Human Breast Ductal Carcinoma Research Articles

Low progesterone receptor levels in high-grade DCIS correlate with HER2 upregulation and the presence of invasive components.

In this study, we investigated pivotal molecular markers in human high-grade breast ductal carcinoma in situ (DCIS). Expression status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2) was measured among various subtypes (Luminal (Lum) A, LumB HER2-, LumB HER2+, HER2-enriched and triple-negative). In total, 357 DCIS cases were classified into respective subtypes, according to the 2013 St. Gallen guidelines. Each subtype was categorized into three subcategories: "Pure" (those without an invasive component), "W/invasive" (those with an invasive component), and "All" (the entire group of the given subtype). ER and PR expression were registered as intervals. Equivocal HER2 immunohistochemistry (IHC) cases (2+) were further investigated using dual-color in situ hybridization. The majority of patients (71%) were over the age of 50. We discovered no significant differences in the proportion of age between the "Pure" and "W/invasive" groups. There was no significant difference in ER/PR expression between "Pure" luminal subtypes of DCIS and "W/invasive" cases. We compared the HER2 IHC scores of "0", "1+", and "2+" among LumA and LumB HER2 subtypes and identified no statistically significant differences between "Pure" and "W/invasive" (p = 0.603). ER and PR expression ≥ 50% cutoff value was present in > 90% of all LumA cases. The incidences of cases with ER expression at cutoff values of < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). The proportion of cases with PR expression < 20% showed significant differences in the various luminal subtypes. In luminal B subtypes, low PR expression (< 20%) was significantly associated with both strong HER2 expression (3+) and the presence of an invasive component (p = 0.0001 and p = 0.0365, respectively). ER and PR expression at ≥ 50% cutoff values were found in more than 90% of LumA cases. Samples with ER < 10% and ≥ 50% in LumA were significantly different compared to other luminal subtypes (p < 0.0001). Low PR expression in high-grade DCIS was strongly associated with HER2 overexpression (3+) and an invasive component (p = 0.0001 and p = 0.0365, respectively).

MTORC1 Mediates Biphasic Mechano-Response to Orchestrate Adhesion-Dependent Cell Growth and Anoikis Resistance.

Cells constantly sense and respond to not only biochemical but also biomechanical changes in their microenvironment, demanding for dynamic metabolic adaptation. ECM stiffening is a hallmark of cancer aggressiveness, while survival under substrate detachment also associates with poor prognosis. Mechanisms underlying this, non-linear mechano-response of tumor cells may reveal potential double-hit targets for cancers. Here, an integrin-GSK3β-FTO-mTOR axis is reported, that can integrate stiffness sensing to ensure both the growth advantage endowed by rigid substrate and cell death resistance under matrix detachment. It is demonstrated that substrate stiffening can activate mTORC1 and elevate mTOR level through integrins and GSK3β-FTO mediated mRNA m6 A modification, promoting anabolic metabolism. Inhibition of this axis upon ECM detachment enhances autophagy, which in turn conveys resilience of tumor cells to anoikis, as it is demonstrated in human breast ductal carcinoma in situ (DCIS) and mice malignant ascites. Collectively, these results highlight the biphasic mechano-regulation of cellular metabolism, with implications in tumor growth under stiffened conditions such as fibrosis, as well as in anoikis-resistance during cancer metastasis.

Oncolytic Activity of Canine Distemper Virus in Human Ductal Breast Carcinoma Cells

Introduction: Oncolytic virotherapy is a novel strategy for cancer treatment in humans and companion animals. Canine distemper virus (CDV) is known to induce apoptosis in tumor cells, thus serving as a potential candidate for oncolytic therapy. However, the mechanism of viral oncolytic activity is less studied and varies depending on the type of cancer and cell lines. Methods: In the present study, the susceptibility of the MCF-7 cell line to CDV infection was assessed using the CDV strain, which was confirmed previously through sequence analysis in the Vero cell line. The impact of CDV infection on cell proliferation and apoptosis was studied by evaluating the expression of four target genes including the myeloid cell leukemia 1 (MCL-1), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), transcription factor (SP1), and DNA (cytosine-5)-methyltransferase 3A (DNMT3A). Results: CDV replication in the cells induced cytopathic effect and decreased in the cell proliferation rates compared to the uninfected control. MCL-1, SP1, and PIK3R1 gene expression was down-regulated, while the expression of DNMT3A was up-regulated 3 days post-infection. The expression levels of the target genes suggest that CDV may be inducing the intrinsic apoptotic pathway in the cancer cell line. Conclusion: Overall, the results strongly propose CDV strain as a potential candidate for cancer therapy after detailed studies.

Abstract 6350: Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy

Abstract BST2, a raft-associated type II transmembrane protein, functions as a restriction factor and inhibits HIV release by cross-linking virions onto infected cell surfaces. It also engages ILT7, a specific inhibitory receptor on plasmacytoid dendritic cells, which are associated with poor prognosis when infiltrate patients’ breast tumors. Two groups have identified BST2 isoforms of different lengths, generated by post-transcriptional modification. Based on these findings and our work on protein interaction of ILT7-BST2, we hypothesized that the expression of BST2 isoforms differed in normal and cancer tissues. To demonstrate that, we generated and characterized anti-long BST2 isoform monoclonal antibodies (mAbs). Results were compared with our previously developed mAb anti-BST2 full length molecule, 26F8, that recognizes a common epitope to all BST2 isoforms. By comparing the amino acid sequences of different isoforms of BST2, we identified a region of 24 residues on the extracellular domain of BST2, restricted to its long isoform. We then generated mAbs by hybridoma technology, immunizing BALB/c mice with the 24 mers peptide-KLH conjugated. Screening and positive selection of several clones were performed following our lab SOP. One of the mAbs, LA5, was selected based on positive ELISA against the immunogen and FACS staining of recombinant HEK293 cells expressing full-length BST2 protein. Pharmacokinetic determined using OCTET platform, revealed a KD of 4.61+/- 0.074 × 10−9 M for LA5. Epitope mapping of LA5 on BST2 overlapping peptides, confirmed specific recognition of full-length BST2 protein and the peptide used as immunogen. LA5 heavy and light chains were sequenced and analysis was performed by IgBlast and IMGTV. Binding of LA5 and 26F8 to the myeloma cell lines MM1 (B-lymphoblast) and U266 (B-lymphocyte) by flow cytometry, indicated 97-100% positive cells with both mAbs, with higher MFI values observed for clone 26F8: 5.8 and 1.8-fold-increase in MM1 and U266 cells respectively. IHC on formalin-fixed paraffin embedded tissues of human ductal breast carcinoma, revealed that LA5 staining was able to identify infiltrating carcinoma cells but not the normal surrounding acinar cells. In contrast, 26F8 staining did not discriminate between BST2+ malignant and surrounding normal acinar cells. Currently, we are analyzing a panel of &amp;gt;105 breast cancer tissue samples of different molecular classifications. Initial data revealed a high-level of expression of BST2 (by 26F8) on the epithelial area of the tumors, lower in the TME (tumor microenvironment-infiltrating immune cells) and almost negative expression in the surrounding fibroblasts rich stroma. Evaluation of IHC staining by LA5 is being processed and yet to be established. These IHC findings indicate a potential diagnostic value of LA5 to distinct between tumor and normal cells in patients’ specimens. Treatment prospects may arise during the process. Citation Format: Laura C. Bover, Ahmed Muhsin, Long T. Vien, Felipe Amaya Manzanares, Zhuang Wu, Janis D. Johnson, Diego Castro Reyes, Myrthala Moreno-Smith, Estefania Labanca, Irene Sorin, Silvia Saucedo, Silvina Gazzaniga, Alicia I. Bravo. Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy [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 6350.

Toxicity of new monophenolic synthetic activator of Keap1/Nrf2/ARE redox-sensitive signaling system &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;

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&lt;sub&gt;50&lt;/sub&gt; 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&lt;sub&gt;50&lt;/sub&gt; 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&lt;sub&gt;50&lt;/sub&gt; = 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&lt;sub&gt;50&lt;/sub&gt; 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.

Comparison of metabolite profiles and cytotoxicity of the black sea cucumber (Holothuria atra) dried with different drying techniques

Holothuria atra is a species of sea cucumber that could be a source of cytotoxic compounds. The present work aimed to determine the effect of different drying techniques on the profile of metabolites and cytotoxicity of sea cucumber extracts. Sea cucumbers were dried in the sun for 3 d, and then oven-dried at a temperature of 40, 50, and 60°C for 24 h. Fresh sea cucumbers without drying served as a control. The dried and fresh sea cucumbers were extracted by maceration for 12 h using ethanol. The metabolite profiling was performed using Fourier-transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC) analyses, whereas the cytotoxicity was assayed by MTT method using the human breast ductal carcinoma cells (T47D). Analyses of FTIR and HPLC showed that the bioactive compounds of the ethanol extract were relatively stable during drying. This was in line with its cytotoxicity against T47D cells, which did not show significant differences between treatments. Based on these results, it was concluded that drying by sun or oven did not damage the bioactive compounds in the ethanolic extract of sea cucumber.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Human Breast Extracellular Matrix Microstructures and Protein Hydrogel 3D Cultures of Mammary Epithelial Cells.

Simple SummaryHuman breast tissue extracellular matrix (ECM) is a microenvironment essential for the survival and biological activities of mammary epithelial cells. The ECM structural features of human breast tissues remain poorly defined. In this study, we identified the structural and mechanical properties of human normal breast and invasive ductal carcinoma tissue ECM using histological methods and atomic force microscopy. Additionally, a protein hydrogel was generated using human breast tissue ECM and defined for its microstructural features using immunofluorescence imaging and machine learning. Furthermore, we examined the three-dimensional growth of normal mammary epithelial cells or breast cancer cells cultured on the ECM protein hydrogel, where the cells exhibited biological phenotypes like those seen in native breast tissues. Our data provide novel insights into cancer cell biology, tissue microenvironment mimicry and engineering, and native tissue ECM-based biomedical and pharmaceutical applications.Tissue extracellular matrix (ECM) is a structurally and compositionally unique microenvironment within which native cells can perform their natural biological activities. Cells grown on artificial substrata differ biologically and phenotypically from those grown within their native tissue microenvironment. Studies examining human tissue ECM structures and the biology of human tissue cells in their corresponding tissue ECM are lacking. Such investigations will improve our understanding about human pathophysiological conditions for better clinical care. We report here human normal breast tissue and invasive ductal carcinoma tissue ECM structural features. For the first time, a hydrogel was successfully fabricated using whole protein extracts of human normal breast ECM. Using immunofluorescence staining of type I collagen (Col I) and machine learning of its fibrous patterns in the polymerized human breast ECM hydrogel, we have defined the microstructural characteristics of the hydrogel and compared the microstructures with those of other native ECM hydrogels. Importantly, the ECM hydrogel supported 3D growth and cell-ECM interaction of both normal and cancerous mammary epithelial cells. This work represents further advancement toward full reconstitution of the human breast tissue microenvironment, an accomplishment that will accelerate the use of human pathophysiological tissue-derived matrices for individualized biomedical research and therapeutic development.

The Cytotoxic Effect of the Extract of Anchusa strigosa (Him Him) Grown in Jordan Against Different Cancer Cell Lines

Anchusa strigosa - prickly alkanet from Boraginaceae grows in roadsides, and fields of a broad range of habitats from mediterranean woodlands, to steppe vegetation, to true desert. It is commonly known as" him him" or "lisan al thawr". Anchusa can withstand hard weather conditions and hence is widely cultivated. The color of its flowers can range from pure white to deep cobalt blue. Various parts of A. strigosa are used in traditional medicine for treating several diseases or symptoms, such as abdominal pain, bronchitis, cough, and diarrhea. The goal of this study was to examine the cytotoxic effect of the crude extract of A. strigosa roots and leaves and their fractions against various tumor cell lines: adenocarcinoma MCF-7, human breast ductal carcinoma T-47D; human breast carcinoma MDA-MB-231; and colorectal carcinomas Caco-2. In conclusion the antiproliferative effect was assessed by SRB assay where it showed that the phytochemical constituents in the leaf part of the plant (A. strigosa) do have more potential in depressing the proliferation rate of the cell lines than the root part.

Comparative study of the influence of imaging resolution on linear retardance parameters derived from the Mueller matrix

Polarization imaging techniques are emerging tools to provide quantitative information of anisotropic structures, such as the density and orientation distribution of fibers in tissue samples. Recently, it is found that when using Mueller matrix polarimetry to obtain the structural features of tissue samples, some information can be revealed by relatively low-resolution polarization parameter images. Thus, to analyze what kinds of anisotropic optical and structural information contained in high-resolution polarization images are preserved in low-resolution ones, here we carry out a comparative study of the influence of imaging resolution on the Mueller matrix derived linear retardance parameters. We measure the microscopic Mueller matrix of human healthy breast duct tissues and ductal carcinoma in situ (DCIS) tissues, which have distinct typical fibrous structures, using objectives with different numerical aperture. Then we quantitatively compare a group of image texture feature parameters of the linear retardance parameters images under high and low imaging resolutions. The results demonstrate that the fibers density information contained in the texture features of linear retardance δ parameter image are preserved well with the decline of imaging resolution. While for the azimuthal orientation parameter θ which closely related to the spatial location, we still need high imaging resolution to obtain quantitative structural information. The study provides an important criterion to decide which information of fibrous structures can be extracted accurately using transmission Mueller matrix microscope with low numerical aperture objectives.

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.

Probing variations of fibrous structures during the development of breast ductal carcinoma tissues via Mueller matrix imaging.

Recently, we developed a label-free method to probe the microstructural information and optical properties of unstained thin tissue slices based on microscopic Mueller matrix imaging technique. In this paper, we take the microscopic Mueller matrix images of human breast ductal carcinoma tissue samples at different pathological stages, and then calculate and analyze their retardance-related Mueller matrix-derived parameters. To reveal the microstructural features more quantitatively and precisely, we propose a new method based on first-order statistical properties of image to transform the 2D images of Mueller matrix parameters into several statistical feature vectors. We evaluate each statistical feature vector by corresponding classification characteristic value extracted from the statistical features of Mueller matrix parameters images of healthy breast duct tissue samples. The experimental results indicate that these statistical feature vectors of Mueller matrix derived parameters may become powerful tools to quantitatively characterize breast ductal carcinoma tissue samples at different pathological stages. It has the potential to facilitate automating the staging process of breast ductal carcinoma tissue, resulting in the improvement of diagnostic efficiency.

Investigation of the cytotoxicity of bioinspired coumarin analogues towards human breast cancer cells

Coumarins possess a wide array of therapeutic capabilities, but often with unclear mechanism of action. We tested a small library of 18 coumarin derivatives against human invasive breast ductal carcinoma cells with the capacity of each compound to inhibit cell proliferation scored, and the most potent coumarin analogues selected for further studies. Interestingly, the presence of two prenyloxy groups (5,7-diprenyloxy-4-methyl-coumarin, 4g) or the presence of octyloxy substituent (coumarin 4d) was found to increase the potency of compounds in breast cancer cells, but not against healthy human fibroblasts. The activity of potent compounds on breast cancer cells cultured more similarly to the conditions of the tumour microenvironment was also investigated, and increased toxicity was observed. Results suggest that tested coumarin derivatives could potentially reduce the growth of tumour mass. Moreover, their use as (combination) therapy in cancer treatment might have the potential of causing limited side effects.Graphic abstract

Parietin as an efficient and promising anti-angiogenic and apoptotic small-molecule from Xanthoria parietina

Lichens that are exclusive symbiotic organisms composed of fungus and alga, are considered as a wealthy source of biologically and pharmacologically active small-molecules thanks to the tight metabolic relationship between symbiotic partners. We herein report cytotoxic, anti-angiogenic and apoptotic profile of a lichen derived small-molecule named parietin. Parietin was isolated from the acetone extract of Xanthoria parietina (L.) Th.Fr (1860), Teloschistaceae, which was gathered from Afyon, Turkey. AlamarBlue™ cell viability, lactate dehydrogenase membrane leakage and PicoGreen™ dsDNA quantitation assays were used to determine the cytotoxic concentrations of parietin on cisplatin-resistant BRCA2-mutated human breast TNM stage IV adenocarcinoma (HCC1428), human breast ductal carcinoma (T-47D), and human umbilical vein endothelial (HUVEC) cells. Additionally, cell adhesion, endothelial tube formation, reactive oxygen species accumulation and active caspase 3 determination assays were employed to identify the anti-angiogenic and apoptotic efficiency of parietin. Low concentrations of parietin such as 50 and 100 µM showed a significant anti-angiogenic and apoptotic activity though the half-maximal inhibitory concentration (IC50) values were higher than 600 µM on the cells. On the other hand, it was observed that parietin shows less cytotoxic and membrane degradative activities on healthy HUVEC cells than the HCC1428 and T-47D breast cancer cells. Parietin seems to be a promising anti-angiogenic and apoptotic lichen metabolite for the further investigations.

Design, synthesis, and biological evaluation of a new class of histone acetyltransferase p300 inhibitors

p300 is an important histone acetyltransferase in epigenetics, and its overexpression is closely related to various diseases such as cancers. C646 is one of the most representative p300 inhibitors and used in various studies of p300. However, its intrinsic drawbacks such as containing potentially toxic groups prevent it from further development. In order to find potent p300 inhibitors with good drug-like properties, C646 was chosen as the lead compound and a series of new p300 inhibitors were designed based on the principle of bioisosterism and reasonable scaffold hopping, and the structure-activity relationship was systematically explored. Ten of them were found to show comparable inhibitory activity as C646. The most potent compound, 1r (IC50 = 0.16 μM), showed better p300 inhibitory activity than C646 with improved drug-like properties. Western blotting experiment confirmed that 1r could reduce the level of H3K27 acetylation more significantly than C646. Further cellular assay indicated that it could inhibit the proliferation of human breast ductal carcinoma cell T47D and human breast cancer cell MCF7 with the IC50 values of 5.08 μM and 22.54 μM, respectively. Docking study of 1r with p300 protein showed the possible reasons for its higher inhibition activity. Thus, compound 1r might be with potential for development as a novel epigenetic agent targeting p300.

Abstract 1616: Adjuvant supplementation of docosahexaenoic acid with 2-deoxyglucose, metformin, and aspirin on cellular metabolism in breast, lung, and prostate tumor cells

Abstract A standard treatment for cancer often involves the use of adjuvant supplementation with targeted therapy to maximize tumor suppression. Recent studies have shown that docosahexaenoic acid (DHA; C22:6 n-3), a long chain omega-3 polyunsaturated fatty acid, acts on an array of molecular oncogenes that are often targeted by anti-tumorigenic drugs. After a careful review of DHA’s mechanistic properties, 2-deoxyglucose (2DG), metformin, and aspirin were identified as drugs that share similar molecular targets as DHA. 2DG and metformin both target cellular metabolism by inhibiting intracellular ATP, albeit different mechanisms. 2DG inhibits glycolysis, while metformin targets the mitochondria. Aspirin inhibits inflammation via irreversible acetylation of platelet COX. The current study investigates the effects of DHA in combination with each drug on numerous cancer subtypes to determine if concurrent treatments can synergistically inhibit tumor proliferation. In BT-474 human breast ductal carcinoma, MDA-MB-231 human breast adenocarcinoma, and A549 human lung adenocarcinoma, DHA was shown to enhance the tumor-inhibiting effects of 2DG via intracellular ATP reduction. In contrast, DHA showed no effect with metformin on the same tumor cell lines. With limited studies localizing DHA’s metabolic effects, these results suggest that DHA may have potential selectivity for ATP inhibition via glycolysis. In response to DHA + aspirin, the PC-3 prostate adenocarcinoma and MDA-MB-231 displayed synergistic inhibition of cell viability, while LNCaP prostate carcinoma showed an additive effect. With studies showing DHA’s ability to inhibit endothelial COX, further studies will be done to determine whether the synergistic effects of DHA + aspirin expand to COX expression as well. Moreover, downstream targets of 2DG and metformin will be studied to determine the extent of DHA enhancement on oncogenic targets, such as mTOR, 4EB-P1, and GRB10. The results of this study provide preliminary evidence for the efficacy of adjuvant supplementation with DHA in combination with anti-tumorigenic drugs. With ongoing analysis, the potential for adjuvant nutritional supplementation will allow for additional treatment strategies for future patients. Citation Format: Irvin V. Ma, Michael Mouradian, Andrew R. Cooper, Erika D. Vicente, Ronald S. Pardini. Adjuvant supplementation of docosahexaenoic acid with 2-deoxyglucose, metformin, and aspirin on cellular metabolism in breast, lung, and prostate tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1616.

Cytotoxicity and apoptosis induction of sea cucumber Holothuria atra extracts

Context: Cancer is one of the major causes of death. Sea cucumbers are marine invertebrate that is widely used and utilized in traditional medicine. One of the most studied of sea cucumbers efficacy is its anticancer property. The anticancer property of sea cucumbers is much related to the content of an active compound called saponin. Objective: The objective of this research is to determine the cytotoxicity and apoptosis induction of sea cucumber Holothuria atra ethanol extract and to study its anticancer active compound. Materials and Methods: Sea cucumber of H. atra was taken from Halmahera waters, North Maluku Indonesia. The extraction was conducted by maceration method using ethanol 96% then continued with liquid-liquid partition and separation using C18 column. The investigation of active compound content was conducted by phytochemical and spectroscopic analysis using liquid chromatography-ion trap-time of a flight-mass spectrophotometer (LC-IT-ToF-MS). The cytotoxicity test was performed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method against human breast ductal carcinoma cell (T47D), human breast adenocarcinoma (MCF7), human colorectal adenocarcinoma cell (WiDr), and human cervix adenocarcinoma cell lines (HeLa). The apoptotic induction test was performed using flow cytometry using Fluorescein isothiocyanate (FITC) Annexin-V-Apoptosis and caspase-3 detection using FITC Active Caspase-3 kit. Results: Phytochemical test showed that ethanol extract of H. atra contained alkaloids, flavonoids, steroids-triterpenoids, phenols, and saponins. The extract showed cytotoxic activity against the 4 cell lines with Inhibition concentration 50 values ranging from 9.6 to 14.3 μg/ml. Flow cytometry analysis showed that the T47D cell population underwent apoptosis after treated with ethanol extract. The extract also activated caspase-3 on the T47D cells. The results of LC-IT-ToF-MS analysis showed that the active fraction from C18 column separation contained saponin and identified as Cucumechinol and Philinopgenin B. Conclusions: The results of this study indicated that H. atra active extract has good cytotoxicity and has potential to be developed as an anticancer agent. Abbreviations used: MNPs: Marine natural products; LC-IT-ToF-MS: Liquid chromatography-ion trap-time of flight-mass spectrophotometer; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; T47D: Human breast ductal carcinoma cell; MCF7: Human breast adenocarcinoma cell; WiDr: Human colorectal adenocarcinoma cell; HeLa: Human cervix adenocarcinoma cell; RPMI: Roswell park memorial institute medium; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate; PBS: Phosphate buffer saline; BD: Becton Dickinson; IC50: Inhibition concentration 50; PS: Phosphatidylserine; PI: Propidium iodide.

Anti-breast cancer and anti-angiogenic potential of a lichen-derived small-molecule: barbatolic acid.

Natural products have been used for centuries as the most potent remedies to cure many diseases including cancer diseases. Angiogenesis is defined as the formation of new capillaries from existing vessels and plays a key role in the tumorigenesis process. Barbatolic acid is a little known lichen-derived small-molecule. In the present study, barbatolic acid was isolated from the acetone extract of Bryoria capillaris, and its anti-breast cancer and anti-angiogenic potential was investigated using human umbilical vein endothelial cells (HUVECs), human breast ductal carcinoma (T-47D) and cisplatin-resistant BRCA2-mutated human breast TNM stage IV adenocarcinoma (HCC1428) cells. AlamarBlue™ cell viability, lactate dehydrogenase cellular membrane degradation and PicoGreen™ dsDNA quantitation assays were performed to determine the cytotoxic potential of barbatolic acid. Anti-angiogenic and anti-migratory activities were investigated using endothelial tube formation assay and scratch wound healing assay, respectively. Half maximal inhibitory concentration of barbatolic acid was found to be higher than 100µM for HUVEC, HCC1428 and T-47D cells. The sub-cytotoxic concentrations such as 25µM, 50µM and 100µM were applied to determine anti-angiogenic and anti-migratory activities. Although the sub-cytotoxic concentrations inhibited endothelial tube formation and cellular migration in a concentration depended manner, barbatolic acid was more effective on the migration of HCC1428 and T-47D breast cancer cells than the migration of HUVECs. Consequently, the findings suggest that barbatolic acid is a promising anti-angiogenic and anti-migratory agent and the underlying activity mechanisms should be investigated by further in vitro and in vivo experiments.

IL-6-mediated cross-talk between human preadipocytes and ductal carcinoma in situ in breast cancer progression

BackgroundThe function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model.MethodsGFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody.ResultsIn the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition.ConclusionsOur findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Epidermal growth factor receptor-mediated regulation of matrix metalloproteinase-2 and matrix metalloproteinase-9 in MCF-7 breast cancer cells

In breast cancer, increased epidermal growth factor receptor (EGFR) expression and phosphorylation have been correlated with increased invasive potential and poor prognosis. Interaction of EGFR with its ligand epidermal growth factor (EGF) activates cellular signalling cascades promoting tumour invasion. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR-mediated regulation of MMP-2 and MMP-9 in breast cancer was investigated using metastatic human breast ductal carcinoma cell line MCF-7. Culture of MCF-7 cells on 1µg/ml EGF-coated culture dishes for 24h led to appreciable increase in MMP-2 and MMP-9 expression and activity. Expression of membrane type-1 matrix metalloproteinase (MT1-MMP) and focal adhesion kinase (FAK), phosphorylation of EGFR and phosphatidylinositol 3' kinase (PI3K), and nuclear translocation of EGFR and cellular migration were also appreciably increased. Targeting EGFR-EGF interactions by treatment of MCF-7 cells with anti-EGFR monoclonal antibodies prior to culture on EGF prevented appreciable upregulation of MMP-2 and MMP-9 expression and activity. Increased expression of MT1-MMP and FAK, phosphorylation of EGFR and PI3K and enhanced cell migration were also inhibited. Treatment of cells with PI3K inhibitor LY294002 prevented upregulation of MMP-2 and MMP-9 indicating that EGFR-mediated signalling for MMP regulation occurs through PI3K. As increased EGFR activity has been observed in highly invasive breast cancers, targeting EGFR-EGF interactions might render such cancers less invasive by inhibiting EGFR-mediated upregulation of MMP-2 and MMP-9 and therefore could be of importance in their clinical management.