MIA PaCa-2 Pancreatic Cancer Cells Research Articles

Biological impact and therapeutic potential of a novel camptothecin derivative (FLQY2) in pancreatic cancer through inactivation of the PDK1/AKT/mTOR pathway

BackgroundCamptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest. Aim of the studyThis study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs. MethodsThe cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2′s antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model. ResultsFLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects. ConclusionThe study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer.

Activated NAD+ biosynthesis pathway induces olaparib resistance in BRCA1 knockout pancreatic cancer cells.

PARP inhibitors have been developed as anti-cancer agents based on synthetic lethality in homologous recombination deficient cancer cells. However, resistance to PARP inhibitors such as olaparib remains a problem in clinical use, and the mechanisms of resistance are not fully understood. To investigate mechanisms of PARP inhibitor resistance, we established a BRCA1 knockout clone derived from the pancreatic cancer MIA PaCa-2 cells, which we termed C1 cells, and subsequently isolated an olaparib-resistant C1/OLA cells. We then performed RNA-sequencing and pathway analysis on olaparib-treated C1 and C1/OLA cells. Our results revealed activation of cell signaling pathway related to NAD+ metabolism in the olaparib-resistant C1/OLA cells, with increased expression of genes encoding the NAD+ biosynthetic enzymes NAMPT and NMNAT2. Moreover, intracellular NAD+ levels were significantly higher in C1/OLA cells than in the non-olaparib-resistant C1 cells. Upregulation of intracellular NAD+ levels by the addition of nicotinamide also induced resistance to olaparib and talazoparib in C1 cells. Taken together, our findings suggest that upregulation of intracellular NAD+ is one of the factors underlying the acquisition of PARP inhibitor resistance.

Abstract 2885: O-carborane-entrapped polymeric Micelles for Proton Boron Capture Therapy

Abstract Introduction: Proton boron capture therapy (PBCT) has the potential to enhance the biological effectiveness of proton therapy, based on proton-boron fusion reactions (11B + p → 3α + 8.7 MeV), which produce 3 α particles. In this study, we formulated o-carborane, a high boron content cage-like compound, into a polymeric micelle (M-carb) as a delivery tool to achieve effective delivery of the boronated compound to cancer cells and validated the enhancement of the biological effectiveness of proton therapy promoted by the uptake of o-carborane-entrapped in the micelle. Methods: O-carborane was effectively entrapped into an amphiphilic block copolymer to yield a stable polymeric micelle. The size and surface charge of the resulting M-carb were characterized by dynamic light scattering (DLS) and the uptake of M-carb in MIA PaCa-2 pancreatic cancer cells was measured by inductively coupled plasma mass spectrometry (ICP-MS). A colony formation assay was employed to assess the impact of M-carb on the survival of MIA PaCa-2 cells irradiated with a proton beam at the entrance and distal Spread-Out Bragg Peak (SOBP) positions at doses ranging from 0, 2, 5, and 8 Gy. The γH2AX foci were quantified by immunofluorescence to assess treatment-induced DNA double-strand breaks. Boron phenylalanine (BPA) was used as a control boron-carrying agent. Results: O-carborane-entrapped polymeric micelles (M-carb) had a diameter of about 60 nm and a relatively narrow size distribution (polydispersity index ~0.15) and possessed a slight negative charge. After 6 h incubated with M-carb at an equivalent concentration of 200 µM boron, MIA PaCa-2 cells were found to have taken up ~45 ng 11Boron per million cells. The colony formation assay demonstrated a significant decrease in the survival rate of MIA PaCa-2 cells after 8 Gy proton irradiation in the presence of 40 ppm of boron (corresponding to 4.61 µM BPA or 0.46 µM carborane) compared to irradiation treatment alone (p<0.05). At 8 Gy, the survival rates were 0.10, 0.07, and 0.04 for the following irradiation conditions: i) irradiation alone; ii) in the presence of BPA; in the presence of M-carb. At 2 and 5 Gy, these survival rate values were 0.78, 0.69, and 0.69 and 0.33, 0.19, and 0.16, respectively. Importantly, M-carb alone at the concentration used in proton irradiation experiments displayed no cytotoxicity. Immunofluorescence assays revealed that at both 2 Gy and 5 Gy, BPA and M-carb significantly increased γH2AX foci compared to irradiation alone (p<0.0001). Conclusion: M-carb was efficiently taken up by MIA PaCa-2 cells. PBCT mediated by M-carb or BPA enhanced the relative biological effectiveness of proton therapy with consequent improvement of its cell-killing efficacy in pancreatic cancer cells. This approach holds promise for advancing proton therapy as a new treatment modality for pancreatic cancer. Citation Format: Qiu-Xu Teng, Guodong Zhang, Haiyan Chen, Filipa Mendes, Narayan Sahoo, Célia Fernandes, António Paulo, Chun Li. O-carborane-entrapped polymeric Micelles for Proton Boron Capture Therapy [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 2885.

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.

Synchrotron radiation-FTIR biomolecular assessment in PANC-1 and MIA PaCa-2 pancreatic cancer cells after treatment with Gd-TiO2 nanoparticles

The increasing incidence rate of pancreatic cancer worldwide, along with high resistance to existing chemotherapies and radiotherapy, demand new approaches for its treatment. Nanoparticle-guided cancer treatment modalities are promising in their anti-cancer efficiency and upgrading potential. We have synthesized and characterized Gd-doped titanium-dioxide nanoparticles (Gd-TiO2 NPs), which showed good biocompatibility with MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. To assess the effect that these NPs have on tumor cells, we used synchrotron radiation Fourier transform infrared spectroscopy and principal component analysis. We elucidated the biochemical changes in the two cell lines induced after their interaction with these NPs. Results demonstrated changes in nucleic acids and carbohydrate metabolism and alterations in the protein conformation in both cell lines. In addition, considerable changes in lipid area were observed in PANC-1 cell line, suggesting that Gd-TiO2 NPs have a prospective to be further studied as systems for pancreatic cancer treatment.

Development of novel pyrimidine nucleoside analogs as potential anticancer agents: Synthesis, characterization, and In-vitro evaluation against pancreatic cancer

The present study proposed modification of 5-FU by conjugation with an acyl chloride and a 5-membered heterocyclic ring to improve its in-vitro cytotoxicity and metabolic stability. 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. The structure of the synthesized compounds was validated using NMR and micro-elemental analysis. The antiproliferative activity of the analogs was determined against MiaPaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells. The analog's stability in human liver microsomes was quantified by HPLC. We found that the XYZ-I-73 (IC50 3.6 ± 0.4 μM) analog was most effective against MiaPaCa-2 cells compared to XYZ-I-71(IC50 12.3 ± 1.7 μM), GemHCl (IC50 24.2 ± 1.3 μM), Irinotecan (IC50 10.1 ± 1.5 μM) and 5-FU (IC50 13.2 ± 1.1 μM). The antiproliferative effects of this analog in Miapaca-2 cells is evident based on it having a 7-fold,3-fold, and 4-fold increased cytotoxic effect over Gem-HCl, Irinotecan, and 5-FU, respectively. On the other hand, XYZ-I-71 exhibited a 2-fold increased cytotoxic effect over Gem-HCl but a comparable cytotoxic effect to 5-FU and Irinotecan in MiaPaCa-2 cells. A similar trend of higher XYZ-I-73 inhibition was observed in PANC-1 and BxPC-3 cultures. For 48-h MiaPaCa-2 cell migration studies, XYZ-I-73 (5 μM) 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 absorbance studies demonstrated significant inhibition of PARP expression of XYZ-I-73 treated cells compared to 5-FU, GemHCl, and XYZ-I-71. Further, BAX and p53 expressions were significantly increased in cells treated with XYZ-I-73 compared to 5-FU, GemHCl, and XYZ-I-71. In-vitro, metabolic stability studies showed that 80 ± 5.9% of XYZ-I-71 and XYZ-I-73 remained intact after 2 h exposure in liver microsomal solution compared to 5-FU. The XYZ-I-73 analog 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.

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.