patients’ prognosis. Two SM-related subtypes (SMRGcluster A and B) were identified in PC. SMRGcluster A had a poorer outcome and more active SM process compared to SMRGcluster B. Immune analysis revealed that SMRGcluster B had higher immune and stromal scores and CD8 + T cell abundance, while SMRGcluster A had a higher tumour purity score and M0 macrophages and activated dendritic cell abundance. PC with SMRGcluster B was more susceptible to gemcitabine, paclitaxel, and oxaliplatin. Then SM-related prognostic model (including ANLN, ANKRD22, and DKK1) was built, which had a very good predictive performance. Single-cell analysis revealed that in PC microenvironment, macrophages, epithelial cells, and endothelial cells had relatively higher SM activity. ANKRD22, DKK1, and ANLN have relatively higher expression levels in epithelial cells. Cell subpopulations with high expression of ANKRD22, DKK1, and ANLN had more active SM activity. In vitro experiments showed that ANKRD22 knockdown can inhibit the proliferation, migration, and invasion of PC cells.ConclusionThis study revealed the important significance of SM in PC and identified SM-associated molecular subtypes and prognostic model, which provided novel perspectives on the stratification, prognostic prediction, and precision treatment of PC patients.

Genome-Wide CRISPR Screen Identifies Genes Involved in Metastasis of Pancreatic Ductal Adenocarcinoma.

Background/Objectives: Early and aggressive metastasis is a major feature of pancreatic ductal adenocarcinoma. Understanding the processes underlying metastasis is crucial for making a difference to disease outcome. Towards these ends, we looked in a comprehensive manner for genes that are metastasis-specific. Methods: A genome-wide CRISPR-Cas9 gene knockout screen with 259,900 single guide RNA constructs was performed on pancreatic cancer cell lines with very high or very low metastatic capacity, respectively. Functional aspects of some of the identified genes were analysed in vitro. The injection of tumour cells with or without a gene knockout into mice was used to confirm the effect on metastasis. Results: The knockout of 590 genes-and, with higher analysis stringency, 67 genes-affected the viability of metastatic cells substantially, while these genes were not vital to non-metastasizing cells. Further evaluations identified different molecular processes related to this observation. One of the genes was MYBL2, encoding for a well-known transcription factor involved in the regulation of cell survival, proliferation, and differentiation in cancer tissues. In our metastasis-focussed study, no novel functional activity was detected for MYBL2, however. Instead, a metastasis-specific transformation of its genetic interaction with FOXM1 was observed. The interaction was synergistic in cells of low metastatic capacity, while there was a strong switch to a buffering mode in metastatic cells. In vivo analyses confirmed the strong effect of MYBL2 on metastasis. Conclusions: The genes found to be critical for the viability of metastatic cells form a basis for further investigations of the processes responsible for triggering and driving metastasis. As shown for MYBL2, unexpected processes of regulating metastasis might also be involved.

Serum from Hypertensive Patients Induces Cancer-Supporting Phenotype of Vascular Endothelium In Vitro

Background/Objectives: Large-scale epidemiological studies have established a bidirectional association between hypertension and cancer. However, the underlying mechanisms explaining this connection remain unclear. In our study, we investigated whether serum from patients with hypertension (HT) could enhance the aggressiveness of cancer cells in vitro through alterations in endothelial cell phenotype. Methods: Experiments were performed using EAhy926 endothelial cells and ovarian (SKOV-3), colorectal (SW480), pancreatic (PSN-1), breast (MCF-7), and lung (A549) cancer cell lines. Results: This study showed that conditioned medium (CM) produced by EAhy926 cells, when exposed to serum from patients with untreated hypertension (HT-CM), promotes the proliferation, migration, and invasion of every cancer cell line tested. In addition, endothelial cells subjected to HT serum promote the adhesion of all cancer cell types except PSN-1. An intensified transendothelial invasion of cancer cells was accompanied by decreased expression of junctional proteins (connexin 43, E-cadherin, occluding, desmoglein) in HT serum-treated endothelial cells. Quantitative analysis of the secretome of endothelial cells subjected to HT serum showed that they secrete increased amounts of CCL2, CXCL1, CXCL8, bFGF, HGF, IL-6, PAI-1, and TGF-β1. Moreover, cancer cells exposed to HT-CM display increased mRNA expression for several pro-cancerogenic agents, including CXCL8, tPA, and VEGF. Conclusions: Our report shows that hypertension may potentiate cancer cell aggressiveness by modulating endothelial cell phenotype. Further tests with antihypertensive drugs are required to assess whether effective treatment of hypertension can mitigate its cancer-promoting potential.

The prognostic significance of twist in pancreatic cancer and its role in cancer promotion through the regulation of the immune microenvironment and EMT mechanisms

ObjectivePancreatic cancer has a poor prognosis due to its high malignancy and rapid progression. The limited immunogenicity of pancreatic cancer (PAAD) contributes to its low responsiveness to immunotherapy, yet its underlying mechanism remains poorly understood. As a cancer promoting gene, Twist participates in EMT in various tumors and promotes tumor progression. The interplay between EMT and the tumor microenvironment (TME) emerges as a pivotal factor influencing tumor immunity and response to immunotherapy. Twist therefore has potential as a biomarker for gauging the outcome of tumour immunotherapy.This research aimed to assess the Twist's prognostic significance in PAAD and its relationship to immunotherapy response.MethodsIn this research, transcriptional data and epigenetic alterations of Twist in pancreatic cancer, along with their impact on the prognosis of PAAD patients, were analyzed using databases. Functional enrichment analysis elucidated the biological role of Twist in PAAD. Subsequently, databases including CIBERSORT and TIDE were employed to investigate the association between Twist expression and immune cell infiltration, immune checkpoint genes, and immunotherapy sensitivity within the pancreatic cancer immune microenvironment.Paraffinized specimens from patients with pancreatic ductal adenocarcinoma confirmed by postoperative pathology were selected for Twist expression verification, and the difference was analyzed by Chi-square test; uncontaminated pancreatic cancer cell lines were used for Twist expression verification, and the differences were analyzed by Student t-test.ResultsTwist mRNA expression was notably upregulated in PAAD, positively correlating with gene methylation levels. Analyses of Kaplan–Meier and Cox regression showed a correlation between better overall survival and lower Twist expression. Functional annotation indicated that Twist-associated differentially expressed genes (DEGs) were involved in EMT regulation and acute inflammation. High expression of Twist leads to a significant reduction in the infiltration of anti-tumor immune cells such as Monocytes, NKcellsactivated, and TcellsCD8, further supporting its creation of a typical immunosuppressive microenvironment in pancreatic cancer. Twist expression is positively correlated with the expression of HARVCR2, LAIR1, LGALS3 and other genes, which may be related to the treatment response to immune checkpoint inhibitors (ICIs). TIDE analysis predicts that patients with high expression of Twist will be insensitive to immunotherapy. Twist is significantly over-expressed in pancreatic cancer cell lines and tissues, and is negatively correlated with E-cadhrin expression, but positively correlated with N-cadherin,Snail, and ZEB1.ConclusionHigh Twist expression in PAAD signifies a grim prognosis. Its elevated levels not only contribute to tumor progression through EMT induction but also exert regulatory control over the immune microenvironment, leading to immunosuppression and diminished effectiveness of immunotherapy.

METTL16 inhibits pancreatic cancer proliferation and metastasis by promoting MROH8 RNA stability and inhibiting CAPN2 expression.

N6-methyladenosine (m6A) modification plays a crucial role in the progression of various cancers, including pancreatic cancer, by regulating gene expression. However, the specific mechanisms by which m6A affects pancreatic cancer metastasis remain unclear. This study aims to elucidate the role of METTL16, an m6A writer gene, in regulating core genes such as CAPN2 and MROH8, influencing tumor growth and metastasis. Transcriptomic data from pancreatic cancer patients in The Cancer Genome Atlas (TCGA) were analyzed to identify m6A-related genes. We performed correlation and survival analyses to uncover core genes influenced by m6A expression. Functional assays, including METTL16 knockdown and overexpression experiments, were conducted in pancreatic cancer cell lines, patient-derived organoids, and animal models. Immunofluorescence, co-immunoprecipitation (Co-IP), and m6A-specific quantitative PCR were used to validate protein interactions and m6A modifications. Chromatin immunoprecipitation (ChIP) analysis was utilized to investigate transcription factor binding at gene promoter regions. METTL16 and METTL3 were identified as key m6A regulators associated with improved prognosis in pancreatic cancer patients (P<0.05). CAPN2, CHMP2B, ITGA3, ITGA6, ITPR1, and RAC1 were identified as core genes linked to m6A expression, all significantly correlated with patient prognosis (P<0.05). METTL16 overexpression significantly inhibited tumor growth and metastasis (P<0.001) by downregulating CAPN2 through an indirect mechanism involving the transcription factor TBP and the gene MROH8. MROH8 negatively regulated CAPN2 by promoting TBP degradation, with METTL16 enhancing MROH8 mRNA stability through m6A modifications (P<0.01). Functional assays demonstrated that METTL16 and YTHDC2 (an m6A reader) collaboratively enhanced MROH8 mRNA stability, thereby inhibiting CAPN2 expression and reducing tumor proliferation and metastasis (P<0.001). This study reveals a novel regulatory axis involving METTL16, MROH8, and TBP that modulates CAPN2 expression, contributing to the suppression of pancreatic cancer progression. The METTL16-MROH8-TBP-CAPN2 pathway offers potential therapeutic targets for pancreatic cancer treatment, highlighting the significance of m6A modifications in tumor regulation. Further clinical validation is needed to confirm these findings in human patients.

Oncolytic Coxsackievirus B3 Strain PD-H Is Effective Against a Broad Spectrum of Pancreatic Cancer Cell Lines and Induces a Growth Delay in Pancreatic KPC Cell Tumors In Vivo

Pancreatic cancer is one of the deadliest cancers globally, with limited success from existing therapies, including chemotherapies and immunotherapies like checkpoint inhibitors for patients with advanced pancreatic ductal adenocarcinoma (PDAC). A promising new approach is the use of oncolytic viruses (OV), a form of immunotherapy that has been demonstrated clinical effectiveness in various cancers. Here we investigated the potential of the oncolytic coxsackievirus B3 strain (CVB3) PD-H as a new treatment for pancreatic cancer. In vitro, PD-H exhibited robust replication, as measured by plaque assays, and potent lytic activity, as assessed by XTT assays, in most pancreatic tumor cell lines, outperforming two other coxsackievirus strains tested, H3N-375/1TS and CVA21. Thus, H3N-375/1TS showed efficient replication and lytic efficiency in distinctly fewer tumor cell lines, while most tumor cells were resistant to CVA21. The oncolytic efficiency of the three OV largely correlated with mRNA expression levels of viral receptors and their ability to induce apoptosis, as measured by cleaved caspase 3/7 activity in the tumor cells. In a syngeneic mouse model with subcutaneous pancreatic tumors, intratumoral administration of PD-H significantly inhibited tumor growth but did not completely stop tumor progression. Importantly, no virus-related side effects were observed. Although pancreatic tumors respond to PD-H treatment, its therapeutic efficacy is limited. Combining PD-H with other treatments, such as those aiming at reducing the desmoplastic stroma which impedes viral infection and spread within the tumor, may enhance its efficacy.

Targeting Human Pancreatic Cancer with a Fluorophore-Conjugated Mucin 4 (MUC4) Antibody: Initial Characterization in Orthotopic Cell Line Mouse Models.

Background/Objectives: Pancreatic cancer is the third leading cause of death related to cancer. The only possible cure presently is complete surgical resection; however, this is limited by difficulty in clearly defining tumor margins. Enhancement of the visualization of pancreatic ductal adenocarcinoma (PDAC) tumor margins using near-infrared dye-conjugated tumor-specific antibodies was pioneered by using anti-CEA, anti-CA19.9, and anti-MUC5AC in orthotopic mouse models of pancreatic cancer. Recently, an antibody to Mucin 4 (MUC4) conjugated to a fluorescent probe has shown promise in targeting colon tumors in orthotopic mouse models. Methods: In the present study, we targeted pancreatic cancer using an anti-MUC4 antibody conjugated to IRDye800 (anti-MUC4-IR800) in orthotopic mouse models. Two pancreatic cancer human cell lines were used, SW1990 and CD18/HPAF. Results: Anti-MUC4-IR800 targeted the two pancreatic cancer cell line tumors in orthotopic mouse models with high tumor-to-pancreas ratios and high tumor-to-liver ratios, with greater targeting seen in SW1990. Conclusions: The present results suggest anti-MUC4-IR800's potential to be used in fluorescence-guided surgical resection of pancreatic cancer.

Combinatorial Anti-Cancer Effect of Polypurine Reverse Hoogsteen Hairpins against KRAS and MYC Targeting in Prostate and Pancreatic Cancer Cell Lines

Introduction: KRAS and MYC are proto-oncogenes that are strictly regulated in healthy cells that have key roles in several processes such as cell growth, proliferation, differentiation, or apoptosis. These genes are tightly interconnected, and their dysregulation can lead to cancer progression. We previously individually targeted these oncogenes using Polypurine Reverse Hoogsteen (PPRH) hairpins, mostly targeting the complementary strand of G-quadruplex-forming sequences. We validated them in vitro in different cancer cell lines with deregulated KRAS and/or MYC. In this work we focused on our understanding of the cooperative dynamics between these oncogenes, by investigating the combined impact of PPRHs targeting KRAS and MYC in pancreatic and prostate cancer cells. Results: The combinations had a modulatory impact on the expression of both oncogenes, with transcriptional and translational downregulation occurring five days post-treatment. Out of the four tested PPRHs, MYC-targeting PPRHs, especially HpMYC-G4-PR-C directed against the promoter, showed a greater cytotoxic and expression modulation effect. When both KRAS- and MYC-targeting PPRHs were applied in combination, a synergistic reduction in cell viability was observed. Conclusion: The simultaneous targeting of KRAS and MYC demonstrates efficacy in gene modulation, thus in decreasing cell proliferation and viability.

Unraveling the hepatoprotective and anti-pancreatic cancer potential of Caralluma europaea: a comprehensive in vivo, in vitro and in silico evidence

Caralluma europaea Guss. (C. europaea) is a medicinal plant used for cancer treatment. However, these treatments may be associated with complications that need to be investigated. This work aims to evaluate not only the chemical composition but also the hepatoprotective and anticancer properties of C. europaea extracts. The chemical constitution of the hydroethanolic extract was explored using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The hydroethanolic extract, flavonoids, and polyphenols-rich extract at 100, 15, and 50 mg/kg, respectively, were administered to acetaminophen-treated rats for seven days. We used Western blotting and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) to determine the protein and the mRNA levels of cancer stemness markers in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 exposed to increasing doses of C. europaea extracts. In silico analysis was used to evaluate the effects of phenolic compounds revealed in C. europaea on caspase-3 and HSP90, and on liver damage on CYP2E1. The primary phenolics detected by GC-MS and HPLC were ferulic acid and benzofurazan. The positive control group showed an increase in AST, ALT, ALP, triglycerides, and VLDL levels. C. europaea extracts demonstrated hepatoprotective effects by ameliorating acetaminophen-induced alterations of biochemical and hispathological parameters. Immunoblotting and RT-qPCR profiling of cancer stemness markers indicated a reduction in the expression levels of Oct-4 and Nanog proteins, as well as a reduction in the mRNA levels of CD133 by 50–60% and Sox2 by 80–90% in pancreatic cancer cells. Molecular docking showed that naringenin presented the highest docking Gscore on CYP2E1 (−8.199) and HSP90 (−7.742). In conclusion, C. europaea extracts could be considered as a safe and promising therapeutic strategy to sensitize pancreatic cancer cells to chemotherapy.

Adipose tissue-derived extracellular vesicles from obese mice suppressed splenocyte-mediated pancreatic cancer cell death.

Obesity is a risk factor for pancreatic cancer and negatively contributes to the immune system. However, the mechanisms by which obesity mediates these actions are still poorly understood. Recent studies have demonstrated that extracellular vesicles (EVs) are key mediators of communication between cells and may influence various aspects of cancer progression. We aim to explore the influence of EVs derived from adipose tissue of obese mice on cytokine production within the interactions between cancer cells and immune cells. We isolated EVs from the adipose tissue of both C57BL6/J mice and Ob/Ob mice. Subsequently, we treated EVs with Panc02 cells, the murine ductal pancreatic cancer cell line, which were co-cultured with splenocytes. Viability and SMAD4 gene expression were examined in Panc02 cells, and cytokine concentrations of IL-6, IL-4, IL-12, and IL-12p70 were measured in the cultured medium. Interestingly, we observed a significant reduction in splenocyte-mediated Panc02 cell death when treated with EVs derived from the adipose tissue of Ob/Ob mice, compared to those from C57BL6/J mice. Additionally, EVs from Ob/Ob mice-derived adipose tissue significantly increased the levels of IL-4, IL-2, and IL-12p70 in the culture media of Panc02 cells co-cultured with splenocytes, compared to EVs from C57BL6/J mice-derived adipose tissue. Adipose tissue-derived EVs from obese mice suppressed splenocyte-mediated Panc02 cell death and upregulated IL-4, IL-2, and IL-12p70 in cultured medium.

An Optimized Liquid Chromatography-Mass Spectrometry Method for Ganglioside Analysis in Cell Lines.

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell-cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography-mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R2 = 0.9961-0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types.

Synthesis of New Fluorinated Tubastatin A Derivatives Based on Cyclopentane/Cyclohexane with Good Anti‐tumor Activity in Vitro

AbstractTwo series of novel fluorinated Tubastatin A derivatives based on cyclopentane or cyclohexane substitution were first time synthesized by 5 steps at high yield. The influence of the cycloalkanes substituted N‐methylpiperidine and/or fluorinated group on the cap group of Tubastatin A for the in vitro anti‐tumor activity of seven different tumor cell lines (human hepatoma cells Bel7402 and HepG2, human nasopharyngeal carcinoma cells CNE2 and SUNE1, human breast cancer cells MDA‐MB‐231 and MCF‐7, and human pancreatic cancer cells SW1990) was investigated. Compared with Tubastatin A, these novel derivatives with fluorinated groups on the benzene ring of the cap group enhance activity, and modification of the N‐methylpiperidine to cycloalkanes of the cap group improves solubility. The most promising compound trifluoromethyl and cyclohexane substituted derivative, N‐hydroxy‐4‐((6‐(trifluoromethyl)‐1,2,3,4‐tetrahydro‐9H‐carbazol‐9‐yl)methyl) benza‐mide (6 h), had a wide range of anti‐tumor cell range, the IC50 value for human pancreatic cancer cell line SW1990 was 1.17 μM. Hence, fluorinated groups and cyclohexane result in a wide range of anti‐tumor cell range and good anti‐cancer activity.