Positive Breast Cancer Cell Lines Research Articles

Oestrogen Represses Noggin Expression by Interfering With BMP/Smad Signalling in ER Positive Breast Cancer.

As an antagonist of bone morphogenetic protein (BMP), Noggin facilitates osteolytic bone metastases from breast cancer. The present study aimed to further dissect its role in oestrogen receptor (ER) positive breast cancer. Noggin expression in ER positive breast cancer cell lines (MCF-7 and T-47D) was determined under conditions of oestrogen deprivation and treatment with 17-β-oestradiol (E2). Activation of Smad1/5/8 in the oestrogen-regulated Noggin was examined using recombinant human BMP7 (rhBMP7) and a BMP receptor inhibitor (LDN-193189). The influence of Noggin on cellular functions was evaluated in MCF-7 and T-47D cell lines. Responses to tamoxifen and chemotherapy drugs were determined in MCF-7 and T-47D cells with Noggin over-expression using MTT assay. Noggin expression was negatively correlated with ERα in breast cancers. Noggin was up-regulated upon oestrogen deprivation, an effect that was eliminated by E2 Furthermore, increased levels of phosphorylated Smad1/5/8 were observed in the oestrogen-deprived MCF-7 and T-47D cells, which was prevented by E2 and LDN-193189, respectively. BMP7-induced Noggin expression and activation of Smad1/5/8 was also prevented by E2 and LDN-193189. Noggin over-expression resulted in an increase in the proliferation of both MCF-7 and T-47D cells. MCF-7 and T-47D cells over-expressing Noggin exhibited a good tolerance to tamoxifen (TAM), DTX, and 5-FU, but the percentage of viable cells was higher compared with the controls. Noggin expression can be repressed by oestrogen through inference with the BMP/Smad signalling. Over-expression of Noggin promotes the proliferation of MCF-7 and T-47D cells, contributing to drug resistance.

Differential effects of docosahexaenoic acid (DHA) and linoleic acid (LA) on miR-101 and miR-342 tumor suppressor microRNAs in Taxol-treated HER2-positive breast cancer cells

Background & AimsDocosahexaenoic acid (DHA) and linoleic acid (LA) have been shown to exhibit anti-proliferative effects against breast cancer cells. However, the mechanisms underlying these effects are not yet fully understood. One potential mechanism is through the regulation of microRNAs (miRs), which are known to play a crucial role in breast cancer development and progression. This study aimed to investigate the expression of miR-342 and miR-101 as tumor-suppressor miRs in the human HER-2 positive breast cancer cell line BT-474 after treatment with DHA, LA, alone or in combination with Taxol, a standard chemotherapy agent. MethodsThe human breast cancer cell line BT-474 was cultured, and the IC50 for Taxol was determined using the MTT assay. Cells were then cultured and treated for 24 hours with 100 μM DHA and 50 μM LA, alone or in combination with the respective IC50 of Taxol. Cells were harvested, and miRNA extraction and cDNA synthesis were performed using standard methods. Expression levels of miRs were analyzed using quantitative real-time PCR (qRT-PCR), and results were normalized against U6 snRNA expression levels. ResultsThe Taxol IC50 for BT-474 cells was 19 nM. According to the data obtained from our study, it was observed that Taxol treatment resulted in the down-regulation of both miR-101 and miR-342 (3.69 (p < 0.0001) and 1.88 fold, (p < 0.0001) respectively). In addition, DHA, LA and DHA+LA caused up-regulation of miR-101 (0.11, 0.05, 0.03 fold (p < 0.0001) respectively) but not miR-342 (decreased by 1.93 (p < 0.0001), 2.89 (p < 0.0001) and 1.19 fold (p = 0.0029) respectively). Notably, treatment with DHA, LA and DHA+LA was able to restore the down-regulated expression of miR-101 (0.25 (p < 0.0001), 0.05 (p = 0.0012) and 0.06 fold (p < 0.0001) respectively) during Taxol treatment. ConclusionOur study demonstrates that DHA and LA can effectively compensate for the reduced expression of miR-101 during Taxol treatment. These findings suggest that dietary fatty acids may play a critical role in modulating the anti-cancer effects of chemotherapy agents. Future studies are needed to investigate the functional aspects of dietary fatty acids on breast cancer development and progression.

Laminin I mediates resistance to lapatinib in HER2-positive brain metastatic breast cancer cells in vitro

The role of extracellular matrix (ECM) prevalent in the brain metastatic breast cancer (BMBC) niche in mediating cancer cell growth, survival, and response to therapeutic agents is not well understood. Emerging evidence suggests a vital role of ECM of the primary breast tumor microenvironment (TME) in tumor progression and survival. Possibly, the BMBC cells are also similarly influenced by the ECM of the metastatic niche; therefore, understanding the effect of the metastatic ECM on BMBC cells is imperative. Herein, we assessed the impact of various ECM components (i.e., Tenascin C, Laminin I, Collagen I, Collagen IV, and Fibronectin) on brain metastatic human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) cell lines in vitro. The highly aggressive TNBC cell line was minimally affected by ECM components exhibiting no remarkable changes in viability and morphology. On the contrary, amongst various ECM components tested, the HER2-positive cell line was significantly affected by Laminin I with higher viability and demonstrated a distinct spread morphology. In addition, HER2-positive BMBC cells exhibited resistance to Lapatinib in presence of Laminin I. Mechanistically, Laminin I-induced resistance to Lapatinib was mediated in part by phosphorylation of Erk 1/2 and elevated levels of Vimentin. Laminin I also significantly enhanced the migratory potential and replicative viability of HER2-positive BMBC cells. In sum, our findings show that presence of Laminin I in the TME of BMBC cells imparts resistance to targeted therapeutic agent Lapatinib, while increasing the possibility of its dispersal and clonogenic survival.

Abstract PO4-24-09: Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer

Abstract Purpose Because more than 60% of breast cancers are hormone-receptor (HR) positive, endocrine therapy plays an important role. Tamoxifen is an antagonist of estrogen receptor-α and main treatment drug for HR-positive breast cancers. However, efficacy is not satisfactory because of the development of resistance. EGFR expression was associated with poor prognosis in HR-positive breast cancers. Loss of ER by EGFR activation induced tamoxifen resistance. And extracellular signal-regulated kinase (ERK) is another important factor for tamoxifen resistance. Activation of ERK signaling in breast cancer is not only associated with augmented tumor growth and metastasis but also with tamoxifen resistance. Therefore, the inhibition of ERK/EGFR signaling associated with tamoxifen resistance could improve the survival rate. Grape seed extract (GSE) is which contains a high content of flavonoid polyphenolic compounds. GSE is known to have various pharmacological effects, including antioxidant, anti-inflammatory, antitumor activity. In this study, we explored the activity of GSE in tamoxifen resistant breast cancer cells by inhibiting EGFR signaling pathway. Method We examined the suppressive effect of GSE on various signaling pathways. To confirm the pharmacological activity of GSE, we treated EGFR positive breast cancer cells with GSE. Levels of mRNA and protein expression were analyzed by real-time PCR and western blot. And cell proliferation and invasiveness were analyzed by the colony-forming assay and the Boyden chamber assay. Results 1. GSE down regulates EGFR and ERK in EGFR positive breast cancer cell line. The cells were treated with the indicated concentrations GSE for 48 hrs. The levels of p- and t-ERK and t-EGFR were measured through Western blotting. The levels of phosphorylated EGFR and ERK were decreased by GSE at the indicated doses for 48hrs. 2. EGF, a ligand of EGFR, upregulates the MMP-9 and GSE suppresses MMP-9. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR with its ligand epidermal growth factor (EGF) regulates Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). After treating EGFR positive breast cancer cells with EGF and TGF, MMP-9 levels were increased. However, MMP-9 level was decreased with GSE as indicated doses for 48hrs. Conclusion Taken together, GSE inhibited the ERK/EGFR and EGF/MMP-9 signaling pathway in EGFR positive breast cancer cells. Therefore, we propose the possibility of GSE as an effective adjuvant for tamoxifen resistant breast cancer. Citation Format: Seung Pil Jung, Eun-Shin Lee, Ji Young You, Jung Hwa Chun, Hyunyou Kim. Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-09.

Abstract 5540: Single cell multiomic analysis of human brain metastasis reveals conserved dormancy population

Abstract This study explores the complex realm of brain metastasis, pivotal in crafting effective anti-tumor strategies. Leveraging single-cell multi-omics sequencing, we profiled the transcriptomes and chromatin accessibility landscapes of tumor cells and their surrounding microenvironment in human brain metastases that originated from across a spectrum of primary cancers. Our investigations have identified distinct, yet uniformly present, tumor cell subpopulations, each defined by unique gene expression patterns, chromatin accessibility, and the interactions between tumor and stromal cells. Notably, one such subpopulation exhibits a dormancy signature, characterized by enhanced interferon response, oxidative phosphorylation, and upregulated HLA class molecules, alongside alterations in circadian rhythms. Our trajectory analysis charts a course from these dormant cells to rapidly proliferating ones, with notable shifts in circadian rhythm genes, underscoring their potential role in brain metastasis. This aligns with recent findings that disruptions in circadian rhythms can modify stem cell characteristics in glioblastomas. This insight led us to focus on a specific circadian rhythm gene, highly active in the dormant cells and located on the HER2 amplicon—an area linked to high brain metastasis relapse rates in patients. We've developed inducible models for both overexpression and knockdown to scrutinize this gene's impact in her2 positive breast cancer cell lines, examining gene expression and proliferation in vitro. Complementing this, we are conducting spatial transcriptomic analysis to evaluate the microenvironmental regulation of tumor dormancy in human brain metastases. This multi-omics approach reveals a nuanced regulatory landscape of brain metastasis, highlighting a novel and often overlooked factor in metastasis progression and brain tumor relapse: the interplay between circadian rhythms and tumor dormancy. This discovery not only deepens our understanding of brain metastases but also opens potential new avenues for therapeutic interventions. Citation Format: Remi Klotz, Yiru Wang, Anu Sunkara, Frank Attenelo, Min Yu. Single cell multiomic analysis of human brain metastasis reveals conserved dormancy population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5540.

Efficacy and delivery strategies of the dual Rac/Cdc42 inhibitor MBQ-167 in HER2 overexpressing breast cancer

Trastuzumab and trastuzumab-based treatments are the standard of care for breast cancer patients who overexpress the human epidermal growth factor receptor 2 (HER2). However, patients often develop resistance to trastuzumab via signaling from alternative growth factor receptors that converge to activate guanine nucleotide exchange factors (GEFs) that in turn activate the Rho GTPases Rac and Cdc42. Since Rac and Cdc42 have been implicated in high tumor grade and therapy resistance, inhibiting the activity of Rac and Cdc42 is a rational strategy to overcome HER2-targeted therapy resistance. Therefore, our group developed MBQ-167, a dual Rac/Cdc42 inhibitor with IC50s of 103 nM and 78 nM for Rac and Cdc42, respectively, which is highly effective in reducing cell and tumor growth and metastasis in breast cancer cell and mouse models. Herein, we created a trastuzumab resistant variant of the SKBR3 HER2 positive breast cancer cell line and show that Rac activation is a central mechanism in trastuzumab resistance. Next, we tested the potential of targeting MBQ-167 to HER2 overexpressing trastuzumab-resistant cell lines in vitro, and show that MBQ-167, but not trastuzumab, reduces cell viability and induces apoptosis. When MBQ-167 was targeted to mammary fatpad tumors established from HER2 overexpressing cells via immunoliposomes functionalized with trastuzumab, MBQ-167 and MBQ-167-loaded liposomes show equal efficacy in reducing the viability of trastuzumab-resistant cells, inhibiting tumor growth in mouse xenografts, and reducing metastasis to lungs and liver. This study demonstrates the efficacy of MBQ-167 as an alternative therapeutic in HER2 overexpressing cancers, delivered either in free form or in liposomes.

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

MUC1 aptamer-conjugated niclosamide-loaded PLGA-PEG nanoparticles attenuate HIF-1 stabilization upon hypoxia in MCF7 breast cancer cells

Hypoxia is related to aggressive phenotype and poor prognosis in breast cancer. HIF-1, a transcription factor (TF), plays an essential role in the adaptation of tumor cells to hypoxia. It is stabilized in hypoxic conditions and activates other TFs and signaling pathways that are associated with the proliferation, survival, and progression of cancer cells in hypoxia. As a result, targeting hypoxia by destabilizing HIF-1 is a promising approach to inhibit cell adaptation to hypoxia. Niclosamide (NIC) has been identified as a HIF-1 inhibitor and it is envisioned that the anti-cancer efficacy of NIC can be improved through targeted drug delivery systems (DDSs) as a therapeutic strategy. Here, MUC1 aptamer (Ap)-targeted PLGA/PEGylated nanoparticles (Ap-PLGA-NPs) were developed for targeted delivery of NIC (Ap-NIC-PLGA-NPs) to MUC1 positive breast cancer cell line in hypoxia environment. The obtained findings showed that targeted inhibition of HIF-1 using fabricated targeted-NIC-loaded NPs increased the rate of apoptosis in breast cancer cells in hypoxic conditions which was further confirmed by geno-/cytotoxicity assays and flow cytometry analysis. Downregulation of HIF-1α, HIF-1β, Eno1, and GluT1 in treated breast cancer cells under hypoxic conditions also indicated a potential role for Ap-NIC-PLGA-NPs in tumor cell adaptation to hypoxia.

Abstract LB_A24: Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways

Abstract The anti-tumor properties of baicalein, a flavonoid compound, have been extensively documented. However, the specific effects of baicalein on ER𝛼 positive breast cancer cell lines, namely T47-D and MCF-7, are less well-known. In this study, we conducted comprehensive in vitro experiments to elucidate the comparative impacts of baicalein on these two cell lines. Our results demonstrate that baicalein exerts robust anti-cancer effects, inducing morphological changes, reducing colony formation, and inhibiting cell proliferation in both T47-D and MCF-7 cells. These effects were more pronounced at higher concentrations. Additionally, baicalein treatment impeded the migratory capabilities of both cell lines, as shown by wound healing assays. Furthermore, our data show that baicalein triggers apoptosis, leading to an increase in early apoptotic, late apoptotic, and dead cells. This apoptotic process appears to be caspase 3/7-dependent. Interestingly, the pro-apoptotic effects were more potent in T47-D cells, accompanied by significant upregulation of pro-apoptotic genes and downregulation of anti-apoptotic markers. Baicalein treatment also induced oxidative stress and DNA damage, evidenced by an increase in reactive oxygen species (ROS) and phosphorylation of ATM and H2A.X. Importantly, baicalein inhibited the PI3K signaling pathway, a key mediator of cancer progression. Genome-wide transcriptomic analysis revealed significant changes in gene expression patterns, with baicalein treatment causing both upregulation and downregulation of several genes. Pathway analysis revealed that cell cycle and ferroptosis pathways were activated in both cell lines upon baicalein treatment. In conclusion, our findings highlight the potential of baicalein as a therapeutic agent against breast cancer, while also revealing nuanced differences in response between the T47-D and MCF-7 breast cancer cell lines. Citation Format: Besa Xhabija, Aidan J. McLaughlin, Ibiere Lovelyn Epelle, Tristan H. Nielson, Elisabeta Ujeniuc, Mariam I. Duhaini. Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A24.

Abstract A044: The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer

Abstract KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A044.

Electrochemical Modification of Polypeptides at Selenocysteine.

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e-SE is highlighted through late-stage C-terminal modification of the FDA-approved cancer drug leuprolide and assembly of a library of anti-HER2 affibody conjugates bearing complex cargoes. Following assembly by e-SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated.

Electrochemical Modification of Polypeptides at Selenocysteine

AbstractMild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e‐SE) platform for the efficient site‐selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e‐SE is highlighted through late‐stage C‐terminal modification of the FDA‐approved cancer drug leuprolide and assembly of a library of anti‐HER2 affibody conjugates bearing complex cargoes. Following assembly by e‐SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated.

Development of High-Loading Trastuzumab PLGA Nanoparticles: A Powerful Tool Against HER2 Positive Breast Cancer Cells.

Trastuzumab, a therapeutic monoclonal antibody directed against HER2, is routinely used to treat HER2-positive breast cancer with a good response rate. However, concerns have arisen in the clinical practice due to adverse side effects. One way to overcome these limitations is to encapsulate trastuzumab in nanoparticles to improve cytotoxic activity, increase intracellular drug concentrations, escape the immune system and avoid systemic degradation of the drug in vivo. A double emulsion method was used to encapsulate trastuzumab into poly(lactic-co-glycolic) nanoparticles, effective for their biocompatibility and biodegradability. These nanocarriers, hereafter referred to as TZPs, were characterised in terms of size, homogeneity, zeta potential and tested for their stability and drug release kinetics. Finally, the TZPs cytotoxicity was assessed in vitro on the HER2 positive SKBR3 breast cancer cell line and compared to free trastuzumab. The TZPs were stable, homogeneous in size, with a reduced zeta potential. They showed higher encapsulation efficiency and drug loading, a prolonged trastuzumab release kinetics that retained its physicochemical properties and functionality. TZPs showed a stronger cytotoxicity and increased apoptosis than similar doses of free trastuzumab in the cell line analysed. Confocal microscopy and flow cytometry assessed TZPs and trastuzumab cellular uptake while Western blot evaluated downstream signalling, overall HER2 content and shedding. TZPs exert more robust effects than free trastuzumab via a dual mode of action: TZPs are taken up by cells through an endocytosis mechanism and release the drug intracellularly for longer time. Additionally, the TZPs that remain in the extracellular space release trastuzumab which binds to the cognate receptor and impairs downstream signalling. This is the sole modality used by free trastuzumab. Remarkably, half dose of TZPs is as efficacious as the highest dose of free drug supporting their possible use for drug delivery in vivo.

Recombinant anti-human epidermal growth factor receptor type 2 single-chain variable fragment-alpha-luffin fusion protein as a putative immunotoxin against human epidermal growth factor receptor type 2-positive breast cancer cells: an experimental research.

Breast cancer is one of the most frequent causes of cancer death in women. The application of immunotoxins to target overexpressed biomarkers on the surface of cancer cells and delivery of the toxin molecules into these cells has attracted too much attention during the last decade. This study was conducted to investigate the possible in-vitro cytotoxic and apoptotic activity of previously designed recombinant immunotoxin compromising anti-HER2 single-chain variable fragment (scFv) and alpha-luffin protein in human epidermal growth factor receptor type 2 (HER2)-positive and HER2-negative breast cancer cell lines. The previously designed recombinant immunotoxin and alpha-luffin protein were expressed in E. coli host cells and purified using Ni-affinity chromatography. The cytotoxicity of the proteins was tested through MTT and apoptosis studies on HER2-positive and HER2-negative breast cancer cell lines. Treatment of SKBR3 and MDA-MB-468 cells with the immunotoxin caused differential cytotoxicity and apoptotic events. Flow cytometry analysis indicated that the immunotoxin could arrest SKBR3 cells at the G0/G1 phase and induce apoptosis and cell death which were not observed in HER2-negative MDA-MB-468 cells. Annexin V/PI staining revealed late apoptotic events in SKBR3 cells treated with the immunotoxin which was different from the early apoptosis induced by the alpha-luffin protein alone. This immunotoxin could be a promising tool in developing new targeted therapeutic agents against HER2-positive cancer cells. Animal experiments are needed before making firmed conclusions.

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.

MELK and Del-1 as druggable targets in TNBC.

e15100 Background: In our previous study, the researchers found that Del-1 was a promising predictive marker for breast cancer. However, the Del-1 downstream targets and biological effectors remain unknown and undruggable in breast cancer. We used RNA-seq to analyze the transcriptome and investigate the mechanism of Del-1 in regulating the course of breast cancer to uncover a druggable target. Methods: To evaluate the expression levels of Del-1 and MELK, mRNA levels in several breast cancer cell lines were analyzed. Due to the heterogeneity of TNBC, eight different TNBC cell lines were analyzed (BT20, TNBC; MDA-MB-468, basal-like 1; HCC-1806, basal-like 2; DU4475, Immunomodulatory; BT549, mesenchymal-like; MDA-MB-231, mesenchymal stem-like; HS578T, mesenchymal stem-like; MDA-MB-453, TNBC Luminal androgen receptor). Total RNA was isolated using RNAiso Plus (TaKaRa, Otsu, Shiga, Japan). Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit. High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA). OTS167 was treated for inhibition of MELK. The effects of MELK on cell proliferation and invasion were determined using MTT and Matrigel Transwell assays. To further investigate the relationship between DEL-1 and MELK, the researchers performed dual inhibition of DEL-1 &amp; MELK. Results: Del-1 and MELK expression were significantly increased in TNBC cell lines compared to luminal or HER 2 positive breast cancer cell lines. Expression of Del-1 and MELK was dramatically enhanced in MB 468, HCC-1806, and MB 231 TNBC cell lines. Three TNBC cell lines were transfected with Del-1-specific siRNA to examine the effect of Del-1 downregulation on breast cancer. Del-1 knockdown HCC-1806 &amp; MDA-MB 231 cells significantly decreased MELK expression, suggesting the possible relation between Del-1 and MELK. OTS 167, a MELK inhibitor, significantly inhibits breast cell proliferation and promotes cell apoptosis (MBA-MB-468, p &lt; 0.001). Furthermore, when the OTS 167 was treated, the cell viability decreased as the dose of the MELK inhibitor increased, and the degree was the greatest in MBA-MB 468, basal-like 1 TNBC cell line. To further investigate the relationship between DEL-1 and MELK, the researchers inhibited DEL-1 and then MELK in MTT assay. Dual inhibition significantly reduced cell viability in MDA-MB468 and MDA-MB 231 cell lines, with the basal-like 1 cell lines showing the most significant effect. Conclusions: In this study, we sought to investigate the association between De1-1 and MELK in TNBC cell lines and tumor tissue. We found that MELK is downstream of del-1 and is a promising target, especially in the basal-like 1 subtype. Further research is warranted.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Abstract 2390: Claudin-4 probe identifies triple negative breast cancer using multispectral optoacoustic tomography

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. The cancer is known for the lack of estrogen and her2 receptors. TNBC has a 5-year survival rate of 77% as for the other breast cancers averaging out at 93%. The CL-750 probes are made to identify the claudin-4 that is expressed in TNBC, making it easy to identify the cancer quickly so we can treat quickly. Methods: TNBC, aggressive pancreatic and kidney cancer cell lines were used to assess the expression of claudin-4 via western blot. Based upon the C-terminal fragment of Clostridium perfingens, we synthesized a 30 aa peptide. CL peptide was conjugated to amine reactive HiLite 750 nm fluorescent dye. The CL-750 probes were purified using dialysis to remove unattached dye. MDA-MB-468 (TNBC), MDA-MB-231 (TNBC), S2VP10 (pancreatic), and 293 (kidney) cells were incubated with CL-750 probes for 30 minutes and evaluated with near infrared-fluorescence using Odyssey imaging software. Using the treated cells were then scraped into agar tissue mimicking phantoms, the probes were assessed for optoacoustic activity using the MSOT (Multispectral optoacoustic tomography). Results: Using western blot to show expression of claudin-4 within cell lines the S2VP10 pancreatic (positive control) showed the most expression of claudin-4, while the 293 kidney cells showed a lesser amount. Both MDA-MB-468 and MDA-MB-231 TNBC cells showed the lowest amount of claudin-4 nonetheless both showed enough to continue with experiments. Cell line S2VP10 showed an extreme amount of binding to probe, while as the probe also bound to both smaller amount of claudin positive breast cancer cell line (MDA-MB-231) and larger amount of claudin breast cancer cell line (MDA-MB-468). Both CL-Short-750 and CL-Long-750 showed signal using MSOT. The CL-Long-750 showed 2x as much signal as CL-Short-750 making the Cl-long-750 probe more efficient in identifying claudin positive cell TNBC (p&amp;lt;0.05). Conclusion: Both CL-750-probes have the capability of identifying claudin-4 positive cell lines, but CL-Long-750 was more effective and had greater binding. Citation Format: Laycee Parsons, William M. MacCuaig, Lacey R. McNally. Claudin-4 probe identifies triple negative breast cancer using multispectral optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2390.

Deinoksantinin HER2 Pozitif Meme Kanserinde Tamoksifenin Antiproliferatif Aktivitesi Üzerindeki Agonistik Etkileri: MDA-MB-453 Üzerine Bir In vitro Çalışma

A good understanding of the pathogenesis of breast cancer, in which in 2020 an estimated 684,996 women across the world died, plays a vital role in the development of treatment methods. In recent years, estrogen receptor (ER), progesterone receptor (PR), and Epidermal Growth Factor receptor-2 (HER2) expression levels have been substantial prognostic markers. The combinational therapy approach is the use of two or more active ingredients or methods. The most important benefit of this treatment method is to reduce the development of drug resistance since the probability of the carcinoma being resistant to more than one drug at the same time is low. Tamoxifen, an estrogen receptor competitive and nonsteroidal drug, has been used for nearly 20 years to treat patients with hormone receptor-positive breast cancer. Deinoxanthin is a xanthophyll derivative purified from the cell wall of a radiation-resistant bacterium, Deinococcus radiodurans. This xanthine derivative has been shown in some studies to have proapoptotic and antiproliferative effects on some types of cancer. Within the study's scope, it aimed to increase the effectiveness of Tamoxifen with deinoxanthin. Using the ACTB gene as a reference to investigate the synergistic effect of Tamoxifen and deinoxanthin on the MDA-MB-453 breast cancer cell line; Expression levels of BAX, CASP-3, BCL-2, and HER2 genes were examined by RT-qPCR method. The ELISA method determined the amounts of BAX, CASP-3, BCL-2, and HER2 proteins and compared them with RT-qPCR results. It was determined that the antiproliferative effect of Tamoxifen on the HER2 positive breast cancer cell line was increased with the combination of deinoxanthin.

Cyclopia extracts act as selective estrogen receptor subtype downregulators in estrogen receptor positive breast cancer cell lines: Comparison to standard of care breast cancer endocrine therapies and a selective estrogen receptor agonist and antagonist.

Breast cancer is the most diagnosed type of cancer amongst women in economically developing countries and globally. Most breast cancers express estrogen receptor alpha (ERα) and are categorized as positive (ER+) breast cancer. Endocrine therapies such as, selective estrogen receptor modulators (SERMs), aromatase inhibitors (AIs), and selective estrogen receptor downregulators (SERDs) are used to treat ER+ breast cancer. However, despite their effectiveness, severe side-effects and resistance are associated with these endocrine therapies. Thus, it would be highly beneficial to develop breast cancer drugs that are as effective as current therapies, but less toxic with fewer side effects, and less likely to induce resistance. Extracts of Cyclopia species, an indigenous South African fynbos plant, have been shown to possess phenolic compounds that exhibit phytoestrogenic and chemopreventive activities against breast cancer development and progression. In the current study, three well characterized Cyclopia extracts, SM6Met, cup of tea (CoT) and P104, were examined for their abilities to modulate the levels of the estrogen receptor subtypes, estrogen receptor alpha and estrogen receptor beta (ERβ), which have been recognized as crucial to breast cancer prognosis and treatment. We showed that the Cyclopia subternata Vogel (C. subternata Vogel) extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, reduced estrogen receptor alpha protein levels while elevating estrogen receptor beta protein levels, thereby reducing the ERα:ERβ ratio in a similar manner as standard of care breast cancer endocrine therapies such as fulvestrant (selective estrogen receptor downregulator) and 4-hydroxytamoxifen (elective estrogen receptor modulator). Estrogen receptor alpha expression enhances the proliferation of breast cancer cells while estrogen receptor beta inhibits the proliferative activities of estrogen receptor alpha. We also showed that in terms of the molecular mechanisms involved all the Cyclopia extracts regulated estrogen receptor alpha and estrogen receptor beta protein levels through both transcriptional and translational, and proteasomal degradation mechanisms. Therefore, from our findings, we proffer that the C. subternata Vogel extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, selectively modulate estrogen receptor subtypes levels in a manner that generally supports inhibition of breast cancer proliferation, thereby demonstrating attributes that could be explored as potential therapeutic agents for breast cancer.