Epithelial Tumor Cell Lines Research Articles

Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models.

Despite the cumulative evidence supporting HER3 as a target for antitumor therapies, no agents targeting HER3 have been approved for cancer treatment. Most of the agents evaluated in preclinical and clinical trials have been specific monoclonal antibodies (MAbs), with few examples of active immunotherapy directed against this receptor. However, some cancer vaccine formats may generate polyclonal antibodies (PAbs) that replicate the diverse effector mechanisms of MAbs, including ligand neutralization and receptor degradation. In this study, we developed a protein subunit-based monovalent vaccine candidate targeting the extracellular domain (ECD) of HER3. Immunization of mice with a formulation targeting murine ErbB3-ECD successfully overcome tolerance to this self-antigen, inducing high titers of ErbB3-specific PAbs. The antitumor potential of this formulation and the induced PAbs was demonstrated in vivo and in vitro in an ErbB3-overexpressing 3LL-D122-derived tumor model. The immunogenicity of the HER3-ECD-based vaccine candidate was confirmed by the induction of high titers of HER3-specific PAbs. Consistent with the initial results, HER3-ECD-targeting PAbs were cytotoxic in several human epithelial tumor cell lines and exerted antitumor effects in vivo. These results support the value of HER3 as a tumor antigen and the effector mechanisms of HER3-specific therapeutic MAbs, while suggesting the potential of the proposed vaccine candidate for the treatment of HER3-expressing carcinomas.

Dual-Purpose Aptamer-Based Sensors for Real-Time, Multiplexable Monitoring of Metabolites in Cell Culture Media.

The availability of high-frequency, real-time measurements of the concentrations of specific metabolites in cell culture systems will enable a deeper understanding of cellular metabolism and facilitate the application of good laboratory practice standards in cell culture protocols. However, currently available approaches to this end either are constrained to single-time-point and single-parameter measurements or are limited in the range of detectable analytes. Electrochemical aptamer-based (EAB) biosensors have demonstrated utility in real-time monitoring of analytes in vivo in blood and tissues. Here, we characterize a pH-sensing capability of EAB sensors that is independent of the specific target analyte of the aptamer sequence. We applied this dual-purpose EAB to the continuous measurement of pH and phenylalanine in several in vitro cell culture settings. The miniature EAB sensor that we developed exhibits rapid response times, good stability, high repeatability, and biologically relevant sensitivity. We also developed and characterized a leak-free reference electrode that mitigates the potential cytotoxic effects of silver ions released from conventional reference electrodes. Using the resulting dual-purpose sensor, we performed hourly measurements of pH and phenylalanine concentrations in the medium superfusing cultured epithelial tumor cell lines (A549, MDA-MB-23) and a human fibroblast cell line (MRC-5) for periods of up to 72 h. Our scalable technology may be multiplexed for high-throughput monitoring of pH and multiple analytes in support of the broad metabolic qualification of microphysiological systems.

Phytochemical profiling and investigation of antioxidant, anti-proliferative, and antibacterial properties in spontaneously grown Sicilian sumac (Rhus coriaria L.) fruits

This study aimed to assess the phytochemical profile and associated functional properties of sumac (Rhus coriaria L.) fruits harvested from wild plants in Sicily.Chemical characterization unveiled exceptionally high levels of polyphenols (10.99 g GAE/100 g DW), including substantial quantities of proanthocyanidins (61.577 mg PACE/100 g DW), and identified 82 phytochemicals belonging to various flavonoid classes. The hydroalcoholic extract from Sicilian sumac exhibited remarkable redox-active properties, providing antioxidant protection in a cell-based model of lipid peroxidation (CAA50: 1.116 μg/mL). Additionally, it displayed significant antiproliferative activity against four human tumor epithelial cell lines with GI50 values ranging from 31.08 to 149.74 μg/mL and robust antibacterial activity against major foodborne pathogens (MIC: 12.5–25.0 mg/mL).Our findings highlight Sicilian sumac fruit as a rich source of phytochemicals that positively contribute to the cellular redox state even when consumed in small quantities. Additionally, its diverse bioactivities indicate potential applications across food and non-food sectors.

Preparation and Properties of Natural Polysaccharide-Based Drug Delivery Nanoparticles.

In recent years, natural polysaccharides have been widely used in the preparation of drug delivery systems. In this paper, novel polysaccharide-based nanoparticles were prepared by layer-by-layer assembly technology using silica as a template. The layers of nanoparticles were constructed based on the electrostatic interaction between a new pectin named NPGP and chitosan (CS). The targeting ability of nanoparticles was formed by grafting the RGD peptide, a tri-peptide motif containing arginine, glycine, and aspartic acid with high affinity to integrin receptors. The layer-by-layer assembly nanoparticles (RGD-(NPGP/CS)3NPGP) exhibited a high encapsulation efficiency (83.23 ± 6.12%), loading capacity (76.51 ± 1.24%), and pH-sensitive release property for doxorubicin. The RGD-(NPGP/CS)3NPGP nanoparticles showed better targeting to HCT-116 cells, the integrin αvβ3 high expression human colonic epithelial tumor cell line with higher uptake efficiency than MCF7 cells, the human breast carcinoma cell line with normal integrin expression. In vitro antitumor activity tests showed that the doxorubicin-loaded nanoparticles could effectively inhibit the proliferation of the HCT-116 cells. In conclusion, RGD-(NPGP/CS)3NPGP nanoparticles have potential as novel anticancer drug carriers because of their good targeting and drug-carrying activity.

Endophytic Fungi from Mangrove Plant Acanthus ilicifolius L.: Antimicrobial, Anticancer, and Species Determination

The current study aimed to screen, identify and evaluate the biological activity of fungi strains from the mangrove plant Acanthus ilicifolius L, which has proven to be a rich source of fungal species. Thirteen fungi were screened from the leaves, stem bark, and roots. Each fungal strain was cultivated in rice as media, and then the secondary metabolites of the fungus were extracted with ethyl acetate. The extracts were tested for antimicrobial and cytotoxic activities. Next, a phylogenetic tree was reconstructed based on the sequence similarities of each strain. The fungal extract AIA1 displayed excellent antimicrobial activity in a concentration of 5 % against some pathogenic bacterial strains with an inhibition zone in the range of 11.87 ± 0.87 to 15.42 ± 0.51 mm. The brine shrimp lethality test (BSLT) showed that strains AIA1 and AIKB3 were toxic against Artemisia salina L with an LC50 value of less than 80 ppm. The MTT assay determined the cytotoxicity against the human ductal breast epithelial tumor (T47D) cell line. Based on molecular identification, AIA1, AIKB3, and AID1 showed maximum similarity (> 95 %) with Pestalotiopsis sp. AIA1, Trichoderma yunnanense AIKB3, and Schizopyllum commune AID1, respectively. The ethyl acetate extracts of fungi strains from the mangrove plant A. ilicifolius showed strong antibacterial and moderate cytotoxic activities, which make them good candidates for secondary metabolite isolation. HIGHLIGHTS This is the first report on exploring the endophytic fungi from the mangrove plant Acanthus ilicifolius collected from Mangrove Forest Tourism Park, West Sumatera, Indonesia Endophytic fungi strains from mangrove plant Acanthus ilicifolius showed good activity on the antimicrobial and cytotoxic assay Three potential endophytic fungi were identified asPestalotiopsis AIA1, Trichoderma yunnanense AIKB3, and Schizopyllum commune AID1 GRAPHICAL ABSTRACT

Sodium Butyrate Induces CRC Cell Ferroptosis via the CD44/SLC7A11 Pathway and Exhibits a Synergistic Therapeutic Effect with Erastin.

Human normal intestinal epithelial cell line FHT and colorectal tumor cell line HCT-116 were treated with NaB alone or in combination with different programmed cell death inhibitors. Cell activity was then assessed with MTT assays and PI staining; ferroptosis with Fe2+, glutathione (GSH), and lipid peroxidation assays; signaling pathway screening with PCR arrays; and CD44, SCL7A11, and GPX4 expression with Western blotting. A CD44-overexpressing HCT-116 cell line was constructed to determine the effect of the overexpression of CD44 on NaB-induced ferroptosis. The synergistic effect of co-treatment with NaB and Erastin was assessed by isobolographic analysis. NaB induced apoptosis and ferroptosis in HCT-116 cells but only induced low-level apoptosis in FHC cells. Moreover, NaB significantly increased intracellular Fe2+ and promoted GSH depletion and lipid peroxidation in HCT-116 cells. Ferroptosis-related qPCR array analysis identified CD44/SLC7A11 as a potential effector molecular of NaB-induced ferroptosis. NaB significantly inhibited the expression of CD44 and SLC7A11 in mouse CRC tissues. A CD44 overexpressed HCT-116 cell line was used to verify that CD44/SLC7A11 was a key signaling pathway that NaB-induced GSH depletion, lipid peroxidation accumulation, and ferroptosis in HCT-116 cells. Examination of whether NaB can increase the effect of ferroptosis agents showed that NaB, in combination with Erastin, a ferroptosis inducer, further promoted HCT-116 cell death and increased changes of ferroptosis markers. Our results suggest that NaB induces ferroptosis in CRC cells through the CD44/SLC7A11 signaling pathway and has synergistic effects with Erastin. These results may provide new insights into CRC prevention and the combined use of NaB and ferroptosis-inducing agents.

Interacting with tumor cells weakens the intrinsic clockwise chirality of endothelial cells.

Endothelial cells (ECs) possess a strong intrinsic clockwise (CW, or rightward) chirality under normal conditions. Enervating this chirality of ECs significantly impairs the function of the endothelial barrier. Malignant tumor cells (TCs) undergo metastasis by playing upon the abnormal leakage of blood vessels. However, the impact of TCs on EC chirality is still poorly understood. Using a transwell model, we co-cultured the human umbilical vein endothelial cells or human lung microvascular endothelial cells and breast epithelial tumor cell lines to simulate the TC-EC interaction. Using a micropatterning method, we assessed the EC chirality changes induced by paracrine signaling of and physical contact with TCs. We found that the intrinsic clockwise chirality of ECs was significantly compromised by the TC's physical contact, while the paracrine signaling (i.e., without physical contact) of TCs causes minimal changes. In addition, ECs neighboring TCs tend to possess a left bias, while ECs spaced apart from TCs are more likely to preserve the intrinsic right bias. Finally, we found the chirality change of ECs could result from physical binding between CD44 and E-selectin, which activates protein kinase C alpha (PKCα) and induces pseudopodial movement of EC toward TC. Our findings together suggest the crucial role of EC-TC physical interaction in EC chirality and that weakening the EC chirality could potentially compromise the overall endothelial integrity which increases the probability of metastatic cancer spread.

The essential oil from Drimys winteri possess activity: Antioxidant, theoretical chemistry reactivity, antimicrobial, antiproliferative and chemical composition

The Mapuche and their ancestors have used D. winteri in traditional medicine. In the present study, the essential oil extract of D. winteri leaves (DW_EO) were characterized chemically and biologically to evaluate its pharmacological activity. In vitro antioxidant activity was assayed, and antitumor activity was evaluated in non-tumor and tumor-cell culture lines. Caenorhabditis elegans was used as a model to evaluate toxicity, and the chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry. The chemical oil composition was characterized principally of five major terpenes: 4 sesquiterpenes γ-Eudesmol (39.7%), β-Caryophyllene (33.7%), Elemol (25.9%), α-Eudesmol (0.3%) and 1 diterpene Kaunene (0.4%). By quantum calculations, it was determined that all oils have the ability to capture and yield electrons, which is consistent with the moderate antioxidant activity of DW_EO detected in vitro. Furthermore, by molecular docking is estimated that these oils can bind to proteins involved in the production of oxygen radicals. Of these proteins, CYP2C9 could bind energetically, reaching binding energy between −6.8 and −9.2 kCal/mol for the 5 terpenes studied, highlighting among these β-Caryophyllen and γ-Eudesmol. DW_EO has effect against H. pylori (MIC 32 μg/ml), S. aureus (MIC 8 μg/ml), E. coli (MIC 32 μg/ml) and C. albicans (MIC 64 μg/ml), β-Caryophyllen and γ -Eudesmol (MIC 64 μg/ml) and could selectively inhibit the proliferation of epithelial tumor cell lines but showed low against C. elegans (0.39–1.56 μg mL−1). Therefore, DW_EO may be used as a source of bioactive compounds in novel pharmacological treatments for medical application, agronomics, sanitation, and food.

Chemical composition, antioxidant, antimicrobial and antiproliferative activity of Laureliopsis philippiana essential oil of Chile, study in vitro and in silico

Chilean Laureliopsis philippiana has been used in traditional medicine by the Mapuche and their ancestors. To evaluate its pharmacological activity, Laureliopsis philippiana leaf essential oil extract (LP_EO) was chemically and biologically characterized in the present study. In vitro antioxidant potential was analyzed, and antitumor activity was evaluated in non-tumor and tumor cell culture lines. Caenorhabditis elegans was used as a model for evaluating toxicity, and the chemical composition of the essential oil was analyzed using gas chromatography–mass spectrometry. The oil contains six major monoterpenes: eucalyptol (27.7 %), linalool (27.6 %), isozaphrol (19.5 %), isohomogenol (12.6 %), α-terpineol (7.7 %), and eudesmol (4.8 %). Based on quantum mechanical calculations, isosafrole and isohomogenol conferred in vitro antioxidant and antimicrobial activity to LP_EO. In addition, LP_EO showed antimicrobial activity against clinical Helicobacter pylori isolates (MIC 64 and MBC > 128 μg·mL−1), Staphylococcus aureus (MIC 32 and MBC > 64 μg·mL−1), Escherichia coli (MIC 8 and MBC 16 μg·mL−1) and Candida albicans (MIC 64 and > 128 μg·mL−1). LP_EO could selectively inhibit the proliferation of epithelial tumor cell lines but showed low toxicity against Caenorhabditis elegans (0.39 to 1.56 μg·mL−1). Therefore, LP_EO may be used as a source of bioactive compounds in novel pharmacological treatments for veterinary and human application, cosmetics, or sanitation.

Pepper Alkaloid Piperine Increases Radiation Sensitivity of Cancer Cells from Glioblastoma and Hypopharynx In Vitro.

In our study, our aim was to examine the cytotoxic and radio-sensitizing effect of the alkaloid piperine, a major pungent of black pepper, on two different human epithelial tumor cell lines in vitro. The growth of the human cell lines T98G (glioblastoma) and FaDu (hypopharyngeal carcinoma) was examined under the influence of piperine in different concentrations. In addition, after combined treatment with ionizing radiation, long-term survival was investigated with a colony formation assay. The proliferation was analyzed using the BrdU-assay, while the DNA repair capacity was examined via the γH2AX assay. Piperine reduced the growth of both cell lines in a concentration-dependent manner as well as a time-dependent one. After combined treatment with piperine and ionizing radiation, an inhibition of clonogenic survival could be proven. A reduced proliferation capacity and an additive effect on DNA damage 24 h after irradiation are possible causal mechanisms, which were also demonstrated for both cell lines. Based on the results presented in this study, piperine was shown to have cytotoxic antitumor activity and a radio-sensitizing effect in micromolar concentrations in the human tumor cells that were tested. Based on these results piperine represents a potential therapeutic option in radio-oncological treatment.

Effects of a phthalate metabolite mixture on both normal and tumoral human prostate cells.

Phthalates represent a group of substances used in industry that have antiandrogenic activity and are found in different concentrations in human urine and plasma. More than 8 million tons of phthalates are used each year, predominantly as plasticizers in polyvinyl chloride (PVC) products. Phthalates are widely used in everyday consumer products and improperly discarded into the environment. Furthermore, in vivo studies carried out in our laboratory showed that a mixture of phthalates, equivalent to the mixture used in this study, deregulated the expression of genes and miRNAs associated with prostatic carcinogenic pathways. Thus, this study was designed to establish an in vitro model to assess pathways related to cell survival, proliferation, apoptosis, and biosynthesis of miRNAs, using both normal and tumoral prostatic epithelial cells exposed to an environmentally relevant mixture of phthalate metabolites. Tumor (LNCaP) and normal (PNT-2) prostatic epithelial cell lines were exposed for 24 and 72 h to vehicle control or the phthalate mixture. The selected metabolite mixture (1000 μmol/L) consisted of 36.7% monoethyl phthalate (MEP), 19.4% mono(2-ethylhexyl) phthalate (MEHP), 15.3% monobutyl phthalate (MBP), 10.2% monoisobutyl phthalate (MiBP), 10.2% monoisononyl phthalate (MiNP), and 8.2% monobenzyl phthalate (MBzP). Gene expression was performed by qRT-PCR and cell migratory potential was measured using cell migration assays. Our results showed that the mixture of phthalates increased cell turnover, oxidative stress, biosynthesis, and expression of miRNAs in LNCaP cells; thus, increasing their cellular expansive and migratory potential and modulating tumor behavior, making them possibly more aggressive. However, these effects were less pronounced in benign cells, demonstrating that, in the short term, benign cells are able to develop effective mechanisms or more resistance against the insult.

Homosalate boosts the release of tumour-derived extracellular vesicles with protection against anchorage-loss property.

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63‐ or CD9‐positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set‐up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell‐based high‐content screening to identify clinically‐approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti‐inflammatory drug found in sunscreens which robustly increased EVs’ release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate‐, but not by Bafilomycin A1‐treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.

Overcoming barriers to solid tumor immunotherapy using natural killer cell therapies designed to mimic intraepithelial group 1 innate lymphoid cells

Abstract Our work aims to design cell-based immunotherapies for maximum solid tumor efficacy by leveraging insights from tissue-resident cells and single-cell RNA sequencing of innate immune cell subsets present within patient tumors. We discovered that by co-culturing peripheral blood natural killer cells (pbNKs) with irradiated epithelial tumor cells, we induced high expression of surface integrins, increased cytotoxicity and IFNg production, decreased sensitivity to TGFb, and significantly enhanced solid tumoroid infiltration. The cells were profiled using CyTOF and we determined that they closely resembled intraepithelial group 1 innate lymphoid cells, so we refer to them as ieILC1-like cells. We quantified the cytotoxicity of ieILC1-like cells against a variety of target cell lines and determined that they are broadly cytotoxic and capable of antibody-dependent cellular cytotoxicity, which was assessed using cetuximab. Tumor-infiltrating capacity was modeled using 3D tumoroids grown from single-cell suspensions of epithelial tumor cell lines in basement membrane extracts; ieILC1-like NK cells or pbNK cells were fluorescently labeled, added to tumoroids, and imaged using confocal microscopy. We stimulated the cells in the presence or absence of overnight TGFb exposure to determine the extent of TGFb-mediated immunosuppression. ieILC1-like cells were more resistant to TGFb and produced significantly more IFNg after stimulation than their pbNK counterparts. Preliminary in vivo work indicates that ieILC1-like cells perform comparably to K562-expanded NK cells. Thus, ieILC1-like NK cells represent a novel class of cell therapy that are more capable of infiltrating solid tumors and resisting their immunosuppressive cues. Supported by NIH R35DE030054.

Expression of immunoglobulins in human epithelial tumors and their potential role in carcinogenesis

The traditional view of immunoglobulin (Ig) production only by B-lymphocytes and plasma cells has been revisited. Non-lymphoid tumor cells can also synthesize and secrete Ig with unidentified specificity. Expression of Ig genes was detected in the cells of malignant neoplasms of epithelial origin, such as breast carcinoma, colorectal cancer, prostate cancer, as well as in epithelial tumor cell lines. mRNA of the IgG1 heavy (H) chain constant region, sterile Iɣ-Cɣ transcript, H and light (L) chains of IgG, V(D)J recombination of H and L chain gene segments, as well as RAG1 (recombination-activating gene 1) and RAG2 enzymes, which are required for V(D)J recombination, were found in cancer cell lines and resected carcinoma tissues. IgG produced by cancer cells can be involved in the invasion and metastasis of these cells through interaction with E-cadherin, as well as with the metastasis-associated protein MTA1. Tumor-derived IgG plays an important role in malignant progression via activation of platelets by interacting with their FcγRIIa receptors and inducing the production of low levels of reactive oxygen species. The level of IgG in malignant neoplasms is positively correlated with proliferation markers, stage of progression, growth and survival of the tumor. These data modernize the current views on the mechanisms of carcinogenesis and create the basis for the search for new diagnostic and prognostic markers in malignant neoplasms, as well as methods of their target therapy. Further in-depth studies of the phenomenon of Ig production by tumor cells will contribute to more effective practical application of the accumulated knowledge in this field.

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents.

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl2(DMSO)2], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and 1H, 13C, 15N and 195Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC50 = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC50 = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).

3D co-cultures of epithelial cells with immune cells as a model of HSV and oncolytic HSV infections

Virus infectivity is commonly investigated with in vitro monolayer cell cultures or in vivo animal models. Ease of growth and manipulation and low cost characterise standard cell culture. Animal models allow investigation of infectivity in the context of tissue structure and environment but are costly and can be limited by species variations. Neither approach can recapitulate the human context, the tissue microenvironment and human immune components. Three-dimensional organotypic raft tissue models can provide most of the advantages of in vitro and in vivo models. We successfully established HSV and oncolytic HSV (HSV1716) infection in 3D raft cultures of epithelial non-tumour (HaCaT) and tumour (SiHa, OVCAR3 and TOV21G) cell lines. Our 3D models allowed the evaluation and quantification of virus replication and the recovery of the virus both in culture media and tissues. We developed a complex 3D co-culture of epithelial cells with human immune cells in order to mimic the tissue microenvironment. This innovation allowed us to study the effect of immune cells in cell killing by HSV1716 in the in vitro tissues. In HSV1716-infected co-culture tissues, immune cells were identified throughout the tissue and some migrated to the areas of infection. The immune activity was identified through increased IL-8 release. Moreover, combining infection with immune cell infiltration increased tumour cell killing in the 3D co-culture model. This new co-culture model could be further developed to identify the role of immune cells in oncolytic viroimmunotherapy and to dissect the involvement of specific single immune cell subpopulations.

Abstract B27: Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA translation through inhibiting the translation of mRNAs into proteins or through degrading mRNAs. Each miRNA potentially targets hundreds of mRNAs, and thus an individual miRNA can regulate entire gene networks. The miR-200 family consists of 5 miRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429) organized into two clusters: the miR-200c/141 cluster and the miR-200b/200a/429 cluster. The miR-200s regulate a variety of cellular functions, with the best characterized being their ability to target mesenchymal transcription factors and thus maintain an epithelial phenotype. Given that miR-200s maintain an epithelial phenotype, we evaluated their role in mesenchymal mammary tumors. All five miR-200s were expressed at significantly lower levels in the murine mesenchymal mammary tumor cell line RJ423 and the human mesenchymal breast cancer cell line MDA-MB-231 compared to epithelial tumor cell lines. To evaluate the function of miR-200s, the miR-200c/141 and miR-200b/200a/429 clusters were re-expressed in the mesenchymal mammary tumors. Re-expression of the miR-200c/141 cluster partially restored an epithelial phenotype in MDA-MB-231 cells while the miR-200b/200a/429 cluster partially restored an epithelial phenotype in RJ423 cells. Re-expression of the miR-200c/141 cluster in MDA-MB-231 cells and re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed tumor growth following intramammary injection and altered the tumor microenvironment. To identify genes regulated by miR-200 expression, RNA sequencing was performed on control and miR-200 re-expressing cells grown in 2D culture and following intramammary injection. Interestingly, the in vivo environment dramatically altered the gene repertoire regulated by the miR-200s. Hierarchical clustering revealed that alterations in gene expression induced by miR-200 re-expression in vitro poorly correlated with the alterations in gene expression induced by miR-200 in vivo. Since miR-200 re-expression significantly decreased tumor growth in vivo, a transgenic model was used to determine whether miR-200s could also impact tumor initiation. Using doxycycline-inducible miR-200 expression in combination with the mammary tumor oncogene, IGF-IR, we found that overexpression of the miR-200b/200a/429 cluster dramatically reduced mammary tumor incidence (100% vs. 19%). These data show the potent impact miR-200s have on mammary tumorigenesis and that gene expression profiling of 2D cultures is a poor predictor of genes regulated by miR-200s in vivo. Citation Format: Katrina L. Watson, Kaitlyn Simpson, Majesta Roth, Courtney Martin, Garret Conquer-Van Heumen, Roger A. Moorehead. Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B27.

Fermented curly kale as a new source of gentisic and salicylic acids with antitumor potential

Abstract This paper demonstrates the role of curly kale fermentation by autochthonous lactic acid bacteria in the formation of bioactive derivatives of phenolic compounds which may have anticancer properties. LC-MS and HPLC analyses were performed and the content of polyphenols in curly kale before and during fermentation were determined. Bioactive properties of salicylic and gentisic acids were tested in experiments on tumor (HT29, SW620) and normal (CCD841CoTr) epithelial cell lines. In this study, gentisic and salicylic acids were thoroughly examined. Gentisic acid has not been detected in Brasssicaceae vegetables so far, and the presence of both these acids was noted in fermented curly kale for the first time. Our results indicated that both salicylic and gentisic acids decreased the number of HT29 cells simultaneously with reduction of the intercellular interactions between these cells. Moreover, immunomodulatory and anti-inflammatory activity of gentisic acid was observed.

Human renal adipose tissue from normal and tumor kidney: its influence on renal cell carcinoma.

Tumor cells can interact with neighboring adipose tissue. We evaluated components present in human adipose explants from normal (hRAN) and kidney cancer (hRAT) tissue, and we evaluated the effects of conditioned media (CMs) from hRAN and hRAT on proliferation, adhesion and migration of tumor and non-tumor human renal epithelial cell lines. In addition, we evaluated the expression of AdipoR1, ObR, CD44, vimentin, pERK and pPI3K on cell lines incubated with CMs. hRAN were obtained from healthy operated donors, and hRAT from patients who underwent a nephrectomy. hRAT showed increased levels of versican, leptin and ObR; and decreased levels of perilipin, adiponectin and AdipoR1, compared to hRAN. Cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRAT-CMs vs. hRAN- or control-CMs. Surprisingly, HK-2, 786-O and ACHN cells showed a significant decrease in cell migration after incubation with hRAN-CMs vs. control-CMs. No difference in proliferation of cell lines was found after 24 or 48 h of treatment with CMs. AdipoR1 in ACHN and Caki-1 cells decreased significantly after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. ObR and CD44 increased in tumor line cells, and vimentin increased in non-tumor cells, after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. We observed an increase in the expression of pERK and pPI3K in HK-2, 786-O and ACHN, incubated with hRAT-CMs. In conclusion, results showed that adipose microenvironment can regulate the behavior of tumor and non tumor human renal epithelial cells.

Radiosensitizing effects of trabectedin on human A549 lung cancer cells and HT-29 colon cancer cells

Background and Purpose Trabectedin is a unique alkylating agent with promising effects against a range of solid tumors. In this study, we aimed to examine the cytotoxic and radiosensitizing effects of trabectedin on two human epithelial tumor cell lines in vitro, and its effects on DNA repair capacity. Methods Cancer cells (A549: human lung cancer cells, HT-29: colon cancer cells) were treated with either trabectedin alone for the determination of their growth, or in combination with radiation for the determination of their metabolic activity, proliferation, and clonogenic survival. Besides, the γH2AX foci assay was performed for the assessment of ionizing radiation-induced DNA damage and to evaluate the influence of trabectedin on DNA damage repair. Results Treatment with trabectedin resulted in a growth-inhibiting effect on both cell lines, with the IC50 values remaining within a low nanomolar range. Analyses of metabolic activity confirmed a cytotoxic influence of trabectedin and a BrdU assay demonstrated an antiproliferative effect. When combined with radiation, incubation with trabectedin was found to enhance the radiosensitivity of the tumor cells. The γH2AX foci assay resulted in an increased number of DNA double-strand breaks (DSBs) in cells treated with trabectedin. Conclusion The results of this study underline the antitumor activity of trabectedin at low nanomolar concentrations. We demonstrated that trabectedin enhanced radiation response in human lung (A549) cancer cells and colon (HT-29) cancer cells. Further studies are necessary to examine trabectedin as a potential candidate for future applications in radiotherapy.