Negative Breast Cancer Cells Research Articles

Phloridzin Docosahexaenoate Inhibits Spheroid Formation by Breast Cancer Stem Cells and Exhibits Cytotoxic Effects against Paclitaxel-Resistant Triple Negative Breast Cancer Cells.

The eradication of cancer stem cells (CSCs) is vital to successful cancer treatment and overall disease-free survival. CSCs are a sub-population of cells within a tumor that are defined by their capacity for continuous self-renewal and recapitulation of new tumors, demonstrated in vitro through spheroid formation. Flavonoids are a group of phytochemicals with potent anti-oxidant and anti-cancer properties. This paper explores the impact of the flavonoid precursor phloridzin (PZ) linked to the ω-3 fatty acid docosahexaenoate (DHA) on the growth of MCF-7 and paclitaxel-resistant MDA-MB-231-TXL breast cancer cell lines. Spheroid formation assays, acid phosphatase assays, and Western blotting were performed using MCF-7 cells, and the cell viability assays, Annexin-V-488/propidium iodide (PI) staining, and 7-aminoactinomycin D (7-AAD) assays were performed using MDA-MB-231-TXL cells. PZ-DHA significantly reduced spheroid formation, as well as the metabolic activity of MCF-7 breast cancer cells in vitro. Treatment with PZ-DHA also suppressed the metabolic activity of MDA-MB-231-TXL cells and led to apoptosis. PZ-DHA did not have an observable effect on the expression of the drug efflux transporters ATP-binding cassette super-family G member 2 (ABCG2) and multidrug resistance-associated protein 1 (MRP1). PZ-DHA is a potential treatment avenue for chemo-resistant breast cancer and a possible novel CSC therapy. Future pre-clinical studies should explore PZ-DHA as a chemo-preventative agent.

Periostin drives extracellular matrix degradation, stemness, and chemoresistance by activating the MAPK/ERK signaling pathway in triple–negative breast cancer cells

BackgroundAdipose tissue, which is mainly composed of adipocytes, is a crucial component of the tumor microenvironment, particularly in breast cancer. Adipocytes surround breast cancer cells and may participate in cell‒cell interactions in the breast microenvironment. However, little is currently known about how adipocytes influence the biological behavior of the surrounding breast cancer cells. Hence, this study sought to investigate the role and underlying mechanisms of periostin in triple–negative breast cancer (TNBC) cells cocultured with adipogenic conditioned medium (ACM) and palmitic acid (PA).MethodsHuman TNBC cell lines (MDA‒MB‒231 and SUM159PT) were treated with ACM and PA, then the expression of periostin, matrix metalloproteinases (MMPs) and stemness–related molecules were assessed by Western blotting and RT‒qPCR. The cellular viability was assessed using CCK‒8 assay. Plasmid transfection, RNA sequencing, and pathway inhibitor were used to explore the specific mechanisms of periostin.ResultsACM and PA elevated the expression of both MMPs and stemness–related molecules in TNBCs. MMPs can promote tumor cell infiltration and migration by degrading the extracellular matrix, and stemness expression increases the development of tumor chemoresistance. Additionally, ACM and PA increased periostin expression, while inhibiting periostin disrupted the involvement of ACM and PA in promoting extracellular matrix degradation, stemness, and chemoresistance in TNBCs. Furthermore, this study found that periostin promoted TNBC progression by activating the MAPK/ERK signaling pathway and that inhibition of MAPK/ERK signaling reduced the phenotype caused by periostin upregulation in TNBCs treated with ACM or PA. Finally, the present results showed that the high expression of POSTN, which encodes periostin, was substantially related to worse survival in TNBC patients.ConclusionsThe results of the study elucidated for the first time how periostin is the key protein secreted in TNBCs in response to the adipocyte–regulated tumor microenvironment, while periostin–neutralizing antibodies may serve as potential therapeutic agents in relation to TNBC progression.

Ultrasensitive determination of surface proteins on tumor-derived small extracellular vesicles for breast cancer identification based on lanthanide-activated signal amplification strategy

Small extracellular vesicles (sEVs) carrying multiple tumor-associated proteins inherited from parental cells play crucial roles in noninvasive breast cancer (BC) diagnosis. However, it is challenging to assess the subtle variations of surface proteins on sEV membranes due to the highly heterogeneous BC. Therefore, a simple and ultrasensitive assay based on lanthanide (Ln3+)-activated luminescence signal amplification was developed to detect multiple surface proteins on BC-derived sEVs. Multiple protein biomarkers on sEVs can be well identified with high sensitivity and specificity through dissolution-amplified luminescence of the NaEuF4 nanoparticle-based nanoprobe. We employ linear discriminant analysis to successfully discriminate triple negative BC cell (MDA-MB-231 cell) derived sEVs from other breast cell lines (MCF-7, SK-BR-3, BT474 and MCF-10A cell). Furthermore, the strategy enables high accuracy for districting the progression stages of BC patients and healthy donors. The simple and sensitive signal amplification strategy exhibits great potential for early clinic diagnosis by precise protein profiling of sEVs.

Cantharidin induces apoptosis of human triple negative breast cancer cells through mir-607-mediated downregulation of EGFR

BackgroundTriple negative breast cancer (TNBC) is a major subtype of breast cancer, with limited therapeutic drugs in clinical. Epidermal growth factor receptor (EGFR) is reported to be overexpressed in various TNBC cells. Cantharidin is an effective ingredient in many clinical traditional Chinese medicine preparations, such as Delisheng injection, Aidi injection, Disodium cantharidinate and vitamin B6 injection. Previous studies showed that cantharidin had satisfactory pharmacological activity on a variety of tumors. In this study, we aimed to study the therapeutic potential of cantharidin for TNBC treatment by targeting EGFR, and expound its novel regulator miR-607.MethodsThe effect of cantharidin on breast cancer in vivo was evaluated by 4T1 mice model. Then the effects of cantharidin on TNBC cells was assessed by the MTT, colony formation, and AnnexinV-PE/7AAD staining. Cantharidin acts on EGFR were verified using the cell membrane chromatography, RT-PCR, Western blotting, MTT, and so on. Mechanistic studies were explored by dual-luciferase report assay, RT-PCR, western blotting, and immunofluorescence staining assay.ResultsCantharidin inhibited TNBC cell growth and induce apoptosis by targeting EGFR. miR-607 was a novel EGFR regulator and exhibited suppressive functions on TNBC cell behaviors. Mechanistic study showed that cantharidin blocked the downstream PI3K/AKT/mTOR and ERK/MAPK signaling pathway.ConclusionOur results revealed that cantharidin may be served as a potential candidate for TNBC treatment by miR-607-mediated downregulation of EGFR.Graphical

Phenotypically sorted highly and weakly migratory triple negative breast cancer cells exhibit migratory and metastatic commensalism

BackgroundIntratumor heterogeneity is a well-established hallmark of cancer that impedes cancer research, diagnosis, and treatment. Previously, we phenotypically sorted human breast cancer cells based on migratory potential. When injected into mice, highly migratory cells were weakly metastatic and weakly migratory cells were highly metastatic. The purpose of this study was to determine whether these weakly and highly migratory cells interact with each other in vitro or in vivo.MethodsTo assess the relationship between heterogeneity in cancer cell migration and metastatic fitness, MDA-MB-231 and SUM159PT triple negative breast cancer cells were phenotypically sorted into highly migratory and weakly migratory subpopulations and assayed separately and in a 1:1 mixture in vitro and in vivo for metastatic behaviors. Unpaired, two-tailed Student’s t-tests, Mann–Whitney tests, ordinary, one-way ANOVAs, and Kruskal–Wallis H tests were performed as appropriate with p < 0.05 as the cutoff for statistical significance.ResultsWhen highly and weakly migratory cells are co-seeded in mixed spheroids, the weakly migratory cells migrated farther than weakly migratory only spheroids. In mixed spheroids, leader–follower behavior occurred with highly migratory cells leading the weakly migratory cells in migration strands. When cell suspensions of highly migratory, weakly migratory, or a 1:1 mixture of both subpopulations were injected orthotopically into mice, both the mixed cell suspensions and weakly migratory cells showed significant distal metastasis, but the highly migratory cells did not metastasize significantly to any location. Notably, significantly more distal metastasis was observed in mice injected with the 1:1 mixture compared to either subpopulation alone.ConclusionsThis study suggests that weakly migratory cells interact with highly migratory cells in a commensal fashion resulting in increased migration and metastasis. Together, these findings indicate that cancer cell subpopulation migration ability does not correlate with metastatic potential and that cooperation between highly migratory and weakly migratory subpopulations can enhance overall metastatic fitness.

Acetyl-cinobufagin suppresses triple-negative breast cancer progression by inhibiting the STAT3 pathway.

The incidence of breast cancer (BC) worldwide has increased substantially in recent years. Epithelial-mesenchymal transition (EMT) refers to a crucial event impacting tumor heterogeneity. Although cinobufagin acts as an effective anticancer agent, the clinical use of cinobufagin is limited due to its strong toxicity. Acetyl-cinobufagin, a pre-drug of cinobufagin, was developed and prepared with greater efficacy and lower toxicity. A heterograft mouse model using triple negative breast cancer (TNBC) cell lines, was used to evaluate the potency of acetyl-cinobufagin. Signal transducer and stimulator of transcription 3 (STAT3)/EMT involvement was investigated by gene knockout experiments using siRNA and Western blot analysis. Acetyl-cinobufagin inhibited proliferation, migration, and cell cycle S/G2 transition and promoted apoptosis in TNBC cells in vitro. In general, IL6 triggered the phosphorylation of the transcription factor STAT3 thereby activating the STAT3 pathway and inducing EMT. Mechanistically, acetyl-cinobufagin suppressed the phosphorylation of the transcription factor STAT3 and blocked the interleukin (IL6)-triggered translocation of STAT3 to the cell nucleus. In addition, acetyl-cinobufagin suppressed EMT in TNBC by inhibiting the STAT3 pathway. Experiments in an animal model of breast cancer clearly showed that acetyl-cinobufagin was able to reduce tumor growth. The findings of this study support the potential clinical use of acetyl-cinobufagin as a STAT3 inhibitor in TNBC adjuvant therapy.

NIR-Triggered SrSnO3 -photosynthesized nanocatalyst: A potent anticancer agent against triple negative breast cancer cells

The major systemic therapeutic option for Triple Negative Breast Cancer (TNBC) is chemotherapy. TNBC often responds well to chemo, however, chemotherapy carries toxic adverse effects and content-limited interventional efficiency. Thereby, a drug-free treatment paradigm is imperative, in which synthetic polymers spontaneously display efficient anticancer efficacy without the use of conventional chemotherapeutic agents. Aiming at the goal to treat TNBC, SrSnO3 nanoparticles were devised as a nanocatalyst to accomplish near infrared (NIR) photocatalytic production of oxidative holes for achieving hole therapy. An inexpensive sol-gel method was employed in this study to synthesize SrSnO3 nanoparticle using strontium chloride hexahydrate and stannous chloride dihydrate. Transmission electron microscopy (TEM), X-ray diffraction (XRD), Field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) analyses were used to characterize the synthesized SrSnO3 nanoparticles while Density Functional Theory (DFT) was used to study the nanoparticle at atomistic level and the band gap was calculated to be 1.84 eV using Perdew−Burke−Ernzerhof (PBE) functionals. The anticancer effect of SrSnO3 nanoparticle was tested against TNBC cell line MDA-MB-231 at different concentration (10–50 μg/mL) with near infrared (NIR) irradiation while cells without any treatment or treatment with only SrSnO3 or NIR served as controls. Under NIR irradiation, the IC50 value was found to be 40 μg/mL achieving 50% cell death with augmented release of reactive oxygen species (ROS) over time. Reduced and oxidized glutathione (GSH and GSSG, respectively) serve as cellular redox buffers and protect cells from ROS mediated damage. Nearly 18.6% reduced glutathione (GSH) was found to be consumed with 5 min of NIR irradiation that was augmented to 66.5% of GSH when exposure time was increased to 10 min suggesting a prooxidant mediated death of cancer cell line. The results suggested SrSnO3 nanoparticle is a potent photocatalyst when triggered by NIR and can enable highly effective and biosafe hole-mediated treatment for TNBC.

Anti-breast cancer activity of bioactive metabolites from Andrographis paniculata through inhibition of PI3K activity in triple negative breast cancer (MDA-MB-231) cells

Andrographis paniculata, known for many therapeutic potentials such as antiviral, anti-microbial, anticancer, antineoplastic and anti-inflammation properties. Flavonoids and diterpenoids of A. paniculate were extensively studied for their anticancer and anti-inflammation properties. Although, extract of Andrographis paniculata have shown for exhibit anti-breast cancer activities, the molecular targets for the medicinal molecules of Andrographis paniculata are not yet reported. This research study is aimed to identify the anti-breast cancer molecules of Andrographis paniculata and to study the underlying mechanisms. In order to identify the active anticancer molecules that are responsible for we used in silico screening and docking studies. The in silico studies correlated with in vitro mechanistic studies to identify the target and their underlining mechanism of action. In this work, we have reported the bioactive compounds of Andrographis paniculata against PI3K using in vitro and molecular docking with in silico ADME studies. Based on their binding affinities to PI3K, the diterpenoid neoandrographolide suggested the excellent binding scores with PI3K in comparison with the reference inhibitor alepsilib. Furthermore, among them two molecules - andrographolide and neoandrographolide has suggested to have high absorption capacity in gastro-intestinal (GI) tract. In vitro validation results of these molecules against PI3K expressing MDA MB-231 cells have showed the neoandrographolide exhibited excellent anticancer effects against the MDA MB-231 cells. Further mechanistic studies have showed the inducting the programmed cell death and arrested the cancer cell proliferation in G-1 phase upon treatment with neoandrographolide in MDA- MB-231 cancer cells with down-regulation of PI3K. In this present investigation, we have identified the neoandrographolide of A. paniculata that had strong interaction with the PI3K which may be necessary as potential anti-cancer activities in the TNBC.

Synthesis, anticancer activity and molecular modeling study of novel substituted triazole linked tetrafluoronaphthalene hybrid derivatives

To create some novel anticancer molecules, a library of novel series of various triazoles linked to the hydroxyl group of 5,6,7,8-tetrafluoronaphthalen-1-ol (3) was designed and synthesized via CuAAC reaction ‘Click Chemistry’ of tetrafluoronaphthalene based terminal alkyne with substituted organic azides. The structural characterizations of the targeted Click products 9–18 were confirmed by FTIR, 1H NMR, 19F NMR, 13C NMR and HRMS spectroscopy. Synthesized compounds were tested in two triple negative breast cancer (TNBC) cell lines to understand their anticancer potentials. According to our findings, compounds 14 and 13 showed high cytotoxicity in BT549 cells at 20 μM and 30 μM, respectively. Moreover, these compounds blocked the migration of BT549 cells. In the MDA-MB-231 cell line, compound 18 exhibited high cytotoxicity and can block cell migration for 24 h. Molecular docking study with synthesized novel compounds was performed by Glide/SP method against SphK1 drug target. Furthermore, molecular dynamics (MD) simulation was carried out for the compounds 12–14 and 18. The compounds 13 and 14 may be potential inhibitor candidates in place of a reference inhibitor. A pharmacophore model was generated with the most potent compound 14, and the approved drugs were screened using the modules of Discovery Studio to find similar drugs. Consequently, this comprehensive study encompassing design, synthesis, in vitro and in silico analyses were correlated with the structure-activity relationship between compounds. The findings have the potential to unveil promising drug candidates for future studies. Communicated by Ramaswamy H. Sarma

Co-expression of galectin-3 and vimentin in triple negative breast cancer cells promotes tumor progression, metastasis and survival.

Lack of druggable targets and complex expression heterogeneity of known targets is common among TNBC subtypes. An enhanced expression of galectin-3 in TNBCs has already been documented. We have observed a tumor progression-dependent galectin-3 expression in TNBCs compared to adjacent epithelium and non TNBCs. To unravel the association of galectin- 3 in tumor progression, aggressiveness and drug resistance in TNBC patients. Galectin-3 expression in 489 breast cancer tissues was correlated with clinicopathological features and the results were validated in cell lines and mouse model by silencing galectin-3 using shRNA and the proteins were profiled by western blot and qRT-PCR. Protein interaction was analyzed by GFP Trap and Mass spectrometry. Galectin-3 expression correlated with tumor stage in TNBC and a lower galectin-3 expression was associated with poor patient survival. The positive correlation between galectin-3, vimentin and CD44 expression, pinpoints galectin-3 contribution to epithelial to mesenchymal transition, drug resistance and stemness. Vimentin was found as an interacting partner of galectin-3. Duplexing of galecin-3 and vimentin in patient samples revealed the presence of tumor cells co-expressing both galectin-3 and vimentin. In vitro studies also showed its role in tumor cell survival and metastatic potential, elementary for tumor progression. In vivo studies further confirmed its metastatic potential. Tumor progression dependent expression pattern of galectin 3 was found to indicate prognosis. Co-expression of galectin-3 and vimentin in tumor cells promotes tumor dissemination, survival and its metastatic capability in TNBCs.

A New Quinone-Based Inhibitor of Mitochondrial Complex I in D-Conformation, Producing Invasion Reduction and Sensitization to Venetoclax in Breast Cancer Cells.

Mitochondrial Complex I plays a crucial role in the proliferation, chemoresistance, and metastasis of breast cancer (BC) cells. This highlights it as an attractive target for anti-cancer drugs. Using submitochondrial particles, we identified FRV-1, an ortho-carbonyl quinone, which inhibits NADH:duroquinone activity in D-active conformation and reduces the 3ADP state respiration dependent on Complex I, causing mitochondrial depolarization, ATP drop, increased superoxide levels, and metabolic remodeling towards glycolysis in BC cells. Introducing methyl groups at FRV-1 structure produced analogs that acted as electron acceptors at the Complex I level or increased the inhibitory effect of FCCP-stimulated oxygen consumption rate, which correlated with their redox potential, but increased toxicity on RMF-621 human breast fibroblasts was observed. FRV-1 was inactive in the naphthoquinone oxidoreductase 1 (NOQ1)-positive BC cell line, MCF7, but the sensitivity was recovered by dicoumarol, a NOQ1 inhibitor, suggesting that FRV-1 is a NOQ1 substrate. Importantly, FRV-1 selectively inhibited the proliferation, migration, and invasion of NQO1 negative BC cell, MDA-MB-231, in an OXPHOS- and ROS-dependent manner and sensitized it to the BH3 mimetic drug venetoclax. Overall, FRV-1 is a novel Complex I inhibitor in D-active conformation, blocking possibly the re-activation to A-state, producing selective anti-cancer effects in NQO1-negative BC cell lines.

Synthesis of Novel Pyrazole-Oxindole Conjugates with Cytotoxicity in Human Cancer Cells via Apoptosis.

A novel series of pyrazole-oxindole conjugates were prepared and characterized as potential cytotoxic agents by FT-IR, NMR and HR-MS. The cytotoxic activity of these compounds was tested in the Jurkat acute T cell leukemia, CEM acute lymphoblastic leukemia, MCF10 A mammary epithelial and MDA-MB 231 triple negative breast cancer cell lines. Among the tested conjugates, 5-methyl-3-((3-(1-phenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6h emerged as the most cytotoxic with a CC50 of 4.36+/-0.2 μM against Jurkat cells. The mechanism of cell death induced by 6h was investigated through the Annexin V-FITC assay via flow cytometry. Reactive oxygen species (ROS) accumulation, mitochondrial health and the cell cycle progression were also evaluated in cells exposed to 6h. Results demonstrated that 6h induces apoptosis in a dose-response manner, without generating ROS and/or altering mitochondrial health. In addition, 6h disrupted the cell cycle distribution causing an increase in DNA fragmentation (Sub G0-G1), and an arrest in the G0-G1 phase. Taken together, the 6h compound revealed a strong potential as an antineoplastic agent evidenced by its cytotoxicity in leukemia cells, the activation of apoptosis and restriction of the cell cycle progression.

Targeted drug-loaded PLGA-PCL microspheres for specific and localized treatment of triple negative breast cancer

The paper presents the results of the experimental and analytical study of targeted drug-loaded polymer-based microspheres made from blend polymer of polylactic-co-glycolic acid and polycaprolactone (PLGA-PCL) for targeted and localized cancer drug delivery. In vitro sustained release with detailed thermodynamically driven drug release kinetics, over a period of three months using encapsulated targeted drugs (prodigiosin-EphA2 or paclitaxel-EphA2) and control drugs [Prodigiosin (PGS), and paclitaxel (PTX)] were studied. Results from in vitro study showed a sustained and localized drug release that is well-characterized by non-Fickian Korsmeyer–Peppas kinetics model over the range of temperatures of 37 °C (body temperature), 41 °C, and 44 °C (hyperthermic temperatures). The in vitro alamar blue, and flow cytometry assays in the presence of the different drug-loaded polymer formulations resulted to cell death and cytotoxicity that was evidence through cell inhibition and late apoptosis on triple negative breast cancer (TNBC) cells (MDA-MB 231). In vivo studies carried out on groups of 4-week-old athymic nude mice that were induced with subcutaneous TNBC, showed that the localized release of the EphA2-conjugated drugs was effective in complete elimination of residual tumor after local surgical resection. Finally, ex vivo histopathological analysis carried out on the euthanized mice revealed no cytotoxicity and absence of breast cancer metastases in the liver, kidney, and lungs 12 weeks after treatment. The implications of the results are then discussed for the development of encapsulated EphA2-conjugated drugs formulation in the specific targeting, localized, and sustain drug release for the elimination of local recurred TNBC tumors after surgical resection.Graphical

Downregulation of ACC expression suppresses cell viability and migration in the malignant progression of breast cancer.

Exploring new diagnostic biomarkers and molecular targets is of great importance in breast cancer treatment. The present study investigated the effects of acetyl-CoA carboxylase (ACC) expression interference on the malignant progression of breast cancer cells. ACC expression was knocked down using a lentiviral vector and this was verified by quantitative polymerase chain reaction and western blotting. MCF-7 and MDA-MB-231 breast cancer cells were randomly allocated into the following groups: Normal breast cancer cells (control), breast cancer cells transduced with a negative control lentiviral vector and breast cancer cells transduced with an ACC knockdown lentiviral vector. Screening for stable transgenic strains was successful. Cell viability, apoptosis and migration were determined using Cell Counting Kit-8, flow cytometry and scratch test, respectively. The protein expression levels of N-cadherin, Vimentin and Bax were detected by western blotting. In addition, a nude mouse model of subcutaneous metastatic tumor was established using MCF-7 breast cancer cells, and tumor volume was assessed. Furthermore, pathological condition and apoptosis were detected using hematoxylin and eosin, and TUNEL staining, respectively. The protein expression levels of N-cadherin, Vimentin and Bax were detected by western blotting. The in vitro experiments showed that knockdown of ACC expression significantly decreased the viability and migration, and increased the apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. In vivo experiments revealed that ACC knockdown effectively reduced the tumor volume in nude mice, and promoted tumor cell apoptosis. Both in vitro and in vivo experiments showed that ACC knockdown can reduce the protein expression levels of N-cadherin and Vimentin, and increase Bax expression. These findings suggested that downregulation of ACC expression may significantly reduce the malignant progression of breast cancer, and could be considered a potential therapeutic target.

In-vitro anticancer evaluation of newly designed and characterized tri/tetra-substituted imidazole congeners- maternal embryonic leucine zipper kinase inhibitors: Molecular docking and MD simulation approaches

Our cascading attempt to develop new potent molecules now involves designing a series of imidazole derivatives and synthesizing two sets of 2,4,5- tri-substituted (4a–4d) and 1,2,4,5-tetra-substituted (6a–6d) imidazole by the principle of Debus-Radziszewski multicomponent synthesis reaction. The structures of the obtained compounds were confirmed by 1H/13C NMR, FT-IR, elemental analysis, purity and the retention time was analyzed by HPLC. Based upon the binding affinity in the molecular docking studies, we have synthesized different imidazole derivatives from which compound 6c have been found to show more anti-proliferative activity by inducing apoptosis at a higher rate than the other compounds corroborating the in-silico prediction. The structure and crystallinity of compound 4d have been confirmed by single XRD analysis. The synthesized molecules were screened for their in vitro anti-cancer properties in triple negative breast cancer cell line (MDA-MB-231), pancreatic cancer cell lines (MIA PaCa-2) and oral squamous cell carcinoma cell line (H357) and results indicated that all the compounds inhibited the cell proliferation in a concentration-dependent manner at different time points. The compounds 4b and 6d were found to be effective against the S. aureus bacterial strain whereas only compound 4d fairly inhibited the fungal strain of T. rubrum with a MIC 12.5 μg/mL. Molecular docking study reveals good interaction of the synthesized compounds with known target MELK involved in oncogenesis having high binding profiles. The lead compound 6c was further analyzed by the detailed molecular dynamics study to establish the stability of the ligand–enzyme complex.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

Design, synthesis and docking studies of novel 4-aminophenol-1,2,4-oxadiazole hybrids as apoptosis inducers against triple negative breast cancer cells targeting MAP kinase

In our study, a series of novel 4-aminophenol benzamide-1,2,4-oxadiazole hybrid analogues have been designed and synthesized by condensing 4-hydroxyphenyl arylamides (3a–c) and 5-chloromethyl-3-aryl-1,2,4-oxadiazoles (6a–d). The structure of the synthesised compounds was verified by various spectroscopic techniques (1H NMR, 13C NMR, IR and LC-MS). All the prepared compounds were subjected to in silico and in vitro antiproliferative study against TNBC cell lines MDA-MB-468 and MDA-MB-231. The investigations revealed that compound 7k significantly promoted apoptosis against MDA-MB-468 and MDA-MB-231 cells with IC50 values of 22.31 µM and 26.27 µM, respectively. Compound 7k interacted with crucial active sites of MAPK and exhibited the highest docking score of −7.06 kcal/mol. Docking was validated with molecular dynamic studies with simulation for 100 ns, depicting various stable interactions with MAPK. Consequently, 7k forms stable H-Bonds and π-π stacking with amino acid residues along with π-cation. Our investigations reveal that the in vitro antiproliferative study of 7k was in good correlation with the in silico studies. Hence, 7k serves as a potential novel lead for the inhibition of TNBCs by downregulating MAPK P38. Communicated by Ramaswamy H. Sarma

In vivo optical imaging of tumor stromal cells with hypoxia-inducible factor activity.

Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b+ F4/80+ macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.

Targeted Destruction of S100A4 Inhibits Metastasis of Triple Negative Breast Cancer Cells.

Most patients who die of cancer do so from its metastasis to other organs. The calcium-binding protein S100A4 can induce cell migration/invasion and metastasis in experimental animals and is overexpressed in most human metastatic cancers. Here, we report that a novel inhibitor of S100A4 can specifically block its increase in cell migration in rat (IC50, 46 µM) and human (56 µM) triple negative breast cancer (TNBC) cells without affecting Western-blotted levels of S100A4. The moderately-weak S100A4-inhibitory compound, US-10113 has been chemically attached to thalidomide to stimulate the proteasomal machinery of a cell. This proteolysis targeting chimera (PROTAC) RGC specifically eliminates S100A4 in the rat (IC50, 8 nM) and human TNBC (IC50, 3.2 nM) cell lines with a near 20,000-fold increase in efficiency over US-10113 at inhibiting cell migration (IC50, 1.6 nM and 3.5 nM, respectively). Knockdown of S100A4 in human TNBC cells abolishes this effect. When PROTAC RGC is injected with mouse TNBC cells into syngeneic Balb/c mice, the incidence of experimental lung metastases or local primary tumour invasion and spontaneous lung metastasis is reduced in the 10-100 nM concentration range (Fisher's Exact test, p ≤ 0.024). In conclusion, we have established proof of principle that destructive targeting of S100A4 provides the first realistic chemotherapeutic approach to selectively inhibiting metastasis.

Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis.

The PI3K/AKT pathway is activated in 43-70% of breast cancer (BC)-patients and promotes the metastatic potential of BC cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for triple-negative breast cancer (TNBC)-patients with brain metastases. The impact of AKT1-knockout (AKT1_KO) and AKT-inhibition using Ipatasertib on MDA-MB-231 BR cells was assessed using in vitro cell proliferation and migration assays. AKT1-knockout in MDA-MB-231BR cells was performed using CRISPR/Cas9. The effect of AKT1-knockout on radiosensitivity of MDA-MB-231BR cell lines was determined via colony formation assays after cell irradiation. To detect genomic variants in AKT1_KO MDA-MB-231BR cells, whole-genome sequencing (WGS) was performed. Pharmacological inhibition of AKT with the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability but did not impact cell migration. Moreover, only MDA-MB-231BR cells were sensitized following Ipatasertib-treatment. Furthermore, specific AKT1-knockout in MDA-MB-231BR showed reduced cell viability in comparison to control cells, with significant effect in one of two analyzed clones. Unexpectedly, AKT1 knockout led to increased cell migration and clonogenic potential in both AKT1_KO clones. RNAseq-analysis revealed the deregulation of CTSO, CYBB, GPR68, CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN leading to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 knockout in MDA-MB-231BR. Collectively, our results demonstrate that Ipatasertib leads to radiosensitization and reduced cell proliferation of MDA-MB-231BR. AKT1-inhibition showed altered gene expression profile leading to modified cell migration, clonogenic survival and radioresistance in MDA-MB-231BR. We conclude, that AKT1-inhibition in combination with radiotherapy contribute to novel treatment strategies for breast cancer brain metastases.