MIA PaCa-2 Pancreatic Cancer Cells Research Articles

Tilia species (linden) exert anti-cancer effects on MIA PaCa-2 cells through the modulation of oxidative stress and inflammation

This study investigated the anti-cancer effects of the chemically characterized Tilia species (linden) on MIA PaCa-2 cells by analyzing various cancer-triggering mechanisms, including oxidative stress and inflammation status. Extracts from the flowers, bracts, and inflorescences of T. cordata, T. platyphyllos, T. rubra, and T. tomentosa were evaluated for antioxidant activity; subsequently, their ability to mitigate inflammation was assessed through in vitro nitrite assays in LPS-induced RAW264.7 cells. The anticancer potentials of the extracts against MIA PaCa-2 pancreatic cancer cells were investigated in 2D (cytotoxic effect) and 3D (effect on spheroid growth) models in vitro. All investigated Tilia species displayed remarkable antioxidant activity and significantly inhibited LPS-induced nitrite, IL-6, and PGE2 production. Extract from T. rubra bracts showed the highest cytotoxic activity against MIA PaCa-2 cells with an IC50 value of 0.16 mg/mL, as well as the most significant delay on spheroid growth, which was further confirmed through the arrest in cell cycle. In the Annexin V cell death assays of T. rubra, cells treated with the flower extract exhibited the highest rate of necrotic population with 66.53%. Overall, our results highlight a potential use for Tilia extracts, particularly T. rubra, in pancreatic cancer treatment by modulating cell death.

Assessing the Efficacy of Mitochondria-Accumulating Self-Assembly Peptides in Pancreatic Cancer: An Animal Study.

Although pancreatic cancer presents with one of the most unfavorable prognoses, its treatment options are very limited. Mitochondria-targeting moieties, considered a new and prominent treatment modality, are expected to demonstrate synergistic anticancer effects due to their distinct mechanism compared to conventional chemotherapeutic approaches. This study evaluated the therapeutic potential of mitochondria-accumulating self-assembly peptides, referred to as Mito-FFs, utilizing both in vitro and in vivo pancreatic cancer models. Cellular viability assays revealed a concentration-dependent decrease in the survival of MIA-PACA2 pancreatic cancer cells upon exposure to Mito-FF treatment (p < 0.05). Subsequent in vitro Mito-FF treatments prompted the use of several molecular analyses, including Real-time PCR, Western blot analysis, and MitoSOX staining, which collectively indicated an upsurge in apoptosis, a concurrent reduction in the antioxidant enzyme expression, and an elevation in mitochondrial ROS levels (p < 0.05). In a murine xenograft model of pancreatic cancer, the intravenous administration of Mito-FF yielded a notable reduction in the tumor volume. Moreover, it upregulated the expression of pro-apoptotic markers, such as cleaved PARP and c-caspase 3, while concurrently downregulating the expression of an anti-apoptotic marker, MCL-1, as evidenced by both Western blot analysis and immunohistochemical staining (p < 0.05). It also resulted in the reduced expression of antioxidant enzymes like HO-1, catalase, and SOD2 within excised tumor tissues, as confirmed using Western blot analysis (p < 0.05). Cumulatively, the findings underscore the significant anticancer efficacy of Mito-FF against pancreatic cancer cells, predominantly mediated through the induction of apoptosis, suppression of antioxidant enzyme expression, and enhancement of mitochondrial ROS levels within the tumor microenvironment.

Ivermectin Combined With Recombinant Methioninase (rMETase) Synergistically Eradicates MiaPaCa-2 Pancreatic Cancer Cells.

Ivermectin was initially utilized as a veterinary medication, demonstrating efficacy against various parasites. Pancreatic cancer is currently one of the most recalcitrant diseases. The aim of the present study was to demonstrate the synergy of the combination of recombinant methioninase (rMETase) and ivermectin to eradicate human pancreatic cancer cells in vitro. MiaPaCa-2 human pancreatic cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with the addition of 10% fetal bovine serum and 1 IU/ml penicillin/streptomycin. Reduction of cell viability by rMETase alone and ivermectin alone and their combination on MiaPaCa-2 cells was determined with the WST-reagent. Four experimental groups were examined in vitro: control group without treatment; ivermectin alone; rMETase alone; ivermectin combined with rMETase. The IC50 of ivermectin for MiaPaCa-2 cells was 5.9 μM. The IC50 of rMETase on MiaPaCa-2 cells was 2.93 U/ml. Ivermectin (5.9 μM) plus rMETase (2.93 U/ml) synergistically greatly reduced the viability of MiaPaCa-2 cells, compared to ivermectin alone (80% reduction vs. 45% reduction, respectively p<0.05). The combination of ivermectin and rMETase effectively eradicated MiaPaCa-2 pancreatic cancer cells. The present results indicate the future clinical potential of the combination of rMETase, currently administered orally to patients as a dietary supplement, and oral ivermectin on pancreatic cancer.

(E)-3-(3-([1,1′-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-ones inducing reactive oxygen species generation through glutathione depletion

The accumulation of reactive oxygen species (ROS) disrupts reduction-oxidation homeostasis, which can result in damage to cancer cells. To identify the compounds generating ROS, compounds containing Michael acceptors were designed because they are suggested to be critical for ROS elevation via glutathione depletion. Twelve (E)-3-(3-([1,1'-biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-ones were synthesized and identified using nuclear magnetic resonance spectroscopy and mass spectrometry. Intracellular ROS levels induced by treatment with the compounds were determined using fluorescence microscopy with the oxidant-sensing fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate. We selected compound 9, which showed the highest activity, and performed further biological experiments, including glutathione depletion and apoptosis assays, using MIA PaCa-2 pancreatic cancer cells. Additionally, the reason why the intracellular ROS level by compound 9 was lower than that of menadione used as a control was explained through in silico docking experiments. Our findings suggest that compound 9 has the potential to act as an anticancer agent by inducing ROS generation through the depletion of intracellular glutathione.

Abstract C035: MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion

Abstract Introduction: Melanoma-associated antigens (MAGEs) are linked to aggressive disease progression and treatment resistance in many cancers, but their role in pancreatic cancer is not well understood. Our preliminary TCGA data analysis suggested that 10% of pancreatic tumors express MAGEA3/6 and potentially help pancreatic cancer cells survive and grow under nutrient stress. This study aims to uncover the molecular mechanisms of MAGEA3/6 function in pancreatic cancer cell metabolism and disease progression under nutrient-poor conditions. Methods: To understand the role of highly homologous genes MAGEA3 and MAGEA6 in pancreatic cancer, we expressed them in highly glycolytic MIA PaCa-2 pancreatic cancer cells. Cells were then exposed to cancer-related nutritional stressors, including glycolysis inhibition by 2-deoxy-D-glucose (2DG) treatment and glutamine depletion. We analyzed the effect of MAGEA6 expression on cell viability and cell metabolism by Alamar Blue viability assay (BioRad), Seahorse (Agilent), Phenotype MicroArray Mammalian analysis (Biolog), transmission electron microscopy (TEM), and RNA sequencing. To confirm our results, we performed loss-of- function studies in MAGEA3/6-positive cells, including PANC1. Results: We found that, compared to control GFP-expressing cells, MAGEA6-expressing MIA PaCa-2 cells were resistant to glycolysis inhibition with 2-DG. In contrast, they were more susceptible to glutamine depletion, suggesting the important role of MAGEA3/6 in metabolic phenotype and adaptations of pancreatic cancer cells. Further, we showed that MAGEA6-expressing cells exhibited increased ATP production and mitochondrial oxidative phosphorylation, especially under 2DG stress. After 48 hours of 2DG treatment, TEM images revealed significant differences in nuclear, mitochondrial, and ribosomal structures in MAGEA6-cells, further suggesting MAGEA6's role in metabolic adaptation. The Phenotype MicroArray assay demonstrated that α-D-Glucose is the primary nutrient source consumed by both GFP and MAGEA6-expressing cells among the 93 nutrients tested. Additionally, A6 cells utilize Pyruvic Acid as an alternative nutrient source more than GFP cells. To uncover molecular underpinning, we performed RNA sequencing and found that MAGEA6 expression affects transcriptome under glutamine depletion and 2DG treatment. Intriguingly, one of the major differentially regulated biological processes was associated with the upregulation of several signaling pathways, including MAPK and AMPK. Conclusion: Our findings suggest that in pancreatic cancer, MAGEA6 recruits stress and growth factor- dependent MAPK and AMPK pathways to overcome glycolysis inhibition, even in glutamine- depleted conditions, providing a growth advantage. This implies that patients with MAGEA6- positive tumors may benefit from combination therapy targeting metabolism and signaling pathways. Citation Format: Sima Tozandehjani, Barbara Breznik, Klementina Fon Tacer, Miloš Vittori, Juan Sebastian Solano, Sara Uhan. MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C035.

Abstract C055: Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element

Abstract Genome-wide association studies (GWAS) have identified independent signals at 5p15.33 across numerous cancers, with protective alleles for one cancer often conferring risk for another. Many of these associations are thought to act via allele-specific alterations in the cis- regulation of target genes. Transcriptomic analyses in multiple tissue types implicate two plausible target genes: TERT and CLPTM1L. However, the mechanisms linking cancer susceptibility alleles to their presumed target genes, along with the observed antagonistic pleiotropy between different cancers remain elusive. With this in mind, we employed a comprehensive approach, integrating statistical fine-mapping of GWAS summary statistics with massively parallel reporter assays (MPRA) and a densely tiled CRISPRi screen performed across eight cell lines, representative of four distinct cancers associated with 5p15.33 alleles – pancreatic, melanoma, lung, and bladder cancer. Integrative screening identified up to 30 variants across multiple signals in the locus with putative allele-specific transcriptional activity. Furthermore, a variable number tandem repeat (VNTR) in intron 9 of CLPTM1L emerged as a potent transcriptional enhancer in CRISPRi screening. Leveraging PacBio sequencing, VNTR alleles of European ancestry samples from the 1000 Genomes Project were genotyped and found to be highly polymorphic, with longer VNTRs linked to the haplotype tagged by rs31490-A (risk increasing for pancreatic cancer). Subsequent imputation into a pancreatic cancer GWAS of 9,040 cases and 12,496 controls revealed an association between longer VNTRs and risk of pancreatic cancer (OR for VNTR in the 75th percentile = 1.17, 95% CI = 1.13-1.22, P = 2.21x10-12). The significance of the association for the lead SNP rs31490 after conditioning on the VNTR genotype dropped to P= 2.20x10-5, suggesting that VNTR alleles merit consideration as part of the credible variant set for pancreatic cancer. Targeted CRISPRi assays suggested VNTR-mediated cis-regulation of both TERT and CLPTM1L in PANC-1 and MIA PaCa-2 pancreatic cancer cell lines, while dual luciferase reporter assays in the same cell lines revealed that VNTR enhancer activity is mediated by Hippo pathway transcription factors. In ongoing experiments, we aim to expand on luciferase reporter assays and DNA-protein binding assays in relevant cell lines with the aim of not only deciphering how longer VNTRs differentially exert their transcriptional effects compared to shorter alleles but also characterizing the divergent directions of effect between different cancer types. These findings underscore the proposition that pancreatic cancer susceptibility at the 5p15.33 locus may be mediated by both SNPs and an uncharted class of variants, yet to be characterized in the post-GWAS landscape. Citation Format: Aidan O'Brien, Hyunkyung Kong, Minal Patel, Katelyn E Connelly, Mai Xu, Irene Collins, Jun Zhong, Jason Hoskins, Michelle Ho, Brenen Papenburg, Mark M Iles, Matthew H Law, Maria-Teresa Landi, Rachael Stolzenber- Solomon, Brian M Wolpin, Alison P Klein, Nick Orr, Stephen J Chanock, Sara Lindström, Marc-Henri Stern, Ludmila Prokunina-Olsson, Jieyon Choi, Kevin M Brown, Laufey T Amundadottir. Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C055.

Abstract C046: Targeting wild-type IDH1 sensitizes homologous recombination proficient pancreatic cancer to PARP inhibition

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related mortality in the United States. While Poly (ADP-ribose) polymerase (PARP) inhibitors show promise for PDAC with Homologous Recombination Deficiency (HRD), this mutation is present in less than 10% of cases. Our research focuses on exploiting a vulnerability in the remaining 90% of HR proficient (HRP) tumors. We identified that wild-type isocitrate dehydrogenase 1 (IDH1) plays a critical role in HR repair. By inhibiting IDH1, we discovered suppression of the homologous recombination (HR) pathway, leading to compromised genomic integrity. This renders cancer cells more susceptible to PARP inhibitor therapy, offering a novel therapeutic approach for HRP PDAC. Methods:We assessed the impact of IDH1 inhibition on HR pathway activity using Western blotting to measure HR repair protein levels and HR reporter assays. The effect of IDH1 inhibition (Ivosidenib) combined with PARP inhibition (Olaparib) on in vitro growth inhibition was assessed through drug combination assays. Additionally, we investigated the molecular mechanisms underlying the synergistic effects of the drug combination. To further evaluate the therapeutic potential of IDH1 inhibition in combination with PARP inhibitors, we examined tumor growth in xenograft models of PDAC in athymic nude mice and survival studies were performed in C57BL/6J mice transplanted with orthotopic murine pancreatic cancer. Results:Wild-type IDH1 blockade induces a BRCA-like phenotype by decreasing α-ketoglutarate (αKG) levels and causing histone hypermethylation. This leads to decreased HR repair efficiency in HRP pancreatic cancer cells (MiaPaCa-2 and Panc-1), resulting in a strong synergistic effect when combined with the DNA-damaging agent Olaparib. The combination treatment significantly reduced cell survival in both short and long-term assays. Furthermore, we discovered that these compounds complemented each other in the DNA damage response, leading to increased γ-H2AX (DNA damage marker) and apoptosis. In vivo studies using murine PDAC models demonstrated that the combination of ivosidenib and olaparib synergistically enhanced anti-tumor activity compared to either single agent alone. Conclusion:These data provide novel insight into the mechanisms underlying in the context of HRP cancer susceptibility to a dual treatment approach involving an IDH1 inhibitor and a PARP inhibitor. This approach holds promise for precision medicine in pancreatic cancer treatment, although further pre- clinical studies are needed for validation and refinement. Citation Format: Mehrdad Zarei, Omid Hajihassani, Hallie J. Graor, Soubhi Tahhan, Alexander W. Loftus, Faith Nakazzi, Semmer A. Ali, Parnian Naji, Luke D. Rothermel, Jonothan R. Brody, Jordan M. Winter. Targeting wild-type IDH1 sensitizes homologous recombination proficient pancreatic cancer to PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C046.

Human bone marrow mesenchymal stem cell-derived exosomes loaded with gemcitabine inhibit pancreatic cancer cell proliferation by enhancing apoptosis.

Pancreatic cancer remains one of the most lethal malignancies, and has limited effective treatment. Gemcitabine (GEM), a chemotherapeutic agent, is commonly used for clinical treatment of pancreatic cancer, but it has characteristics of low drug delivery efficiency and significant side effects. The study tested the hypothesis that human bone marrow mesenchymal stem cell (MSC)-derived exosomes loaded with GEM (Exo-GEM) would have a higher cytotoxicity against human pancreatic cancer cells by enhancing their apoptosis. To investigate the cytotoxicity of MSC-derived Exo-GEM against pancreatic cancer cells in vitro. Exosomes were isolated from MSCs and characterized by transmission electron microscopy and nanoparticle tracking analysis. Exo-GEM through electroporation, sonication, or incubation, and the loading efficiency was evaluated. The cytotoxicity of Exo-GEM or GEM alone against human pancreatic cancer Panc-1 and MiaPaca-2 cells was assessed by MTT and flow cytometry assays. The isolated exosomes had an average size of 76.7 nm. The encapsulation efficacy and loading efficiency of GEM by electroporation and sonication were similar and significantly better than incubation. The cytotoxicity of Exo-GEM against pancreatic cancer cells was stronger than free GEM and treatment with 0.02 μM Exo-GEM significantly reduced the viability of both Panc-1 and MiaPaca-2 cells. Moreover, Exo-GEM enhanced the frequency of GEM-induced apoptosis in both cell lines. Human bone marrow MSC-derived Exo-GEM have a potent cytotoxicity against human pancreatic cancer cells by enhancing their apoptosis, offering a promising drug delivery system for improving therapeutic outcomes.

Combination of S-1 and the oral ATR inhibitor ceralasertib is effective against pancreatic cancer cells.

In our previous study, we found that the Chk1 inhibitor prexasertib enhances the antitumour effect of the oral anticancer drug S-1 against pancreatic cancer cells. In this study, we investigated the effect of combining S-1 and ceralasertib, an oral inhibitor of ATR, which is located upstream of Chk1. Ceralasertib is currently being investigated in multiple clinical trials for various cancers. The cell-proliferation inhibitory effect was measured by MTT assay, using the pancreatic cancer cell lines BxPC-3, SUIT-2, PANC-1, and MIA PaCa-2, while apoptosis was measured by flow cytometry using PI/Annexin staining. The mechanism underlying the combined effect was analysed using western blotting, and the antitumor effect was analysed using a mouse xenograft model. MTT assay revealed that the combination of S-1 and ceralasertib had a synergistic effect, leading to the suppression of cell proliferation. Measurement with PI/Annexin staining revealed that the combination of S-1 and ceralasertib induced apoptosis more efficiently than either drug alone. Western blotting results showed that ceralasertib inhibited S-1-induced activation of ATR and Chk1. The average estimated tumour volume after 3weeks of administration was 601 mm3 in the S-1 group, 580 mm3 in the ceralasertib group, and 298 mm3 in the combination group. The combination of S-1 and ceralasertib demonstrated a high antiproliferative effect in inhibiting tumour growth in vitro.

Targeting Pancreatic Cancer with Novel Nicolaioidesin C Derivatives: Molecular Mechanism, In Vitro, and In Vivo Evaluations.

Pancreatic cancer, one of the deadliest cancers with the lowest 5-year survival rate, often develops resistance to gemcitabine-based chemotherapies. The hypovascular nature of pancreatic tumors forces cancer cells to adapt to nutrient-depleted tumor microenvironments. Conventional anticancer agents targeting rapidly dividing cancer cells under nutrient-rich conditions are largely ineffective against adapted pancreatic cancer cells. Thus, targeting cancer cells under nutrient starvation, termed the "antiausterity strategy", may be effective for pancreatic cancer. This study examined nicolaioidesin C (Nic-C) derivatives as antiausterity agents. Among the 32 derivatives, Nic-15 (4n) exhibited superior cytotoxicity against MIA PaCa-2 and PANC-1 pancreatic cancer cells, inhibited MIA PaCa-2 cell migration and colony formation, and modulated the PI3K/Akt/mTOR pathway, while reducing the ER stress markers induced by gemcitabine. Nic-15 was found to inhibit tumor growth and enhance the efficacy of gemcitabine in an in vivo xenograft model. Nic-15 in combination with gemcitabine may be an effective strategy for the treatment of pancreatic cancer.

Plumbagin's Antiproliferative Mechanism in Human Cancer Cells: A Copper-Dependent Cytotoxic Approach.

Cancer is a serious global health problem, causing the loss of millions of lives each year. Plumbagin, a compound derived from the medicinal plant Plumbago zeylanica, has shown promise in stopping the growth of tumor cells both in laboratory settings and in living organisms. Many plant-based compounds exert their effects through copper's ability to produce reactive oxygen species (ROS). This study aimed to understand how plumbagin, dependent on copper, induces cell death (apoptosis) in human cancer cells through various experiments. The results demonstrate that plumbagin hinders the growth of pancreatic cancer cells PNAC-1 and MIA PaCa-2 by utilizing the copper naturally present in the cells. Unlike metal chelators that remove iron and zinc (desferrioxamine mesylate and histidine), a specific copper chelator called neocuproine lessens the cell death caused by plumbagin. When ROS scavengers are used, plumbagin-induced apoptosis is inhibited, indicating that ROS plays a role in initiating cell death. The study also proves that plumbagin prevents copper from leaving cancer cells by suppressing the expression of specific genes (CTR1 and ATP7A). It is confirmed that plumbagin targets the nuclear copper, leading to signals that promote oxidative stress and, ultimately, cell death. These findings provide valuable insights into the potential of plumbagin as a substance to combat cancer, highlighting the importance of understanding how copper behaves within cancer cells.

Role of Mitochondrial and Cytosolic Folylpolyglutamate Synthetase in One-Carbon Metabolism and Antitumor Efficacy of Mitochondrial-Targeted Antifolates.

Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange among serine, glycine, and formate. In both the cytosol and mitochondria, folates exist as polyglutamates, with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT)2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-day]pyrimidine antifolate that targets SHMT2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens, and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ∼19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (∼30-fold) more than cFPGS (∼4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the nonclassical SHMT2/SHMT1 inhibitor SHIN1. These results suggest a critical role of mFPGS levels in determining antitumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. SIGNIFICANCE STATEMENT: AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT)2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347. Mitochondrial FPGS levels play important roles in determining the antitumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.

Delivery of piperlongumine via hyaluronic acid/phenylboronic acid-mediated dual targetable polymersome for enhanced anticancer functionality against pancreatic tumor

Pancreatic cancer cells highly resistance to conventional chemo drugs, resulting low survival rates. The aim of the study was to design and develop dual targeting polymersomes (DTPS) loaded with phyto alkaloid agent i.e., piperlongumine (PL) for effective pancreatic cancer treatment. Here, hyaluronic acid (HA) was functionalized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPEPEG-NH2), poly(ethylene glycol) bis (amine) (PEG), and phenylboronic acid (PBA) moieties. The designed DTPS could selectively recognize CD44/sialic acid (SA) and deliver PL to MIA PaCa-2 pancreatic cancer cells, facilitated via HA-CD44 and PBA-SA interactions. Drug release and stability results implied sustained PL release profile and pH sensitivity. DTPS could be more efficiently bound with SA than other sugars based on fluorescence spectroscopy. The anticancer efficacy of designed polymersomes was tested with H6C7 normal pancreas cells and SA/CD44-overexpressed MIA PaCa-2 pancreatic cancer cells. DTPS showed both SA and CD44-mediated higher cellular uptake while single-targeted polymersomes showed CD44-mediated cellular uptake. The PL-loaded DTPS efficiently uptake by MIA PaCa-2 cancer cells, causing up to 80% cell growth inhibition, reduced cell spheroids volume and increased dead cells by 58.3%. These results indicate that the newly developed DTPS can effectively serve as a pH-responsive drug delivery system for efficient treatment of cancer.

Ultrasound-stimulated Microbubbles for Treatment of Pancreatic Cancer Cells with Radiation and Nanoparticles: In vitro Study.

This study aims to investigate the radiation enhancement effects of ultrasound-stimulated microbubbles (USMB) with X-rays and nanoparticles on pancreatic cancer cells in vitro. Sonazoid™ microbubbles were used for USMB treatment with a commercially available ultrasound unit. The characterization of the microbubbles before and after ultrasound exposure with different mechanical parameters was evaluated microscopically. Two pancreatic cancer cell lines, MIAPaCa-2 and PANC-1, were treated with different concentrations of microbubbles in combination with 150 kVp X-rays and hydrogen peroxide-modified titanium dioxide nanoparticles. Cell viability was evaluated using a water-soluble tetrazolium dye and a colony formation assay. In addition, intracellular reactive oxygen species (ROS) induced by the combined treatment were assessed. The number of burst microbubbles increased with ultrasound's higher mechanical index and the exposure time. A significant radiation enhancement effect with a significant increase in ROS levels was observed in MIAPaCa-2 cells treated with USMB and 6 Gy X-rays, whereas it was not significant in PANC-1 cells treated with the same. When a higher concentration of USMB was applied with X-rays, no radiation enhancement effects were observed in either cell line. Moreover, there was no radiation enhancement effect by USMB between cells treated with and without nanoparticles. The results indicate that USMB treatment can additively enhance the therapeutic efficacy of radiation therapy on pancreatic cancer cells, while the synergistic enhancement effects are likely to be cell type and microbubble concentration dependent. In addition, USMB did not improve the efficacy of nanoparticle-induced radiosensitization in the current setting.

Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46nM and 72nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4h in mice.

Elimination of radiation-induced senescent cancer cells and stromal cells invitro by near-infrared photoimmunotherapy.

Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as anovel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. NIR-PIT eliminates both senescent cancer and stromal cells invitro suggesting it may be a novel strategy for tumor treatment.

Combination Treatment of Biochanin A and Atorvastatin Alters Mitochondrial Bioenergetics, Modulating Cell Metabolism and Inducing Cell Cycle Arrest in Pancreatic Cancer Cells.

Pancreatic cancer is an aggressive type of cancer, with a dismally low survival rate of <5%. FDA-approved drugs like gemcitabine have shown little therapeutic success, prolonging survival by a mere six months. Isoflavones, such as biochanin A and daidzein, are known to exhibit anti-cancer activity, whereas statins reportedly have anti-proliferative effects. This study investigated the effects of combination treatment of biochanin A and atorvastatin on pancreatic cancer cells. Pancreatic cancer cells AsPC-1, PANC-1, and MIA PaCa-2 were procured from ATCC. The cell viability studies were carried out using MTT & cell count assays. Flow cytometry was used to study cell apoptosis whereas cell metabolism studies were carried out using the Seahorse Mito stress test and XF-PMP assay. The effects of treatment on cell signaling pathways & cell cycle associated proteins were investigated using western blot whereas invasiveness of cancer cells was evaluated using gelatin zymography. The combination treatment decreased the survival and enhanced pro-apoptotic responses compared to single treatments in the pancreatic cancer cells. In PANC-1 cells, the combination treatment decreased invasiveness, reduced expression of activated STAT3 and expression of critical mediators of cell cycle progression. Furthermore, the combination treatment induced a differential inhibition of respiratory complexes in the pancreatic cancer cells. The combination treatment of biochanin A and atorvastatin exerts enhanced anti-cancer effects, inducing apoptosis, down-regulating cell cycle associated proteins and invasiveness in pancreatic cancer cells and merits further investigation for new, improved treatments for pancreatic cancer.

Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

Statins, the drugs used for the treatment of hypercholesterolemia, have come into the spotlight not only as chemoadjuvants, but also as potential stem cell modulators in the context of regenerative therapy. In our study, we compared the in vitro effects of all clinically used statins on the viability of human pancreatic cancer (MiaPaCa-2) cells, non-cancerous human embryonic kidney (HEK 293) cells and adipose-derived mesenchymal stem cells (ADMSC). Additionally, the effect of statins on viability of MiaPaCa-2 and ADMSC cells spheroids was tested. Furthermore, we performed a microarray analysis on ADMSCs treated with individual statins (12 μM) and compared the importance of the effects of statins on gene expression between stem cells and pancreatic cancer cells. Concentrations of statins that significantly affected cancer cells viability (< 40 μM) did not affect stem cells viability after 24 h. Moreover, statins that didn´t affect viability of cancer cells grown in a monolayer, induce the disintegration of cancer cell spheroids. The effect of statins on gene expression was significantly less pronounced in stem cells compared to pancreatic cancer cells. In conclusion, the low efficacy of statins on non-tumor and stem cells at concentrations sufficient for cancer cells growth inhibition, support their applicability in chemoadjuvant tumor therapy.

ZNT5-6 and ZNT7 play an integral role in protein N-glycosylation by supplying Zn2+ to Golgi α-mannosidase II

The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.

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

Camptothecin (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 25years 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. This 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. The 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. FLQY2 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. The 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.