Exploring the role of estrogen and progestins in breast cancer: A genomic approach to diagnosis.

Breast cancer (BC) is the most common cancer among women and a major cause of death from cancer. The role of estrogen and progestins, including synthetic hormones like R5020, in the development of BC has been highlighted in numerous studies. In our study, we employed machine learning and advanced bioinformatics to identify genes that could serve as diagnostic markers for BC. We thoroughly analyzed the transcriptomic data of two BC cell lines, T47D and UDC4, and performed differential gene expression analysis. We also conducted functional enrichment analysis to understand the biological functions influenced by these genes. Our study identified several diagnostic genes strongly associated with BC, including MIR6728, ENO1-IT1, ENO1-AS1, RNU6-304P, HMGN2P17, RP3-477M7.5, RP3-477M7.6, and CA6. The genes MIR6728, ENO1-IT1, ENO1-AS1, and HMGN2P17 are involved in cancer control, glycolysis, and DNA-related processes, while CA6 is associated with apoptosis and cancer development. These genes could potentially serve as predictors for BC, paving the way for more precise diagnostic methods and personalized treatment plans. This research enhances our understanding of BC and offers promising avenues for improving patient care in the future.

Carbon Nanotube-Mediated Delivery of PTEN Variants: In Vitro Antitumor Activity in Breast Cancer Cells.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a crucial tumor suppressor protein with frequent mutations and alterations. Although protein therapeutics are already integral to numerous medical fields, their potential remains nascent. This study aimed to investigate the impact of stable, unphosphorylated recombinant human full-length PTEN and its truncated variants, regarding their tumor suppression activity with multiwalled-carbon nanotubes (MW-CNTs) as vehicles for their delivery in breast cancer cells (T-47D, ZR-75-1, and MCF-7). The cloning, overexpression, and purification of PTEN variants were achieved from E. coli, followed by successful binding to CNTs. Cell incubation with protein-functionalized CNTs revealed that the full-length PTEN-CNTs significantly inhibited cancer cell growth and stimulated apoptosis in ZR-75-1 and MCF-7 cells, while truncated PTEN fragments on CNTs had a lesser effect. The N-terminal fragment, despite possessing the active site, did not have the same effect as the full length PTEN, emphasizing the necessity of interaction with the C2 domain in the C-terminal tail. Our findings highlight the efficacy of full-length PTEN in inhibiting cancer growth and inducing apoptosis through the alteration of the expression levels of key apoptotic markers. In addition, the utilization of carbon nanotubes as a potent PTEN protein delivery system provides valuable insights for future applications in in vivo models and clinical studies.

Comparative EPR Studies on the Influence of Genistein on Free Radicals in Non-Irradiated and UV-Irradiated MCF7, T47D and MDA-MB-231 Breast Cancer Cells.

The antioxidant activity and the association of genistein with carcinogenesis are widely documented. Few studies directly measure the number of free radicals generated in cells, either during the action of factors stimulating their formation, e.g., ultraviolet (UV), or after exposure to antioxidants. The most suitable method for analysing free radicals is electron paramagnetic resonance (EPR) spectroscopy. The EPR method detects a paramagnetic centre with a single electron. Antioxidants neutralize free radicals, therefore, EPR analysis of antioxidant efficacy is as valuable and important as studying the paramagnetic centres of radicals. The aim of the study was to determine the influence of genistein on free radicals basal level and after UV exposure in breast cancer cell lines MCF7, T47D and MDA-MB-231 cell lines. The impact of genistein on cell viability was investigated at concentrations of 0.37 µM, 3.7 µM, 37 µM and 370 µM. Genistein at a concentration of 370 µM revealed a cytotoxic effect on the cells of all three tested breast cancer lines. Genistein at a concentration of 0.37 µM showed no significant effect on the cell viability of all tested breast cancer lines. Therefore, cell proliferation and antioxidant properties were examined using genistein at a concentration of 0.37 µM and 37 µM. X-band (9.3 GHz) EPR spectra of three different types of breast cancer cells (ER-positive, PR-positive and HER-2 negative: MCF7 and T47D and triple-negative MDA-MB-231) were compared. UV irradiation was used as a factor to generate free radicals in cells. The effect of free radical interactions with the antioxidant genistein was tested for non-UV-irradiated (corresponding to the basal level of free radicals in cells) and UV-irradiated cells. The levels of free radicals in the non-irradiated cells studied increased in the following order in breast cancer cells: T47D < MDA-MB-231 < MCF7 and UV-irradiated breast cancer cells: MDA-MB-231 < MCF7 < T47D. UV-irradiation altered free radical levels in all control and genistein-cultured cells tested. UV irradiation caused a slight decrease in the amount of free radicals in MCF7 cells. A strong decrease in the amount of free radicals was observed in UV-irradiated MDA-MB-231 breast cancer cells. The amount of free radicals in T47D cancer cells increased after UV irradiation. Genistein decreased the amount of free radicals in non-irradiated and UV-irradiated MCF7 cells, and only a weak effect of genistein concentrations was reported. Genistein greatly decreased the amount of free radicals in UV-irradiated T47D cancer cells cultured with genistein at a concentration of 3.7 µM. The effect of genistein was negligible in the other samples. Genistein at a concentration of 3.7 µM decreased the amount of free radicals in non-irradiated MDA-MB-231 cancer cells, but genistein at a concentration of 37 µM did not change the amount of free radicals in these cells. An increase in the amount of free radicals in UV-irradiated MDA-MB-231 cancer cells was observed with increasing genistein concentration. The antioxidant efficacy of genistein as a potential plant-derived agent supporting the treatment of various cancers may be determined by differences in signalling pathways that are characteristic of breast cancer cell line subtypes and differences in activation of oxidative stress response pathways.

Nanoplastics increase in vitro oestrogenic activity of neurotherapeutic drugs.

Environmental pollution with plastic nanoparticles (PNPs) has rendered hazard assessment of unintentional human exposure to neurotherapeutic drugs through contaminated water and food ever more complicated. Due to their small size, PNPs can easily enter different cell types and cross different biological barriers, while their high surface-to-volume ratio enables higher adsorption of chemicals. This is how PNPs take the role of a Trojan horse as they enhance bioaccumulation of many different pollutants. One of the health concerns related to water pollution with neurotherapeutic drugs is endocrine disruption, already evidenced for the anticonvulsant drug carbamazepine (Cbz) and antidepressant fluoxetine (Flx). Our study aimed to evaluate endocrine disrupting effects of Cbz and Flx in mixtures with polystyrene nanoparticles (PSNPs) using the in vitro luciferase assay to measure oestrogen receptor activity in T47D-KBluc cells treated with Cbz-PSNPs or Flx-PSNPs mixtures and compare it with the activities observed in cells treated with individual mixture components (Cbz, Flx, or PSNPs). Dose ranges used in the study were 0.1-10 mg/L, 1-100 µmol/L, and 0.1-10 µmol/L for PSNPs, Cbz, and Flx, respectively. Our findings show that none of the individual components activate oestrogen receptors, while the mixtures induce oestrogen receptor activity starting with 0.1 mg/L for PSNPs, 10 µmol/L for Cbz, and 0.5 µmol/L for Flx. This is the first study to evidence that PSNPs increase oestrogen receptor activity induced by neurotherapeutic drugs at their environmentally relevant concentrations and calls for urgent inclusion of complex mixtures in health hazard assessments to inform regulatory response.

Near infrared-responsive quinacrine-gold hybrid nanoparticles deregulate HSP-70/P300-mediated H3K14 acetylation in ER/PR+ breast cancer stem cells.

Aim: This study aimed to determine if quinacrine-gold hybrid nanoparticles (QAuNPs) + near-infrared (NIR) deregulate HSP-70/P300 complex-mediated H3K14 acetylation in estrogen receptor/progesterone receptor (ER/PR+) breast cancer stem cells (CSCs). Materials & methods: Various cells and mouse-based systems were used as models. Results: QAuNP + NIR treatment reduced the nuclear translocation of HSP-70, affected the histone acetyltransferase activity of P300 and specifically decreased H3K14 acetylation in ER/PR+ breast CSCs. Finally, HSP-70 knockdown showed a reduction in P300 histone acetyltransferase activity, decreased H3K14 acetylation and inhibited activation of the TGF-ß gene. Conclusion: This study revealed that QAuNP + NIR irradiation inhibits oncogenic activation of the TGF-ß gene by decreasing H3K14 acetylation mediated through the HSP-70/P300 nuclear complex in ER/PR+ breast CSCs.

Cryptobrachytone C from Cryptocarya pulchrinervia (Kosterm) Leaves on Proliferation, Apoptosis, Migration and Clonogenicity of MCF-7 and T47D Cell Lines.

Cryptocarya pulchrinervia is an Indonesian indigenous plant that grows in Sumatra, Kalimantan and Papua. One of the new compounds extracted from this plant was cryptobrachytone C, which was known to be cytotoxic against cancer cells of Murine leukemia P388 with IC50 10.52 µM. In this study, the cytotoxicity and anticancer properties of cryptobrachytone C on proliferation, apoptosis, migration and clone formation of MCF-7 and T47D breast cancer cell lines were examined, which had not previously been done before. The cytotoxicity of the compound was measured using an MTT (3- (4,5-dimethylthiazol-2- yl) -2,5-di-phenyl-tetrazolium bromide) assay. The cell proliferation was measured using a BrdU assay, and the cell apoptosis was measured using annexin-V FITC, while the cell migration was measured using a transwell filter. The cytotoxic test result demonstrated that cryptobrachytone C was cytotoxic against MCF-7 cells with IC50 12.94 ± 0.32 µM but not against T47D cells with IC50 65.33 ± 2.33 µM nor against normal MRC-5 cells with IC50 122.57 ± 19.84 µM. The cell proliferation assay showed that cryptobrachytone C at IC50 concentration had antiproliferative properties against MCF-7 cancer cell lines (p < 0.05) but did not significantly reduce T47D cell proliferation (p < 0.07). Although the results of the cell apoptosis test showed that cryptobrachytone C could induce the apoptosis of the MCF-7 and T47D cells, it was insignificant (p > 0.05). The cell migration test showed that cryptobrachytone at IC50 concentrations could inhibit the migration of the MCF-7 and T47D cells. The clonogenic test showed that cryptobrachytone C at IC50 concentration can induce the inhibition of the formation of MCF-7 and T47D cell colonies. The cryptobrachytone C anti-cancer character was more signi icant on the MCF-7 cell line compared to the T47D. This study showed that cryptobrachytone C was cytotoxic and had potential as an anti-cancer compound against MCF-7 and T47D breast cancer cell lines.

The Potential of Eleutherine bulbosa in Inducing Apoptosis and Inhibiting Cell Cycle in Breast Cancer: A Network Pharmacology Approach and In Vitro Experiments.

OBJECTIVE: The objective of this study was to evaluate the potential and mechanisms of phytochemicals in Eleutherine bulbosa (EBE) in inducing apoptosis and inhibiting the cell cycle in breast cancer through a network pharmacology approach and in vitro validation. METHODS: This research employed a literature review approach to identify active anti-cancer compounds and utilized a network pharmacology approach to predict the mechanisms of action of EBE compounds in breast cancer. In addition, in vitro experiments were conducted using MTT method to evaluate the effects of EBE on the cytotoxicity of T47D cells, and the flow cytometry method was employed to determine the impact of EBE on apoptosis and the cell cycle. RESULTS: The network pharmacology analysis revealed that EBE had an impact on 42 genes involved in breast cancer, including 23 important target genes implicated in the pathophysiology of breast cancer. Pathway analysis using the KEGG database showed a close association between EBE and crucial signaling pathways in breast cancer, including P53 signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, apoptosis and cell cycle. In vitro experiments demonstrated that EBE exhibited moderate anti-cancer activity. Furthermore, EBE demonstrated significant potential in inducing apoptosis in breast cancer cells, with a percentage of apoptotic cells reaching 93.6%. Additionally, EBE was observed to disrupt the cell cycle, leading to a significant increase in the sub G1 and S phases, and a significant decrease in the G2-M and G1 phases. CONCLUSION: EBE has the potential to be an anti-cancer agent through various mechanisms, including apoptosis induction and cell cycle inhibition in breast cancer cells. These findings provide new insights into the potential of EBE as an alternative treatment for breast cancer.

Safflower (Carthamus tinctorius Linn.) Inhibits Cell Proliferation and Induces Apoptotic in Breast Cancer Cell Lines T47D.

<b>Background and Objective:</b> Safflower (<i>Carthamus tinctorius</i> Linn.) is one of the medicinal plants that contain secondary metabolites that have the potential to as anti-cancer by inducing apoptosis. This study aims to determine the content of secondary metabolite compounds and the induction activity of apoptosis from ethanol extract of safflower in the T47D breast cancer cell line. <b>Materials and Methods:</b> Safflower was extracted using 96% ethanol and assayed for phytochemical screening, cytotoxic tests by cell counting kit-8 to determine inhibitory concentration and apoptosis induction activity by flow cytometry to determine the ability of samples induce the programmed cell cancer in death. The data collected was analyzed with the PRISM GraphPad version. <b>Results:</b> The ethanol extract of safflower contains flavonoid compounds, alkaloids, saponins, tannins and terpenoids. The results of the anticancer activity test showed an IC₅₀ value of 479 µg mL¹ and the best percentage of apoptosis at a concentration of 200 µg mL¹ was 16.61% at the beginning of apoptosis and 10.52% at the end of apoptosis. <b>Conclusion:</b> The safflower can be developed as a breast anticancer agent that works through the induction of apoptosis to improve the effectiveness of breast cancer treatment.

Enhancing Ezetimibe Anticancer Activity Through Development of Drug Nano-Micelles Formulations: A Promising Strategy Supported by Molecular Docking.

Background: Ezetimibe, initially recognized as a cholesterol-lowering agent, has recently attracted attention due to its potential anticancer properties. We aimed to explore an innovative approach of enhancing the drug anticancer activity through the development of drug nano-formulations. Materials and Methods: Fifteen different nano-micelles formulations were prepared utilizing D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and pluronic F127. The prepared formulations were characterized for size, polydispersity index (PDI), zeta potential, and entrapment efficiency (EE). The formulations were morphologically characterized using light and transmission electron microscopies and the drug-binding mode with the active site was investigated using the molecular docking. Cell viability against MCF-7 and T47D was studied. Apoptosis and cell cycle were assessed. Results: The prepared formulations were in the nano-size range (34.01 ± 2.00-278.34 ± 9.11 nm), zeta potential values were very close to zero, and the TPGS-based micelles formulations showed the highest ezetimibe EE (94.03 ± 1.71%). Morphological study illustrated a well-defined, spherical nanoparticles with a uniform size distribution. Molecular docking demonstrated good interaction of ezetimibe with Interleukin-1 Beta Convertase through multiple hydrogen bonding, covalent bond, and hydrophobic interaction. TPGS-based nano-micelle formulation (F5) demonstrated the lowest IC50 against MCF-7 (4.51 µg/mL) and T47D (8.22 µg/mL) cancer cells. When T47D cells were treated with IC50 concentrations of F5, it exhibited significant inhibition with late apoptosis (43.9%), a response comparable to T47D cells treated with an IC50 dose of ezetimibe. Cell cycle analysis revealed that both ezetimibe and F5-treated T47D cells exhibited an increase in the subG1 phase, indicating reduced DNA content and cell death. Conclusion: These findings suggest that F5 could serve as a proficient drug delivery system in augmenting the cytotoxic activity of ezetimibe against breast cancer.

Cell-cycle arrest and mitochondria-dependent apoptosis induction in T-47D cells by the capsular polysaccharide from the marine bacterium Kangiella japonica KMM 3897.

In this study, we reported the in vitro mechanisms of antiproliferative activity of capsular polysaccharide derived from marine Gram-negative bacteria Kangiella japonica KMM 3897 in human breast сarcinoma T-47D cells. Flow cytometric and Western blot analysis revealed that capsular polysaccharide effectively suppressed T-47D cell proliferation by inducing G0/G1 phase arrest and mitochondrial-dependent apoptosis. Moreover, polysaccharide influenced the ERK1/2 and p38 signaling pathways. The results of this study would enrich our understanding of the molecular mechanism of the anti-cancer activity of sulfated polysaccharides from marine Gram-negative bacteria.

Meta-analysis of integrated ChIP-seq and transcriptome data revealed genomic regions affected by estrogen receptor alpha in breast cancer.

BACKGROUND: The largest group of patients with breast cancer are estrogen receptor-positive (ER+) type. The estrogen receptor acts as a transcription factor and triggers cell proliferation and differentiation. Hence, investigating ER-DNA interaction genomic regions can help identify genes directly regulated by ER and understand the mechanism of ER action in cancer progression. METHODS: In the present study, we employed a workflow to do a meta-analysis of ChIP-seq data of ER+ cell lines stimulated with 10 nM and 100 nM of E2. All publicly available data sets were re-analyzed with the same platform. Then, the known and unknown batch effects were removed. Finally, the meta-analysis was performed to obtain meta-differentially bound sites in estrogen-treated MCF7 cell lines compared to vehicles (as control). Also, the meta-analysis results were compared with the results of T47D cell lines for more precision. Enrichment analyses were also employed to find the functional importance of common meta-differentially bound sites and associated genes among both cell lines. RESULTS: Remarkably, POU5F1B, ZNF662, ZNF442, KIN, ZNF410, and SGSM2 transcription factors were recognized in the meta-analysis but not in individual studies. Enrichment of the meta-differentially bound sites resulted in the candidacy of pathways not previously reported in breast cancer. PCGF2, HNF1B, and ZBED6 transcription factors were also predicted through the enrichment analysis of associated genes. In addition, comparing the meta-analysis results of both ChIP-seq and RNA-seq data showed that many transcription factors affected by ER were up-regulated. CONCLUSION: The meta-analysis of ChIP-seq data of estrogen-treated MCF7 cell line leads to the identification of new binding sites of ER that have not been previously reported. Also, enrichment of the meta-differentially bound sites and their associated genes revealed new terms and pathways involved in the development of breast cancer which should be examined in future in vitro and in vivo studies.

A New Diterpenoid of Indonesian Scoparia dulcis Linn: Isolation and Cytotoxic Activity against MCF-7 and T47D Cell Lines.

Scoparia dulcis Linn plays an important role in treatment because it contains active compounds that are proven to have a variety of activities, including cytotoxicity on various cancer cells. The objective of this study is to isolate and identify the cytotoxic compounds in the ethyl acetate fraction of Scoparia dulcis, observe cell cycle inhibition and induction of apoptosis in vitro, and carry out molecular studies using in silico studies. A new diterpene compound was isolated from the ethyl acetate fraction of Scoparia dulcis L. of Indonesian origin. Chromatographic methods were used to isolate the compound, spectroscopic methods were used to elucidate its structure, and these data were compared with those reported in the literature. The compound was tested for its cytotoxic activity against two breast cancer cells (MCF-7 and T47D). The results of the isolated compound showed a cytotoxic effect on MCF-7 and T47D breast cancer cells at IC50 70.56 ± 1.54 and <3.125 ± 0.43 µg/mL, respectively. The compound inhibited the growth of MCF-7 and T47D breast cancer cells and the accumulation of cells in the G1 phases, and it induced apoptosis. Based on a spectroscopic analysis, the isolated compound was identified as 2α-hydroxyscopadiol, which is a new diterpenoid. A docking study revealed that the isolate's hydroxyl groups are essential for interacting with crucial residues on the active sites of the ER and PR and caspase-9. The isolate inhibits ER and PR activity with binding energies of -8.2 kcal/mol and -7.3 kcal/mol, respectively. In addition, the isolate was also able to induce apoptosis through the activation of the caspase-9 pathway with an affinity of -9.0 kcal/mol. In conclusion, the isolated compound from S. dulcis demonstrated anticancer activity based on in vitro and in silico studies.

Complete pathological response of hormone receptor positive invasive breast cancer in a patient with multiple myeloma treated with ixazomib.

Multiple myeloma is a hematological cancer characterized by relapse after treatment and poor prognosis. Ixazomib, a second-generation protease inhibitor, is one of the most recently available treatments for relapsed or refractory multiple myeloma, while it has also shown good potential as antitumoral agent in preclinical solid tumor models such as breast cancer cell lines. Here we report the case of a 68-year-old female with multiple myeloma and an incidental cT1b (9 mm) hormone receptor positive breast cancer lesion that showed a complete pathological response to a three-month combination therapy with Ixazomib, bendamustine and dexamethasone and no signs of disease relapse during the later follow-up. This is the first case report describing such clinical outcome in breast cancer following Ixazomib, bendamustine and dexamethasone combination therapy. To investigate the potential antitumoral activity of Ixazomib in breast cancer, we performed in vitro experiments using two hormone receptor positive breast cancer cell lines. We assessed the synergism between Ixazomib and bendamustine and the antiproliferative effect of Ixazomib. We found no synergistic interaction between the two drugs, while Ixazomib alone showed an antiproliferative effect against tumoral cells, suggesting that this drug has been responsible for tumor regression in our case.

Comparative Evaluation of Lipofectamine and Dendrimer for Transfection of Short RNA Into Human T47D and MCF-10A Cell Lines.

Purpose: Non-viral transfection approaches are extensively used in cancer therapy. The future of cancer therapy lies on targeted and efficient drug/gene delivery. The aim of this study was to determine the transfection yields of two commercially available transfection reagents (i.e. Lipofectamine 2000, as a cationic lipid and PAMAM G5, as a cationic dendrimer) in two breast cell lines: cancerous cells (T47D) and non-cancerous ones (MCF-10A). Methods: We investigated the efficiencies of Lipofectamine 2000 and PAMAM G5 for transfection/delivery of a labeled short RNA into T47D and MCF-10A. In addition to microscopic assessments, the cellular uptakes of the complexes (fluorescein tagged-scrambled RNA with Lipofectamine or PAMAM dendrimer) were quantified by flow cytometry. Furthermore, the safety of the mentioned reagents was assessed by measuring cell necrosis through the cellular PI uptake. Results: Our results showed significantly better efficiencies of Lipofectamine compared to PAMAM dendrimer for short RNA transfection in both cell types. On the other hand, MCF-10A resisted more than T47D to the toxicity of higher concentrations of the transfection reagents. Conclusion: Altogether, our research demonstrated a route for comprehensive epigenetic modification of cancer cells and depicted an approach to efficient drug delivery, which eventually improves both short RNA-based biopharmaceutical industry and non-viral strategies in epigenetic therapy.

BRK confers tamoxifen-resistance in breast cancer via regulation of tyrosine phosphorylation of CDK1.

Tamoxifen (Tam) has been the first-line therapy for estrogen receptor-positive breast cancer since its FDA-approval in 1998. Tam-resistance, however, presents a challenge and the mechanisms that drive it have yet to be fully elucidated. The non-receptor tyrosine kinase BRK/PTK6 is a promising candidate as previous research has shown that BRK knockdown resensitizes Tam-resistant breast cancer cells to the drug. However, the specific mechanisms that drive its importance to resistance remain to be investigated. Here, we investigate the role and mechanism of action of BRK in Tam-resistant (TamR), ER+, and T47D breast cancer cells using phosphopeptide enrichment and high throughput phopshoproteomics analysis. We conducted BRK-specific shRNA knockdown in TamR T47D cells and compared phosphopeptides identified in these cells with their Tam-resistant counterpart and parental, Tam-sensitive cells (Par). A total of 6492 STY phosphosites were identified. Of these sites, 3739 high-confidence pST sites and 118 high-confidence pY sites were analyzed for significant changes in phosphorylation levels to identify pathways that were differentially regulated in TamR versus Par and to investigate changes in these pathways when BRK is knocked down in TamR. We observed and validated increased CDK1 phosphorylation at Y15 in TamR cells compared to BRK-depleted TamR cells. Our data suggest that BRK is a potential Y15-directed CDK1 regulatory kinase in Tam-resistant breast cancer.

Cowanin induces apoptosis in breast cancer cells via Bcl-2 signaling pathway.

OBJECTIVES: This study aimed to investigate the effect of cowanin the mechanism of cowanin toward cell death and BCL-2 protein (antiapoptotic) expression of T47D breast cancer. METHODS: The cell death was evaluated by double staining, namely acridine orange and propidium iodide, and then observed under a fluorescence microscope. Meanwhile, the BCL-2 protein expression was determined by western blotting with measurement of protein area and protein density. RESULTS: The result found T47D breast cancer cells were viable, apoptosis, and necrosis after treatment with cowanin. The average viable cells, apoptosis, and necrosis percentages were 54.13 %, 45.43 %, and 0.44 %, respectively. Statistical analysis showed cowanin could significantly induce death in T47D breast cancer cells by apoptosis (p<0.05). It was also revealed that cowanin and positive control (doxorubicin) treatment had a significantly decreased protein area and protein density (p<0.05). CONCLUSIONS: It can be concluded that cowanin can induce death in T47D breast cancer cells by apoptosis and affect the expression of Bcl-2 protein in T47D breast cancer cells.

Comparison of Three Low-Molecular-Weight Fluorescent Probes for Measuring Free Zinc Levels in Cultured Mammary Cells.

Free zinc is a critical regulator in signal transduction and affects many cellular processes relevant to cancer, including proliferation and cell death. Acting as a second messenger, altered free intracellular zinc has fundamental effects on regulating enzymes such as phosphatases and caspases. Therefore, the determination of free intracellular zinc levels is essential to assess its influence on the signaling processes involved in cancer development and progression. In this study, we compare three low-molecular-weight fluorescent probes, ZinPyr-1, TSQ, and FluoZin-3, for measuring free zinc in different mammary cell lines (MCF10A, MCF7, T47D, and MDA-MB-231). In summary, ZinPyr-1 is the most suitable probe for free Zn quantification. It responds well to calibration based on minimal fluorescence in the presence of the chelator TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) and maximal fluorescence by saturation with ZnSO4, resulting in the detection of free intracellular zinc in breast cancer subtypes ranging from 0.62 nM to 1.25 nM. It also allows for measuring the zinc fluxes resulting from incubation with extracellular zinc, showing differences in the zinc uptake between the non-malignant MCF10A cell line and the other cell lines. Finally, ZinPyr-1 enables the monitoring of sub-cellular distributions by fluorescence microscopy. Altogether, these properties provide a basis for the further exploration of free zinc in order to realize its full potential as a possible biomarker or even therapeutic target in breast cancer.

Synergistic actions of Alpelisib and Melatonin in breast cancer cell lines with PIK3CA gene mutation.

AIMS: Breast cancer (BC) presents high mortality rate and about 25-46 % have mutation in the PIK3CA gene. Alpelisib is a PI3K inhibitor that acts on p110α, which is a subunit of the PI3K protein. The melatonin shown important anti-neoplastic effects and may increase the effectiveness of chemotherapy. This study evaluated the synergistic action of Alpelisib and Melatonin in BC lines carrying the H1047R mutation in PIK3CA, relative to the cellular dynamics and the PI3K/AKT/mTOR pathway. MAIN METHODS: MDA-MB-468 (triple-ernegative), MDA-MB-453 (H1047R PIK3CA, HER2+) and T-47D cells (H1047R PIK3CA, ER+/PR+) were divided into four treatment groups: control; Melatonin (1 mM); Alpelisib (1 µM); and Alpelisib (1 µM) + Melatonin (1 mM). Cell viability and migration were investigated using the MTT assay and Transwell assay, respectively. Protein expression of PI3K, p-AKT, mTOR, HIF-1α, and caspase-3, was verified using immunocytochemistry. KEY FINDINGS: MTT assay revealed that MDA-MB-453 and T-47D showed reduction in cell viability in all groups, especially in the MDA-MB-453 treated with Melatonin + Alpelisib. MDA-MB-468 presents reduction in cell migration only with Melatonin, while in the lines with mutation, the treatment of Melatonin + Alpelisib caused inhibition of cell migration. PI3K, p-AKT, mTOR and HIF-1α were inhibited after treatment with Melatonin + Alpelisib in MDA-MB-453 and T-47D lines. The expression of caspase-3 increased in all groups in MDA-MB-453 and T-47D cells, being the increase more pronounced in the Melatonin + Alpelisib group. SIGNIFICANCE: These results indicate that the combined use of Melatonin and Alpelisib may be more effective in inhibiting BC in women carrying the PIK3CA gene mutation than either treatment alone.

Tunable femtosecond laser suppresses the proliferation of breast cancer in vitro.

Worldwide, the most frequently diagnosed cancer is female breast cancer, and it poses a serious global health threat. Traditional cancer therapies are associated with various side effects, so developing better therapies for breast cancer is necessary, such as laser therapy which could be a promising treatment option. The aim of the current study was to investigate the femtosecond laser irradiation effect on breast cancer using T47D cell line as an in vitro model. Cells were seeded at a density of 5 × 104 cells/well in 96-well plates and incubated overnight. After that, the cells were exposed to femtosecond laser irradiation at various wavelengths falling in the UV, visible, and IR ranges for 3, 5, or 10 min and at a constant power of 100 mW. Cell viability was measured directly and 24 h after femtosecond laser irradiation using MTT assay. When using different femtosecond laser irradiation parameters, especially the 380 and 400 nm femtosecond laser irradiation, there was significant inhibition of breast cancer cell growth, either directly or 24 h after femtosecond laser exposure. Also, 420 and 440 nm significantly affected the viability of the cells. It was also observed that increasing exposure time enhances the observed effect, so 10 min exposure time was the best time of exposure. However, 700, 720, 750, and 780 nm did not significantly affect the cells viability with different exposure times. It was possible to conclude from the aforementioned results that femtosecond laser irradiation exerted a significant anticancer effect against T47D cells. Consequently, the femtosecond laser could be used successfully for breast cancer management.

Comparison of Radiation Response between 2D and 3D Cell Culture Models of Different Human Cancer Cell Lines.

Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines (PC-3, LNCaP and T-47D) irradiated with varying doses (1, 2, 4, 6, 8 or 20 Gy) of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays (e.g., clonogenic assay, resazurin assay) and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models.