Non-tumorigenic Cell Line Research Articles

Abstract 4784: Multiomic landscape of evolution of Warburg effect identifies drivers of breast cancer

Abstract Early breast carcinogenesis is characterized by an intermittently hypoxic, acidic and nutrient-deprived microenvironment, eventually selecting for the invasive, aerobically glycolytic phenotype known as Warburg Effect (WE), a universal cancer hallmark. We have reproduced the emergence of WE in vitro, by subjecting the nontumorigenic breast epithelial cell line MCF10A to multiple intermittent cycles of media starvation followed by recovery in growth media. Clonal expansion of surviving cells demonstrated increased lactate production in aerobic conditions (WE). A comprehensive molecular characterization, through WES and single cell multiomics, was performed to study the relationship between mutations, CNA, cytogenetic abnormalities, transcriptional dysregulation, and epigenetic modifications. Mutational analysis of 6 WE clones identified 249 shared mutations, including HRAS(G12V), BRAF(P523L) and ALK(Q180X). COSMIC mutational signature 18 (ROS-induced) was identified as the main DNA lesion across all WE clones, suggesting oxidative stress as a main source of mutagenesis associated with WE development. Non-synonymous mutations in FUS, HDAC3, EZH2, MAZ, KMTD2, MET, and other oncogenes were found in the clones, consisting in secondary events in the WE transformation. Geneset enrichment analysis identified over-expression of MYC/E2F targets, MTORC1 signaling, glycolysis and cell cycle pathways, as well as under-expression of EMT/cell adhesion pathways, in WE clones. CNA, based on a consensus from WES, scRNAseq (inferCNV) and scATACseq (epiAneufinder), confirmed amplification of MYC, E2F1 and MYBL2, as well as Gain(19q) and Gain(20) in all WE clones, as well as loss of TP63 in 5 out of 6 WE clones. Integrative single cell transcriptome/chromatin accessibility analysis (ArchR) identified decreased accessibility and expression of EMT-related genes, as a consequence of differential expression of pioneer transcription factors (i.e. reduced expression of TP63, due to deletion, led to silencing of TP53/63/73 targeted genes), as well as well-established EMT regulators (ZEB1, SNAI1/2, NFKB1), among others. We propose that the WE phenotype emerges after intermittent nutrient-derived oxidative stress induces increased expression of HRAS-regulated genes, acutely increasing mutation rate and transcriptionally inducing an EMT-like state. Once HRAS(G12V), MYC/E2F1 amplification and TP53 deletions are fixed in the population, the clones will maintain the ROS production and EMT-like state, even in optimum growth media conditions, completing the transition from an acute response to a chronic-inheritable phenotype known as WE. These findings reinforce the importance of microenvironmental stress driving early carcinogenesis, bridges the gap between environment and genomic-driven causes of malignant transformation and provides testable hypotheses of causal events in connection with WE. Citation Format: Rafael Renatino Canevarolo, Praneeth Reddy Sudalagunta, Joon-Hyun Song, Erez Persi, Mehdi Damaghi, Ariosto Siqueira Silva. Multiomic landscape of evolution of Warburg effect identifies drivers of breast cancer. [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 4784.

Abstract 1226: SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC) and accounts for majority of the deaths from renal cancer. SETD2, the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is frequently mutated in ccRCC. Studies have shown that SETD2 mutations are often subclonal and the mutation rate dramatically increases from 25% in primary ccRCC to more than 60% in metastatic ccRCC, suggesting SETD2 loss plays an important role in renal cancer metastasis. However, mechanisms underpinning how SETD2 mutation drives invasion and metastasis in ccRCC is largely unknown. Here, by performing RNA-seq and ATAC-seq in a non-tumorigenic renal epithelial cell line isogenic for SETD2 loss-of-function, we found that SETD2 inactivation induces dramatic changes in the transcriptome and drastically enhances chromatin accessibility, which allows binding of a variety of transcription factors. We also discovered that SETD2 loss induces a TGFβ-independent EMT program, which is a major pathway linked to metastasis. Correlation of RNA-seq with ATAC-seq identifies SOX2, OCT2 and PRRX1 as key transcription factors promoting the SETD2 loss-driven EMT program. Furthermore, we found that this distinct EMT program is in part triggered by secreted factors, which suggests that SETD2 mutation promotes a metastasis-permissive tumor microenvironment through paracrine signaling. In summary, our findings reveal that SETD2 is a key regulator of EMT phenotypes that may explain the role of SETD2 mutations in ccRCC metastasis and further uncover potential therapeutic targets for SETD2-mutant ccRCC patients. Citation Format: Tianchu Wang, Ryan Wagner, Ryan Hlady, Sungho Kim, Xiaoyu Pan, Thai Ho, Keith Robertson. SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner [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 1226.

4-Nerolidylcatechol (4-NC) and Docetaxel Synergize in Controlling Androgen- independent Prostate Cancer Cells.

Effective cancer treatment still challenges medicine since the strategies employed so far are not sufficiently safe and capable of specifically eliminating tumor cells. Prostate cancer (PCa) is a highly incident malignant neoplasm, and the outcome of patients, especially those with advanced castration-resistant PCa (CRPC), depends directly on the efficacy of the therapeutic agents, such as docetaxel (DOC). This study investigated the synergistic potentiation of 4-nerolidylcatechol (4-NC) with DOC in inhibiting androgen-independent PCa cells. The cytotoxic effect of 4-NC was evaluated against non-tumorigenic (RWPE-01) and PCa cell lines (LNCaP and PC-3), and the antiproliferative potential of 4-NC was assessed by flow cytometry and colony formation. The Chou-Talalay method was applied to detect the synergistic effect of 4-NC and DOC, and the mechanism of anticancer activities of this combination was investigated by analyzing players in epithelial-mesenchymal transition (EMT). 4-NC significantly reduced the viability of PC-3 cells in a dose-dependent manner, decreasing colony formation and proliferation. The combination of 4-NC and DOC was synergistic in the androgen-independent cells and allowed the reduction of DOC concentration, with increased cytotoxicity and induction of apoptosis when compared to compounds alone. Furthermore, when 4- NC was co-administered with DOC, higher expression levels of proteins associated with the epithelial phenotype were observed, controlling EMT in PC-3 cells. Collectively, these data demonstrated, for the first time, that the combination of 4-NC with reduced doses of DOC could be especially valuable in the suppression of oncogenic mechanisms of androgen-independent PCa cells.

Glyphosate and Aminomethylphosphonic Acid (AMPA) Modulate Glutathione S-Transferase in Non-Tumorigenic Prostate Cells.

Glyphosate (GLY) was developed in the early 1970s and has become the most used broad-spectrum herbicide in the world so far. Its main metabolite is aminomethylphosphonic acid (AMPA), and the accumulation of GLY and its derivative compounds raises some concerns regarding possible health outcomes. In this study, we aimed to evaluate the effects of GLY and AMPA on prostate cell lines by evaluating cell viability, proliferation, gene and protein expression, and cellular pathways involved in the response to oxidative stress. Our results indicated that GLY and AMPA reduced the cell viability of tumorigenic and non-tumorigenic prostate cell lines only at higher concentrations (10 mM GLY and 20 mM AMPA). In contrast, both compounds increased the clonogenicity of non-tumorigenic PNT2 cells, mainly at concentrations below the IC50 (5 mM GLY and 10 mM AMPA). Moreover, treatment of non-tumorigenic cells with low concentrations of GLY or AMPA for 48 h increased GSTM3 expression at both mRNA and protein levels. In contrast, the treatments decrease the GST activity and induced an increase in oxidative stress, mainly at lower concentrations. Therefore, both compounds can cause cellular damage even at lower concentrations in non-tumorigenic PNT2 cells, mainly affecting cell proliferation and oxidative stress.

Mechanical profile of human keratinocytes expressing HPV-18 oncogenes

During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.

Anticancer effect and safety of doxorubicin and nutraceutical sulforaphane liposomal formulation in triple-negative breast cancer (TNBC) animal model

Female breast cancer is the most deadly cancer in women worldwide. The triple-negative breast cancer subtype therapies, due to the lack of specific drug targets, are still based on systemic chemotherapy with doxorubicin, which is burdened with severe adverse effects. To enhance therapeutic success and protect against systemic toxicity, drug carriers or combination therapy are being developed. Thus, an innovative liposomal formulation containing doxorubicin and the main nutraceutical, sulforaphane, has been developed. The anticancer efficacy and safety of the proposed liposomal formulation was evaluated in vivo, in a 4T1 mouse model of triple-negative breast cancer, and the mechanism of action was determined in vitro, using triple-negative breast cancer MDA-MB-231 and non-tumorigenic breast MCF-10A cell line. The elaborated drug carriers were shown to efficiently deliver both compounds into the cancer cell and direct doxorubicin to the cell nucleus. Incorporation of sulforaphane resulted in a twofold inhibition of tumor growth and the potential of up to a fourfold reduction in doxorubicin concentration due to the synergistic interaction between the two compounds. Sulforaphane was shown to increase the accumulation of doxorubicin in the nuclei of cancer cells, accompanied by inhibition of mitosis, without affecting the reactive oxygen species status of the cell. In normal cells, an antagonistic effect resulting in less cytotoxicity was observed. In vivo results showed that sulforaphane incorporation yielded not only cardioprotective, but also nephro- and hepatoprotective effects. The results of the research revealed the prospects of applying sulforaphane as a component of liposomal doxorubicin in triple-negative breast cancer chemotherapy.

Synthesis and characterization of novel Schiff base-silicon (IV) phthalocyanine complex for photodynamic therapy of breast cancer cell lines

BackgroundPhotodynamic therapy is an alternative anticancer treatment approach that promises high therapeutic efficacy. In this study, it is aimed to investigate the PDT-mediated anticancer effects of newly synthesized silicon phthalocyanine (SiPc) molecules on MDA-MB-231, MCF-7 breast cancer cell lines, and non-tumorigenic MCF-10A breast cell line. MethodsNovel bromo substituted Schiff base (3a), its nitro homolog (3b), and their silicon complexes (SiPc-5a and SiPc-5b) were synthesized. Their proposed structures were confirmed by FT-IR, NMR, UV–vis and MS instrumental techniques. MDA-MB-231, MCF-7 and MCF-10A cells were illuminated at a light wavelength of 680 nm for 10 min, giving a total irradiation dose of 10 j/cm2. MTT assay was used to determine the cytotoxic effects of SiPc-5a and SiPc-5b. Apoptotic cell death was analyzed using flow cytometry. Changes in the mitochondrial membrane potential were determined by TMRE staining. Intracellular ROS generation was observed microscopically using H2DCFDA dye. Colony formation assay and in vitro scratch assay were performed to analyze the clonogenic activity and cell motility. Transwell migration and matrigel invasion analyzes were conducted to observe changes in the migration and invasion status of the cells. ResultsThe combination of SiPc-5a and SiPc-5b with PDT exhibited cytotoxic effects on cancer cells and triggered cell death. SiPc-5a/PDT and SiPc-5b/PDT decreased mitochondrial membrane potential and increased intracellular ROS production. Statistically significant changes were detected in cancer cells' colony-forming ability and motility. SiPc-5a/PDT and SiPc-5b/PDT reduced cancer cells' migration and invasion capacities. ConclusionThe present study identifies PDT-mediated antiproliferative, apoptotic, and anti-migratory characteristics of novel SiPc molecules. The outcomes of this study emphasize the anticancer properties of these molecules and suggest that they may be evaluated as drug-candidate molecules for therapeutic purposes.

Ubiquitin-Specific Proteases as Potential Therapeutic Targets in Bladder Cancer—In Vitro Evaluation of Degrasyn and PR-619 Activity Using Human and Canine Models

The inhibition of ubiquitin-specific proteases (USPs) is a novel and promising direction in the development of molecularly targeted therapies in oncology. The aim of the present study was to examine whether Degrasyn could be a potential therapeutic agent against bladder cancer (BC). Also, we aimed to determine whether Degrasyn is more effective in terms of anti-cancer activity compared to the non-selective DUB inhibitor PR-619. To facilitate the translational value of the obtained results, our experiments were performed using both human and canine in vitro models of BC. Human T24 (urothelial grade III BC) and SV-HUC-1 (non-tumorigenic urothelial cell line), as well as canine K9TCC-PU-NK and RDSVS-TCC1 (both derived from invasive grade III urothelial bladder tumors) cell lines, were used in the present study. Cell proliferation was determined using the MTT assay and Ki-67 proliferation assay, and the level of apoptosis induced by Degrasyn and PR-619 was evaluated by Annexin V-FITC staining and caspase 3/7 activation assay. Western blot was used to assess DNA damage and key proteins involved in apoptosis. Degrasyn inhibited the proliferation of all BC cell lines in a concentration- and time-dependent manner. Lower concentrations of Degrasyn were more potent against human and canine BC cell lines compared to PR-619. Degrasyn induced caspase-dependent apoptosis and triggered DNA damage. PR-619 did not show a significant pro-apoptotic effect. Our results demonstrate that Degrasyn significantly impairs the growth of in vitro models of human and canine BC. Selective USP inhibition with Degrasyn seems to be more effective in reducing BC cell proliferation and inducing apoptosis and DNA damage than non-selective USP inhibition with PR-619.

Cytotoxic Effect of Phenylethanoid Glycosides Isolated from Plantago lanceolata L.

The aim of the study is to investigate whether the bioactive compounds isolated from P. lanceolata inflorescences, namely, phenylethanoid glucosides, acteoside, plantamajoside, and a flavonoid, isorhamnetin-3-O-rutinoside-4'-O-glucoside, possessed cytotoxic activity against the selected cancer cell lines. The potential antitumor effects of two phenylethanoid glycosides and one flavonoid were evaluated via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on seven human carcinoma cell lines (MCF-7, MDA-MB-231, Caco-2, HepG2, OVCAR-3, U138-MG, U251-MG) and one nontumorigenic mammary epithelial cell line (MCF-12A). For the first time, acteoside was studied in ovarian cancer cell line OVCAR-3, and plantamajoside in all cell lines except breast adenocarcinoma MDA-MB-281 and hepatocarcinoma HepG2. The phenylethanoid glycosides showed stronger cytotoxic activity than that of the glycoside flavonoid. Acteoside and plantamajoside, at concentrations of 200 and 300 μM, respectively, had a highly toxic effect on the selected two cancer cell lines of breast adenocarcinoma MDA-MB-231 and MCF-7, ovarian cancer cell line OVCAR-3, glioblastoma cell line U138-MG, and hepatocarcinoma cell line HepG2. Both glycosides were significantly less cytotoxic towards nontumorigenic cell line MCF-12A; the effect appeared at a concentration of 400 μM. For the first time, the activity of acteoside and plantamajoside was compared in one parallel investigation. The results are discussed against a broad background of existing knowledge on biological effects, their mechanisms, and structure-activity relationships. Phenylethanoids may be potential compounds with cytotoxic activity against the selected cancer types.

One-pot two-component synthesis of halogenated xanthone, 3-o substituted xanthone, and prenylated xanthone derivatives as aromatase inhibitors

This study describes the synthesis, characterization, and biological activities of halogenated xanthone, 3-O substituted xanthone, and prenylated xanthone derivatives. The molecular structures of these compounds were identified using 1H NMR, 13C NMR, 2D NMR, FTIR and DIMS. All synthesized compounds were evaluated against two cancer cell lines: breast adenocarcinoma MCF-7 and MDA-MB-231 by application of the MTT assay. With this approach, xanthones 9 and 13 were identified as putative aromatase activating agents through the inhibition of interactions with MCF-7 and MDA-MB-231. The selected compounds, 9 and 13, were further examined for toxicity against the African Green Monkey Kidney cell (VERO) and Non-tumorigenic Lung Epithelial cell lines (BEAS-2B). In addition, in-silico docking studies were conducted to estimate the binding poses and residues implicated in probable MCF-7 and MDA-MB-231 interactions.

Development of a 7-miRNA prognostic signature for patients with bladder cancer

Bladder carcinoma (BC) represents one of the most prevalent malignant cancers, while predicting its clinical outcomes using traditional indicators is difficult. This study aimed to develop a miRNA signature for the prognostic prediction of patients with BC. MiRNAs that expressed differentially were identified between 413 BC and 19 non-tumor patients, whose prognostic values were evaluated using univariate and multivariate Cox regression analyses. The independent prognostic factors were screened out and were used to establish a signature. The risk score of the signature was calculated. Receiver operating characteristic (ROC) curves and Kaplan-Meier curves were used to verify the predictive performance of the miRNA signature and the risk score. A nomogram was constructed which integrated with the miRNA signature and clinical parameters. Experiments were performed. 7 prognosis related miRNAs were selected as independent risk factors, and a 7-miRNA signature was constructed, with an area under ROC (AUC) of 0.721. The 7-miRNA-signature based risk score acts as an independent prognostic factor, with satisfactory predictive performance (AUC = 0.744). Increased miR-337-3p expressions were detected in tumor samples and BC cell lines than in non-tumorigenic tissues and cell lines. Experiments suggested that miR-337-3p induces the proliferation, migration, and invasion of BC cells. The constructed 7-miRNA signature is a promising biomarker for predicting the prognosis of patients with BC, and miR-337-3p may act as a candidate therapeutic target in BC treatments.

Synthesis, biological evaluation, and computational studies of some novel quinazoline derivatives as anticancer agents

A series of quinazolinone derivatives (7a–7h) were synthesized as antiproliferative agents. All compounds, were synthesized through three steps method and structurally evaluated by FTIR, 1H-NMR, 13CNMR and Mass spectroscopy. Their cytotoxic activities were assessed using MTT protocol against three humans cancerous (MCF-7, A549 and 5637) and normal (MRC-5) cell lines. In addition, molecular docking and simulation studies of the synthesized compounds were performed to assessment their orientation, interaction mode against EGFR as plausible mechanism of quinazoline compounds as anticancer agents. The synthesized compounds mostly showed moderate activity against the three studied cell lines. They also indicated an appropriate selectivity against tumorigenic and non-tumorigenic cell line. The molecular docking results also confirmed biological activity. Most of the compounds fulfilled Lipinski rule. Collectively, these compounds with further modification can be considered as potent antiproliferative agents.

A 50 Hz magnetic field influences the viability of breast cancer cells 96 h after exposure

BackgroundThe exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) results in various biological responses. Some studies have suggested a possible cancer-enhancing effect, while others showed a possible therapeutic role. This study investigated the effects of in vitro exposure to 50 Hz ELF-MF for up to 24 h on the viability and cellular response of MDA-MB-231 and MCF-7 breast cancer cell lines and MCF-10A breast cell line.Methods and resultsThe breast cell lines were exposed to 50 Hz ELF-MF at flux densities of 0.1 mT and 1.0 mT and were examined 96 h after the beginning of ELF-MF exposure. The duration of 50 Hz ELF-MF exposure influenced the cell viability and proliferation of both the tumor and nontumorigenic breast cell lines. In particular, short-term exposure (4–8 h, 0.1 mT and 1.0 mT) led to an increase in viability in breast cancer cells, while long and high exposure (24 h, 1.0 mT) led to a decrease in viability and proliferation in all cell lines. Cancer and normal breast cells exhibited different responses to ELF-MF. Mitochondrial membrane potential and reactive oxygen species (ROS) production were altered after ELF-MF exposure, suggesting that the mitochondria are a probable target of ELF-MF in breast cells.ConclusionsThe viability of breast cells in vitro is influenced by ELF-MF exposure at magnetic flux densities compatible with the limits for the general population and for workplace exposures. The effects are apparent after 96 h and are related to the ELF-MF exposure time.

Cell Adhesion Molecules Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model.

Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell–cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells.

A Different Trend of Heat Shock Proteins 90 mRNA and Protein Inhepatocellular Carcinoma Cell Line-Secreted Extracellular Vesicles

Primary Hepatocellular Carcinoma (HCC) does not usually show any symptoms at the early stage and the use of biomarkers is necessary to aid in diagnosis. Recently Extracellular Vesicles (EVs), submicron membranebound structures secreted from different cell types containing a wide variety of bioactive molecules, have increased the attention in many cancers, including HCC, becoming an auspicious candidate as biomarkers and therapy in the scenario of limited diagnostic and treatment option. Many indications have shown that heat shock proteins (Hsps) are important modulators in treatment resistance and invasion of HCC becoming attractive therapeutic targets. In particular, Hsp90a/β isoforms have been found to play critical roles in regulating the proliferation, apoptosis, and metastasis of tumor cells, suggesting for these proteins a role as targets for modern anticancer therapies. The study aimedto verify the presence of Hsp90a/β in EVs secreted by an HCC tumor cell line (HepG2) and by a non-tumorigenic hepatocyte cell line (WRL68), both at protein and mRNA levels, and to analyze their expression variations. The result showed that Hsp90s are transported by the EVs as protein but not at the mRNA level. To build new therapeutic targets using EVs or other organelles as performed on exosomes in recent studies, it is essential to evaluate the action at the pre or post-transcriptional level given their different behavior in transporting proteins or mRNA.

Fusion of the β2-adrenergic receptor with either Gαs or βarrestin-2 produces constitutive signaling by each pathway and induces gain-of-function in BEAS-2B cells.

The β2AR is a prototypical G protein-coupled receptor (GPCR) known to orchestrate different cellular responses by the stimulation of specific signaling pathways. The best-established signaling pathways for the β2AR are the canonical Gs pathway and the alternative β arrestin 2 (βarr2) pathway. Exploring each pathway separately remains a challenging task due to the dynamic nature of the receptor. Here, we fused the β2AR with its cognate transducers, Gαs and βarr2, using short linkers as a novel approach for restricting the conformation of the receptor and preferentially activating one of its two signaling pathways. We characterized the behavior of our fusion proteins β2AR-Gαs and β2AR-βarr2 in HEK293 cells by measuring their constitutive activity, transducer recruitment, and pharmacological modulation. Our fusion proteins show (a) steric hindrance from the reciprocal endogenous transducers, (b) constitutive activity of the β2AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by β2AR ligands. Based on these characteristics, we further explored the possibility of a gain-of-function mechanism in the human lung non-tumorigenic epithelial cell line, BEAS-2B cells. This immortalized human bronchial epithelial cell line has immunomodulatory properties through cytokine release mediated by β2AR stimulation. Our findings suggest that each signaling pathway of the β2AR is biased toward either the Th1 or Th2 inflammatory response suggesting a role in regulating the immune phenotype of respiratory diseases. Our data imply that our fusion proteins can be used as tools to isolate the function elicited by a single signaling pathway in physiologically relevant cell types.

Biomolecular Interactions of Cytotoxic Ruthenium Compounds with Thiosemicarbazone or Benzothiazole Schiff Base Chelates.

Herein we illustrate the formation and characterization of new paramagnetic ruthenium compounds, trans-P-[RuCl(PPh3 )2 (pmt)]Cl (1) (Hpmt=1-((pyridin-2-yl)methylene)thiosemicarbazide), trans-P-[RuCl(PPh3 )2 (tmc)]Cl (2) (Htmc=1-((thiophen-2-yl)methylene)thiosemicarbazide) and a diamagnetic ruthenium complex, cis-Cl, trans-P-[RuCl2 (PPh3 )2 (btm)] (3) (btm=2-((5-hydroxypentylimino)methyl)benzothiazole). Agarose gel electrophoresis experiments of the metal compounds illustrated dose-dependent binding to gDNA by 1-3, while methylene blue competition assays suggested that 1 and 2 are also DNA intercalators. Assessment of the effects of the compounds on topoisomerase function indicated that 1-3 are capable of inhibiting topoisomerase I activity in terms of the ability to nick supercoiled plasmid DNA. The cytotoxic activities of the metal complexes were determined against a range of cancer cell lines versus a non-tumorigenic control cell line, and the complexes were, in general, more cytotoxic towards the cancer cells, displaying IC50 values in the low micromolar range. Time-dependent stability studies showed that in the presence of strong nucleophilic species (such as DMSO), the chloride co-ligands of 1-3 are rapidly substituted by the former as proven by the suppression of the substitution reactions in the presence of an excess amount of chloride ions. The metal complexes are significantly stable in both DCM and an aqueous phosphate buffer containing 2 % DMSO.

Amygdalin as a chemoprotective agent in co-treatment with cisplatin.

Amygdalin is a naturally occurring glycoside used in traditional Chinese medicine and is known to have anti-cancer properties. Even though the anti-cancer properties of amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate a possible chemo-protective role of amygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug cisplatin. Human non-tumorigenic MCF12F epithelial cell line, human fibroblasts cells, human breast cancer MCF7 and MDA-MB-231 cells were treated with cisplatin in a dose- and time-depended manner in the absence or presence of amygdalin. When MCF12F cells and fibroblasts underwent pre-treatment with amygdalin followed by cisplatin treatment (24 h amygdalin + 24 h cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. As attested by flow cytometry, combination treatment was associated with decreased the percentage of late apoptotic cells compared with monotherapy (fold-change of decrease = 1.6 and 4.5 for 15 and 20 μΜ, respectively). Also, the proteins expression of PUMA, p53, phospho-p53 and Bax decreased, when a combination treatment was used vs. cisplatin alone, while the proapoptotic proteins Bcl-2 and Bcl-xL exhibited an increased tendency in the presence of amygdalin. Moreover, the levels of pro-apoptotic genes PUMA, p53, and BAX mRNA were significantly downregulated (∼83%, ∼66%, and ∼44%, respectively) vs. cisplatin alone, while the mRNA levels of anti-apoptotic genes BCl-2 and Bcl-XL were upregulated (∼44.5% and ∼51%, respectively), vs. cisplatin alone after 24 h of combination treatment. The study on the Combination index (CI) assay indicated that amygdalin could be possibly considered as an antagonist to cisplatin (2.2 and 2.3) for MCF12F and fibroblast cells, respectively. In contrast, for the breast cancer MCF7 and MDA-MB-231 cells, amygdalin and cisplatin indicated a synergistic effect (0.8 and 0.65), respectively. Our present findings suggest that amygdalin has chemo-modulatory effect when used in co-treatment with cisplatin and is able to protect normal breast cells as well as the fibroblasts during chemotherapy treatment, indicating a strong selective chemoprotective ability and may contribute to a better quality of life for cancer patients.

Transcriptional level evaluation of osteopontin/miRNA-181a axis in hepatocellular carcinoma cell line-secreted extracellular vesicles

Recent evidence suggested the role of secreted extracellular vesicles (EVs) in the intracellular signalling within the liver becoming a promising candidate as biomarker in hepatocellular carcinoma (HCC). Osteopontin (OPN) seems to play a relevant role both for early diagnosis of HCC than on the mechanisms that drive oncogenesis but, to date, information on the expression levels of OPN in EVs secreted by HCC tumor cell line are missing. The study aimed to verify, by transcriptional and proteomic study, the presence of OPN in EVs secreted by tumorigenic (HepG2) and non-tumorigenic hepatocyte cell line (WRL68), and to analyse the expression variations of OPN, its isoforms and miRNA-181a in both these EVs. “In silico analysis” was also performed via the Gene expression Profiling Interactive analysis (GEPIA) and Hepatocellular Carcinoma Database (HCCDB). An up-regulation of OPN in EVs secreted by HepG2 with respect to WRL68 was found in line with the results obtained by the “in silico analysis”. The study demonstrates, for the first time, the OPN isoforms and its modulator miRNA-181a expression in EVs secreted by both cell lines, highlighting high levels of OPN isoforms in EVs secreted by HepG2 and identifying OPN as a promising biomarker for HCC diagnosis.

ANKHD1 contributes to the malignant phenotype of triple-negative breast cancer cells.

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, has been identified as a regulator of signaling pathways and cellular processes of relevance in carcinogenesis. However, the role of ANKHD1 in breast cancer remains unclear. The aim of the present study was to characterize the expression pattern and involvement of ANKHD1 in the malignant phenotype of breast cancer cell lines and to investigate the clinical relevance of ANKHD1 in a breast cancer context. Gene and protein expressions were assessed in the cell lines by quantitative reverse transcription PCR and Western blot analysis, respectively, and ANKHD1 silencing through siRNA transfection was conducted for further in vitro functional assays. The expression of ANKHD1 was identified in non-tumorigenic breast epithelium and breast cancer cell lines, but differences in cellular localization were found among the neoplasia subtypes. ANKHD1 silencing reduced the viability, clonogenicity, and migration of triple-negative breast cancer (TNBC) cells. Bioinformatics analyses demonstrated that patients with triple-negative basal-like 2 and mesenchymal breast cancer subtypes had high ANKHD1 expression associated with poor recurrence-free survival. Therefore, these data indicate that ANKHD1 relevance in breast cancer varies among its subtypes, indicating the importance of ANKHD1 in TNBC.