Human Mammary Carcinoma Cell Line Research Articles

Glycosylated Porphyrin Derivatives for Sonodynamic Therapy: ROS Generation and Cytotoxicity Studies in Breast Cancer Cells.

Sonodynamic therapy (SDT) is a promising alternative to photodynamic therapy for achieving site-specific cytotoxic therapy. Porphyrin derivative molecules have been reported extensively in photodynamic therapy. We have previously shown that the glycosylation of porphyrin-based sonosensitizers can enhance their cellular uptake. However, the sonodynamic potential of these water-soluble glycosylated porphyrins has not been investigated. In this study, we characterized the sonodynamic response of two water-soluble glycosylated porphyrin derivatives. Ultrasound (US) exposure was performed (1 MHz frequency, intensities of 0.05-1.1 W/cm2) for 0-3 min in continuous mode. Reactive oxygen species (ROS) generation was quantified via ultraviolet-visible (UV-vis) spectrophotometry. MTT assay was used to quantify cytotoxicity caused by sonodynamic effects from these derivatives in the human mammary carcinoma (SUM-159) cell line in vitro. ROS generation from the porphyrin derivatives was demonstrated at a concentration of 15 μM. No significant cytotoxic effects were observed with the sonosensitizer alone or US exposure alone over the tested range of intensities and duration. The free base porphyrin derivative caused 60-70% cell death, whereas the zinc-porphyrin derivative with Zn metal conjugation caused nearly 50% cytotoxicity when exposed at 0.6 W/cm2 intensity for 3 min. These studies demonstrate the potential of anticancer SDT with soluble glycosylated porphyrins.

Abstract A046: Heterogenous nuclear ribonucleoprotein E1 regulates leukemia inhibitory factor receptor (LIFR) expression through FAM3C in mammary epithelial cells

Abstract Leukemia inhibitory factor receptor (LIFR) signals through signal transducer and activator of transcription 3 (STAT3), a transcription factor that is associated with poor prognosis due to its overexpression and/or hyperactivity in 40% of breast cancer cases. LIFR has been defined as either a promoter or suppressor of metastasis in mammary carcinoma, and recently we have shown that its expression is significantly upregulated following loss of the RNA-binding protein heterogenous nuclear ribonucleoprotein E1 (hnRNP E1, “E1”). We have further shown that E1 is a regulator of the epithelial-to-mesenchymal transition (EMT) and of differentiation in mammary epithelium. Mechanistically, E1 acts as a tumor suppressor by binding to and inhibiting translation of mRNAs involved in EMT and cellular self-renewal, including the cytokine FAM3C. We have demonstrated that FAM3C binds to LIFR in the extracellular space and activates STAT3. Herein, we characterize induction of LIFR expression in response to FAM3C/LIFR signaling, and show that E1 upregulates LIFR transcription through FAM3C, involving initiation at the LIFR promoter locus. Induction of LIFR expression in mammary epithelial cells has been shown to be associated with tumor initiation, breast cancer stem cells, and metastasis. Additionally, our bioinformatic analysis reveals a signature of transcriptional regulation associated with FAM3C/LIFR interaction. Comparison between RNA-sequencing data from our mouse model of mammary carcinoma and human mammary carcinoma cell lines reveals common biological pathways and potential therapeutic targets. Future directions will be aimed at investigating the effect of modulation of LIFR expression during tumor dissemination in vivo. Citation Format: William S. Streitfeld, Annamarie C. Dalton, Philip H. Howe. Heterogenous nuclear ribonucleoprotein E1 regulates leukemia inhibitory factor receptor (LIFR) expression through FAM3C in mammary epithelial cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A046.

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

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

Effect of cucurbitacin on malignant biological behavior of breast cancer cells, and its possible underlying mechanism

Purpose: To study the influence of cucurbitacin on malignant biological behavior of mammary carcinoma cells, and the likely mechanism involved.Methods: Human mammary carcinoma cell line MDA-MB-436 was selected for cell culture and treated with different concentrations of cucurbitacin. The effect of cucurbitacin on cell activity, cell colonyformation capacity, cell invasion, migration potential, matrix metalloproteinase-9 (MMP-9) activity, and levels of vascular endothelial growth factor A (VEGFA), epithelial calcium adhesion (E-cadherin), and neurogenic calcium adhesion (N-cadherin) were measured. Moreover, levels of wave protein (vimentin), phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated signaling transduction, and transcription activation factor 3 (p-STAT3) and phosphorylated protein kinase B (p-Akt) were determined.Results: With increase in cucurbitacin dose, there was significant decrease in cell viability, cell colony ratio, cell invasion and migration capacity, and expression levels of MMP-9, VEGFA, e-cadherin, ncadherin, vimentin, P-EGFR, P-STAT3 and p-Akt (p < 0.05).Conclusion: Cucurbitacin inhibits the proliferation, invasion, and migration of breast cancer cells by down-regulating the expressions of EGFR/STAT3/Akt signaling-related proteins, and inhibiting epithelial-mesenchymal transition transformation.

A Systematic Investigation of Trigonelline, Diosgenin, Protodioscin, and Dioscin from Trigonella foenum-graecum (fenugreek seeds) has Anti-cancer and Antioxidant Properties Without Side Effects

Systematic review and meta-analysis criteria were used to plan and conduct this systematic review (PRISMA). Different literature of reviews explicit that Fibers, flavonoids, polysaccharides, and saponins are the principal chemical components of Trigonella foenum-graecum. Some of the components may have mitogenic actions, which result in immune-competent cells being restored. Some of these ingredients are also antioxidants and may have immune-stimulant characteristics. In vitro, the extract from the seeds of the fenugreek seed was found to be cytotoxic to numerous cancers, including T-cell lymphoma, but not to normal cells. Breast, pancreatic, and prostate cancer cell lines showed growth inhibition after being treated with a specific amount of the extract. Diosgenin suppressed MCF-7 cell proliferation in a dose-dependent manner, according to in vitro tests. Diosgenin's effect on a human mammary carcinoma cell line, as well as its therapeutic potential for N-nitroso-N-methyl urea-induced breast cancer. Fenugreek extracts and their isolated constituents have been revealed to have anti-cancer activities in several investigations. Trigonelline, Diosgenin, Protodioscin, and Dioscin were found to be the most potent anticancer ingredients in fenugreek. Furthermore, fenugreek seeds are high in many essential chemicals, which show asignificant role in the suppression and inhibition of various malignancies. In addition, studies on the estrogenic effects of fenugreek seeds in vitro and in vivo have demonstrated its potential as an HRT alternative.

Chemical Composition and Cytotoxic Activity of the Fractionated Trunk Bark Essential Oil from Tetraclinis articulata (Vahl) Mast. Growing in Tunisia.

The aim of the present research was to determine the chemical composition and the cytotoxic effects of Tetraclinis articulata trunk bark essential oil (HEE) obtained by steam distillation and five fractions obtained by normal phase silica chromatographic separation. Chemical analysis allowed the identification of 54 known compounds. Relatively high amounts of oxygenated sesquiterpenes (44.4–70.2%) were detected, mainly consisting of caryophyllene oxide (13.1–26.6%), carotol (9.2–21.2%),14-hydroxy-9-epi-(E)-caryophyllene (3.2–15.5%) and humulene epoxide II (2.6–7.2%). The cytotoxic activity against human mammary carcinoma cell lines (MDA-MB-231) and colorectal carcinoma cell lines (SW620) of the essential oil and its fractions were assessed. All the samples displayed moderate to weak activity compared to 5-fluorouracil. The colorectal carcinoma cell line was relatively more sensitive to the essential oil and its fractions compared to the breast cancer cell line, showing IC50 values from 25.7 to 96.5 μg/mL. In addition, the essential oil and its fraction E.2 revealed a cytotoxic activity against colorectal carcinoma cell line, with IC50 values lower than 30 μg/mL. This is the first report on the chemical composition and cytotoxic activity of the trunk bark essential oil of T. articulata.

Spatiotemporal Mislocalization of Nuclear Membrane-Associated Proteins in γ-Irradiation-Induced Senescent Cells.

Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence nor changes in the LINC protein distribution before irradiation. However, the cytological changes following irradiation were more pronounced in shRNA knockdown cells compared to original cell lines. We conclude that mislocalization of LINC complex proteins is a significant characteristic of cellular senescence phenotypes and may influence complex events at the nuclear membrane, including trafficking and heterochromatin attachment.

Biological evaluation of novel thiomaltol-based organometallic complexes as topoisomerase II\u03b1 inhibitors

Topoisomerase IIα (topo2α) is an essential nuclear enzyme involved in DNA replication, transcription, recombination, chromosome condensation, and highly expressed in many tumors. Thus, topo2α-targeting has become a very efficient and well-established anticancer strategy. Herein, we investigate the cytotoxic and DNA-damaging activity of thiomaltol-containing ruthenium-, osmium-, rhodium- and iridium-based organometallic complexes in human mammary carcinoma cell lines by means of several biological assays, including knockdown of topo2α expression levels by RNA interference. Results suggest that inhibition of topo2α is a key process in the cytotoxic mechanism for some of the compounds, whereas direct induction of DNA double-strand breaks or other DNA damage is mostly rather minor. In addition, molecular modeling studies performed for two of the compounds (with Ru(II) as the metal center) evinces that these complexes are able to access the DNA-binding pocket of the enzyme, where the hydrophilic environment favors the interaction with highly polar complexes. These findings substantiate the potential of these compounds for application as antitumor metallopharmaceuticals.Graphic abstract

Crystal engineering of an adenine-decavanadate molecular device towards label-free chemical sensing and biological screening.

Due to the inherent geometrical interdependencies of nucleic acid structures, the ability to engineer biosensors that rely on the specific interactions of these compounds is of considerable importance. Additionally, sensing or screening in a label-free fashion is a capability of these structures that can be readily achieved by exploiting the fluorescent component. In this work, the [AdH]6[V10O28].4(H2O) (1) supramolecular structure is introduced using adenine and decavanadate moieties that allow probing of selectivity to specific nucleic acid binding events by optical changes. The structure of (1) is an alternating organic-inorganic hybrid architecture of cationic adeninium (AdH+) ribbons and anionic decavanadate (DV)-water sheets. The luminescent screening and anticancer activity of compound (1) on the two human mammary carcinoma cell lines MDA-MB-231 and MCF7 were investigated using fluorescent microscopy and MTT assays, respectively. It was found that compound (1) is cell permeable with no toxicity below 12.5 µM concentration and moderate cytotoxicity at concentrations as high as 200 µM in human breast cancer cell lines, making it a useful tool to study the cell nucleus in real time.

Absence of integrin \u03b13\u03b21 promotes\xa0the progression of HER2-driven breast cancer in vivo

BackgroundHER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3β1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3β1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis.MethodsTo investigate the role of α3β1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3β1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3β1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence.ResultsTumor onset in mice was independent of the presence of α3β1. In contrast, the depletion of α3β1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3β1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow.ConclusionDownregulation of α3β1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3β1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3β1 in breast cancer.

Abstract 2899: Antitumor activity of CIGB-300, a peptide CK2 inhibitor, in breast cancer models

Abstract CK2 is a serine-threonine kinase that has been involved in growth, proliferation and cell apoptosis. CK2 has a constitutive expression and it has more than 300 substrates. In recent years, CK2 became an interesting target for anticancer therapies. Inhibition of CK2 showed antitumor activity in different types of cancer. Because breast cancer is one of the main tumor types in which CK2 is overexpressed, we focus on the effect of CK2 inhibition as a modulator of key features of breast cancer cell biology. The aim of the present study was to evaluate the role of inhibition of CK2 by CIGB-300 in murine and human mammary carcinoma cell lines. For this purpose, we evaluated the action of CIGB-300 on viability, apoptosis, cell cycle, clonogenic capacity and migration using three different breast tumor cell lines, MCF7 (positive for estrogen, progesterone and HER2-neu receptors), MDA MB 231 (representative of triple negative tumors) and F3II, a murine mammary carcinoma. As compared the potential of CK2 inhibition in these models we included another chemical inhibitor of the enzyme, CX-4945. Our results showed that CIGB-300 reduced the viability of MDA MB 231 (IC50=120 μM) and MCF-7 (IC50=140 μM) (p<0.05, ANOVA). We observed that in both human and murine cell lines CIGB-300 exerts a pro-apoptotic action (TUNEL assay, p<0.05, χ2) and arrest cell cycle. We also found that the inhibition of CK2 by CIGB-300 decreased the clonogenic capacity and migration of F3II, MCF-7 and MDA-MB-231 (p<0.05, ANOVA). In vivo studies using the syngeneic F3II breast cancer model in BalB/c mice showed a decrease in the number of lung metastases in mice treated systemically with CIGB-300 after primary tumor surgery (Mann Whitney test, p<0.05). However, CK2 inhibition did not affect the growth of local recurrences. Also, intravenous administration of CIGB-300 decreased lung metastasis development of F3II cells. These results demonstrate an antimetastatic effect of CIGB-300 in a systemic inoculation model after surgical removal of primary tumors. In this work we demostrated the antitumor effect of CIGB-300 in breast cancer models such as those reported in myeloid leukemia, lung and cervix and head cancer. Citation Format: Maria F. Gottardo, Carla S. Capibianco, Johanna E. Sidabra, Yasser Perera, Silvio Perea, Daniel F. Alonso, Hernan G. Farina. Antitumor activity of CIGB-300, a peptide CK2 inhibitor, in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2899.

Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells.

Rab GTPases recruit peripheral membrane proteins and can define organelle identity. Rab18 localizes to the endoplasmic reticulum (ER) but also to lipid droplets (LDs), where it has been implicated in effector protein recruitment and in defining LD identity. Here, we studied Rab18 localization and function in a human mammary carcinoma cell line. Rab18 localized to the ER and to LD membranes on LD induction, with the latter depending on the Rab18 activation state. In cells lacking Rab18, LDs were modestly reduced in size and numbers, but we found little evidence for Rab18 function in LD formation, LD turnover on cell starvation, or the targeting of several proteins to LDs. We conclude that Rab18 is not a general, necessary component of the protein machinery involved in LD biogenesis or turnover.

Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner.

Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells “home” to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1–141) and PTH(1–34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule.

Breast-cancer extracellular vesicles induce platelet activation and aggregation by tissue factor-independent and -dependent mechanisms

IntroductionCancer-associated thrombosis is one of the major causes of worse prognosis among tumor-bearing patients. Extracellular vesicles derived from cancer cells, which can be divided mainly into microvesicles and exosomes, can participate in several tumor progression phenomena. Tumor-derived microvesicles positive for tissue factor (TF) have been associated with thrombotic risk in certain cancer types. Cancer cell-derived exosomes, however, have not. In this study we evaluated the capacity of extracellular vesicles (EVs, containing both microvesicles and exosomes) derived from breast-cancer cell lines in promoting platelet activation, aggregation and plasma coagulation, in experiments that access both TF-dependent and -independent activities. Materials and methodsEVs were isolated from the conditioned media of two human mammary carcinoma cell lines: MDA-MB-231 (highly invasive) and MCF-7 (less invasive). TF-independent EV/platelet interaction, platelet P-selectin exposure and aggregation were evaluated. Western blotting, plasma clotting and platelet aggregation in the presence of plasma were performed for the measurement of TF-dependent activity in EVs. ResultsInteraction between MDA-MB-231 EVs and washed platelets led to increased platelet P-selectin exposure and platelet aggregation compared to MCF-7 EVs. MDA-MB-231 EVs had higher TF protein levels and TF-dependent procoagulant activity than MCF-7 EVs. Consequently, TF-dependent platelet aggregation was also induced by MDA-MB-231 EVs, but not by MCF-7 EVs. ConclusionOur results suggest that MDA-MB-231 EVs induce TF-independent platelet activation and aggregation, as well as TF-dependent plasma clotting and platelet aggregation by means of thrombin generation. In this context, aggressive breast cancer-derived EVs may contribute to cancer-associated thrombosis.

ShRNA-mediated downregulation of α-N-Acetylgalactosaminidase inhibits migration and invasion of cancer cell lines.

Objective(s):Extracellular matrix (ECM) is composed of many kinds of glycoproteins containing glycosaminoglycans (GAGs) moiety. The research was conducted based on the N-Acetylgalactosamine (GalNAc) degradation of ECM components by α-N-acetylgalactosaminidase (Nagalase) which facilitates migration and invasion of cancer cells. This study aims to investigate the effects of Naga-shRNA downregulation on migration and invasion of cancer cell lines.Materials and Methods:In this study, MCF-7 cell line (human mammary carcinoma cell line) and A2780 (human ovarian carcinoma cell line) were used. The level of normalized Naga expression and Nagalase protein were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay/western blotting, respectively. Migration and invasion were determined using transwell assays, and statistical analysis was carried out by ANOVA test.Results:Response to transduction by shRNA compared to the control group, migrative and invasive properties of the transfected cells were significantly inhibited.Conclusion:These results indicate that Nagalase may have an important role in migration and invasion of cancer cells and can be considered as a candidate for further studies.

Molecular characterization of breast cancer cell lines by clinical immunohistochemical markers.

Breast cancer is the leading cause of cancer mortality in females worldwide. Studies based on gene expression profiles have identified different breast cancer molecular subtypes, such as luminal A and B cells, cancer cells that are estrogen receptor (ER) and/or progesterone receptor (PR) positive, human epidermal growth factor 2 (HER2)-enriched cells, cancer cells that exhibit an overexpression of the oncogene HER2, and triple-negative cells, cancer cells that are negative for ER, PR and HER2 expression. Immunohistochemistry is the most common type of method used for the identification of these molecular subtypes, through the identification of specific cell receptors. The present study aimed to evaluate the ER, PR and HER2 receptor expression in human breast cancer cell lines, and to classify the corresponding molecular subtype comparing two alternative methods. In the present study, a panel of human mammary carcinoma cell lines: BT-20; Hs578T; MCF-7; MCF-7/AZ; MDA-MB-231; MDA-MB-468; SKBR3; and T47D were used. Immunohistochemical and immunocytochemistry assays were used to characterize the breast cancer subtypes of these cell lines according to the expression of ER, PR and HER2 receptors. The results revealed the molecular characterization of this panel of breast cancer cell lines, using the differential expression of classical and clinically used markers in concordance with previous studies. In addition, these data are important for additional in vitro studies of these specific receptors.

Antiproliferative and apoptotic effect ofPleurotus ostreatuson human mammary carcinoma cell line (michigan cancer foundation-7)

Aim: The aim of this study was to explore the antiproliferative and apoptotic effect of Pleurotus ostreatus ethanolic extract (POEet) on human mammary carcinoma cell line (Michigan cancer foundation-7 [MCF-7]). Methods: Gas chromatography-mass spectrometry (GC-MS) was performed to isolate and quantify the active constituent of POEet chloroform fraction. Cytotoxic property of POEet on MCF-7 and Vero cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We further evaluated the apoptotic effect by measuring the mitochondrial membrane potential (ΔΨm), dual staining (acridine orange/ethidium bromide), comet assay, DNA fragmentation, and oxidant/antioxidant status in MCF-7 cells treated with and without POEet. Results: GC-MS analysis identified 30 phytochemical constituents from the POEet chloroform fraction. MTT assay revealed a greater degree of cytotoxicity at almost all doses of POEet, with IC30 value (164.59 µg/mL) and IC50 value (1024.02 µg/mL) greatly inhibiting the cell growth. In addition, POEet could modulate the levels of oxidant/antioxidant status and induce a potent loss mitochondrial membrane potential, DNA damage, and apoptosis in MCF-7 cells. Conclusion: The study demonstrates a potent anticancer property of P. ostreatus against human mammary carcinoma cells which might be of value in nutraceutical industry. Further investigations are essential to establish it as a treatment against breast cancer.

The role of new eudesmane-type sesquiterpenoid and known eudesmane derivatives from the red alga Laurencia obtusa as potential antifungal–antitumour agents

A new eudesmane sesquiterpenoid, eudesma-4(15),7-diene-5,11-diol (1) along with the known trinor-sesquiterene, teuhetenone (2), and a seco-eudesmane sesquiterpene, chabrolidione B (3), have been isolated from the Red Sea red alga Laurencia obtusa. The chemical structures were elucidated on the basis of extensive spectroscopic analysis. The antifungal and cytotoxic activities of the isolated metabolites were tested against several fungi, yeast and human mammary carcinoma cell line (MCF-7). Compounds 1 and 3 showed a much better activity [minimum inhibitory concentration (MIC): 2.9 μM] than that of amphotericin B (MIC: 4.6 μM). Interestingly, compound 2, the least active antifungal compound, retained the high anticancer activity against MCF-7 (22 μM) in comparison with cisplatin (59 μM), which was determined by employing lactate dehydrogenase assay. Compounds 1–3 are recorded here for the first time from algal flora. The chemotaxonomic importance of the isolated metabolites was discussed.

Using a sequence of estrogen response elements as a DNA aptamer for estrogen receptors in vitro.

Estrogen receptors (ERs) are overexpressed in approximately 70% of breast cancer cases, and they play an important role in tumorigenesis. ERs are strong predictive factors for measuring responses to hormonal therapies. Aptamers are short and single stranded oligonucleotides that are able to recognize target molecules with high affinity. In the present study, we selected and synthesized an oligonucleotide, which has a similar sequence to estrogen response element in the Xenopus Vitellogenin A2 gene. The synthesized oligonucleotide was evaluated by using immunostaining of paraffin-embedded breast cancer tissues and treating MCF-7 human mammary carcinoma cell line in vitro. We found that the synthesized oligonucleotide had a high binding affinity to ER similar to estradiol. Using a specific anti-ER antibody as a standard control, we showed that the synthesized oligonucleotide specifically recognized and immunostained tumor cells of breast cancer without cross-reaction with normal tissues. The overall agreement of ER detection between the anti-ER antibody and the ER aptamer was 97.1% (kappa value=0.943; 95% CI=0.879-1.006; p<0.002). Similar to tamoxifen or fulvestrant, the oligonucleotide also had an inhibitory effect on cell proliferation of MCF-7 cell line in a dose- and time-dependent fashion but had no cytotoxic effect on human normal mammary epithelial cells. Therefore, the synthesized oligonucleotide may be used as an aptamer for immunostaining of paraffin-embedded tissue sections for breast cancer diagnosis, as well as a potential ER antagonist in the treatment of breast cancer.

Inhibitory and apoptosis-inducing effects of Newcastle disease virus strain AF2240 on mammary carcinoma cell line.

Breast cancer is the malignant tumour that developed from cells of the breast and is the first leading cause of cancer death among women worldwide. Surgery, radiotherapy, and chemotherapy are the available treatments for breast cancer, but these were reported to have side effects. Newcastle disease virus (NDV) known as Avian paramyxovirus type-1 (APMV1) belongs to the genus Avulavirus in a family Paramyxoviridae. NDV is shown to be a promising anticancer agent, killing tumour cells while sparing normal cells unharmed. In this study, the oncolytic and cytotoxic activities of NDV AF2240 strain were evaluated on MDA-MB-231, human mammary carcinoma cell line, using MTT assay, and its inhibitory effects were further studied using proliferation and migration assays. Morphological and apoptotic-inducing effects of NDV on MD-MB-231 cells were observed using phase contrast and fluorescence microscopes. Detection of DNA fragmentation was done following terminal deoxyribonucleotide transferase-mediated Br-dUTP nick end labeling staining (TUNEL) assay, which confirmed that the mode of death was through apoptosis and was quantified by flow cytometry. Furthermore, analysis of cellular DNA content demonstrated that the virus caused an increase in the sub-G1 phase (apoptotic peak) of the cell cycle. It appears that NDV AF2240 strain is a potent anticancer agent that induced apoptosis in time-dependent manner.