T47D Breast Cancer Cells Research Articles

Discovery of novel tris-1,2,3-triazole-based hybrids as VEGFR2 inhibitors with potent anti-proliferative and cytotoxicity through apoptosis induction.

The discovery of novel anti-cancer drugs motivated us to synthesize a new series of triple 1,2,3-triazole-based arm scaffolds featuring distinct un functionalized alkyl and/or aryl side chains with possible anti-cancer action using the click chemistry approach under both conventional and green microwave irradiation (MWI) methods. The Cu(I) catalyzed cycloaddition reaction of targeted tris-alkyne with un functionalized aliphatic and aromatic azides has been adopted as an efficient approach for synthesizing the desired click adducts. Microwave irradiation improved the synthetic processes, resulting in higher yields and faster reaction times. Spectroscopic techniques (FT-IR, 1H, 13C NMR andCHN analysis) were used for the elucidation of the resulting click structures. The newly synthesized tris-1,2,3-triazoles exhibited promising cytotoxicity, particularly compounds 26 and 28, with IC50 values of 22.18µM and 20.3µM against A549 and CaCo-2 cells, respectively. While they had IC50 values of 23.06µM and 21.91µM against T-47D and CaCo-2 cells, respectively. Both compounds exhibited promising anti-proliferative activity through the wound healing assay. Additionally, both compounds induced total apoptotic cell death by 68.3% and 58.5%, respectively, compared to untreated cells (7.7%). Furthermore, they induced necrotic cell death by 1.4% and 10.5%, respectively, compared to 0.1% in the untreated cells. For the molecular target, compounds 26 and 28 exhibited potent VEGFR2 inhibition with IC50 values of 35.5nM and 27.8nM, respectively, and this was highlighted through the molecular docking findings. Tris-1,2,3-triazoles (26 and 28) exhibited promising cytotoxicity and anti-proliferative against T-47D breast cancer cells through apoptosis and VEGFR2 inhibition using both enzyme kit and western blotting protein expression assays. Molecular docking study highlighted the binding affinity of tested compounds towards the VEGFR2 protein. Accordingly, tris-1,2,3-triazoles (26 and 28) can be further developed as more potent anti-cancer agents.

Insulin-like growth factor binding protein-6 modulates proliferative antagonism in response to progesterone in breast cancer.

Breast cancer is one of the most diagnosed cancers worldwide. The insulin-like growth factor (IGF) system promotes proliferation and survival in breast cancer cells and is regulated by 6 insulin-like growth factor binding proteins (IGFBPs). The IGFBPs sequester IGFs to prolong their half-life and attenuate binding to insulin-like growth factor 1 receptor (IGF1R). While IGFBP-6 has been studied in some cancers it has not been studied extensively in hormone receptor positive breast cancer. Survival analysis using available databases indicated that high IGFBP-6 levels improve overall survival in progesterone receptor positive breast cancers. IGFBP-6 is transcriptionally induced by progesterone in T47D breast cancer cells resulting in increased intracellular and extracellular IGFBP-6 protein. Knockdown of IGFBP-6 resulted in reduced proliferative antagonism when estradiol stimulated T47D cells were cotreated with progesterone and protein levels of both progesterone receptor isoforms (PR-A and PR-B) were decreased following knockdown of IGFBP-6. P21(Cip1/Waf1), which is progesterone responsive, was not induced in response to progesterone following knockdown of IGFBP-6. Cyclin E2, a cell cycle regulator, is induced by progesterone only when IGFBP-6 is knocked down. Stable overexpression of IGFBP-6 in MCF-7 cells resulted in an increase in Epidermal Growth Factor Receptor (EGFR) and this expression was further enhanced when cells were cotreated with progesterone and estradiol. These results indicate that IGFBP-6 is a regulator of progesterone action, and that PR is required for the observed protective effects of IGFBP-6 in breast cancer.

Deubiquitinating enzymes in breast cancer: in silico analysis of gene expression and metastatic correlation

Breast cancer, the most prevalent cancer in females, is a heterogeneous disease with various molecular subtypes, which presents challenges in diagnosis and treatment. Ubiquitination is one of the most critical post-translational protein modifications, that plays regulatory roles in numerous cellular processes including cell cycle progression, DNA replication & repair, apoptosis, transcription regulation, protein localization, trafficking and signal transduction. This modification can be reversed by deubiquitinases, or DUBs, a superfamily of cysteine proteases and metalloproteases that cleave ubiquitin-protein bonds. Dysregulation of DUBs has been associated to various diseases including cancer, making them promising targets for cancer therapy. We leveraged publicly available breast cancer datasets and employed various bioinformatics tools to identify differentially expressed DUBs in breast cancer. Our analysis identified six genes (COPS5, EIF3H, MINDY1, MINDY2, PSMD14 and USP26) with significant differential expression and survival implications. We further validated our findings experimentally and found upregulation of COPS5, EIF3H and MINDY 1 in MCF-7 and T47D breast cancer cell lines using qPCR analysis. To identify the role of these genes, EIF3H and COPS5, in disease progression, we constructed a protein-protein interaction (PPI) network with genes associated with metastasis and explored their correlation at the gene expression level in breast cancer patients. Together, this comprehensive study sheds light on DUB gene expression patterns in breast cancer with the potential to identify novel targets for therapeutic interventions.

Abstract PR002: Hypoxia-induced epigenetic changes coordinate transcription start site selection and translational control through remodeling of 5’UTRs

Abstract Adaptation to hypoxia enhances cancer cell malignancy and negatively impacts patient outcomes. Reprogramming of mRNA translation is an essential component of the hypoxia-adaptive response. During hypoxia, cells undergo a global decrease in protein synthesis. However, translation of subsets of transcripts encoding stress-response, survival and stemness factors is increased. This selective regulation is thought to be governed largely via the interactions between specific mRNA features and remodeling of the translational apparatus. However, our understanding of the mechanisms that govern translational perturbations under hypoxia remains incomplete. Here, we interrogated the effects of hypoxia on global changes in histone methylation and transcription start site (TSS) selection, coupled with monitoring corresponding alterations in total mRNA levels and translation efficiencies on a genome-wide scale in T47D breast cancer cells and H9 human embryonic stem cells. This revealed widespread epigenetic alterations leading to TSS switching and extensive remodeling of 5’UTRs, and allowed us to map the impact of specific 5’UTR features on translation efficiency under hypoxia. Pyruvate Dehydrogenase Kinase 1 (PDK1), a key enzyme in orchestrating metabolic adaption to hypoxia, undergoes TSS switching leading to increased availability of efficiently translated 5’UTR isoforms, and we show that this effect is coordinated at an epigenetic level. Therefore, our findings uncover a mechanism driving translational reprogramming under hypoxia, whereby alterations in translational apparatus are orchestrated with epigenetic perturbations that result in 5’UTR remodeling of the transcriptome. Citation Format: Kathleen Watt, Bianca Dauber, Krzysztof J Szkop, Laura Lee, Predrag Jovanovic, Shan Chen, Laia Masvidal, Kristofersson Tandoc, Ranveer Palia, Ivan Topisirovic, Ola Larsson, Lynne-Marie Postovit. Hypoxia-induced epigenetic changes coordinate transcription start site selection and translational control through remodeling of 5’UTRs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR002.

P21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2.

Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK.

Evaluation of Xanthone and Cinnamoylbenzene as Anticancer Agents for Breast Cancer Cell Lines through In Vitro and In Silico Assays

Breast cancer is a severe global disease for women as the number of deaths increases annually. Therefore, attempts to find new anticancer agents are critical and inevitable. In this work, we report the investigation on the anticancer activity of xanthone and cinnamoylbenzene compounds against two breast cancer cell lines, i.e., T47D and MCF-7, through experimental in vitro and theoretical in silico assays. Xanthone and cinnamoylbenzene exhibit anticancer activity with a half-maximal inhibitory concentration (IC50) of 136.7–194.3 and 235.8–262.4 µg/mL against T47D and MCF-7 cancer cells, respectively. Cinnamoylbenzene generates less cytotoxicity to normal Vero cells with a selectivity index of 1.095–2.102. The molecular docking studies agree with the experimental data in which cinnamoylbenzene is more active against T47D with an IC50 of 136.7 µg/mL due to Topoisomerase II inhibition through π-π stacked interactions with Adenine12 and Guanine13 nitrogen bases. Meanwhile, xanthone is more active against MCF-7 with an IC50 of 235.8 µg/mL due to EGFR inhibition through van der Waals interaction and hydrogen bond with Glutamic acid767 and Methionine769 amino acid residues, respectively. Additionally, the pharmacokinetic parameters of xanthone and cinnamoylbenzene are predicted through absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, and they show better suitability than doxorubicin as the commercial anticancer drug.

“Sustainable synthesis of Camellia sinensis-mediated silver nanoparticles (CsAgNP) and their anticancer mechanisms in breast cancer cells”

The present investigation focuses on synthesizing eco-friendly and cost-effective silver nanoparticles (CsAgNP) utilizing Camellia sinensis ethanolic extract (CsE) as a reducing agent and investigating the potential enhancement in its anticancer efficacy as compared to CsE. The CsAgNP formation was confirmed through the color change from pale green to dark brown and further validated using UV–visible spectroscopy in the 400-450 nm range. The optimal CsAgNP synthesis parameters include 1:4 ratio of CsE: 1 mM AgNO3, 60 min of duration and 50 °C reaction temperature. The morphology and the size of nanoparticles were estimated using AFM, SEM and TEM where the results showed a smooth topography with a size <100 nm. The CsAgNP crystalline form was confirmed through SAED pictures and silver's presence confirmed through EDX analysis. FTIR study ascertained the capping agents and distortion in functional groups compared to CsE. The anticancer potency of CsAgNP and crude extract (CsE) was assessed against the T-47D breast cancer cells by MTT assay. CsAgNP displayed strong activity towards T-47D cells (IC50 8 μg/ml) compared to CsE and relatively low activity towards the normal HEK-293 cells. Further, fluorescence microscopy and flow cytometry data revealed that the CsAgNP promotes apoptosis and also induces G2-M phase cell cycle arrest. Furthermore, CsAgNP treatment decreases p53 and Bcl-2 protein expression, while increasing Bax, Cytochrome c and Caspase-3 levels, indicating mitochondrial-mediated apoptotic pathway activation. Thus, our research aims to investigate the potential of using Camellia sinensis to synthesize CsAgNP, a potent drug delivery system, to enhance anticancer effectiveness and advance cancer therapy in the future.

Sodium arsenite-induced DNA methylation alterations exacerbated by p53 knockout in MCF7 cells

Epigenetic alterations are ubiquitous across human malignancies. Thus, functional characterization of epigenetic events deregulated by environmental pollutants should enhance our understanding of the mechanisms of carcinogenesis and inform preventive strategies. Recent reports showing the presence of known cancer-driving mutations in normal tissues have sparked debate on the importance of non-mutational stressors potentially acting as cancer promoters. Here, we aimed to test the hypothesis that the presence of mutations in p53, a commonly mutated gene in human malignancies, may influence cellular response to an environmental non-mutagenic agent, potentially involving epigenetic mechanism. We used the CRISPR-Cas9 system to generate knockouts of p53 in MCF7 and T47D breast cancer cell lines and characterized DNA methylome changes by targeted pyrosequencing and methylome-wide Infinium MethylationEPIC BeadChip arrays after exposure to sodium arsenite, a well-established human carcinogen with documented effects on the epigenome. We found that the knockout of p53 alone was associated with extensive alterations in DNA methylation content, with predominant CpG hypermethylation concurrent with global demethylation, as determined by LINE-1 repetitive element pyrosequencing. While exposure to sodium arsenite induced little to no effects in parental cell lines, mutant cells, upon treatment with sodium arsenite, exhibited a markedly altered response in comparison to their wild-type counterparts. We further performed genome regional analyses and found that differentially methylated regions (DMRs) associated with exposure to sodium arsenite map to genes involved in chromatin remodeling and cancer development. Reconstitution of wild-type p53 only partially restored p53-mutant-specific differential methylation states in response to sodium arsenite exposure, which may be due to the insufficient reconstitution of p53 function, or suggestive of a potential exposure-specific epigenetic memory. Together, our results revealed wide-spread epigenetic alterations associated with p53 mutation that influence cellular response to sodium arsenite exposure, which may constate an important epigenetic mechanism by which tumour promoting agents synergize with driver mutations in cancer promotion.

Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells

ABSTRACT The estrogen signalling pathway is highly dynamic and primarily mediated by estrogen receptors (ERs) that transcriptionally regulate the expression of target genes. While transcriptional functions of ERs have been widely studied, their roles in RNA biology have not been extensively explored. Here, we reveal a novel biological role of ER alpha (ERα) in mRNA 3’ end processing in breast cancer cells, providing an alternative mechanism in regulating gene expression at the post-transcriptional level. We show that ERα activates poly(A) specific ribonuclease (PARN) deadenylase using in vitro assays, and that this activation is further increased by tumour suppressor p53, a factor involved in mRNA processing. Consistent with this, we confirm ERα-mediated activation of nuclear deadenylation by PARN in samples from MCF7 and T47D breast cancer cells that vary in expression of ERα and p53. We further show that ERα can form complex(es) with PARN and p53. Lastly, we identify and validate expression of common mRNA targets of ERα and PARN known to be involved in cell invasion, metastasis and angiogenesis, supporting the functional overlap of these factors in regulating gene expression in a transactivation-independent manner. Together, these results show a new regulatory mechanism by which ERα regulates mRNA processing and gene expression post-transcriptionally, highlighting its contribution to unique transcriptomic profiles and breast cancer progression.

Synthesis, structures, and cytotoxicity insights of organotin(IV) complexes with thiazole-appended pincer ligand

Diorganotin complexes of the compositions [Me2Sn(L)] (1), [n-Bu2Sn(L)] (2), [Ph2Sn(L)]⋅C6H6 (3), [Bz2Sn(L)]⋅C6H6 (4) and [n-Oct2Sn(L)] (5) were synthesized by reacting R2SnO (R = Me, n-Bu, Ph, Bz or n-Oct) with the N2,N6-di(thiazol-2-yl)pyridine-2,6-dicarboxamide (H2L, where H2 denotes the two acidic protons) in refluxing toluene. Additionally, the mono-n-butyltin complex [n-BuSn(HL)Cl2]·H2O (6) was synthesized from n-BuSnCl3 and H2L in acetonitrile. Compounds were characterized by FT-IR, 1H, 13C and 119Sn NMR spectroscopy, while their solid-state structures were examined using single-crystal X-ray diffraction studies. In diorganotin compounds 1–5, the dianionic tridentate ligands (Npy, N−, N−) act as κ-N3 chelators. In 6, the L moiety (O, Npy, N−) acts as a κ-ON2 tridentate chelator, with involvement of one of the carboxamide oxygen atoms. The coordination polyhedron around the Sn(IV) ion is completed either by two axial Sn-R ligands in compounds 1–5 or by n-Bu and Cl ligands in compound 6, giving rise to distorted trigonal bipyramid or octahedral structures, respectively. The tin NMR results show that the penta-coordinated structures of compounds 1–5 and the hexacoordinated structure of compound 6, observed in the solid-state, are retained in solution. The in vitro antitumor activities of 1–5 were tested on T-47D breast cancer cells. Of these, diphenyltin compound 3 showed the highest anti-proliferative effect, with an IC50 of 10 ± 1.60 μM. Compound 3 exhibited selective toxicity, potentially inducing apoptosis via reactive oxygen species generation and nuclear changes, indicating promise as a breast cancer treatment. This study is the first to explore thiazole-appended organotin compounds for cytotoxicity.

Gastrin-releasing peptide receptor as theranostic target in estrogen-receptor positive breast cancer: A preclinical study of the theranostic pair [55Co]Co- and [177Lu]Lu-DOTA-RM26

BackgroundPatients with advanced metastatic estrogen receptor-positive breast cancer often develop resistance to standard treatments, leading to uncontrolled progression. Thus, innovative therapies are urgently needed. The gastrin-releasing peptide receptor (GRPR) is overexpressed in various cancers, including breast cancer, making it an interesting theranostic target. RM26, a GRPR-targeting antagonist, has demonstrated promising in vivo kinetics in prostate cancer models. This study evaluated the theranostic capabilities of [55Co]Co−/[177Lu]Lu-DOTA-RM26 in vitro in estrogen receptor-positive breast cancer cells and assessed the diagnostic potential of [55Co]Co-DOTA-RM26 in vivo in a breast cancer mouse model. MethodsWe analyzed the binding specificity of [57Co]Co-/[177Lu]Lu-DOTA-RM26 in T47D breast cancer cells, using [57Co]Co-DOTA-RM26 as a surrogate for [55Co]Co-DOTA-RM26. The therapeutic efficacy of increasing [177Lu]Lu-DOTA-RM26 concentrations was determined via viability assay in vitro. Ex vivo biodistribution of [57Co]Co-DOTA-RM26 (17.2 ± 2.7 kBq, 33 ± 5.2 pmol/mouse) was investigated in 12 mice (n= 4/group) with orthotopic breast cancer tumors. The mice were sacrificed at 4 and 24 h post-injection (pi), including a blocking group (20 nmol of unlabeled [Tyr4]-Bombesin) at 4 h pi. For imaging, two tumor-bearing mice underwent [55Co]Co-DOTA-RM26 PET/CT, 4 and 24 h pi (2.8 ± 0.2 MBq, 167.5 ± 0.5 pmol/mouse), with or without GRPR blocking. ResultsIn vitro studies revealed high, specific binding of [57Co]Co-DOTA-RM26 (43 ± 1 % of total added activity per 106 cells (%IA/106)) and [177Lu]Lu-DOTA-RM26 (37 ± 4 %IA/106). The activity was predominantly localized at the cell surface: 71 ± 3 % and 80 ± 6 % for [57Co]Co-DOTA-RM26 and [177Lu]Lu-DOTA-RM26, respectively. [177Lu]Lu-DOTA-RM26 significantly reduced cell viability at all activity concentrations >0.625 MBq/mL (p < 0.0001), with cell viability below 1 % at concentrations ≥5 MBq/mL. Biodistribution data (n = 12) indicated a high, specific tumor uptake of [57Co]Co-DOTA-RM26, surpassing all other tissues significantly at both time points, 3.7 ± 0.6 % of the injected activity per gram (%IA/g) 4 h pi and 0.98 ± 0.05 %IA/g 24 h pi. The kidneys showed the second-highest uptake (2.0 ± 0.1 %IA/g 4 h pi), followed by the pancreas (1.4 ± 0.4 %IA/g 4 h pi). PET/CT imaging with [55Co]Co-DOTA-RM26 supported the biodistribution data and, distinctly visualized the tumor 24 h pi and showed an improved tumor-to-background compared to the earlier time points. Effective GRPR blocking significantly reduced tumor uptake in the PET images 24 h pi. ConclusionThese findings suggest that the theranostic pair [55Co]Co−/[177Lu]Lu-DOTA-RM26 holds significant promise as a theranostic agent for estrogen receptor-positive breast cancer.

Exosomal Delivery Enhances the Antiproliferative Effects of Acid-Hydrolyzed Apiaceae Spice Extracts in Breast Cancer Cells.

Breast cancer remains a leading cause of death worldwide. The Apiaceae plant family includes many culinary spices that have been shown to have medicinal properties. Many phytochemicals exhibit potent bioactivities but often suffer from poor uptake and oral bioavailability. Bovine milk and colostrum exosomes are a compelling drug delivery platform that could address this issue; these natural nanoparticles can be loaded with hydrophilic and lipophilic small molecules and biologics, resulting in lower doses needed to inhibit cancer growth. Ethanolic extracts of eight Apiaceae spices were examined for phytochemical content and antiproliferative potential. Acid hydrolysis (AH) was employed to remove glycosides, asses its impacts on extract efficacy, and evaluate its effects on exosome loading and subsequent formulation efficacy. Antiproliferative activity was assessed through MTT assays on T-47D, MDA-MB-231, and BT-474 breast cancer cells; all extracts exhibited broad antiproliferative activity. AH enhanced the bioactivity of cumin, caraway, and fennel in T-47D cells. Celery, cumin, anise, and ajwain showed the highest activity and were assayed in exosomal formulations, which resulted in reduced doses required to inhibit cellular proliferation for all extracts except AH-cumin. Apiaceae spice extracts demonstrated antiproliferative activities that can be improved with AH and further enhanced with exosomal delivery.

Uncaria nervosa Elmer, a new herbal source for betulinic acid and ursolic acid: Metabolites profiling, isolation, and in vitro cytotoxicity studies against T47D breast cancer

Abstract* Background Uncaria nervosa Elmer is an Indonesian herbal plant that is traditionally used for breast cancer. The results of phytochemical screening contained alkaloids, flavonoids, and terpenoids in the ethanol extract of this plant. Based on literature searches, reports regarding the bioactive compounds responsible for breast cancer have not been found. Further research is needed to understand the potential of Uncaria nervosa Elmer as a breast cancer treatment and to identify the specific compounds responsible for its effects Methods This study aims to determine the metabolite profiling of ethanol extract, the isolation, characterization of bioactive compounds, and their bioactivity in T47D breast cancer cells. The research began by extracting the leaves by maceration using 70% ethanol, and then solid phase extraction was carried out using the solid phase extraction (SPE) method. In this study, the sorbent used was polyamide. The extract was analyzed using a tandem analysis technique based on LCMS using the MZmine and SIRIUS platforms. Isolation was carried out using column chromatography, and preparative recycling HPLC. Bioactive compounds were characterized using UV, HPLC, NMR, and 2D NMR, as well as bioactivity tests using the MTT method. Results The results show that the extract contained N-[(1,3-dimethyl-2,6-dioxo-7-prop-2-ynylpurin-8-yl) amino] formamide, N-(3-phenylbutyl)hexan-2-amine, 1,1-Dichloro-1-nitrosopropane, ceratodictyol, betulinic acid, ursolic acid, 7-methyl-N-[6-[(7-methyl-6-oxooctanoyl) amino] hexyl]-6-oxononanamide, Nervisterol and 3,5,10-tris (acetyloxy)-2-hydroxy-4,14,16,16-tetramethyl-8-methylidene-13-oxo-15oxatetracyclo [9.4.1.01,14.04,9] hexadecan-7-yl 3-phenylprop-2-enoate. The ethanol extract of Uncaria nervosa Elmer leaves contains nine compounds consisting of alkaloids, terpenoids, and fatty acid. The bioactive compounds that were successfully isolated were betulinic acid, and ursolic acid, with IC50 values of ˃100 and 14,70±4,50 μg/ml, respectively. These compounds were reported in this plant for the first time. Conclusion Betulinic acid, and ursolic acid have been successfully isolated from leaves Uncaria nervosa Elmer, and ursolic acid have moderate cytotoxic activity on T47D breast cancer cells.

Zinc transporter ZnT5 is associated with epithelial mesenchymal transition via SMAD1 in breast cancer.

Zinc levels in breast cancer tissues have been reported to be higher than those in normal tissues. In addition, the expression levels of zinc transporters, including ZnT5 and ZnT6, are reportedly higher in breast cancer than in normal breast tissues. ZnT5 and ZnT6 also contribute to heterodimer formation and are involved in several biological functions. However, the functions of ZnT5 and ZnT6 heterodimers in breast cancer remain unknown. Therefore, we first investigated the immunolocalization of ZnT5 and ZnT6 in pathological breast cancer specimens and in MCF-7 and T-47D breast cancer cells. Next, we used small interfering RNA to assess cell viability and migration in ZnT5 knockdown MCF-7 and T-47D cells. Immunohistochemical analysis showed that the number of ZnT5-positive breast cancer cells was inversely correlated with the pathologic N factor status. ZnT5 knockdown had no effect on cell viability in the presence of 100 μM ZnCl2 in MCF-7 and T-47D cells. In a wound healing assay, 100 μM ZnCl2 treatment inhibited cell migration of MCF-7 and T-47D cells, whereas ZnT5 knockdown promoted cell migration, decreased E-cadherin expression and increased vimentin, slug and matrix metalloproteinase 9 expression. Antibody arrays showed that ZnT5 knockdown increased the expression of SMAD1, and that dorsomorphin treatment inhibited the promotion of migratory ability induced by ZnT5 knockdown. The results of this study revealed that both ZnT5 may be involved in less aggressive breast cancer subtypes, possibly through inhibition of cell migration.

Enhancing photodynamic and radionuclide therapy by small interfering RNA (siRNA)-RAD51 transfection via self-emulsifying delivery systems (SNEDDS)

Background aimsGene-silencing by small interfering RNA (siRNA) is an attractive therapy to regulate cancer death, tumor recurrence or metastasis. Because siRNAs are easily degraded, it is necessary to develop transport and delivery systems to achieve efficient tumor targeting. Self-nanoemulsifying systems (SNEDDS) have been successfully used for pDNA transport and delivery, so they may be useful for siRNA. The aim of this work is to introduce siRNA-RAD51 into a SNEDDS prepared with Phospholipon-90G, Labrafil-M1944-CS and Cremophor-RH40 and evaluate its efficacy in preventing homologous recombination of DNA double-strand breaks caused by photodynamic therapy (PDT) and ionizing radiation (IR). MethodsThe siRNA-RAD51 was loaded into SNEDDS using chitosan. Transfection capacity was estimated by comparison with Lipofectamine-2000. ResultsSNEDDS(siRNA-RAD51) induced gene silencing effect on the therapies evaluated by cell viability and clonogenic assays using T47D breast cancer cells. ConclusionsSNEDDS(siRNA-RAD51) shown to be an effective siRNA-delivery system to decrease cellular resistance in PDT or IR.

Naphthalene Derivates from Bawang Tiwai Bulb Eleutherine Bulbosa in Borneo

Naphthalene derivative compounds named Eleutherol (1) and Eleutherol C (2) have been isolated from Bawang Tiwai Bulb Eleutherine bulbosa. We tested its cytotoxic activity against T47D breast cancer cells in vitro after isolating compounds 1 and 2 from the EtOH extract using conventional chromatography methods. Their chemical structures were elucidated based on spectroscopic analysis, including IR, HR-TOFMS, 1D, and 2D NMR, and by comparison to those related spectra previously reported. Compounds 1 and 2 were tested for their cytotoxic effects against T47D breast cancer cells and showed moderate cytotoxicity against T47D breast cancer cells with IC50 values of 117.15 and 80.21 µM, respectively, compared with cisplatin 24.07 µM.

Estrogen receptor alpha (ER-α) antagonistic activity of phytoconstituents from Potentilla atrosanguinea and Potentilla fulgens in breast cancer

The Potentilla genus has long been used traditionally as food and a folklore medicine. In the present study, aerial parts of two Potentilla species, Potentilla fulgens and Potentilla atrosanguinea, of western Himalayan origin, were studied for their anti-breast cancer activity. Ethyl acetate (PAA-EA, PFA-EA), methanolic (PAA-ME, PFA-ME) and hydro-methanolic extract (PAA-HM, PFA-HM) of the plants were tested for their antiproliferative activities against MCF-7 and T-47D breast cancer cell lines. The extracts showed good antiproliferative activity against ER-α dominant breast cancer cell line T-47D, having IC50 values 6.19 ± 0.01 to 33.23 ± 0.04 μg/ml. Eight compounds were isolated, characterized, and quantified from ethyl acetate and methanolic extracts by column chromatography, 1D, 2D-NMR, HRMS and TLC densitometric analysis. Two compounds (4 and 6) have shown better antiproliferative activity than standard bazedoxifene and were further evaluated for their ER-α binding affinity via—fluorescence polarization-based competitive binding assay. The antiestrogenic properties of both compounds were assessed using western blotting. Compounds 4 and 6 were found to have significant affinity for the ER-α and managed to decrease its expression by 38 and 54% respectively. Compounds 4 and 6 also had good stability and reactivity as measured by minimal fluctuations in molecular dynamic simulation analysis, a good dock score in molecular docking, and a respectable HOMO-LUMO energy gap in DFT calculations. Compounds 4 and 6 have shown reliable results and can be used in the development of natural product-based anti-breast cancer agents.

Extended Synaptotagmins 1 and 2 Are Required for Store-Operated Calcium Entry, Cell Migration and Viability in Breast Cancer Cells.

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1-STIM1 interaction at ER-PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells.

Unravelling the apoptosis induction potential of Amomum cardamomum seed: A combination in silico and in vitro approach

Introduction: Breast cancer remains a prevalent global malignancy and necessitates a treatment regimen, which is often accompanied by substantial side effects. To address this challenge, alternative therapies with fewer adverse effects are urgently needed. Amomum cardamomum has displayed promising anticancer potential. This study aimed to investigate the impact of A. cardamomum seed on T47D breast cancer cell viability and the ability to induce apoptosis, utilizing in silico and in vitro approaches. Methods: The samples were extracted utilizing the maceration method using ethanol 96% solvent. In addition, the bioactive constituents were identified through phytochemicals and GC/MS analysis. Cell viability was assessed through MTT assay at various concentrations with 24 and 48-hour incubation periods and compared with the control cells. Apoptosis patterns were visualized by Immunofluorescence assay and analyzed utilizing ImageJ software. In silico analyses included three distinct tests, namely pharmacokinetics analysis (ADMET), bioactivity prediction (PASS), and molecular docking. Results: The A. cardamomum seed extract inhibited the growth of the cells with an IC50 value of 97.28 μg/mL in 48 hours of the incubation period. Immunofluorescence assay exhibited that the extract induced apoptosis in over 50% of T47D cells. In silico approaches identified bicyclogermacrene, Germacrene-D, and δ-cadinene as potential JAK3, BRAF v600e, and MMP9 protein inhibitors. These compounds exhibited stronger binding affinities to critical amino acids than control ligands. Conclusion: This research presents compelling evidence that the A. cardamomum extract has anticancer activity against breast cancer by preventing growth and inducing apoptosis.

Functional characterization of PI3K C2 domain mutations detected in breast cancer circulating tumor cells and metastatic cells

BackgroundIn breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor. MethodsPIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging. ResultsWestern blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC50 MK2206: 148 nM, IC50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction. ConclusionsOur results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations.