MIA PaCa-2 Cell Lines Research Articles

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 1978: The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer

Abstract Introduction: Constitutive activation of KRAS through gain-of-function mutations, such as KRASG12C, is thought to reduce sensitivity to HER-targeted TKIs like neratinib (N), lapatinib (L) and tucatinib (T). Sotorasib (S) is a KRASG12C specific inhibitor clinically approved for the treatment of advanced KRASG12C-mutated non-small cell lung cancer. One identified resistance mechanism to sotorasib is the increase of multiple phosphorylated receptor tyrosine kinases, including EGFR and HER2. The objective of this study was to assess the in vitro efficacy of the single agents N, L, T and S, and the combination of N and S in HER2-amplified, KRASG12C-mutated cancer cell lines. Methods: The antiproliferative effects of N, L, T and S were assessed in the HER2-amplified and KRASG12C-mutated KYSE-410 (esophageal) and NCI-H2030 (lung) cancer cell lines, and KRASG12C-mutated MiaPaca-2 (pancreatic) cell line by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between N and S, matrix assays were performed and analyzed using Combenefit software. To further determine the efficacy of the drugs, changes in signaling pathways were assessed by Western blotting. Results: N displayed nanomolar IC50 values (< 100 nM) in all three cell lines, while nanomolar IC50 values with L and T were only achieved in KYSE-410 (IC50 = 426.84 nM and IC50 = 363.38 nM respectively). S was most potent in MiaPaca-2 (IC50 = 6.04 nM), followed by NCI-H2030 (IC50 = 270.60 nM) and KYSE-410 (IC50 = 5.46 µM). Matrix assays showed that NS was broadly additive in MiaPaca-2 cells, and synergistic in both NCI-H2030 and KYSE-410 cell lines. N and NS numerically decreased HER2 levels compared to control in NCI-H2030 after 24h of treatment (6.4-fold, p = 0.58 and 2.5-fold, p = 0.79 respectively) but not in KYSE-410. KRAS levels were significantly increased (1.89-fold, p ≤ 0.05) after treatment with NS in NCI-H2030. No significant effects were observed on the activation of HER2 and EGFR in either cell line at 24h. In NCI-H2030, NS had a significantly stronger inhibitory effect on pERK compared to N (p ≤ 0.0001) and S (p = 0.012) alone. In KYSE-410, pERK levels were also slightly decreased with NS, but not significantly different from N (p = 0.12). However, pAkt levels in KYSE-410 were significantly reduced by N (1.3-fold, p = 0.009) and NS (1.7-fold, p ≤ 0.0001) compared to control, with NS showing a significantly greater decrease than N (p = 0.026). It thus appears that the synergy between N and S occurs through a stronger inhibition of ERK activity in NCI-H2030, while it occurs through a stronger inhibition of Akt activity in KYSE-410. Conclusion: NS is additive or synergistic in the three KRASG12C-mutated cancer cell lines examined. Further work is planned to investigate the basis of the synergy observed. Citation Format: Myra E. Castel, Neil T. Conlon, Naomi Walsh, Georg F. Bischof, John P. Crown, Denis M. Collins. The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated 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 1978.

Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells

Abstract Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations. Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle. Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors. Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions. Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in 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 7080.

Abstract 4161: Identification of pancreatic adenocarcinoma using urokinase plasminogen activator probe detected using optoacoustic imaging

Abstract Purpose: Diagnosing and treating pancreatic cancer remains a challenge. The hypovascularized nature of pancreatic cancer renders modern chemotherapeutics and cancer detection contrast agents ineffective. A promising approach to the challenges is the adoption of a novel imaging technique known as Multispectral Tomography (MSOT). We hypothesize that a urokinase plasminogen activator (UPA) probe will enhance the detection of orthotopic pancreatic cancer xenografts. Methods: Pancreatic tumor cell lines (Panc1, MiaPaca-2, S2VP10, and S2C9) were assessed for UPA-receptor expression using western blot analysis. UPA peptide was synthesized and to the HiLyte-750 NIR dye. Flow cytometry and immunofluorescence were utilized to examine the specificity and activity of this UPA-750. The UPA-750 binding efficiency was assessed through a competitive inhibition assay with a UPAR-blocking antibody using fluorescence microscopy. Then SCID mice (5 mice/group) received orthotopic implantation of S2C9 (1.5 x 105) or MiaPaCa2 (2.0 × 106) pancreatic tumor cells. UPA-750 or 750 dye alone was IV injected into mice after the tumors reached 3mm. Biodistribution and accumulation of UPA-750 were observed through MSOT images taken at 2-hour intervals over 8 hours and at 24 hours. UPA-750 in the tumor, liver, and kidney was confirmed through ex vivo fluorescent imaging. Results: UPAR expression was observed in every pancreatic cancer cell line, with the greatest relative abundance in S2P9 cells (3.0X), followed by S2VP10 (2.5X), Panc1 (1.77X), and MiaPaca-2 (1.3X). Application of the UPAR blocking antibody on S2CP9, S2VP10, MiaPaCa2, and SKOV3p1 (positive control) cell lines resulted in the inhibition of cellular binding of the UPA-750, as evidenced by fluorescence microscopy. UPA-probe was successfully observed in pancreas tumor slices with peak intensity at 4-hrs of imaging by MSOT. Ex vivo NIR fluorescence imaging confirmed UPA-750 signal in pancreatic tumors but not in liver or kidney. Conclusion: UPA-probe exhibits potential as a marker for pancreatic cancer. Citation Format: Vung Kim, Phillip Chuong, Ryan Bynum, William Grizzle, Lacey R. McNally. Identification of pancreatic adenocarcinoma using urokinase plasminogen activator probe detected using optoacoustic imaging [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 4161.

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.

Enhanced Anti-tumor Effect of Flavopiridol in Combination With Gemcitabine in Pancreatic Cancer.

The efficacy of current chemotherapies for pancreatic ductal adenocarcinoma (PDAC) is still unsatisfactory. Flavopiridol inhibits multiple cyclin-dependent kinases, causing cell cycle arrest and inducing cancer cell apoptosis. This study aimed to evaluate the anti-tumor effect of flavopiridol and gemcitabine in PDAC in vitro and in vivo. PANC-1 and MIA PaCa-2 cell lines were treated with gemcitabine and flavopiridol alone, in combination, and sequentially, and cell proliferation, apoptosis, and the cell cycle were evaluated. Proteins related to cell cycle progression (cyclin A, CDK2, E2F-1, and p53) were quantified using western blotting. A xenograft mouse model was generated, and the effects of gemcitabine and flavopiridol, administered alone or in combination, were evaluated by measuring tumor volume and apoptosis degree using the TUNEL assay. Sequential administration of gemcitabine and flavopiridol suppressed PDAC cell proliferation and induced apoptosis. Flavopiridol treatment led to an increase in the number of cells in the S and a decrease in those in the G0/G1 phases. Gemcitabine increased and decreased the number of S- and G2/M-phase cells, respectively. Furthermore, flavopiridol treatment decreased cyclin A and CDK2 expression and increased E2F-1 expression. In a xenograft mouse model, the combined administration of gemcitabine and flavopiridol demonstrated the most significant reduction in tumor volume and induction of apoptosis. Flavopiridol potentiates the anti-tumor activity of gemcitabine by inducing cell cycle arrest and apoptosis. Its synergistic inhibition of PDAC cell proliferation, when combined with gemcitabine, positions flavopiridol as a promising candidate for cancer treatment.

Intracellularly delivered human lactoferrin functions as an activator of Na+/H+ exchanger 7

Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.

Evaluation of gelatin-based hydrogels for colon and pancreas studies using 3D in vitro cell culture.

Biomimetic 3D models emerged some decades ago to address 2D cell culture limitations in the field of replicating biological phenomena, structures or functions found in nature. The fabrication of hydrogels for cancer disease research enables the study of cell processes including growth, proliferation and migration and their 3D design is based on the encapsulation of tumoral cells within a tunable matrix. In this work, a platform of gelatin methacrylamide (GelMA)-based photocrosslinked scaffolds with embedded colorectal (HCT-116) or pancreatic (MIA PaCa-2) cancer cells is presented. Prior to cell culture, the mechanical characterization of hydrogels was assessed in terms of stiffness and swelling behavior. Modifications of the UV curing time enabled a fine tuning of the mechanical properties, which at the same time, showed susceptibility to the chemical composition and crosslinking mechanism. All scaffolds displayed excellent cytocompatibility with both tumoral cells while eliciting various cell responses depending on the microenvironment features. Individual and collective cell migration were observed for HCT-116 and MIA PaCa-2 cell lines, highlighting the ability of the colorectal cancer cells to cluster into aggregates of different sizes governed by the surrounding matrix. Additionally, metabolic activity results pointed out to the development of a more proliferative phenotype within stiffer networks. These findings confirm the suitability of the presented platform of GelMA-based hydrogels to conduct 3D cell culture experiments and explore biological processes associated with colorectal and pancreatic cancer.

Scaffold hopping based designing of selective ALDH1A1 inhibitors to overcome cyclophosphamide resistance: synthesis and biological evaluation.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is an isoenzyme that catalyzes the conversion of aldehydes to acids. However, the overexpression of ALDH1A1 in a variety of malignancies is the major cause of resistance to an anti-cancer drug, cyclophosphamide (CP). CP is a prodrug that is initially converted into 4-hydroxycyclophosphamide and its tautomer aldophosphamide, in the liver. These compounds permeate into the cell and are converted as active metabolites, i.e., phosphoramide mustard (PM), through spontaneous beta-elimination. On the other hand, the conversion of CP to PM is diverted at the level of aldophosphamide by converting it into inactive carboxyphosphamide using ALDH1A1, which ultimately leads to high drug inactivation and CP resistance. Hence, in combination with our earlier work on the target of resistance, i.e., ALDH1A1, we hereby report selective ALDH1A1 inhibitors. Herein, we selected a lead molecule from our previous virtual screening and implemented scaffold hopping analysis to identify a novel scaffold that can act as an ALDH1A1 inhibitor. This results in the identification of various novel scaffolds. Among these, on the basis of synthetic feasibility, the benzimidazole scaffold was selected for the design of novel ALDH1A1 inhibitors, followed by machine learning-assisted structure-based virtual screening. Finally, the five best compounds were selected and synthesized. All synthesized compounds were evaluated using in vitro enzymatic assay against ALDH1A1, ALDH2, and ALDH3A1. The results disclosed that three molecules A1, A2, and A3 showed significant selective ALDH1A1 inhibitory potential with an IC50 value of 0.32 μM, 0.55 μM, and 1.63 μM, respectively, and none of the compounds exhibits potency towards the other two ALDH isoforms i.e. ALDH2 and ALDH3A1. Besides, the potent compounds (A1, A2, and A3) have been tested for in vitro cell line assay in combination with mafosfamide (analogue of CP) on two cell lines i.e. A549 and MIA-PaCa-2. All three compounds show significant potency to reverse mafosfamide resistance by inhibiting ALDH1A1 against these cell lines.

Divergent synthesis of fractionated Cannabis sativa extract led to multiple cannabinoids C-&O-glycosides with anti-proliferative/anti-metastatic properties

Here, we present an interesting, previously unreported method for fractionating a particular class of cannabinoids from the crude leaf extract of Cannabis sativa using HP-20 resins. In this study, we report a novel method of divergent synthesis of fractionated Cannabis sativa extract, which allows the generation of multiple cannabinoids C- and O-glycosides which react with the glycosyl donor 2,3,4,6-tetra-O-acetyl-d-mannosyl trichloroacetimidate (TAMTA) to create eight C- and O-β-d-cannabinoids glycosides (COCG), which are separated by HPLC and whose structures are characterized by 1D, 2D NMR, and mass spectrometry. These glycosides exhibit improved anti-proliferative and anti-metastatic effects against numerous cancer cell lines in vitro and are more water-soluble and stable than their parent cannabinoids. The in vitro testing of the pure cannabinoids (1–4) and their C- & O-glycosides (1a-4a) and 1b-4b exhibited anti-proliferative and anti-metastatic activities against a panel of eight human cancer cell lines in contrast to their respective parent molecules. Different cancer cell lines' IC50 values varied significantly when their cell viability was compared. In addition to the others, compounds 2a, 3a, 4a, and 2b, 3b were highly potent, with IC50values ranging from 0.74 µM (3a) to 51.40 µM (4a).Although2a(1.42 µM) and3a(0.74 µM) exhibited lower IC50values in the MiaPaca-2 cell line than4a(2.58 µM). But, in addition to the comparable anti-clonogenic activity of4ain MiaPaca-2 and Panc-1 cells, it manifested remarkable anti-invasive activity than either 2a or 3a.In contrast to 2a, 2b, 3a, and 3b and their respective parent compounds,4ahad substantial anti-invasive/anti-metastatic capabilities and possessed anti-proliferative activity.The effects of 4a treatment on MiaPaca-2 and Panc-1 cells include a dose-dependent increase in the expression of E-cadherin and a significant decrease in the expression of Zeb-1, Vimentin, and Snail1. Our results demonstrate that divergent synthesis of fractionated Cannabis sativa extract is a feasible and efficient strategy to produce a library of novel cannabinoid glycosides with improved pharmacological properties and potential anticancer benefits.

Abstract C175: The potential effect of KRAS inhibitors on tumor microenvironments

Abstract KRAS is one of the most common driver oncogenes among various cancer types. Researches aiming to develop KRAS inhibitors has been surging recently and the first clinical KRAS inhibitor sotorasib was approved for non-small cell lung cancer in the US and Japan. As KRAS activity has been reported to have certain effects on tumor microenvironments, KRAS inhibitor could potentially induce collateral outcomes on tumor microenvironment. Therefore, I have analyzed the potential influences of resistance to KRAS inhibitors on the tumor microenvironment. At first, I have established pancreatic cancer cell line MIA-Paca2 that acquired resistance to sotorasib, MIA/AMG-R, by continuously exposing them to sotorasib (1 µmol/L). An RNA sequencing analysis were performed with MIA/AMG-R and revealed that a gene set related to hypoxia were upregulated. When non-small cell lung cancer cell line NCI-H358 cells were exposed to sotorasib (1 µmol/L) for 4 hrs, the protein levels of HIF-1A decreased while that in MIA Paca-2 cells were unaffected. To evaluate at which steps of protein synthesis from gene transcription to through protein degradation of HIF-1A, NCI-H358 cells were treated with transcription inhibitor actinomycin D, translation inhibitor cycloheximide or proteasome inhibitor MG-132. The treatment of transcription inhibitor actinomycin D on NCI-H358 cells for 6 hrs did not lead to the decrease of HIF-1A protein level however, translation inhibitor cycloheximide induced drastic decrease in HIF-1A protein. Although exposure of NCI-H358 cells to MG-132 increased the expression level of HIF-1A, the combinational treatment of sotorasib and MG-132 still led to slight decrease in HIF-1A protein level. Also, MG-132 failed to recover the protein expression level decreased by cycloheximide treatment. In total, the cellular expression level of HIF-1A is possibly dependent on translation activity and KRAS inhibitor potentially affect the HIF-1A expression through the regulation of its translation. Conclusively, KRAS inhibitors could potentially be used to reprogram the hypoxic tumor microenvironments besides its primary effect against the downstream signaling of KRAS. Citation Format: Noritaka Tanaka, Takeharu Sakamoto. The potential effect of KRAS inhibitors on tumor microenvironments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C175.

Tetrahydrobenzothiophene derivatives ameliorate Mia PaCa-2 cell progression and induces apoptosis via inhibiting EGFR2 tyrosine kinase signal

A series of new thiophene analogues with acarbonitrile-basedmoiety were designed and synthesized via structural optimization. The conjugates were assessed for their in-vitro cytotoxic activity against a human pancreatic cancer cell line (Mia PaCa-2) and among them compound 5b showed IC50 value of 13.37 ± 2.37 μM. The compounds 5b (20 µM & 25 µM) and 7c (30 & 35 µM) also showed reduced clonogenicity, enhanced ROS and decreased mitochondrial membrane potential in Mia PaCa-2 cells. Treatment with these compounds also increased apoptotic population as evident with the double staining assay. Among the evaluated series, compounds 5b, 5g, 7c, and 9a attained a greater inhibitory potency than first generation’s reversible EGFR inhibitor, Gefitinib. EGFR2 enzyme inhibitory studies revealed that 5b efficiently and arbitrarily suppressed the development of EGFR2 dependent cells and inhibited the enzymatic activity with an IC50 value of 0.68 µM; interestingly, the most effective molecule 5b with N-methyl piperazine substitution, has 1.29-fold greater potency than well-known EGFR inhibitor Gefitinib and increased Gefitinib's anti-growth impact with 2.04 folds greater against Mia PaCa-2. The in-vitro studies were validated with in-silico docking studies wherein compounds 5b and 7c exhibited binding energies of −8.2 and −7.4 Kcal/mol respectively. The present study reveals that tetrahydrobenzothiophene based analogues could be a promising lead for the evolution of potent chemo preventives over pancreatic cancer.

Anticancer activity, phytochemical investigation and molecular docking insights of Citrullus colocynthis (L.) fruits

Cancer disease is regarded as one of the most significant public health issues, regardless of economic standards. Medicinal plants are now regarded as a natural source of anticancer medicines due to their antioxidant and anti-mutagenic actions. Cucurbitaceae is considered to be one of the most economically significant families. One family species is Citrullus colocynthis (L.), which has a high concentration of many active secondary chemical metabolites. Various C. colocynthis plant extracts showed cytotoxicity against some cancer cells. This study aims to identify the C. colocynthis fruit components and determine whether they have anticancer action against MIA PaCa-2 and A431 cells. High-Performance Liquid Chromatography/Quadrupole Time of Flight/Mass Spectrometry (HPLC/QTOF/MS); the technique was accustomed to investigate the compounds of the ethyl acetate (EtOAc) fruit extract. Anticancer activity was investigated on both MIAPaCa-2 and A-431 cell lines. DPPH assay for antioxidant activity was carried out. Molecular modelling was employed to help understand the molecular basis for the observed anticancer activity. 24 compounds were tentatively identified by comparing the extract’s fragmentation pattern in positive mode against reference compounds spectra and literature. The EtOAc extract of C. colocynthis had effective positive results on cancer cells (MIAPaCa-2 and A-431) and was characterized by slight or no harmful effect on normal (healthy) cells. For the DPPH assay, EtOAc and BuOH extracts exhibited high antioxidant activity (86 and 76%, respectively) compared with the oxidative potential of the standard compound (Caffeic acid, 98%). One of the major cucurbitacin derivatives that LC/MS tentatively identified in the EtOAc extract was Cucurbita-5(10),6,23-triene-3β,25-diol. During this study, docking experiments and MD simulations were carried out, which suggested the anti-pancreatic cancer activity of C. colocynthis extract to be attributed to EGFR inhibition by Cucurbita-5(10),6,23-triene-3β,25-diol. Therefore, expansion of this type of research should be encouraged in the hope of obtaining natural therapeutics for cancerous tumors in the future, having the advantage of being cheaper, safer, and with fewer side effects.

Anti-Growth, Antioxidant, and Hepatoprotective Properties of Spirulina Platensis Extract

Spirulina platensis (S. platensis), a filamentous cyanobacterium often referred to as a blue-green alga, is recognized for its antioxidant, immunomodulatory, and anti-inflammatory properties. This study aimed to examine the growth-suppressing effects of S. platensis ethanolic extract on PANC-1 and MIA PaCa-2 human pancreatic cancer cells. Additionally, the in vivo hepatoprotective and antioxidant properties of S. platensis were explored. The ethanolic extract of S. platensis was lyophilized and dissolved in DMSO. Subsequently, PANC-1 and MIA PaCa-2 cell lines were exposed to concentrations ranging from 0.1-1000 µg/ml. Cell viability was assessed using sulforhodamine B viability assay. For in vivo evaluation of hepatoprotective and antioxidant properties, S. platensis was administered by gavage at a dose of 50 mg/kg/day over a four-week period. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured in the heart and liver of rats. The control group that received S. platensis showed a significant elevation in the activity of SOD and GSH-Px enzymes in the heart and liver tissues. There was a significant reduction in the ALT and AST enzyme levels. The extract notably hindered the growth of both the examined cell lines. Future research can focus on studying the effects of S. platensis extracts in conjunction with various chemotherapeutic agents or its effect on different cancer cell lines to better understand its anti-growth attributes.

NRF2 Suppression Enhances the Susceptibility of Pancreatic Cancer Cells, Miapaca-2 to Paclitaxel.

Pancreatic cancer is one of the most deadly diseases, with a very high metastasis and low survival rate. High levels of NRF2 have been detected in numerous malignancies, including head, neck, lung, and colon cancers, promoting the expansion and survival of cancer cells and chemical resistance to stressful conditions and affecting the response to treatment. To evaluate the possibility that modulation of NRF2 expression could be effective in treating pancreatic cancer cells, we explored the effect of knockdown of the NRF2 gene by NRF2-specific siRNA and its influence in combination with paclitaxel on pancreatic cancer cells. Miapaca-2 cell line, due to the high expression of the NRF2 gene, was selected for this study. Then, Miapaca-2 cells in different groups were treated with NRF2 siRNA and paclitaxel separately and in combination. After that, cell viability was measured by MTT assay and apoptosis induction by Annexin V-FITC/PI staining test. Cell cycle and autophagy were examined by flow cytometry, and cell migration was assessed by wound-healing assay. Finally, the expression of genes involved in apoptosis, Bax, Caspase-3, Caspase-9, and genes related to migration pathway, MMP-2, and MMP-9 in different groups were measured using qRT-PCR. Combined use of NRF2-specific siRNA with paclitaxel significantly reduced NRF2 gene expression in pancreatic cancer cells. NRF2 siRNA transfection significantly reduced cell viability. In addition, paclitaxel combination therapy with NRF2 siRNA strengthens the anti-tumor effects, such as inhibiting cell migration and provoking apoptosis, and autophagy and the cell cycle arrest in the G2 phase. NRF2 suppression augmented the expression of Bax, Caspase-3, and Caspase-9 genes and lowered the expression of Bcl-2, MMP-2, and MMP-9 genes, which play crucial roles in the pathways of apoptosis and cell migration, respectively. NRF2 siRNA enhances the susceptibility of Miapaca-2 cells to paclitaxel in pancreatic cancer cells. Thereby, suppressing NRF2 in combination with paclitaxel can be a new and efficacious treatment approach in treating pancreatic cancer.

Anticancer effect of umbelliferone on MKN-45 and MIA PaCa-2 cell lines

In this study, the anticancer activity of umbelliferone (7-hydroxycoumarin-UMB) was investigated in MKN-45 human gastric cancer and MIA PaCa-2 human pancreatic cancer cells. The cytotoxic effect of UMB on MKN-45 and MIA PaCa-2 cells was determined by WST-8 cell viability assay; the effect on colony formation and migration potential by colony forming assay and wound healing/cell migration assay. Apoptotic effect of UMB was determined by measuring the change in mitochondrial membrane potentials, reactive oxygen species levels, and Caspase-3 activities in cells. Anticancer drugs cisplatin and gemcitabine were used as positive controls in experiments, and NIH/Swiss 3 T3 mouse embryonic fibroblast cells were used as a healthy cell group.The results of this study showed that umbelliferone had a significant cytotoxic effect in MKN-45 and MIA PaCa-2 cells, especially after 72 h treatment, while its cytotoxic effect in NIH/3 T3 cells was low. Furthermore, UMB reduces significantly the potential of cells to colonize and migrate; it has been determined that it causes apoptosis by decreasing the mitochondrial membrane potential, increasing intracellular ROS levels and Caspase-3 activity. UMB was found to have more anticancer effect on MIA PaCa-2 cells compared to MKN-45 cells. This showed that UMB has a cell-selective effect.

Treatment of colorectal, gastric, and pancreatic cancers by silver nanoparticles green-formulated using a medicinal plant

As an emerging new class, herbal nanoparticles and especially herbal silver nanoparticles hold great potential in the field of cancer biology. Because of cancer-specific targeting, the consequently attenuated side effects and the massive anti-cancer features render nanoparticle therapeutics desirable platforms for clinically relevant drug development. In the current research, silver nanoparticles were green-formulated by the Ocimum basilicum aqueous extract. Characterization of AgNPs was studied with different techniques. The anticancer potential of silver NPs was determined using the MTT test. The exposed cells with silver NPs were determined about anticancer and toxicity potentials on HUVEC (normal) and cancer cells i.e. pancreatic cancer (PANC-1 and MIA PaCa-2), gastric cancer (MKN45 and KATO III), and colorectal carcinoma (HT-29 and Ramos.2G6.4C10). In the DPPH assay, the IC50 of AgNPs and butylated hydroxytoluene (BHT) were 125 and 62 µg/mL, respectively. Malignant cell lines viability is reduced in the AgNPs presence. The IC50 of AgNPs were 238, 219, 211, 203, 154 and 151 µg/mL against HT-29, Ramos.2G6.4C10, MKN45, KATO III, PANC-1, and MIA PaCa-2 cell lines, respectively. The above results confirm the excellent antioxidant and anti-colorectal carcinoma, anti-gastric cancer, and anti-pancreatic cancer effects of Ag NPs.

Insights into the Anti-inflammatory, Analgesic, and Anticancer Potentials of the Standardized Extracts From Three Cistus L. Species

IntroductionThe popular over-the-counter product of Cistus creticus extract is used in lozenge form. However, the healing potential of other Cistus species as food supplements has not been evaluated comparatively so far. The rationale of this study is to investigate two other Cistus species (C. salviifolius and C. laurifolius) as a substitute for C. creticus. MethodsThe phenolic profile of the hydroalcoholic extracts from the aerial parts of three plants has been evaluated comparatively by high-performance thin-layer chromatography and validated high-performance liquid chromatographic methods. Further biological activity potentials of the extracts as an antioxidant, anti-inflammatory, analgesic, and anticancer were investigated by in vitro techniques. ResultsHigh-performance thin-layer chromatography fingerprinting allows distinguishing Cistus spp. from each other. The highest quercitrin and tiliroside contents were found in C. creticus. C. salviifolius had the highest hyperoside content and exerted the highest antioxidant activity. Furthermore, C. laurifolius, with the highest quercetin amount, exerted the highest anti-inflammatory activity by reducing nitrite levels. The IL-6 level was reduced concentration-dependently by all Cistus spp. In the analgesic activity test, C. laurifolius extract provided the highest decline in PGE2 level. All extracts exerted expressive anticancer activity against pancreatic cancer cell lines MIA PaCa-2 in both 2D and 3D models concentration-dependently. The strongest effect on spheroid size change was caused by C. laurifolius (1 mg/ml) with a 98% reduction. ConclusionAs a result, apart from C. creticus, other Cistus species may also have the potential to be evaluated as food supplements for various healing benefits.

The Combination of PD-L1 and CTLA-4 Suppression Significantly Decreased the Expression Levels of Cancer Stem Cell Factors in the Pancreatic Cancer Cell Line

Background: It is thought that a limited number of aberrant cancer stem cells (CSCs), which encourage carcinogenesis, tumor metastasis, and treatment resistance, are the cause of pancreatic cancer, a disease with a high mortality rate. Increasing evidence shows that CSCs use immunosuppressive properties to avoid immune system identification. In Pancreatic cancer, the therapeutic consequences of the relationship between the expression of immune checkpoints such as programmed death receptor ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the presence of CSCs are poorly known. As a result, in the present investigation, we examined how the expression levels of CSCs were affected by PD-L1 and CTLA-4 knockdown using specific siRNA. Methods: Independently and together, PD-L1 and CTLA-4 siRNA were transfected into MIA PaCa-2 cells. RNA extraction and cDNA synthesis were then performed. Finally, using qRT-PCR, the gene expression levels of CSCs markers, including Nanog, CD133, CD44, and Oct-4, in transfected groups were measured. Results: In the MIA PaCa-2 cell line, siRNA-mediated inhibition of PD-L1 and CTLA-4 decreased the expression of CSC factors. Moreover, combined suppression of these two stemness-related immune checkpoints significantly decreased Nanog, CD133, CD44, and Oct-4 expression compared to inhibition of PD-L1 and CTLA-4 separately. Conclusion: Considering that the combination of PD-L1 and CTLA-4 suppression using siRNA significantly decreased the expression levels of CSCs factors, this approach may be regarded as an effective therapy for this cancer.

Determination of the effects of fusaric acid, a mycotoxin, on cytotoxicity, gamma-H2AX, 8-hydroxy-2 deoxyguanosine and DNA repair gene expressions in pancreatic cancer cells

Pancreatic cancer has a poor prognosis and is an important public health problem for developing countries. Oxidative stress plays an important role in cancer initiation, progression, proliferation, invasion, angiogenesis and metastasis. For this reason, one of the important strategic targets of new cancer therapeutics is to drive cancer cells into apoptosis through oxidative stress. In nuclear and mitochondrial DNA, 8-hydroxy-2′-deoxyguanosine and gamma-H2AX (γ-H2AX) are used as important oxidative stress biomarkers. Fusaric acid (FA) is a mycotoxin that mediates toxicity produced by Fusarium species and exhibits anticancer effects in various cancers via inducing apoptosis, cell cycle arrest, or other cellular mechanisms. The aim of this study was to determine the effects of fusaric acid on cytotoxic and oxidative damage in MIA PaCa-2 and PANC-1 cell lines. In this context, dose and time dependent cytotoxic effect of fusaric acid was determined by XTT method, mRNA expression levels of genes related to DNA repair were determined by RT-PCR, and its effect on 8-hydroxy-2′-deoxyguanosine and γ-H2AX levels was revealed by ELISA assay. According to XTT results, fusaric acid inhibits cell proliferation in MIA PaCa-2 and Panc-1 cells in a dose- and time-dependent manner. IC50 doses were determined as 187.74 μM at 48 h in MIA PaCa-2 cells and 134.83 μM at 48 h in PANC-1 cells, respectively. γ-H2AX and 8-OHdG changes were not found significant in pancreatic cancer cells. The mRNA expression levels of DNA repair-related genes NEIL1, OGG1, XRCC and Apex-1 change with exposure to fusaric acid. This study contributes to the therapeutic approaches to be developed for pancreatic cancer and demonstrates the potential of fusaric acid as an anticancer agent.