MIA PaCa-2 Pancreatic Cancer Cells Research Articles

Abstract 3356: Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice

Abstract New therapeutic targets are needed for pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death worldwide. Phospholipase D (PLD) is a lipid-signaling enzyme that appears to play a critical role in cell proliferation, survival/apoptosis, and downstream oncogenic transformation. Elevated PLD1 or PLD2 (the two classical mammalian isoforms) activity has been shown to promote tumor progression in various cancer types, however, the role and mechanisms of PLD in pancreatic cancer are not yet understood. Thus, the objective is to determine the role of PLD1 regulation in pancreatic carcinogenesis through genetic and pharmacological inhibition. To evaluate the role of PLD1 in tumor growth, we subcutaneously transplanted human MIA PaCa-2 pancreatic cancer cells expressing endogenous PLD1 levels (Ctr KD cells), or MIA PaCa-2 cells in which PLD1 was knocked down (Pld1 KD cells), into immunodeficient nude mice. After 18 days post implantation, tumors that arose from Pld1-KD cells presented a significantly smaller tumor size (p<0.05) and had reduced levels of Bcl-xL and Bcl-2, compared to controls (Ctr KD). Then, to assess the role of PLD1 in the tumor microenvironment, we implanted KPC pancreatic cancer cells into C57BL/6 wild-type (WT) or C57BL/6 PLD1 knockout (Pld1−/−) mice with and without treatment with gemcitabine. Fourteen days after tumor implantation, the growth of KPC tumors was attenuated in Pld1−/− mice by 49% (p<0.05). Treatment with gemcitabine reduced tumor growth by 51.6% (p<0.05) in WT mice and by 79.5% (p<0.03) in Pld1−/− mice. Moreover, we evaluated the effects of PLD1 genetic ablation by crossing LSL-KrasG12D/+; Ptf1Cre/+ (KC) mice into PLD1 knockout (Pld1−/−) mice to generate KC; Pld1−/− mice. At 8 months of age, KC; Pld1−/− mice had significantly smaller pancreases (p<0.05) compared to KC mice. Interestingly, PLD1 ablation increased phosphorylation of ERK and c-Jun in KC; Pld1−/− mice compared to KC mice. Finally, to assess whether pharmacological inhibition of PLD can inhibit pancreatic carcinogenesis, KC mice received daily intraperitoneal injections of a small molecule inhibitor of PLD1 and PLD2 (FIPI) at a dosage of 3 mg/kg of body weight for a duration of 5 weeks. Treatment with FIPI reduced acinar-to-ductal metaplasia (p<0.05) and reduced cell proliferation (p<0.01), when compared to vehicle-treated KC controls. Additionally, FIPI treatment was safe demonstrating no significant differences in body weight between the groups and no significant alteration in the serum levels of liver enzymes, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. In conclusion, these findings reveal PLD1 plays a critical role in pancreatic carcinogenesis and might represent a novel therapeutic target. Citation Format: Hala Amer Addassi, Karen Matsukuma, Gerardo G. Mackenzie. Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3356.

Abstract 7291: Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients

Abstract Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP dependent transmembrane efflux pump that confers to drug resistance. Overexpression of MRP1 has been observed in both pancreatic ductal adenocarcinoma (PDAC) and breast cancer tissues. Numerous proteomics studies have revealed the presence of MRP1 in small extracellular vesicles (sEVs) released from cancer cells. Cancer-derived sEVs are known to be released into the circulation, thus becoming attractive candidates for non-invasive biomarker development. However, whether the MRP1 level in plasma sEVs is indicative of human cancer has not been evaluated. This study examined the MRP1 expression in plasma sEVs derived from patients with PDAC, breast and colon cancer, and age- and gender-matched healthy subjects. The patient plasma samples were obtained primarily from the NCI-sponsored Cooperative Human Tissue Network and Stephenson Cancer Center. Healthy plasma samples were provided by the Oklahoma Blood institute. Human pancreatic cancer cell lines PANC-1, MIA PaCa-2 and BxPC-3, and the pancreatic ductal cell line hTERT-HPNE were tested for MRP1 expression prior to examination of plasma samples. Cellular and plasma sEVs were isolated using the double filtration followed by polymer precipitation method. Western blot was performed to assess the expression level of MRP1 and proteomics was applied to confirm the detection of a truncated form of MRP1 in plasma sEVs. MRP1 expression was detected in the sEVs derived from all three pancreatic cancer cell lines but absent in the sEVs from the HPNE line. Overexpression of a truncated MRP1 protein was detected in plasma sEVs derived from patients with early stage (stage I-IIA) and late stage (III-IV) PDAC, compared with that from matched healthy subjects (n=17 for each group). Similar findings were evident in plasma sEVs from patients with breast cancer (n=7), while the results remained inconclusive in plasma sEVs from patients with colon cancer (n=7). Protein structure analysis indicated that the truncated form of MRP1 likely consists of the regions between the QCRL-1 motif and the C-terminus of the protein. Proteomic analysis detected the peptides derived from the C-terminus region, consistent with the structural analysis. Furthermore, the truncated form of MRP1 can only be detected using antibodies against the epitopes in the C-terminus and QCRL-1 region of MRP1 protein. In summary, we have detected a truncated form of MRP1 protein in plasma sEVs derived from cancer patients, and the level of its expression is significantly elevated in patients with PDAC and breast cancer. Citation Format: Kritisha Bhandari, Jeng Shi Kong, Chao Xu, Ajay Jain, Wei-Qun Ding. Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7291.

Abstract 4495: Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells

Abstract Background: The identification of novel compounds that specifically target cancer cells while sparing normal tissues is crucial. The orphan tyrosine kinase-like receptor 1 (ROR1) is one such target expressed exclusively in various human malignancies, including pancreatic cancer, but not in normal adult tissues. Inhibiting ROR1 signaling shows promise in halting pancreatic cancer cell proliferation and invasion. In this study, lead compounds CSF1R-IN-1 and MCULE-8715589337 were found as potential inhibitors of the ROR1 and were further evaluated for its potential effect on pancreatic cancer cell viability. Method: We curated a compound library of 6 million from PubChem, 2 million lead-like compounds from ZINC15, and 2 million compounds from diverse vendors. Employing Glide’s HTVS followed by XP, we refined the selection based on docking scores lower than the reference molecule, Ponatinib. Compounds were shortlisted by pre-simulation MMGBSA calculations with Schrödinger Prime MM-GBSA module, focusing on compounds with energies below -100 kcal/mol. Then we performed MD simulation for 100ns. Post-simulation MMGBSA on the last 1 ns of simulation assessed change in ligands’ binding energies. The selected leads underwent further validation utilizing two pancreatic cancer cell lines, MiaPaCa-2 and PANC-1. Half-maximal inhibitory concentration (IC50) was assessed using MTS cell viability assay. Result: Comparing pre- and post-simulation MMGBSA energies, the reference compound showed increased, while CSF1R-IN-1 and MCULE-8715589337 exhibited decreased energies. The reference ligand's RMSD showed stability with fluctuations of 3 to 3.5 Å, whereas CSF1R-IN-1 and MCULE-8715589337 had stable and lower RMSD values (approximately 3 Å and 2.4 Å). Test compounds, CSF1R-IN-1 and MCULE-8715589337 showed better molecular interactions with ROR1 compared to reference. The reference compound formed hydrogen bonds with ILE555 and ASP633, along with pi-pi stacking with PHE552. CSF1R-IN-1 forms a hydrogen bond with ILE555 and ASP633, pi-pi stacking with PHE552, and interaction with SER632 through a water bridge. Additionally, compound MCULE-8715589337 forms hydrogen bonds with ASP633, SER632, LYS506, and GLU523. CSF1R-IN-1 notably inhibited proliferation in MiaPaCa-2 cells (IC50: 1.29 μM) while no efficacy in PANC-1 cells (IC50: 78.41 μM). MCULE-8715589337 showed relatively similar IC50 values of 39.06 μM and 37.73 μM in MiaPaCa-2 and PANC-1 cells, respectively. Conclusion: The reduction in MMGBSA energies, low RMSD values, and a higher number of interactions highlight the better characteristics of both compounds compared to the reference. Our findings show CSF1R-IN-1 and MCULE-8715589337 have efficacy in pancreatic cancer cells, but further studies are required to validate and to understand the mechanism of action. Citation Format: Indrakant Kumar Singh, Shradheya RR Gupta, Pooja Mittal, Shivani Soni, Jae Ho Lo, Francesca Battaglin, Yan Yang, Lesly Torres-Gonzalez, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Heinz Josef Lenz. Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4495.

Abstract 6001: Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells

Abstract Background: Myeloid cell leukemia 1 (MCL-1) is an anti-apoptotic member of the BCL-2 protein family which is involved in intrinsic (mitochondrial) pathway of apoptosis and is deregulated in several cancers. Current MCL-1 specific inhibitors have limitations in terms of efficiency, tolerability, and off-target effects and challenges associated with shallow binding groove of MCL-1. In this study, dihydroergotamine mesylate (DHE), an approved anti-migraine drug, was found as a potential ligand of the MCL-1 BH3-binding pocket and was further evaluated for its potential MCL-1 inhibition and effect on pancreatic cancer cells. Methods: PubChem Database was used for library preparation and virtual screening (OpenBabel and AutoDock Vina). The 55 crystal structures available for MCL-1 in Protein Data Bank (PDB) were clustered using principal component analysis with Bio3D R-package to select representative PDBs for further screening. Consensus voting and k-means clustering was used to shortlist compounds for Molecular dynamics (MD) simulations. MD simulations (100 nanoseconds) by GROMACS were employed to measure stability of ligands in MCL-1 binding pocket. DHE efficacy on cell proliferation was evaluated with MTS and clonogenic assays and on apoptosis using the FITC/Annexin V flow cytometry assay in MiaPaCa-2 and PANC-1 pancreatic cancer cell lines. Effect of DHE treatment on MCL-1 gene and protein expression was analyzed using qRT-PCR and western blotting, respectively. Results: Computational screening identified DHE (PubChem ID: 12308974) as a top hit exhibiting consistent interactions within the MCL-1 BH3-binding pocket. Notably, DHE displayed stable RMSD and formed four hydrogen bonds throughout MD simulations. In vitro results showed that DHE significantly reduced viability of MiaPaCa-2 and PANC-1 pancreatic cancer cells with IC50 values of 31 μM and 47 μM, respectively. DHE significantly (P<0.001) suppressed colony formation by pancreatic cancer cells in a dose-dependent (10μM, 20μM, and 40μM) manner. The percentage of Annexin V-positive and Propidium iodide-negative fraction in MiaPaCa-2 cells treated with DHE also increased in a dose-dependent manner. DHE did not have significant effect on MCL-1 mRNA expression. However, western blotting demonstrated MCL-1 protein levels were significantly reduced in both pancreatic cancer cell lines upon DHE treatment. Conclusion: Our findings showed DHE suppresses the pancreatic cancer cells viability and proliferation, and significantly increased early apoptotic cells. DHE treatment had no effect on MCL-1 at the transcription level but significantly reduced the protein, suggesting posttranslational modifications, and thus future mechanism of action studies is required. The present study reports that the anti-migraine drug DHE might be a novel therapeutic agent for pancreatic cancer and needs to be explored further. Citation Format: Pooja Mittal, Jae Ho Lo, Shivani Soni, Francesca Battaglin, Shradheya RR Gupta, Lesly Torres-Gonzalez, Yan Yang, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Jian Yu, Lin Zhang, Joshua Millstein, Indrakant Kumar Singh, Heinz Josef Lenz. Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6001.

Abstract 1825: Synthesis and biological evaluation of novel 5-FU analogs against pancreatic cancer

Abstract Purpose: 5-Fluorouracil (5-FU) is one of the standard chemotherapy drugs used to treat pancreatic cancer. However, treatment only extends survival modestly, and disease recurrence is typical due to systemic instability, drug resistance, severe adverse effects, and limited benefits in patients with locally advanced tumors. These shortcomings have necessitated new strategies to develop safer and more effective anticancer agents. The study’s objective was to synthesize, characterize, and evaluate the cytotoxic effects of novel 5-FU analogs in pancreatic cancer cells. Methods: The analogs, XYZ-I-71 and XYZ-I-73, were synthesized by introducing a tetrahydrofuran ring on 5-fluorocytosine (a precursor of 5-FU) and conjugation with octanoyl chloride and lauroyl chloride, respectively. They were characterized by NMR, HPLC, micro-elemental analysis, and mass spectrometry. Cytotoxicity and cell migration studies were performed to determine the effectiveness of the analogs against MiaPaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells. PARP apoptosis study was conducted in MiaPaca-2 cells using the analogs and 5-FU. The metabolic stability of analogs was determined by using human liver microsomes and quantified by HPLC. Results: Analysis of the 1H-NMR spectra displayed amide bonds at 7.80 ppm and 7.73 ppm, confirming the conjugation of octanoyl and lauroyl chloride to 5-fluorocutosine, respectively. The purity of both analogs was 99.6%. We found that the XYZ-I-73 (IC50 3.6 ± 0.4μM) analog was most effective against MiaPaCa-2 cells compared to XYZ-I-71(IC50 12.3±1.7μM), GemHCl (IC50 24.2 ± 1.3 μM), Irinotecan (IC50 10.1 ± 1.5 μM) and 5-FU (IC50 13.2 ± 1.1 μM). For PANC-1 cell growth inhibition, XYZ-I-73 (IC50 3.9 ± 0.5) was again observed as the most effective agent compared to XYZ-I-71(IC50 8.7±0.9 μM), GemHCl (IC50 10.07±0.9), 5-FU (IC50 20.43±1.2) and Irinotecan (IC50 11.6 ±1.1). A similar pattern of XYZ-I-73 (IC50 5.9±0.7) anticancer activity against BxPC-3 cells was found to be higher than XYZ-I-71(IC50 7.7 ± 0.8), GemHCl (IC50 10.95±0.9), 5-FU (IC50 14±1.1) and Irinotecan (IC50 9.5±1.0). For 24-hour MiaPaCa-2 cell migration studies, XYZ-I-73 (5µM) analog significantly reduced migration (# of migrated cells, 168±2.9) followed by XYZ-I-71(315±2.1), Gem-HCl (762±3.1) and 5-FU (710±3.2). PARP studies demonstrated significant inhibition of PARP expression of XYZ-I-73 treated cells compared to 5-FU and XYZ-I-71. Further, BAX and p53 expressions were significantly increased in cells treated with XYZ-I-73 compared to 5-FU and XYZ-I-71. Metabolic stability studies showed that 80 ± 5.9 % of XYZ-I-71 and XYZ-I-73 remained intact after 2-hour exposure in liver microsomal solution compared to 5-FU. Conclusion: XYZ-I-73 demonstrated a remarkable cytotoxic effect and improved in vitro metabolic stability over the selected standard drugs and may have potential anticancer activity against pancreatic cancer. Citation Format: Esther Frimpong, Raviteja Bulusu, Joy Okoro, Andriana Inkoom, Nkafu Ndemazie, Sherise Rogers, Xue Zhu, Bo Han, Edward Agyare. Synthesis and biological evaluation of novel 5-FU analogs against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1825.

Abstract 4677: Characterizing resistant and metastatic phenotypes to pharmacological ascorbate in pancreatic cancer

Abstract Background and purpose: Pharmacological ascorbate (P-AscH) is a prooxidant that oxidizes to produce high levels of hydrogen peroxide. It is currently being investigated as an anti-cancer neoadjuvant for pancreatic cancer (PC). In phase I clinical trials, P-AscH has generated encouraging results in terms of efficiency and safety. However, 50% of patients do not respond to P-AscH suggesting that resistance occurs in a subset of patients. We have generated PC cell lines resistant to P-AscH to characterize mechanisms of resistance. Methodology: PANC-1 and MIA PaCa-2 PC cell lines were treated with a stepwise progression of increasing concentrations of P-AscH until 10-fold IC50 concentration was fully tolerated (AscR, ascorbate resistant) as determined by clonogenic survival. Once resistance was achieved, various redox enzyme protein expression and activity were measured to determine their role in resistance to P-AscH. Proliferation and cell cycle analysis were conducted on resistant and sensitive cells. An RNA sequencing screen was conducted as an unbiased approach to identify potential candidates crucial for resistance. Finally, phenotypes associated to increased metastatic potential, including collagen invasion and in vivo tumor colonization were explored for characterization. Results: PANC-1 and MIA PaCa-2 AscR cells developed unique mechanisms to tolerate high levels of P-AscH. PANC-1 AscR cells almost exclusively rely on catalase to detoxify H2O2 generated during P-AscH treatments, while MIA PaCa-2 AscR cells do not. It is interesting to note that not every redox enzyme was upregulated in response to P-AscH treatments, including GPx1/4. The AscR cell lines also differed in growth rates. PANC-1 AscR cells developed a faster proliferation rate, whereas MIA PaCa-2 AscR cells significantly decreased their growth. This is consistent with a shortened G1/G0 cell cycle phase and an accumulation in G2/M, respectively. The RNA sequencing screen revealed a metastatic signature in association with resistance to P-AscH, including upregulation of the PI3K/AKT axis. We observed a significant increase in collagen matrix invasion, as well as metastatic colonization in vivo. Additionally, we have observed increased expression of active AKT in both AscR cell lines, indicating a potential mechanism for the increased metastatic phenotype. Conclusion: Our findings demonstrate distinct mechanisms for AcsR cells to tolerate high doses of P-AscH. Although catalase has demonstrated to be a key factor in controlling redox flux, it may not be necessary for all cell types. Not only can P-AscH resistance alter redox enzyme expression, but we have also observed substantial increase in metastatic potential to drive invasion and tumor colonization. In summary, our data indicate a potential route for PC to tolerate high levels of P-AscH and may explain how some patients do not respond to this treatment regimen. Citation Format: Amanda N. Pope, Brianne R. O'Leary, Juan Du, Garry R. Buettner, Michael D. Henry, Joseph J. Cullen. Characterizing resistant and metastatic phenotypes to pharmacological ascorbate in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4677.

Efficient Assessment of Tumor Vascular Shutdown by Photodynamic Therapy on Orthotopic Pancreatic Cancer Using High-Speed Wide-Field Waterproof Galvanometer Scanner Photoacoustic Microscopy.

To identify the vascular alteration by photodynamic therapy (PDT), the utilization of high-resolution, high-speed, and wide-field photoacoustic microscopy (PAM) has gained enormous interest. The rapid changes in vasculature during PDT treatment and monitoring of tumor tissue activation in the orthotopic pancreatic cancer model have received limited attention in previous studies. Here, a fully two-axes waterproof galvanometer scanner-based photoacoustic microscopy (WGS-PAM) system was developed for in vivo monitoring of dynamic variations in micro blood vessels due to PDT in an orthotopic pancreatic cancer mouse model. The photosensitizer (PS), Chlorin e6 (Ce6), was utilized to activate antitumor reactions in response to the irradiation of a 660 nm light source. Microvasculatures of angiogenesis tissue were visualized on a 40 mm2 area using the WGS-PAM system at 30 min intervals for 3 h after the PDT treatment. The decline in vascular intensity was observed at 24.5% along with a 32.4% reduction of the vascular density at 3 h post-PDT by the analysis of PAM images. The anti-vascularization effect was also identified with fluorescent imaging. Moreover, Ce6-PDT increased apoptotic and necrotic markers while decreasing vascular endothelial growth factor (VEGF) expression in MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. The approach of the WGS-PAM system shows the potential to investigate PDT effects on the mechanism of angiographic dynamics with high-resolution wide-field imaging modalities.

Epi-Magnolin, a tetrahydrofurofuranoid lignan from the oleo-gum resin of Commiphora wightii, as inhibitor of pancreatic cancer cell proliferation, in-vitro and in-silico study

Five known furofuran lignans, dia-sesamin (1), 5-methoxysesamin (2), epi-magnolin (3), kobusin (4) and yangambin (5) were isolated for the first-time from the oleo-gum resin of Commiphora wightii. This is the first report on the 13C NMR assignments for epi-magnolin (3). Each of the isolated compounds was evaluated for its ability to inhibit MIA PaCa-2 pancreatic cancer cell line. Among them, epi-magnolin (3) displayed potential activity (IC50 = 29 nM) compared to colchicine (IC50 = 56 nM). 3D-flexible alignment revealed that epi-magnolin (3) has great matching with the tubulin polymerization inhibitor, colchicine. Meanwhile, docking studies exhibited that compounds 1–5 displayed good binding free energies against colchicine binding site (CBS) of tubulin with binding modes that were highly comparable to that of colchicine. Compounds 2, 3, and 5 showed superior binding free energies than colchicine (-24.37 kcal/mol). epi-Magnolin (3) showed the highest binding score against CBS. MD simulation studies confirmed the stability of epi-magnolin (3) in the active site for 200 ns. Furthermore, four online servers (Swiss ADME, pkCSM pharmacokinetics, AdmetSAR, and ProTox-II) were utilized to predict the ADMET parameters. The in-silico pharmacokinetics predictions reveled that epi-magnolin (3) has significant oral bioavailability and drug-like capabilities. Communicated by Ramaswamy H. Sarma

Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.

Nicotine promotes epithelial to mesenchymal transition and gemcitabine resistance via hENT1/RRM1 signalling in pancreatic cancer and chemosensitizing effects of Embelin-a naturally occurring benzoquinone

Pancreatic cancer is lethal due to poor prognosis with 5-year survival rate lesser than 5 %. Gemcitabine is currently used to treat pancreatic cancer and development of chemoresistance is a major obstacle to overcome pancreatic cancer. Nicotine is a known inducer of drug resistance in pancreatic tumor micro-environment. Present study evaluates chemoresistance triggered by nicotine while treating with gemcitabine and chemosensitization using Embelin. Embelin is a naturally occurring benzoquinone from Embelia ribes possessing therapeutic potency. To develop nicotine-induced chemo-resistance, pancreatic cancer cells PANC-1 and MIA PaCa-2 were continuously treated with nicotine followed by exposure to gemcitabine. Gemcitabine sensitivity assay and immunoblotting was performed to assess the chemo-resistance. Antiproliferative assays such as migration assay, clonogenic assay, Mitochondrial Membrane Potential (MMP) assay, dual staining assay, comet assay, Reactive Oxygen Species (ROS) assay, cell cycle analysis and immunoblotting assays were performed to witness the protein expression involved in chemoresistance and chemosensitization. Epithelial to mesenchymal transition was observed in nicotine induced chemoresistant cells. Gemcitabine sensitivity assay revealed that relative resistance was increased to 6.26 (p < 0.0001) and 6.45 (p < 0.0001) folds in resistant PANC-1 and MIA PaCa-2 compared to parental cells. Protein expression studies confirmed resistance markers like hENT1 and dCK were downregulated with subsequent increase in RRM1 expression in resistant cells. Embelin considerably decreased the cell viability with an IC50 value of 4.03 ± 0.08 μM in resistant PANC-1 and 2.11 ± 0.04 μM in resistant MIA PaCa-2. Cell cycle analysis showed Embelin treatment caused cell cycle arrest at S phase in resistant PANC-1 cells; in resistant MIA PaCa-2 cells there was an escalation in the Sub G1. Embelin upregulated Bax, γH2AX, p53, ERK1/2 and hENT1 expression with concomitant down regulation of Bcl-2 and RRM1. Bioactive molecule embelin, its combination with gemcitabine could provide new vistas to overcome chemo resistance in pancreatic cancer.

Identification of a Novel, Potent, and Orally Bioavailable Guanidine-Based SHP2 Allosteric Inhibitor from Virtual Screening and Rational Structural Optimization for the Treatment of KRAS Mutant Cancers.

Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a highly attractive therapeutic target for treating Kirsten rat sarcoma viral oncogene (KRAS) mutant cancers. In this work, a series of guanidine-based SHP2 allosteric inhibitors were discovered via virtual screening and rational structural optimization. Notably, lead compound 23 with potent SHP2 inhibitory activity (IC50 = 17.7 nM) effectively inhibited the proliferation, migration, and invasion of MIA PaCa-2 pancreatic cancer cells. Furthermore, compound 23 featured great in vivo pharmacokinetic properties (AUCpo = 4320 nM·h; F = 66.3%) and exhibited significant antitumor efficacy in the MIA PaCa-2 xenograft mouse model. This demonstrates that compound 23 is a potential lead compound for the development of SHP2 allosteric inhibitors to treat KRAS mutant cancers. Moreover, these guanidine-based scaffolds may provide an opportunity to mitigate the potential safety risks of the alkyl amine motif predominately incorporated in current SHP2 allosteric inhibitors.

Combination effects of amino acid transporter LAT1 inhibitor nanvuranlat and cytotoxic anticancer drug gemcitabine on pancreatic and biliary tract cancer cells

BackgroundCytotoxic anticancer drugs widely used in cancer chemotherapy have some limitations, such as the development of side effects and drug resistance. Furthermore, monotherapy is often less effective against heterogeneous cancer tissues. Combination therapies of cytotoxic anticancer drugs with molecularly targeted drugs have been pursued to solve such fundamental problems. Nanvuranlat (JPH203 or KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), has novel mechanisms of action to suppress the cancer cell proliferation and tumor growth by inhibiting the transport of large neutral amino acids into cancer cells. This study investigated the potential of the combined use of nanvuranlat and cytotoxic anticancer drugs.MethodsThe combination effects of cytotoxic anticancer drugs and nanvuranlat on cell growth were examined by a water-soluble tetrazolium salt assay in two-dimensional cultures of pancreatic and biliary tract cancer cell lines. To elucidate the pharmacological mechanisms underlying the combination of gemcitabine and nanvuranlat, we investigated apoptotic cell death and cell cycle by flow cytometry. The phosphorylation levels of amino acid-related signaling pathways were analyzed by Western blot. Furthermore, growth inhibition was examined in cancer cell spheroids.ResultsAll the tested seven types of cytotoxic anticancer drugs combined with nanvuranlat significantly inhibited the cell growth of pancreatic cancer MIA PaCa-2 cells compared to their single treatment. Among them, the combined effects of gemcitabine and nanvuranlat were relatively high and confirmed in multiple pancreatic and biliary tract cell lines in two-dimensional cultures. The growth inhibitory effects were suggested to be additive but not synergistic under the tested conditions. Gemcitabine generally induced cell cycle arrest at the S phase and apoptotic cell death, while nanvuranlat induced cell cycle arrest at the G0/G1 phase and affected amino acid-related mTORC1 and GAAC signaling pathways. In combination, each anticancer drug basically exerted its own pharmacological activities, although gemcitabine more strongly influenced the cell cycle than nanvuranlat. The combination effects of growth inhibition were also verified in cancer cell spheroids.ConclusionsOur study demonstrates the potential of first-in-class LAT1 inhibitor nanvuranlat as a concomitant drug with cytotoxic anticancer drugs, especially gemcitabine, on pancreatic and biliary tract cancers.

Orally bioavailable styryl derivative of rohitukine-N-oxide inhibits CDK9/T1 and the growth of pancreatic cancer cells

The chromone alkaloid is one of the classical pharmacophores for cyclin-dependent kinases (CDKs) and represents the first CDK inhibitor to reach clinical trials. Rohitukine (1), a chromone alkaloid isolated from Dysoxylum binectariferum inspired the discovery of several clinical candidates. The N-oxide derivative of rohitukine occurs naturally, with no reports on its biological activity. Herein, we report isolation, biological evaluation, and synthetic modification of rohitukine N-oxide for CDK9/T1 inhibition and antiproliferative activity in cancer cells. Rohitukine N-oxide (2) inhibits CDK9/T1 (IC50 7.6 μM) and shows antiproliferative activity in the colon and pancreatic cancer cells. The chloro-substituted styryl derivatives, 2b, and 2l, inhibit CDK9/T1 with IC50 values of 0.17 and 0.15 μM, respectively. These derivatives display cellular antiproliferative activity in HCT 116 (colon) and MIA PaCa-2 (pancreatic) cancer cells with GI50 values of 2.5–9.7 μM with excellent selectivity over HEK293 (embryonic kidney) cells. Both analogs induce cell death in MIA PaCa-2 cells via inducing intracellular ROS production, reducing mitochondrial membrane potential, and inducing apoptosis. These analogs are metabolically stable in liver microsomes and have a decent oral pharmacokinetics in BALB/c mice. The molecular modeling studies indicated their strong binding at the ATP-binding site of CDK7/H and CDK9/T1.

Gemcitabine‑fucoxanthin combination in human pancreatic cancer cells.

Gemcitabine is a chemotherapeutic agent for pancreatic cancer treatment. It has also been demonstrated to inhibit human pancreatic cancer cell lines, MIA PaCa-2 and PANC-1. The aim of the present study was to investigate the suppressive effect of fucoxanthin, a marine carotenoid, in combination with gemcitabine on pancreatic cancer cells. MTT assays and cell cycle analysis using flow cytometry were performed to study the mechanism of action. The results revealed that combining a low dose of fucoxanthin with gemcitabine enhanced the cell viability of human embryonic kidney cells, 293, while a high dose of fucoxanthin enhanced the inhibitory effect of gemcitabine on the cell viability of this cell line. In addition, the enhanced effect of fucoxanthin on the inhibitory effect of gemcitabine on PANC-1 cells was significant (P<0.01). Fucoxanthin combined with gemcitabine also exerted significant enhancement of the anti-proliferation effect in MIA PaCa-2 cells in a concentration dependent manner (P<0.05), compared with gemcitabine treatment alone. In conclusion, fucoxanthin improved the cytotoxicity of gemcitabine on human pancreatic cancer cells at concentrations that were not cytotoxic to non-cancer cells. Thus, fucoxanthin has the potential to be used as an adjunct in pancreatic cancer treatment.

Magnetic resonance/fluorescence dual-modality contrast agents targeting αvβ6-overexpressing tumors based on A20FMDV2 peptide as a ligand

Pancreatic ductal adenocarcinoma (PDAC) is a malignant digestive system tumor with a poor late-stage prognosis. This study aimed to identify new methods for the early detection of PDAC. The nanoprobe A20FMDV2-Gd-5-FAM was developed using A20FMDV2 (N1AVPNLRGDLQVLAQKVART20-NH2, A20FMDV2) as the ligand and characterized using dynamic light scattering, transmission electron microscopy, Fourier transform infrared analysis, and UV absorption spectroscopy. The binding of pancreatic cancer cells AsPC-1, MIA PaCa-2, and normal human pancreatic H6C7 cells (HPDE6-C7) to the probe was verified using laser confocal microscopy, and the biocompatibility of the probe was evaluated in vivo. In vivo magnetic resonance and fluorescence imaging were also performed on nude mice with subcutaneous pancreatic tumor xenografts to verify the bimodal imaging performance of the probe. The probe exhibited good stability and biocompatibility and an enhanced relaxation rate (25.46 ± 1.32 mM−1 s−1) than Gd-DTPA. Confocal laser scanning microscopy results revealed that the A20FMDV2-Gd-5-FAM probe could be successfully ingested and internalized, and infrared analysis results demonstrated that the probe was linked successfully. Finally, magnetic resonance T1WI imaging and intravital fluorescence imaging demonstrated the specific signal enhancement of the probe at the tumor site. In conclusion, the bimodal molecular probe A20FMDV2-Gd-5-FAM showed a stable magnetic resonance and fluorescence bimodal imaging performance and is a promising new approach for diagnosing early-stage cancers with a high integrin αvβ6 expression.

Abstract 1658: ADT-007 binds RAS and inhibits RAS signaling

Abstract RAS is a critically important oncogenic protein that is mutated in approximately 1/3 of cancers resulting in aberrant activation of downstream signaling, which drives malignant transformation. Current molecular targeted therapeutics, and several in development, inhibit only specific mutant alleles (G12C, G12D). In addition, compounds which directly inhibit RAS via proteolytic degradation or inhibit RAS activation by SOS1 are in preclinical development and are referred to as Pan-KRAS inhibitors. Previously investigators have reported that the NSAID, sulindac, can selectively inhibit RAS mutant tumorigenesis by a cyclooxygenase (COX)-independent mechanism. Sulindac, and more potent analogs have also previously been reported to inhibit RAS-mediated transformation and directly bind RAS. Here we describe an ultra-potent non-COX inhibitory derivative of sulindac, ADT-007, which binds to and inhibits RAS nucleotide binding and RAS-effector association. ADT-007 binding to KRAS was evaluated by Micro-Tag cell target engagement and by Cellular Thermal Shift Assay (CETSA) which demonstrated a potency of target engagement (EC50) value in the subnanomolar range. Consistent with molecular docking studies, HSQC NMR spectroscopy using recombinant KRAS revealed that ADT-007 interacted with KRAS after Mg2+ chelation to obtain a nucleotide free (NF) state, resulting in chemical shift changes and signal attenuation of residues in the P-loop and nucleotide binding domain. Similarly, biochemical assays confirmed that ADT-007 prevented MANT-GTP binding to recombinant NF KRAS but did not compete with bound GTP. Functional assays also showed that KRAS binding to RAF-RBD(GST) was inhibited by ADT-007. The compound inhibited constitutive RAS activation (RAF-RBD pulldown) in serum starved MiaPaCa-2 pancreatic cancer cells harboring a KRAS-G12C mutation and demonstrated Pan-RAS inhibition in serum- or EGF-stimulated cells. Further, ADT-007 inhibited AKT phosphorylation and EGF-stimulated downstream ERK1/2 phosphorylation. Finally, ADT-007 demonstrated RAS-selective growth inhibition in isogenic pancreatic and colorectal cancer cell pairs (BxPC-3, HT29). Together, these experiments support further development of ADT-007 and related analogs for treatment of RAS-driven cancers. Citation Format: Adam B. Keeton, Xi Chen, Jacob Valiyaveettil, Chung-Hui Huang, Tyler E. Mattox, Khalda Fadlalla, Jeremy B. Foote, Donald J. Buchsbaum, Kristy L. Berry, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Gregory Gorman, Lori Coward, Yulia Y. Maxuitenko, Forrest T. Smith, Gary A. Piazza. ADT-007 binds RAS and inhibits RAS signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1658.

Functionalisation of extracellular vesicles with cyclic-RGDyC potentially for glioblastoma targeted intracellular drug delivery

With the intrinsic ability to cross the blood-brain barrier, small extracellular vesicles (sEVs) hold promise as endogenous brain-targeted drug delivery nano-platforms for glioblastoma (GBM) treatment. To increase GBM targetability, this study aimed to functionalise sEVs with cyclic arginine-glycine-aspartic acid-tyrosine-cysteine (cRGDyC), a ligand for integrin (αvβ3) that is overexpressed in GBM cells. Firstly, the intrinsic cellular uptake of sEVs derived from GBM U87 and pancreatic cancer MIA PaCa-2 cells was investigated on the donor cells. To obtain functionalised sEVs (cRGDyC-sEVs), DSPE-mPEG2000-maleimide was incubated with the selected (U87) sEVs, and cRGDyC was subsequently conjugated to the maleimide groups via a thiol-maleimide coupling reaction. The GBM cell targetability and intracellular trafficking of cRGDyC-sEVs were evaluated on U87 cells by fluorescence and confocal microscopy, using unmodified sEVs as a reference. The cytotoxicity of doxorubicin-loaded vesicles (Dox@sEVs, Dox@cRGDyC-sEVs) was compared with a standard liposome formulation (Dox@Liposomes) and free Dox. Both U87 and MIA PaCa-2 cell-derived sEVs displayed tropism with the former being >4.9-fold more efficient to be internalised into U87. Therefore, the U87-derived sEVs were chosen for GBM-targeting. Approximately 4000 DSPE-mPEG2000-maleimide were inserted onto each sEV with cRGDyC conjugated to the maleimide group. The cell targetability of cRGDyC-sEVs to U87 cells improved 2.4-fold than natural sEVs. Despite their proneness to be colocalised with endosomes/lysosomes, both Dox@sEVs and Dox@cRGDyC-sEVs showed superior cytotoxicity to U87 GBM cells compared to Dox@Liposomes, particularly Dox@cRGDyC-sEVs. Overall, U87-derived sEVs were successufully conjugated with cRGDyC via a PEG linker, and cRGDyC-sEVs were demonstrated to be a potnetial integrin-targeting drug delivery vehicle for GBM treatment.Graphic abstract.

İnsan Pankreas Kanserinde Borik Asit Uygulaması ile Bazı IncRNA’ların ve İnvazyonun Baskılanması

Objective: It was aimed to investigate the effects of boric acid on some lncRNAs and invasion in PANC-1 and MIA PaCa-2 pancreatic cancer cells. Materials and Methods: The effects of boric acid on cell viability and invasion were investigated using XTT test and invasion chambers, respectively. lncRNA H19 and UCA1 expressions were evaluated in pancreatic cancer using TCGA data. Its effects on expressions of these lncRNAs and invasion genes were determined by qRT-PCR analysis. Results: The IC50 doses of boric acid were calculated as 14.25 mM in PANC-1 cells and 15.71 mM in MIA PaCa-2. TCGA data showed that H19 and UCA1 expressions were elevated in pancreatic cancer. H19 and UCA1 lncRNA levels were decreased with boric acid treatment. In addition, boric acid increased CDH1 and TIMP1 in both cell lines. However, it suppressed CDH2 expression. Boric acid increased TIMP2 in PANC-1 cells and TIMP3 expression in MIA PaCa-2 cells. In the invasion test, boric acid significantly suppressed invasion in both cells. Conclusions: Boric acid suppressed H19 and UCA1 expressions, which were found to be high in pancreatic cancer. In addition, it showed an anti-invasive effect by changing the expressions of genes that are important in invasion.

Boric Acid Shows ER Stress and Apoptosis Mediated Anticancer Activity in Human Pancreatic Cancer MIA PaCa-2 and PANC-1 Cells

Amaç: Bu çalışmada borik asitin insan pankreas kanseri MIA PaCa-2 ve PANC-1 hücrelerinde endoplazmik retikulum (ER) stresi ve apoptoz aracılı antikanser etkisinin araştırılması amaçlanmıştır. Gereçler ve Yöntem: Borik asitin pankreas kanseri hücrelerinin canlılığı üzerine etkisi ve IC50 değeri XTT testi ve CompuSyn version 1.0 yazılımı kullanılarak hesaplanmıştır. Apoptotik, anti-apoptotik ve ER stresi ile ilişkili genlerin ifadesi belirlenmiştir. Borik asitin bu hücrelerin koloni oluşum kapasitesi üzerine etkisi ise koloni oluşum testi ile değerlendirilmiştir. Bulgular: Borik asit zaman ve doz bağımlı olarak her iki hücre hattında da hücre canlılığını baskılamıştır. XTT testi sonucunda MIA PaCa-2 ve PANC-1 hücrelerinde borik asitin IC50 dozlarının sırasıyla 15707,5 ve 14248,8 μM olduğu bulunmuştur. Borik asitin her iki hücre hattında da apoptoz ile ilişkili genlerden BAX, CASP3, CASP8, CYCS ve FAS genlerinin ifadelerini anlamlı derecede arttırdığı gözlenmiştir. PANC- 1 hücrelerinde CASP9 ve FADD genlerinin ifadeleride anlamlı derecede yükselmiştir. Borik asitin her iki hücre hattında da ER stresi ile ilişkili ATF4, HSP47 ve XBP1 genlerinin ifadesini istatistiksel olarak anlamlı derecede arttırdığı görülmüştür. Ayrıca borik asit muamelesi sadece PANC-1 hücrelerinde ATF6, CHOP ve EIF2A ifadelerini anlamlı derecede arttırmıştır. MIA PaCa-2 hücrelerinde ise borik asit GRP78 gen ifadesinin istatistiksel olarak artmasına neden olmuştur. Koloni oluşum testi sonuçları borik asitin her iki hücre hattında da koloni oluşum kapasitelerinin anlamlı derecede baskılandığını göstermiştir. Sonuç: Borik asit her iki insan pankreas kanseri hücrelerinde hücre canlılığı ve koloni oluşumunu azaltmış olup apoptoz ve ER stresi ile ilişkili genlerin ifadesini değiştirmiştir. Bu bulgular, borik asitin insan pankreas kanseri hücrelerinde ER stresi ve apoptoz aracılı antikanser etkisini göstermektedir.

PW06 Triggered Fas-FADD to Induce Apoptotic Cell Death In Human Pancreatic Carcinoma MIA PaCa-2 Cells through the Activation of the Caspase-Mediated Pathway.

Pancreatic cancer has higher incidence and mortality rates worldwide. PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one] is a carbazole derivative containing chalcone moiety which was designed for inhibiting tumorigenesis in human pancreatic cancer. This study is aimed at investigating PW06-induced anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro. The results showed PW06 potent antiproliferative/cytotoxic activities and induced cell morphological changes in a human pancreatic cancer cell line (MIA PaCa-2), and these effects are concentration-dependent (IC50 is 0.43 μM). Annexin V and DAPI staining assays indicated that PW06 induced apoptotic cell death and DNA condensation. Western blotting indicated that PW06 increased the proapoptotic proteins such as Bak and Bad but decreased the antiapoptotic protein such as Bcl-2 and Bcl-xL. Moreover, PW06 increased the active form of caspase-8, caspase-9, and caspase-3, PARP, releasing cytochrome c, AIF, and Endo G from mitochondria in MIA PaCa-2 cells. Confocal laser microscopy assay also confirmed that PW06 increased Bak and decreased Bcl-xL. Also, the cells were pretreated with inhibitors of caspase-3, caspase-8, and caspase-9 and then were treated with PW06, resulting in increased viable cell number compared to PW06 treated only. Furthermore, PW06 showed a potent binding ability with hydrophobic interactions in the core site of the Fas-Fas death domains (FADD). In conclusion, PW06 can potent binding ability to the Fas-FADD which led to antiproliferative, cytotoxic activities, and apoptosis induction accompanied by the caspase-dependent and mitochondria-dependent pathways in human pancreatic cancer MIA PaCa-2 cells.