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Archives of Razi Institute Feb 2023In the migration and metastasis of cancer cells, it is necessary to rotate the focal adhesion (FA). MAP4K4 plays a vital role in the formation of cytoskeletal...
In the migration and metastasis of cancer cells, it is necessary to rotate the focal adhesion (FA). MAP4K4 plays a vital role in the formation of cytoskeletal regeneration, but its role in regulating FA dynamics and cancer cell migration is not well understood. This present aimed to investigate the role of MAP4K4 in regulating FA dynamics and cell migration in the human breast cancer cell line. For this purpose, different variants, including MAP4K4 (wild type), partial active mutation kinase (MAP4K4-T178D), mutant with inactivated or reduced activity kinase (MAP4K4-T178A) and inactive kinase mutation (MAP4K4-K54R) was used in the evaluation. GFP-paxillin was also used as a marker in basal breast cancer cells (MDA-MB-231) in determining FA dynamics. Time-lapse and confocal microscopes were used to record FA dynamics and cell migration. The present study's findings showed that cells expressing MAP4K4-K54R, MAP4K4-T178D and MAP4K4-T178A type slowly down the FA turnover rates and had much larger FAs than those expressing WT MAP4K4 in MDA-MB-231 breast cancer cell line. Furthermore, inhibiting MAP4K4 strongly inhibited FA formation and reduced cell migration speed. In conclusion, MAP4K4 regulates FA dynamics and cancer cell migration, most probably through activating FA proteins and cytoskeleton.
Topics: Humans; Cell Movement; Focal Adhesions; Intracellular Signaling Peptides and Proteins; MCF-7 Cells; MDA-MB-231 Cells; Mutation; Neoplasms; Protein Serine-Threonine Kinases
PubMed: 37312710
DOI: 10.22092/ARI.2022.358953.2340 -
BMC Research Notes Apr 2023The toxicology of herbicides, which are currently in use is under-explored. One highly used but under investigated herbicide is pendimethalin. Here we mined...
Comparison of transcriptome alterations induced by pendimethalin or its commercial formulation Stomp Aqua in human MCF-7, MCF-10 A and MCF-12 A mammary epithelial cells.
OBJECTIVE
The toxicology of herbicides, which are currently in use is under-explored. One highly used but under investigated herbicide is pendimethalin. Here we mined high-throughput data from the US National Toxicology Program (NTP) to identify whether pendimethalin possesses an estrogenic capability in human cells. We also evaluated effects of pendimethalin and its reference commercial formulated herbicide Stomp Aqua on the transcriptome profile of three human mammary epithelial cell lines, cancerous MCF-7 and non-cancerous MCF-10 A and MCF-12 A to see whether this compound could have endocrine disrupting effects and if co-formulants present in the commercial formulation could amplify its toxicity.
RESULTS
The data mined from the US NTP database suggests that pendimethalin activates estrogen receptors at a concentration of approximately 10?M. MCF-7, MCF-10A and MCF-12A cells were exposed to 10 ?M pendimethalin and Stomp Aqua at an equivalent concentration. Transcriptome analysis showed changes in gene expression patterns implying that pendimethalin affected ubiquitin-mediated proteolysis and the function of the spliceosome. The formulated pendimethalin product Stomp Aqua gave comparable effects suggesting pendimethalin was responsible for the observed transcriptome alterations. Given the lack of information on the exposure to this pesticide, our study prompts the need for biomonitoring studies, especially under occupational use scenarios, to understand if low level exposure to pendimethalin could have endocrine disrupting effects on populations exposed to this compound. A deeper understanding of the exposure and mechanisms of action of this endocrine-disrupting pesticide is needed.
Topics: Humans; Transcriptome; MCF-7 Cells; Pesticides; Herbicides; Epithelial Cells
PubMed: 37098576
DOI: 10.1186/s13104-023-06327-w -
Biomolecules Apr 2021subsp. is an important medicinal plant in several countries, including Turkey. This study aimed to evaluate the cytotoxicity of a crude extract of subsp. against... (Comparative Study)
Comparative Study
subsp. is an important medicinal plant in several countries, including Turkey. This study aimed to evaluate the cytotoxicity of a crude extract of subsp. against different breast cell lines to determine invasion, adhesion, and lipid peroxidation. The cytotoxic effects on MCF-7 breast cancer and MCF-12A as the immortalized cell line were examined by the XTT assay. Invasion and adhesion studies were performed according to the manufacturer's kit procedure to IC values for 48 h. Lipid peroxidation was measured in the MCF-7 cell. A bioinformatics analysis was conducted to unravel the mechanism of action underlying antiproliferative effects, as well. According to XTT results, the tested extract showed a time- and a concentration-dependent cytotoxic effect. The most effective concentration was 100.5 µg/mL (48 h), which was selected for biological activities, such as apoptotic activity, invasion, adhesion, and lipid peroxidation assays. The extract caused tumoral cell death, and it did not have a cytotoxic effect on healthy human breast cells. Duplication times and measurement of CI analyses of cells were performed using the real-time cell analysis system xCELLigence. Finally, the bioinformatics analysis indicated the prominent role of quercetin as an extract component exerting a key role in the observed antiproliferative effects. This was supported by the micromolar/submicromolar affinity of quercetin towards proto-oncogene serine/threonine-protein kinase (PIM-1) and hematopoietic cell kinase (HCK), both involved in breast cancer. Altogether, our findings proposed that the extraction of the plant can be an effective strategy to isolate biomolecules with promising cytotoxic effects against breast cancer cells.
Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Fabaceae; Female; Humans; Inhibitory Concentration 50; MCF-7 Cells; Plant Extracts; Proto-Oncogene Mas
PubMed: 33946222
DOI: 10.3390/biom11050671 -
Scientific Reports Oct 2017Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension...
Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension cultures, but remains poorly understood in substrate-dependent cells. Here, we used electron, confocal and time-lapse microscopy in combination with pharmacological inhibition of intracellular components to study the kinetics of entosis using two human substrate-dependent tumor cultures, A431 and MCF7. In total, we identified and characterized five consecutive stages of entosis, which were common for both examined cell lines. We further demonstrated that actin filaments in the entotic as well as invading cells were crucial for entosis. Microtubules and the Golgi apparatus of entotic cells provided membrane expansion required for internalization of the invading cell. Depolymerization of microfilaments and microtubules, and disintegration of the Golgi complex inhibited entosis. We confirmed the presence of adhesive junctions and discovered the formation of desmosomes between the invading and entotic cells. The internalized cell was shown to be degraded due to the lysosomal activation in both cells whereas the disintegration of the Golgi apparatus did not affect the process. Thus, in the substrate-dependent cultures, entosis requires microfilaments, microtubules and the Golgi complex for cell invasion, but not for internalized cell degradation.
Topics: Actin Cytoskeleton; Entosis; Epithelial Cells; Golgi Apparatus; Humans; Kinetics; Lysosomes; MCF-7 Cells; Microscopy, Confocal; Microscopy, Electron; Microtubules; Neoplasms; Time-Lapse Imaging
PubMed: 28970591
DOI: 10.1038/s41598-017-12867-6 -
BMC Research Notes Aug 2021Breast cancer cell growth and proliferation requires lipids for energy production, cell membrane synthesis, or as signaling molecules. Lipids can be delivered to cells...
OBJECTIVES
Breast cancer cell growth and proliferation requires lipids for energy production, cell membrane synthesis, or as signaling molecules. Lipids can be delivered to cells by lipoprotein lipase (LPL), an extracellular lipase that hydrolyzes triacylglycerols and phospholipids from lipoproteins, that is expressed by adipose tissue and some breast cancer cell lines. Studies have shown that lipoprotein hydrolysis products induce pro-inflammatory cytokine secretion by endothelial cells. Thus, our objective was to determine if hydrolysis products generated by LPL from total lipoproteins can also promote pro-inflammatory cytokine secretion from breast cancer cells.
RESULTS
Using cytokine arrays, we found that MDA-MB-231 cells increased secretion of seven cytokines in response to treatment with lipoprotein hydrolysis products. In contrast, MCF-7 cells showed decreased secretion of two cytokines. Expanding the analysis to additional cell lines by ELISA, we found increased secretion of TNF-α and IL-6 by MDA-MB-468 cells, and increased secretion of IL-4 by MDA-MB-468 and SKBR3 cells. The changes to cytokine secretion profiles of the breast cancer cell types examined, including the non-cancerous MCF-10a breast cells, were independent of increased cell metabolic activity. These results provide information on how lipoprotein hydrolysis products within the tumor microenvironment might affect breast cancer cell viability and progression.
Topics: Breast Neoplasms; Cytokines; Endothelial Cells; Female; Humans; Hydrolysis; Lipoprotein Lipase; Macrophages; Triple Negative Breast Neoplasms; Tumor Microenvironment
PubMed: 34404457
DOI: 10.1186/s13104-021-05728-z -
International Journal of Nanomedicine 2018Nanotechnology has gained important interest, especially in the development of new therapies; the application of gold nanoparticles (AuNPs) in the treatment and...
BACKGROUND
Nanotechnology has gained important interest, especially in the development of new therapies; the application of gold nanoparticles (AuNPs) in the treatment and detection of diseases is a growing trend in this field. As cancer represents a serious health problem around the world, AuNPs are studied as potential drugs or drug carriers for anticancer agents. Recent studies show that AuNPs stabilized with chitosan (CH) possess interesting biological activities, including potential antitumor effects that could be selective to cancer cells.
MATERIALS AND METHODS
In this study, we synthesized sodium citrate-AuNPs and CH-capped AuNPs of 3-10 nm, and analyzed their cytotoxicity in cervical (HeLa) and breast (MCF-7) cancer cells, and in peripheral blood mononuclear cells (PBMCs). Then, we evaluated the clonogenic potential, cell cycle, nuclear alterations, caspase dependence, and reactive oxygen species (ROS) production in HeLa and MCF-7 cells after chitosan gold nanoparticles (CH-AuNPs) exposure.
RESULTS
Our data showed that CH-AuNPs are cytotoxic in a dose-dependent manner in the cancer cell lines tested, while they induce low cytotoxicity in PBMCs. Sodium citrate gold nanoparticles did not show cytotoxic effects. In both HeLa and MCF-7 cell lines, CH-AuNPs inhibit clonogenic potential without inducing cell cycle arrest or nuclear alterations. The cell death mechanism is specific for the type of cancer cell line tested, as it depends on caspase activation in HeLa cells, whereas it is caspase independent in MCF-7 cells. In all cases, ROS production is mandatory for cell death induction by CH-AuNPs, as ROS inhibition with N-acetyl cysteine inhibits cell death.
CONCLUSION
Our results show that CH-AuNPs are selective for HeLa and MCF-7 cancer cells, rather than normal PBMCs, and that ROS production seems to be a conserved feature of the cell death mechanism induced by CH-AuNPs. These results improve the knowledge of CH-AuNPs and open the way to the design of new pharmacological strategies using these agents against cancer.
Topics: Antineoplastic Agents; Caspases; Cell Cycle Checkpoints; Cell Death; Cell Survival; Chitosan; Dose-Response Relationship, Drug; Female; Gold; HeLa Cells; Humans; Leukocytes, Mononuclear; MCF-7 Cells; Metal Nanoparticles; Reactive Oxygen Species
PubMed: 29910612
DOI: 10.2147/IJN.S165289 -
International Journal of Molecular... Nov 2021Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular...
Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular communication, enabling them to share information and act as a synchronized syncytium. This cellular communication has been considered a strong tumor suppressor, but it is now recognized that some type of Cxs can be pro-tumorigenic. For example, Cx46 expression is increased in human breast cancer samples and correlates with cancer stem cell (CSC) characteristics in human glioma. Thus, we explored whether Cx46 and glioma cells, can set up CSC and epithelial-to-mesenchymal transition (EMT) properties in a breast cancer cell line. To this end, we transfected MCF-7 cells with Cx46 attached to a green fluorescent protein (Cx46GFP), and we determined how its expression orchestrates both the gene-expression and functional changes associated with CSC and EMT. We observed that Cx46GFP increased Sox2, Nanog, and OCT4 mRNA levels associated with a high capacity to form monoclonal colonies and tumorspheres. Similarly, Cx46GFP increased the mRNA levels of n-cadherin, Vimentin, Snail and Zeb1 to a higher migratory and invasive capacity. Furthermore, Cx46GFP transfected in MCF-7 cells induced the release of higher amounts of VEGF, which promoted angiogenesis in HUVEC cells. We demonstrated for the first time that Cx46 modulates CSC and EMT properties in breast cancer cells and thus could be relevant in the design of future cancer therapies.
Topics: Breast Neoplasms; Cell Line, Tumor; Connexins; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; MCF-7 Cells; Nanog Homeobox Protein; Neoplastic Stem Cells; SOXB1 Transcription Factors; Vascular Endothelial Growth Factor A; Zinc Finger E-box-Binding Homeobox 1
PubMed: 34830485
DOI: 10.3390/ijms222212604 -
STAR Protocols Sep 2022Capillary electrophoresis mass spectrometry (CE-MS) can measure the intracellular amount of highly polar and charged metabolites; liquid chromatography mass spectrometry...
Capillary electrophoresis mass spectrometry (CE-MS) can measure the intracellular amount of highly polar and charged metabolites; liquid chromatography mass spectrometry (LC-MS) can quantify hydrophobic metabolites. A comprehensive metabolome analysis requires independent sample preparation for LC-MS and CE-MS. Here, we present a protocol to prepare for sequentially analyzing the metabolites from one sample. Here we describe the steps for breast cancer cell lines, MCF-7 cells, but the protocol can be applied to other cell types.
Topics: Cell Line; Cells, Cultured; Mass Spectrometry; Metabolome; Metabolomics
PubMed: 35819883
DOI: 10.1016/j.xpro.2022.101531 -
Journal of Experimental & Clinical... Nov 2017Claudin-6 (CLDN6), a member of CLDN family and a key component of tight junction, has been reported to function as a tumor suppressor in breast cancer. However, whether...
BACKGROUND
Claudin-6 (CLDN6), a member of CLDN family and a key component of tight junction, has been reported to function as a tumor suppressor in breast cancer. However, whether CLDN6 plays any role in breast cancer chemoresistance remains unclear. In this study, we investigated the role of CLDN6 in the acquisition of chemoresistance in breast cancer cells.
METHODS
We manipulated the expression of CLDN6 in MCF-7 and MCF-7/MDR cells with lv-CLDN6 and CLDN6-shRNA and investigated whether CLDN6 manipulation lead to different susceptibilities to several chemotherapeutic agents in these cells. The cytotoxicity of adriamycin (ADM), 5-fluorouracil (5-FU), and cisplatin (DDP) was tested by cck-8 assay. Cell death was determined by DAPI nuclear staining. The enzyme activity of glutanthione S-transferase-p1 (GSTP1) was detected by a GST activity kit. Then lv-GSTP1 and GSTP1-shRNA plasmids were constructed to investigate the potential of GSTP1 in regulating chemoresistance of breast cancer. The TP53-shRNA was adopted to explore the regulation mechanism of GSTP1. Finally, immunohistochemistry was used to explore the relationship between CLDN6 and GSTP1 expression in breast cancer tissues.
RESULTS
Silencing CLDN6 increased the cytotoxicity of ADM, 5-FU, and DDP in MCF-7/MDR cells. Whereas overexpression of CLDN6 in MCF-7, the parental cell line of MCF-7/MDR expressing low level of CLDN6, increased the resistance to the above drugs. GSTP1 was upregulated in CLDN6-overexpressed MCF-7 cells. RNAi -mediated silencing of CLDN6 downregulated both GSTP1 expression and GST enzyme activity in MCF-7/MDR cells. Overexpresssion of GSTP1 in CLDN6 silenced MCF-7/MDR cells restored chemoresistance, whereas silencing GSTP1 reduced the chemoresistance due to ectopic overexpressed of CLDN6 in MCF-7 cells. These observations were also repeated in TNBC cells Hs578t. We further confirmed that CLDN6 interacted with p53 and promoted translocation of p53 from nucleus to cytoplasm, and both the expression and enzyme activity of GSTP1 were regulated by p53. Clinicopathologic analysis revealed that GSTP1 expression was positively associated with CLDN6 in human breast cancer samples.
CONCLUSION
High expression of CLDN6 confers chemoresistance on breast cancer which is mediated by GSTP1, the activity of which is regulated by p53. Our findings provide a new insight into mechanisms and strategies to overcome chemoresistance in breast cancer.
Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cisplatin; Claudins; Cytoplasm; Doxorubicin; Drug Resistance, Neoplasm; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Humans; MCF-7 Cells; Tumor Suppressor Protein p53
PubMed: 29116019
DOI: 10.1186/s13046-017-0627-9 -
Molecules (Basel, Switzerland) Dec 2023Urinary tract infection is an infectious disease that requires immediate treatment. It can occur in any age group and involves both genders equally. The present study...
Urinary tract infection is an infectious disease that requires immediate treatment. It can occur in any age group and involves both genders equally. The present study was to check the resistance of some antibiotics and to assess the antibacterial potential of three extracts of three plants against notorious bacteria involved in urinary tract infections. Along with assessing the antibacterial activity of plant extracts, we checked for the anticancer potential of these extracts against the cancer cell lines MCF-7 and A2780. Cancer is the leading cause of mortality in developed countries. Determinations of total flavonoid content, total phenolic content, total alkaloid content, total tannin content, total carotenoid content, and total steroid content were performed. The disk diffusion method was used to analyze the antibacterial activity of plant extracts. Ethanolic extract of showed sensitivity (25-28 mm) against bacteria, whereas chloroform and hexane extracts showed resistance against all bacteria except (25 mm). Ethanolic extract of L. showed sensitivity (22-25 mm) against bacteria, whereas chloroform and hexane extracts showed resistance. Ethanolic extract of L. showed sensitivity (8-16 mm) against all bacteria except , whereas chloroform and hexane extracts showed resistance. Positive controls showed variable zones of inhibition (2-60 mm), and negative control showed 0-1 mm. The antibiotic resistance was much more prominent in the case of hexane and chloroform extracts of all plants, whereas ethanolic extract showed a sensitivity of bacteria against extracts. Both cell lines, MCF-7 and A2780, displayed decreased live cells when treated with plant extracts.
Topics: Male; Female; Humans; Pistacia; Olea; Hexanes; Cell Line, Tumor; MCF-7 Cells; Chloroform; Ovarian Neoplasms; Anti-Bacterial Agents; Plant Extracts; Staphylococcus; Bacteria; Microbial Sensitivity Tests
PubMed: 38138636
DOI: 10.3390/molecules28248148