Ductal Carcinoma Cell Line Research Articles

64Cu2+ Complexes of Tripodal Amine Ligands' In Vivo Tumor and Liver Uptakes and Intracellular Cu Distribution in the Extrahepatic Bile Duct Carcinoma Cell Line TFK-1: A Basic Comparative Study.

Copper (Cu) is a critical element for cancer cell proliferation and considerably accumulates in the nucleus. 64Cu2+ is an anticancer radiopharmaceutical that targets the copper requirement of cancer cells. However, intravenously injected 64Cu2+ ions primarily accumulate in the liver. Ligand complexation of 64Cu2+ may be a promising method for increasing tumor delivery by reducing liver uptake. In this study, we used three tripodal amine ligands [tris(2-aminoethyl)amine (Tren), diethylenetriamine (Dien), and tris(2-pyridylmethyl)amine (TPMA)] to enclose 64Cu2+ ions and compared their in vivo tumor and liver uptakes using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1. We examined intracellular Cu distribution using microparticle-induced X-ray emission (micro-PIXE) analysis of these compounds. 64Cu2+-Tren and 64Cu2+-Dien showed higher tumor uptake than 64Cu2+-TPMA and 64Cu2+ ions in TFK-1 tumors. Among the three 64Cu2+ complexes and 64Cu2+ ions, liver uptake was inversely correlated with tumor uptake. Micro-PIXE analysis showed that in vitro cellular uptake was similar to in vivo tumor uptake, and nuclear delivery was the highest for 64Cu2+-Tren. Conclusively, an inverse correlation between tumor and liver uptake was observed using three 64Cu2+ complexes of tripodal amine ligands and 64Cu2+ ions. These results provide useful information for the future development of anticancer 64Cu radiopharmaceuticals.

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts.

The current study's objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves'-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape.

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan-Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell-cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

Increase delivery, cytotoxicity, stability and induction of apoptosis in cisplatin by establishing a new complex with methotrexate

Cisplatin, as an effective anti-cancer drug, is widely used to treat breast cancer. However, undesirable side effects and drug resistance have partially limited its use for some patients. One of the causes of side effects is the lack of differentiation between cancer cells and normal cells, which could be reduced by targeted drug delivery. Therefore, we decided to replace methotrexate, as a targeting ligand, with chlorine atoms of cisplatin and evaluate its effect on cytotoxicity and induction of apoptosis in T47D (invasive ductal carcinoma) cell lines. FTIR and 13C NMR methods proved the coordination of methotrexate to cisplatin and the formation of the eight-membered ring between them. The results of the MTT assay showed that the cis-[Pt(NH3)2MTX] complex showed a time- and dose-dependent cytotoxic effect at both periods of 48 and 72 h. Although the IC50 of the new complex was higher when compared to cisplatin (78 vs. 33 μM), it completely inhibited the cell viability of the T47D cells after 72 h of exposure. The results of the real-time PCR test approved that the cis-[Pt(NH3)2MTX] complex can induce the intrinsic apoptosis pathway by increasing the level of Bak1 proteins, while cisplatin does not affect any of the studied genes. Molecular docking confirmed that four amino acids of folate receptors such as ASP 81, SER 174, SER 101, and TRP 138 were involved in binding to the cis-[Pt(NH3)2MTX] complex. Finally, the atoms-in-molecules method and molecular electrostatic potential confirmed that the new complex has more stability than cisplatin via the coexistence of the Intra-HB and Inter-HB interactions.

Abstract 4801: Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma

Abstract Hypoxia is a feature of the pancreatic ductal adenocarcinoma (PDA). Although gemcitabine (GEM) is widely used in chemotherapy for PDA, drug resistance restricts its clinical effectiveness. We have found that hypoxia contributes to chemoresistance, however the mechanism is still unclear. The aim of this study is to clarify the mechanism and to develop a new therapeutic strategy. First, we examined the effect of hypoxia to GEM sensitivity, stemness and energy metabolism in three human pancreatic ductal carcinoma cell lines. The inhibition rate by GEM was lower under CoCl2-induced chemical hypoxia than normoxia. Reactive oxygen species (ROS) after GEM was increased under normoxia but not under hypoxia. Flux analysis showed that both oxidative phosphorylation (OXPHOS) and glycolysis were suppressed under hypoxia, indicating a quiescent state. Next, we investigated the relationship between hypoxia and stemness. Hypoxia increased stem cell markers mRNA (C-Myc, Oct-3/4, CD24, CD44, CD133) expression and tumorsphere formation efficiency (TFE). In addition, hypoxia and GEM administration reduced sphere size but increased the number of spheres and TFE. This indicated that hypoxia and GEM suppressed proliferation, however promoted stemness. Second, to examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced OXPHOS and decreased mitochondrial ROS levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. In addition, tetramethylrhodamine-stained mitochondrial membrane potential levels decreased, suggesting mitochondrial impairment. This indicated that long hypoxia and treatment with GEM induced mitochondrial impairment and acquisition of stemness, causing chemoresistance. We further investigated the effect of lauric acid (LAA), a medium-chain fatty acid, on GEM resistance. We have previously reported that LAA causes reprogramming of energy metabolism and induces cell death in cancer cells. Treatment with LAA restored GEM sensitivity in both GEM-resistant cell lines. Flux analysis showed that both OXPHOS and glycolysis were promoted, indicating displacement from quiescent state by LAA. These results suggest that decreased energy production, decreased mitochondrial ROS levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of OXPHOS by LAA could be a tool to overcome GEM resistance. Citation Format: Tadataka Takagi, Hiroki Kuniyasu, Masayuki Sho, Rina Tani, Shiori Mori, Shingo Kishi, Yukiko Nishiguchi, Yudai Hojo. Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4801.

Abstract PD10-02: PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms

Abstract Breast cancer is a leading cause of female mortality and despite advancements in diagnostics and personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. The druggable estrogen receptor (ER, ESR1), overexpressed in two-thirds of all breast cancer, evolves in 30% of tumors exposed to endocrine therapy consequently resulting in treatment resistance. A more recently discovered mechanism of ER mediated endocrine resistance is ER fusion proteins. ER fusions, found predominately in metastatic endocrine resistant disease, harbor ESR1 exons 1-6 fused to an in-frame gene partner due to an ESR1 intron 6 translocation break. Our lab has demonstrated that ER fusion proteins, which lack the C-terminal ligand-binding domain (LBD), recapitulate phenotypes of ER proteins harboring endocrine-resistant point mutations occurring in the LBD. Our research goals aim to 1) determine fusion prevalence and emergence, 2) understand fusion mechanisms of resistance and 3) explore alternative treatment options for our patients. The promiscuous nature of fusion partners hinders fusion detection by traditional sequencing and thus our research team has developed a novel ER fusion detection method, EnRich. The EnRich probe set (4,324 probes in total) is composed of two separate probe pools targeting at 2x tiling intronic and exonic ESR1, the upstream promoter region and select oncogenic genes. To optimize the EnRich pipeline, DNA was extracted from a frozen tumor sample and a PDX model harboring ESR1-DAB2 and ESR1-LPP fusions, respectively. ESR1-DAB2 and ESR1-LPP were accurately detected by quantifying discordant paired-end or split reads mapped to chromosome 6. In addition, liquid biopsies from 15 patients with ER positive advanced breast cancer were also assessed through EnRich to uncover unidentified ESR1 structural variants. Two patient samples were detected to harbor ESR1 fusions (ESR1-CCDC170, ESR1-AKAP12 and ESR1-YAP1) and were further validated in corresponding mRNA. These recurrent and novel fusions were supported with more than 10 reads each, indicating that the EnRich pipeline is an effective and accurate sequencing approach to understanding ER fusion prevalence. Our lab, furthermore, has studied ER fusion proteins mechanistically. We have stably overexpressed ER fusions in transgenic breast cancer cell lines engineered with shRNA targeting the endogenous wildtype ER (ESR1-WT). In this ESR1-WT depleted cellular context, we found that ER fusions (notably ESR1-SOX9 and ESR1-YAP1) demonstrate ER hyperactivation through an estrogen response element (ERE) assay compared to ESR1-WT and a truncated exon 1-6 ESR1 (ESR1∆CTD). Importantly, fusion ERE activity was robust in the absence of the ER ligand, estradiol, as well as in the presence of endocrine therapies, implying ER fusion proteins function in a ligand independent, endocrine resistant mechanism. ER fusion positive cell lines were also enriched in oncogenic phenotypes such as enhanced 3D growth, cell survival via colony formation, and migration in a wound scratch assay. These enhanced metastatic potentials of the ER fusions were observed in both invasive ductal and lobular carcinoma cell lines, albeit at varying magnitudes depending on the 3’ fusion partner and phenotype being assessed. Although the ER fusions harbored unique characteristics due to the C-terminal partner, transcriptomic profiling revealed that enhanced EMT and KRAS signaling signatures were shared among all fusions when compared to the ESR1-WT and ESR1∆CTD cell lines, which may serve as future exploitable drug targets. Comprehensive detection and functional evaluation of ER fusion proteins will provide clinicians and patients with better understanding of tumor endocrine-resistant prevalence and discovery of more effective treatment options. Citation Format: Megan E. Yates, Tiantong Liu, Jagmohan Hooda, Sichun Yang, Riyue Bao, Jennifer M. Atkinson, ADRIAN V. LEE, Steffi Oesterreich. PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-02.

Phytochemical research of the anticancer potential of Aloe turkanensis

Abstract. Adem FA, Yenessew A, Yusuf AO, Wanjohi JM. 2022. Phytochemical research of the anticancer potential of Aloe turkanensis. Asian J Nat Prod Biochem 20: 75-88. The number of people diagnosed with cancer is rising worldwide, particularly in Sub-Saharan Africa. The search for new cancer treatments continues to benefit greatly from nature as a rich supply of promising chemicals. The anticancer effects of quinones have made them a popular medicinal class among natural chemicals (e.g., daunomycin and doxorubicin). Aloe turkanensis Christian, like other members of the genus Aloe, is a good place to get quinones. Dried and powdered A. turkanensis rhizomes and leaves were cold percolated in a mixture of dichloromethane and methanol (1:1). The crude extracts significantly decreased the viability of the human extrahepatic bile duct cancer cell line (TFK-1). Twelve chemicals were isolated from the crude extracts through chromatographic separations on silica gel, Sephadex LH-20, and preparative TLC. Spectroscopic techniques such as UV, 1H, and 13C NMR, COSY, NOESY, HMBC, and HSQC were used to determine the structures of the isolated compounds. Two naphthoquinones [3,5,8-trihydroxy-2-methylnaphthalene-1,4-dione (1) and 5,8-dihydroxy-3-methoxy-2-methylnaphthalene-1,4-dione (2)], seven anthraquinones [chrysophanol (3), aloesaponarin I (4), aloesaponarin II (5), laccaic acid D methyl ester (6) helminthosporin (8) aloe-emodin (10) and ?-L-11-O-rhamnopyranosylaloe-emodin (11)], a pre-anthraquinone [aloesaponol I (7)] a pyrone derivative [feralolide (9)] and a benzoic acid derivative [3,4-dihydroxybenzoic acid (12)] were the chemicals that made up these substances. This study reported again that the Aloe genus produced the naphthoquinones [3,5,8-trihydroxy-2-methylnaphthalene-1,4-dione (1) and 5,8-dihydroxy-2-methoxy-2-methylnaphthalene-1,4-dione (2)]. In addition, this study made the first report of 3,5,8-trihydroxy-2-methylnaphthalene-1,4-dione (1) from the Asphodelaceae family. Human extrahepatic bile duct carcinoma (TFK-1) and liver cancer (HuH7) cell lines were used to test the extracted compounds for in-vitro anticancer activity. The anthraquinone aloe-emodin (10) and the naphthoquinone 5,8-dihydroxy-3-methoxy-2-methylnaphthalene-1,4-dione (2) showed significant inhibition against TFK-1 cell lines using the 3- [4,5-dimethylthiazol-2-yl]-2-,5-diphenyltetrazolium bromide (MTT) assay, with IC50 values of 6.0 and Aloesaponol I (7), a pre-anthraquinone, inhibited the development of TFK-1 cells with IC50 values of 10.0 µg/mL and HuH7 cells with 88.0 µg/mL. Both ?-L-11-O- rhamnopyranosyl aloe-emodin (11) and aloesaponarin II (4), an anthraquinone, decreased TKF-1 cell line viability with IC50 values of 23.0 and 34.0 µg/mL, and HuH7 cell line viability with IC50 values of 47.0 and 55.0 µg/mL, respectively. IC50 values of 46.0 µg/mL for helminthosporin (8) showed considerable inhibition of TFK-1 cell growth, although it did not affect HuH7 cells at those concentrations. Extrahepatic bile duct (TFK-1) and liver (HuH7) cancer cell lines are sensitive to these chemicals identified here for the first time. Before these chemicals can be established as potentially effective anticancer medicines, more research on normal cell lines and their mechanism of action is required.

ONC201-Induced Mitochondrial Dysfunction, Senescence-like Phenotype, and Sensitization of Cultured BT474 Human Breast Cancer Cells to TRAIL.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

Toxicity of new monophenolic synthetic activator of Keap1/Nrf2/ARE redox-sensitive signaling system <i>in vitro</i> and <i>in vivo</i>

One of the promising areas of modern pharmacology is the development of «indirect antioxidants» capable of activating redox-sensitive signaling systems, primarily the Keap1/Nrf2/ARE system. Among its chemical inductors is the hydrophilic monosubstituted monophenol (3’-tert-butyl-4’-hydroxyphenyl)sodium propylthiosulfonate (TS-13) in development. The aim of the study was to investigate TS-13 antiproliferative activity against tumor cell line BT-474 in vitro and acute oral toxicity in mice in vivo. Material and methods. The relationship between TS-13 concentration and proliferative activity of human breast ductal carcinoma cell line BT-474 was determined using the MTT test, the IC<sub>50</sub> was calculated and compared to the previously obtained for MCF-7 line; results were correlated with the functional properties of cells based on gene expression (in silico GSEA). In vivo acute toxicity was studied in 50 female C57Bl/6J mice, who received a TS-13 solution in distilled water at various doses by intragastric gavage. LD<sub>50</sub> obtained experimentally and predicted in silico using the GUSAR web service were compared. Results and discussion. TS-13 inhibited the proliferation of BT-474 cells in a concentration-dependent manner (exponential approximation, IC<sub>50</sub> = 59.5 μM) and was 2.2 times more toxic than for MCF-7 cells. This may be due to functional differences between the BT-474 and MCF-7 lines, as evidenced by the GSEA results. The LD<sub>50</sub> value established in the in vivo experiment was 936 mg/kg body weight, the obtained value satisfactorily corresponds to the predicted in silico (561 mg/kg), although in reality the compound turned out to be somewhat less toxic than could be expected based on its structure. Conclusions. A study of the acute toxicity of the new water-soluble monophenol TC-13 allows the classification of it as slightly toxic (toxicity rating level 4) according to the Hodge – Sterner scale) or as moderately hazardous (hazard class 3) according to GOST 12.1.007-76.

The Use of the QbD Approach to Optimize the Co-Loading of Simvastatin and Doxorubicin in Liposomes for a Synergistic Anticancer Effect.

The present study aimed to optimize a liposomal formulation co-encapsulating simvastatin (SIM) and doxorubicin (DOX) that has future perspectives in anticancer therapy. The optimization process was performed by implementing the Quality by Design concept and by considering the results of a previous screening study. Failure Mode and Effects Analysis was used for the identification of the potential critical factors, i.e., phospholipid, SIM and DOX concentration, which were assessed in an optimization experimental design with the purpose of designing an optimal formulation. The optimal formulation, meeting the established quality profile, was additionally characterized in terms of the release profile and antiproliferative effects. During dissolution studies, a novel chronoamperometric method was used for the simultaneous quantification of SIM and DOX. The obtained data confirmed the similarity of this method with a validated HPLC method. The anticancer potential of the optimal formulation was tested against two human cancerous cell lines, namely T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line. The results highlighted that the antiproliferative effect of the optimal formulation is concentration dependent and favors a synergistic effect of the two drugs.

Gemcitabine Resistance in Pancreatic Ductal Carcinoma Cell Lines Stems from Reprogramming of Energy Metabolism

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis because it is often detected at an advanced stage, and drug resistance interferes with treatment. However, the mechanism underlying drug resistance in PDAC remains unclear. Here, we investigated metabolic changes between a parental PDAC cell line and a gemcitabine (GEM)-resistant PDAC cell line. We established a GEM-resistant cell line, MIA-G, from MIA-PaCa-2 parental (MIA-P) cells using continuous therapeutic-dose GEM treatment. MIA-G cells were also more resistant to 5-fluorouracil in comparison to MIA-P cells. Metabolic flux analysis showed a higher oxygen consumption rate (OCR) in MIA-G cells than in MIA-P cells. Notably, OCR was suppressed by GEM treatment only in MIA-G cells. GEM treatment increased mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) in MIA-P cells, but not in MIA-G cells. Glutamine uptake and peroxidase levels were elevated in MIA-G cells. The antioxidants N-acetyl-L-cysteine and vitamin C increased the sensitivity to GEM in both cell lines. In MIA-G cells, the expression of the mitochondrial transcription factor A also decreased. Furthermore, rotenone reduced the sensitivity of MIA-P cells to GEM. These findings suggest that the suppression of oxidative phosphorylation contributes to GEM resistance by reducing ROS production. Our study provides a new approach for reducing GEM resistance in PDAC.

One-pot preparation of nano-scaled magnetic-pectin particles (Fe3O4@pectin NPs): cytotoxicity, antioxidant, and anti-liver cancer properties

This work described the one-pot synthesis of orange pectin encapsulated Fe3O4 nanoparticles (Fe3O4@Pectin NPs) which is prepared by co-precipitation of Fe(II/(III) ions in alkaline solution mediated by pectin. This process led to formation of magnetic nanoparticles within the network of pectin. Physicochemical characterization of the as-synthesized Fe3O4@Pectin NPs was carried out through Fourier transformed infrared spectroscopy (FT-IR), electron microscopy (SEM and TEM), energy dispersive X-ray spectroscopy (EDX), vibrating sample magnetometer (VSM) and X-ray diffraction (XRD). The in vitro cytotoxic and anti-liver cancer effects of biologically synthesized Fe3O4@Pectin NPs against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cancer cell lines were assessed. The anti-liver cancer properties of the Fe3O4@Pectin NPs could significantly remove pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cancer cell lines in a time and concentration-dependent manner by MTT assay. The IC50 of the Fe3O4@Pectin NPs were 8, 13, 10, and 7 µg/mL against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cancer cell lines. The antioxidant activity of Fe3O4@Pectin NPs was determined by DPPH method. The Fe3O4@Pectin NPs showed the high antioxidant activity according to the IC50 value. It seems that the anti-human liver cancer effect of recent nanoparticles is due to their antioxidant effects.

Green formulation of Ag nanoparticles by Hibiscus rosa-sinensis: Introducing a navel chemotherapeutic drug for the treatment of liver cancer

An eco-friendly biosynthesized Ag NPs immobilized Hibiscus rosa-sinensis extract has been introduced. The as-prepared nanoparticles were characterized using UV–Vis, SEM, and FT-IR analysis. In the FT-IR test, the presence of many antioxidant compounds with related bonds caused the excellent condition for reducing of silver in the silver nanoparticles. In UV–Vis, the clear peak in the wavelength of 428 nm indicated the formation of silver nanoparticles. The synthesized nanoparticles had very low cell viability and high anti-liver cancer activities dose-dependently against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cell lines without any cytotoxicity on the normal cell line (HUVEC). The synthesized nanoparticles inhibited half of the DPPH molecules in the concentration of 78 µg/mL. Perhaps notable anti-liver cancer activities of the synthesized nanoparticles against common liver cancer cell lines are linked to their antioxidant activities.

Iron nanoparticles green-formulated by Coriandrum sativum leaf aqueous ‎extract: Investigation of its anti-liver cancer effects

IntroductionRecently, scientists have ‎understood that metallic nanoparticles especially iron nanoparticles have ‎excellent anticancer properties‎. A green, productive, and environmentally method was developed for the valuable study and the effective preparation of the biosynthesis of iron nanoparticles using aqueous extracts from the leaf of Coriandrum sativum as a result of reducing and stabilizing factor. The simplicity of the reaction, heterogeneous system, and easy work up are the benefits of the present method.Material and methodsThe as-prepared nanoparticles (FeNPs) was characterized using UV-Vis, SEM, and FT-IR. It has ‎been shown that the iron nanoparticles have spherical shape and uniform size.ResultsThe synthesized nanoparticles had very low cell viability and high anti-liver cancer activities ‎dose-dependently against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal ‎carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) ‎cell lines without any cytotoxicity on the normal cell line (HUVEC). The synthesized ‎nanoparticles inhibited half of the DPPH molecules in the concentration of 132 µg/mL. Perhaps ‎notable anti-liver cancer activities of the synthesized nanoparticles against common liver cancer ‎cell lines are linked to their antioxidant activities. ‎ConclusionsOur results point out that the FeNPs from Coriandrum sativum extract are apposite stabilizing ‎agents, which serve as an effective anticancer agent against liver cancer cell lines.‎

NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines.

Simple SummaryPotassium channels permit the selective passage of K+ ions across the cell me brane and are important for setting the membrane potential and for the transmission of electrical signals in all cells. Ca2+ activated K+ channels provide a means to couple intracellular calcium signaling to changes of the membrane potential. Among these, the large-conductance Ca2+-activated K+ channel, known as BK, has been proposed to be involved in several cancer-associated processes. In the present work, we tested various BK channel activators for anti-cancer effects in melanoma and pancreatic duct carcinoma cell lines. Only one of the activators (NS-11021) had effects on cancer-associated processes. However, the compound, as a side-effect, also increased the intracellular Ca2+ concentration independently of BK channel activation. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects.Potassium channels have emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large-conductance Ca2+-activated K+ channel, often referred to as BK channel, is involved in several cancer-associated processes. Here, we investigated the effects of different BK activators, NS-11021, NS-19504, and BMS-191011, in IGR39 (primary melanoma cell line) and Panc-1 (primary pancreatic duct carcinoma cell line), highly expressing the channel, and in IGR37 (metastatic melanoma cell line) that barely express BK. Our data showed that NS-11021 and NS-19504 potently activated BK channels in IGR39 and Panc-1 cells, while no effect on channel activation was detected in IGR37 cells. On the contrary, BK channel activator BMS-191011 was less effective. However, only NS-11021 showed significant effects in cancer-associated processes, such as cell survival, migration, and proliferation in these cancer cell lines. Moreover, NS-11021 led to an increase of intracellular Ca2+ concentration, independent of BK channel activation, thus complicating any interpretation of its role in the regulation of cancer-associated mechanisms. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects in the melanoma and PDAC cell lines examined. Importantly, our results raise an alarm flag regarding the use of presumably specific BK channel openers as anti-cancer agents.

Evaluation of antiangiogenic and antiproliferative potential of ethanolic extracts of Andrographis echioides using in vitro and in ovo assays.

Andrographis echioides is a prevalently used medicinal herb in South Asian countries. Scientific researches with the extracts of A. echioides revealed its antipyretic, anti-inflammatory, antimicrobial, ulceroprotective, and hepatoprotective properties. This study was done to elucidate antiproliferative and antiangiogenic potential of ethanolic extracts of A. echioides (EEAE) by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and chorioallantoic membrane (CAM) assay. EEAE was prepared using Soxhlet apparatus with ethanol after being sun-dried and powdered. MCF 7 (human invasive breast ductal carcinoma) cell lines retaining attributes of differentiated mammary epithelium with both estrogen and progesterone receptors were treated with EEAE, and antiproliferative effect was seen using Mosmann method of MTT assay using 5-fluorouracil (5-FU) as a comparator. The evaluation of antiangiogenic potential of EEAE was done by comparing mean vessel density (MVD) in chick CAM after treatment with EEAE, thalidomide, and vascular endothelial growth factor (VEGF) using CAM assay, an in ovo assay. EEAE displayed antiproliferative activity from low to high concentrations with MTT assay. The IC50 of EEAE and 5-FU was 62.5 and 15.6 μg/ml, respectively (P < 0.05). The exhibition of its antiangiogenic activity increased proportionately with increasing concentration. VEGF increased MVD by 45.94%; thalidomide decreased it by 53.76%. There was a decrease of MVD by 5.91%, 20.46%, and 35.95% at concentrations of 25, 50, and 100 μg of EEAE, respectively. EEAE possessed significant antiangiogenic and antiproliferative activity, making them a promising substrate in the development of a novel anticancer drug and can be successfully used in the therapy of various cancers after establishment of the anticancer effects in animal models and subsequently in clinical trials.

Platelet-derived extracellular vesicles regulate cell cycle progression and cell migration in breast cancer cells

Platelets have been extensively implicated in the progression of cancer and platelet-derived extracellular vesicles (PEVs) are gaining growing attention as potential mediators of the platelet-cancer interplay. PEVs are shed from platelet membrane in response to extracellular stimuli and carry important biological signals for intercellular communication. In this study we demonstrate that PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. PEVs were massively internalized by the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not by the MCF-7 cell line. In SKBR3 cells, PEVs decreased mitochondrial dehydrogenase activities and altered cell cycle progression without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the distribution in the different phases of the cell cycle. In all the analyzed breast cancer cells, PEVs triggered a sustained increase of intracellular Ca2+, but only in MDA-MB-231 cells, this was associated to the stimulation of selected signaling proteins implicated in migration, including p38MAPK and myosin light chain. Importantly, inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results demonstrate that PEVs are versatile regulators of cancer cell behavior and elicit a variety of different responses depending on the specific breast cancer cell subtype.

Abstract P3-10-06: Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor-positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p&amp;lt;0.01, HR=3.16, CI 1.24-8.03). Using a two ILC cell lines (SUM44PE and MDA-MB134VI) we found that ILC cells were relatively resistant to the anti-estrogen therapies tamoxifen and fulvestrant compared to those derived from IDC. Next, we tested whether the ILC cell lines were sensitive to BET protein inhibition using the pan-BET family inhibitor JQ1. Interestingly, while JQ1 inhibited cell growth in both ILC cell lines tested, apoptosis was only induced in SUM44PE cells, while MDA-MB134VI cells exhibited G1 arrest. Dynamic BH3 profiling was used to dissect the underlying anti-apoptotic dependencies in each ILC cell type and showed that in the JQ1-resistant MDA-MB134VI cells, survival was dependent on sustained Bcl-Xl expression and transcriptional rewiring inducing Fibroblast Growth Factor Receptor 1 (FGFR1). This increase in FGFR1 was also evident in invasive ductal carcinoma (IDC) cell lines that also did not respond to JQ1. The combination of JQ1 and FGFR1 inhibitor, or JQ1 and the Bcl2/Bcl-Xl/Bcl-W inhibitor navitoclax (ABT-263), were highly effective at inhibiting growth in both 2D and 3D models of ILC and IDC. Interestingly, we found in the RATHER cohort of 61 ILC patients that 20% had FGFR1 amplification suggesting that this therapeutic approach may have utility in a large number of ILC patients. Conclusions: We provide evidence that BETi either alone or in combination with FGFR1 inhibitors or BH3 mimetics may be a useful therapeutic strategy for recurrent ILC patients. Citation Format: Darran P O'Connor, Louise Walsh, Kathryn Haley, Bruce Moran, Brian Mooney, Stephen Madden, Sudipto Das, Oscar Rueda, Catriona Dowling, Damir Vareslija, Suet-Feung Chin, Sabine Linn, Leonie Young, Karin Jirstrom, John P Crown, Rene Bernards, Carlos Caldas, William M Gallagher, Triona Ni Chonghaile. Combination of BET and FGFR inhibition as a rational therapeutic strategy for invasive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-06.

Lipidome Profiles of Glioblastoma and Ductal Carcinoma Cell Lines

Lipidome Profiles of Glioblastoma and Ductal Carcinoma Cell Lines

PODXL2 maintains cellular stemness and promotes breast cancer development through the Rac1/Akt pathway.

The cluster of differentiation 34 (CD34) family, which includes CD34, podocalyxin-like protein 1 (PODXL), and PODXL2, are type-I transmembrane sialomucins and markers of hematopoietic stem cells (HSCs) and vascular-associated tissues. CD34 family proteins are expressed by endothelial cells and hematopoietic precursors. PODXL is well known to be associated with invadopodia formation and to promote the epithelial-mesenchymal transition, tumor migration and invasion. PODXL expression was correlated with poor survival of cancer patients. However, the role of PODXL2 in cancer has been less fully explored. To reveal the novel role of PODXL2 in breast cancer, the present study evaluated PODXL2 levels in relation to clinical outcomes of cancer patients by performing a bioinformatics analysis using the Oncomine database, Kaplan-Meier plots, and the CCLE database. Empirical validation of bioinformatics predictions was conducted utilizing the short hairpin (sh)-RNA silencing method for PODXL2 in the BT474 invasive ductal breast carcinoma cell line. The bioinformatics analysis revealed that PODXL2 overexpression was correlated with poor survival of breast cancer patients, suggesting an oncogenic role of PODXL2 in breast carcinoma. In a validation experiment, knockdown of PODXL2 in BT474 cells slightly influenced cell proliferation, suppressed migration, and inhibited expressions of downstream molecules, including Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphorylated (p)-Akt (S473), and p-paxillin (Y31) proteins. In addition, knockdown of PODXL2 reduced expression levels of cancer stem cell (CSC) markers, including Oct-4 and Nanog, and the breast CSC marker aldehyde dehydrogenase 1 (ALDH1). Collectively, our present study demonstrated that PODXL2 plays a crucial role in cancer development and could serve as a potential prognostic biomarker in breast cancer patients.