SKBR3 Breast Cancer Cells Research Articles (Page 1)

To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer. qRT-PCR and Western blotting were used to detect the expression of lncRNA NEAT1, miR-133b, and PD-L1 in breast cancer SKBR3 cells and paclitaxel-resistant SKBR3 cells (SKBR3-PR). The effects of transfection with si-NEAT1 and miR-133b mimics on MRP, MCRP and PD-L1 expressions and cell proliferation, migration and apoptosis were investigated using qRT-PCR, Western blotting, scratch and Transwell assays, and flow cytometry. Rescue experiments were conducted using si-NEAT1 and miR-133b inhibitor. Human THP-1 macrophages were cultured in the presence of conditioned media (CM) derived from SKBR3 and SKBR3-PR cells with or with si-NEAT1 transfection for comparison of IL-4-induced macrophage polarization by detecting the surface markers. LDH@si-NEAT1 nanocarriers were constructed, and their effects on MRP, MCRP and PD-L1 expressions and cell behaviors of the tumor cells were examined. THP-1 cells were treated with the CM from LDH@si-NEAT1-treated tumor cells, and the changes in their polarization were assessed. SKBR3-PR cells showered significantly upregulated NEAT1 and PD-L1 expressions and lowered miR-133b expression as compared with their parental cells. Transfection with si-NEAT1 and miR-133b mimics inhibited viability, promoted apoptosis and enhanced MRP and BCRP expressions in SKBR3-PR cells. NEAT1 knockdown obvious upregulated miR-133b and downregulated PD-L1, MRP and BCRP expressions. The CM from SKBR3-PR cells obviously promoted M2 polarization of THP-1 macrophages, which was significantly inhibited by CM from si-NEAT1-transfected cells. Treatment with LDH@si-NEAT1 effectively inhibited migration and invasion, promoted apoptosis, and reduced MRP, BCRP and PD-L1 expressions in the tumor cells. The CM from LDH@si-NEAT1-treated SKBR3-PR cells significantly downregulated Arg-1, CD163, IL-10, and PD-L1 and upregulated miR-133b expression in THP-1 macrophages. LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.

The combination of trastuzumab and chemotherapeutic drugs improves the prognosis of patients with metastatic disease and reduces the mortality. However, trastuzumab resistance has limited the remarkable improvement of this drug. The carboxyl-terminal modulator protein (CTMP) is involved in the regulation of various cancers through positive or negative regulation of Akt. In the HER2-positive SkBR3 breast cancer cell line, CTMP overexpression increases Akt phosphorylation at Thr308 and Ser473. Therefore, CTMP might mediate trastuzumab resistance. The main objective of the paper is to explore the role of CTMP in trastuzumab efficacy in HER2 + metastatic breast cancer (MBC) patients. Ninety-six patients received trastuzumab in combination with chemotherapy or hormonal therapy until disease progression. The overall responses of all the patients were assessed as follows: complete response (n = 5), partial response (n = 36), stable disease (n = 24), and progressive disease (n = 31). Immunohistochemistry (IHC) staining was carried out to identify CTMP expression in formalin-fixed paraffin-embedded (FFPE) archival tissue blocks. 58 cases had high CTMP expression levels and 38 cases had low CTMP expression levels. The Mann-Whitney U test showed that CTMP expression was markedly higher in trastuzumab non-responders than in trastuzumab responders (P = 0.039). In addition, high CTMP expression was a strong and independent predictor of shorter recurrence-free survival in patients with metastatic breast cancer, as determined by the Kaplan-Meier method. Based on the results, further examination of CTMP in HER2-enriched (MBC) tissue samples could be helpful in predicting patients at risk of tumor progression and trastuzumab resistance.

Through network pharmacological prediction and in vitro experimental verification, the mechanism of action of Lianqiao Jinbei Decoction (LJD) in inhibiting HER2-positive breast cancer cells was clarified, providing experimental evidence for its treatment of HER2-positive breast cancer. Network pharmacology method was used to construct the potential target network of LJD in the treatment of HER2+ breast cancer. After cell culture in vitro, the proliferation of HER2+ SK-BR3 breast cancer cells was investigated using CCK-8 technique. The apoptotic potential of SK-BR3 cells was detected by flow cytometry, and the migration of SK-BR3 cells was detected by cell scratch assay. The expression of HER2 protein in SK-BR3 breast cancer cells was detected by ELISA. HER2 was identified as the central gene and quercetin, β-sitosterol, and luteolin were the primary active ingredients using network pharmacology analysis. Serum-containing LJD medication can stop SK-BR3 cells from proliferating (P<0.05). Serum-containing LJD drugs at high, medium, and low concentrations may induce SK-BR3 cell death (P<0.05). LJD serum at high, medium, and low concentrations reduced the migration of SK-BR3 cells (P<0.05). The expression of HER2 protein was decreased by LJD high, medium, and low concentration drug-containing serum (P<0.05). Regarding treating HER2-positive breast cancer, LJD has a multi-component, multi-target, and multi-pathway mode of action. The primary target of LJD's activity is the HER2 protein. Serum-containing LJD medication can prevent SK-BR3 cells from proliferating and migrating while encouraging their apoptosis. This effect may be attained by preventing HER2 protein expression.

ABSTRACT Sufficient MHC-I expression on cancer cells is essential for the recognition and killing of cancer cells by immune effector cytotoxic T-lymphocyte (CTL). An important mechanism of cancer immune escape is loss or down-regulation of MHC-I. This is frequently associated with reduced expression of NOD-like receptor (NLR) caspase recruitment domain containing protein 5 (NLRC5), genetically and epigenetically. NLRC5, a regulator of MHC-I, has been identified as a potential target of miR-4319 due to its complementary binding site for miR-4319, according to prediction by TargetScan (http://www.targetscan.org/). Inhibition of miR-4319 by IFN-γ (known as MHC-I increasing agent) to upregulate NLRC5 with upregulation of MHC-I in MHC-I-deficient breast cancer cells, however, remains unclear. After treatment with IFN-γ, miR-4319 was detected with qRT-PCR; NLRC5 protein was detected with western-blot; and MHC-I mRNA and protein were detected with qRT-PCR and western-blot, respectively. It was found statistically that miR-4319 was lower and NLRC5 protein was higher in groups of 50 U/ml and 100 U/ml IFN-γ, and MHC-I mRNA and protein were higher in all groups of different concentrations of IFN-γ, except for HLA-A protein in 25 U/ml IFN-γ group, with dose dependent tendency, compared with the control group. IFN-γ inhibits miR-4319 and upregulates NLRC5, thereby enhancing expression of MHC-I in SKBR3 breast cancer cells, while limitations include the absence of functional rescue experiments and in vivo validation. Along with direct cytotoxicity on tumor cells, IFN-γ’s immunomodulatory effect strengthens tumor immunogenicity, counteracts immune evasion mechanisms, and potentially improves the efficacy of cancer immunotherapy.

Resistance of cancer cells to immune-mediated killing poses a significant challenge in optimizing therapeutic strategies and minimizing adverse effects during chemotherapy, radiotherapy, and immunotherapy. Consequently, exploring novel biomarkers are critically needed to assess and monitor cancer treatment response, detect toxic side effects early, and guide individualized therapy. We conducted this research to enhance ourunderstand challenges posed by drug resistance in breast cancer treatment where the impact of pretreatment of BT-474 and SKBR-3 breast cancer cell lines with doxorubicin hydrochloride (doxorubicin) and paclitaxel (taxol) on complement-dependent cytotoxicity (CDC) andopsonization was opserveded. Both cell lines were treated withdifferent concentrations of each drug for 24 and 48 h, followed by evaluating expression of membrane-bound complement regulatory proteins (mCRPs), factor H (fH) binding, iC3b opsonization, and cell killing by CDC. Pretreatmentwith chemotherapeutics led to variable upregulation of mCRPs (CD46, CD55, CD59) and increased fH binding, contributing to reduced cancer cell lysis at both time points. Conversely, iC3b opsonization was enhanced after 48 h of exposure to either drug. Though this work is limited to HER2-positive breast cancer cell lines, our data suggest that though this work is limited to HER2-positive breast cancer cell lines, short-term exposure to chemotherapeutics enhanced their resistance to CDC. However, since they are efficiently opsonized with the iC3b, this could increase their vulnerability to immunological attacks by CDCC) and ADCC. Such unique contribution illuminates a previously unnoticed shift in the immune elimination landscape following chemotherapy and opens doors for enhancing combination immunotherapies.

BackgroundImmune evasion is a characteristic hallmark of cancer. Immunotherapies aim to activate and support the body's immune system to recognize and fight tumor cells. Induction of immunogenic cell death (ICD) and the associated activation of danger signaling pathways can increase the immunogenicity of tumor cells. Therapeutic ICD stimuli activate endoplasmic reticulum stress pathways and apoptosis leading to the cellular expression of damage-associated molecular patterns (DAMPs).The aim of our in vitro study was to investigate whether mistletoe extracts induce characteristics of immunogenic tumor cell death in cancer cell lines.MethodsThree human breast cancer cell lines and one murine melanoma cell line (SKBR3, MDA-MB-231, MCF-7, and B16F10) were treated with aqueous, fermented Viscum album extract (VAE: Iscador Qu spec.) and taxol or tunicamycin as positive controls, respectively. To investigate whether VAE induces ribotoxic stress, we measured the ER stress regulators p-eIF2a, ATF4, and CHOP by Western blot. Cell surface exposure of DAMPs (calreticulin, heat shock proteins hsp70 and hsp90), apoptosis and induction of mitochondrial reactive oxygen species (ROS) were assessed by flow cytometry. HMGB1 and ATP were quantified by ELISA and chemiluminescence assay, respectively.ResultsTreatment with VAE resulted in phosphorylation of eIF2α in all cancer cell lines tested and increased calreticulin (CRT) exposure on the surface of pre-apoptotic SKBR3 breast cancer and B16F10 mouse melanoma cells. VAE exerted a concentration-dependent effect in all cell lines, resulting in a significantly increased exposure of three DAMPs (CRT, hsp70 and hsp90) on the surface of early apoptotic cells. Furthermore, VAE elevated mitochondrial ROS production and the release of ATP. HMGB1 release was not induced by VAE.ConclusionsIn this in vitro study, we demonstrated for the first time the potential of a mistletoe extract to induce surrogate markers of immunogenic cancer cell death. This is a primary step in investigating the potential of VAEs to contribute to ICD-induced tumor-specific immune activation.

Purification and characterization of lectin from Phyllanthus reticulatus (PRL) plant fruit inducing cytotoxic effect on T47D and SKBR3 breast cancer cell lines.

Angiogenesis plays an important role in progression of tumors including breast cancer, which accounts for the vast majority of women's malignant tumors globally, to meet the excessive requirement of oxygen and nutrition for growth, metastasis, and invasion of the tumor. Therefore, targeting tumor angiogenesis has turned into a significant target for cancer therapy. Erbin has a significant effect on the initiation and progression of cancer, including breast cancer, but its role in inhibiting vascular endothelial cell proliferation and angiogenesis by breast cancer cells remains unclear. In this study, human SKBR3 and MCF-7 breast cancer cells were used and transfected with the plasmid and siRNA for overexpression and silence of Erbin, respectively. Western blot, qRT-PCR, CLEIA, CCK-8 and Matrigel Tube Formation Assay were used for the proteins detection, mRNAs detection, detection of VEGF in the culture supernatants, detection of cell proliferation and detection of the angiogenic ability of HUVECs in vitro, respectively. It was shown that the expression of both Erbin protein and mRNA in SKBR3 cells was lower compared to that in MCF-7 cells (p < 0.05). While the expression of VEGF protein was higher in SKBR3 cells than that in MCF-7 cells (p < 0.05). Furthermore, the VEGF protein and mRNA in the cells, VEGF protein in the culture supernatant, HUVEC proliferation in the conditioned medium at 16 h and 24h, the total length of tube formation in the conditioned medium, and pSTAT3 protein in the cells, were downregulated by transfection of Erbin gene in SKBR3 cells and upregulated (excluding HUVEC proliferation at 16 h) by transfection of Erbin siRNA in MCF-7 cells compared with their NC cells (p < 0.05). It can be concluded that Erbin, with inhibiting the STAT3 pathway, suppresses the proangiogenic effects of breast cancer cells, thereby suggesting its potential as a therapeutic target for breast cancer.

IntroductionCancer cells reprogram metabolic pathways to meet energy demands and sustain rapid growth, a hallmark of malignancy. Identifying molecular signatures underlying these changes can aid in early detection and inform targeted therapies. miR-526b has been shown to promote migration, invasion, angiogenesis, and metastasis, yet its role in dysregulated glucose metabolism remains underexplored.MethodsWe used MCF7 (Luminal A) and SKBR3 (HER2-Enriched) breast cancer cell lines, which exhibit distinct metabolic characteristics, to study miR-526b’s impact on metabolic marker expression, ATP production, oxygen consumption rate, and extracellular acidification. Cells were treated with glycolysis inhibitor 2 Deoxy-D-Glucose (2DG) or ox-phos inhibitor Oligomycin (OM) to measure dependence on glycolysis or oxidative phosphorylation. Stable transfection was used to overexpress miR-526b in MCF7 and SKBR3 cell lines, and miRNA inhibitors were used to inhibit miR-526b in MCF7-COX2 cells, comparing its effects across subtypes. Targeted inhibition of EP4 with a specific antagonist (EP4A) RQ-15986 (CJ-042794) was done in aggressive MCF7-COX2 cells to test the involvement of COX-2/EP4.ResultsSKBR3 exhibits an enhanced glycolytic phenotype, while MCF7 demonstrates increased ox-phos metabolism. Overexpression of miR-526b amplified these inherent metabolic properties, increasing ATP production and proliferation in both cell lines. miR-526b enhanced ox-phos activity in MCF7, reducing sensitivity to glycolysis inhibition, whereas it amplified glycolytic metabolism in SKBR3, reducing sensitivity to ox-phos inhibition. Overexpression of COX-2 in MCF7 replicated the metabolic effects of miR-526b. Inhibition of miR-526b in MCF7-COX2 cells enhances HK2 and GLUT1 expression, but did not significantly alter cell proliferation or cell viability. Targeting the COX-2/EP4 axis with a selective EP4A reversed the transcriptomic changes induced by miR-526b, but did not reduce the increased proliferation observed in MCF7-COX2.ConclusionmiR-526b enhances inherent metabolic characteristics of breast cancer cell lines, increasing ATP production, proliferation, and resistance to metabolic inhibitors. Targeting the COX-2/EP4 axis mitigated some of the effects induced by miR-526b, but it did not normalize cell behavior, highlights the complex regulation of glucose metabolism in breast cancer and underscores the need for combination therapy strategies.Graphical

Discovery of CZY43 as a new small-molecule degrader of pseudokinase HER3.

The anticancer properties of recombinant α-luffin (LUF) are wellestablished. However, the cytotoxic effects of encapsulating LUF within niosomes on the SKBR3 breast cancer cell line have yet to be explored. Our study aimed to investigate whether this encapsulation strategy could improve cytotoxic effects. Alpha-luffin was expressed, purified, and refolded. Then, this protein was utilized to craft an optimal formulation, guided by experimental design. In this work, we have explored various physicochemical properties, including particle size, polydispersity index, zeta potential, morphology, entrapment efficiency, drug release and kinetics, storage stability, and FTIR spectroscopy. Additionally, we have assessed the cellular uptake and cytotoxic effect of the optimized niosome formulation on the SKBR3 breast cancer cell line. The optimized niosome exhibited a mean diameter of 315±6.4 nm (DLS). Successful encapsulation of LUF into regularly shaped, spherical niosomes was achieved, with an encapsulation efficiency of 73.45±2.4%. Notably, Niosomal LUF (NLUF) exhibited significantly increased cytotoxicity against SKBR3 cells. These findings suggest that niosomes loaded with LUF hold promise as a potential treatment strategy for breast cancer.

HER2-positive breast cancer is an aggressive subtype characterized by the overexpression of the HER2 receptor, a transmembrane glycoprotein critical for tumor progression. Current therapies often face challenges like drug resistance and systemic toxicity, necessitating the development of advanced drug delivery systems. This study aimed to fabricate and determine the cytotoxicity of pH-sensitive PLA nanoparticles dual-loaded with docetaxel and each of the small molecule tyrosine kinase inhibitors (STKIs) (tucatinib, neratinib, lapatinib) in HER2-positive breast cancer cells. Nanoparticles were synthesized by a dispersion polymerization method using an acidlabile crosslinking agent, and PEG and lactide macromonomers. They were characterized for structure (TEM), surface morphology (SEM), particle size, polydispersity index, zeta potential, and drug loading capacity. Cytotoxicity was assessed in vitro on SKBR3 and MCF7 breast cancer cell lines, with IC50 values compared across formulations. The nanoparticles were spherical with nanoscale sizes and negative zeta potential values. In vitro studies demonstrated enhanced antiproliferative effects of the drug-loaded nanoparticles, with synergistic activity observed between docetaxel and the STKIs. The drug concentrations were halved in combination formulations and resulted in better cytotoxicity compared to single-drug treatments, particularly against SKBR3 cells. The IC50 values were lower in SKBR3 cells than in MCF7 cells, highlighting the role of HER2 expression in the activity of TKIs. The pH-sensitive PLA nanoparticles effectively co-delivered docetaxel and STKIs and demonstrated enhanced efficacy and reduced drug dosages in HER2-positive breast cancer models. This study provides a foundation for further exploration of nanoparticle-based combination therapies with potential applications in treating other aggressive cancer types.

ObjectiveThis study aimed to investigate the synergistic effects of the chemotherapy drug Carfilzomib (CFZ) and Pistachio hull extract on the SK-BR3 breast cancer cell line.MethodsIn this experimental study, we evaluated the effect of Pistachio hull extract and CFZ as standalone treatments on cell viability using the MTT assay at 24- and 48-hours post-treatment. Following this, we conducted combination therapy analyses to assess the potential synergistic relationship between Pistachio hull extract and CFZ after 24- and 48-hours of treatment on both the SK-BR3 breast cancer cell line and the MCF10A normal cell line. We utilized real-time PCR to measure the expression levels of MDR1, MRP1, NF-κB p65, and Caspase3 genes. Additionally, the NF-κB p65 transcription factor was evaluated using ELISA after 24- and 48-hours.ResultsThe MTT assay revealed IC50 values of 2.014 mg/mL and 1.031 mg/mL in the SK-BR3 cell line, and 3.265 mg/mL and 2.994 mg/mL in the MCF10A cell line at 24- and 48-hours post-treatment with Pistachio hull extract. CFZ concentrations of 0.181 × 10− 3 mg/mL and 0.0057 × 10− 3 mg/mL in the SK-BR3 cell line, as well as 5.54 × 10− 3 mg/mL and 2.51 × 10− 3 mg/mL in the MCF10A cell line, inhibited growth by up to 50%. The analysis of combination therapy indicated a synergistic effect between the two treatments after both 24- and 48-hours of exposure. Real-time PCR results demonstrated significant alterations in the expression of MDR1, MRP1, NF-κB p65, and Caspase3 genes, along with changes in NF-κB p65 protein levels in both cell lines following treatment with Pistachio hull extract, CFZ, or their combination compared to the control group (p < 0.05).ConclusionThe findings highlight the effectiveness of CFZ as a proteasome inhibitor when used in conjunction with Pistachio hull extract in breast cancer cell lines. Therefore, both CFZ and Pistachio hull extract, whether administered alone or in combination, represent promising molecular targets for breast cancer treatment.

BackgroundTP53 variant classification benefits from the availability of large-scale functional data for missense variants generated using cDNA-based assays. However, absence of comprehensive splicing assay data for TP53 confounds the classification of the subset of predicted missense and synonymous variants that are also predicted to alter splicing. Our study aimed to generate and apply splicing assay data for a prioritised group of 59 TP53 predicted missense or synonymous variants that are also predicted to affect splicing by either SpliceAI or MaxEntScan.MethodsWe conducted splicing analyses using a minigene construct containing TP53 exons 2 to 9 transfected into human breast cancer SKBR3 cells, and compared results against different splice prediction methods, including correlation with the SpliceAI-10k calculator. We additionally applied the splicing results for TP53 variant classification using an approach consistent with the ClinGen Sequence Variant Interpretation Splicing Subgroup recommendations.ResultsAberrant transcript profile consistent with loss of function, and for which a PVS1 (RNA) code would be assigned, was observed for 42 (71%) of prioritised variants, of which aberrant transcript expression was over 50% for 26 variants, and over 80% for 15 variants. Data supported the use of SpliceAI ≥ 0.2 cutoff for predicted splicing impact of TP53 variants. Prediction of aberration types using SpliceAI-10k calculator generally aligned with the corresponding assay results, though maximum SpliceAI score did not accurately predict level of aberrant expression. Application of the observed splicing results was used to reclassify 27/59 (46%) test variants as (likely) pathogenic or (likely) benign.ConclusionsIn conclusion, this study enhances the integration of splicing predictions and provides splicing assay data for exonic variants to support TP53 germline classification.Graphical abstract

This study aims to evaluate Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA) expression across various breast cancer subtypes, its prognostic significance, and the impact of SDHA knockdown on breast cancer cell functions. To assess SDHA expression in breast cancer, we utilized multiple publicly available databases. Prognostic significance was also evaluated using relevant databases. Methylation status, and enrichment analysis were performed using the GSCA database. The mutational status of SDHA was examined using cBioPortal, and its relationship with immune infiltration and drug sensitivity was assessed. Functional assays, including cell proliferation, colony formation, wound healing, and SDHA knockdown, were performed using MCF-7 and SKBR3 breast cancer cell lines. Our results showed that SDHA was significantly overexpressed in breast cancer tissues compared to normal tissues. High SDHA expression was correlated with worse survival in breast cancer patients. Pathological stage analysis revealed that SDHA expression increased as the disease progressed, with lower methylation levels in tumor tissues suggesting epigenetic regulation of its expression. Functionally, SDHA knockdown in MCF-7 and SKBR3 cells led to significant reductions in cell proliferation, colony formation, and migration, highlighting its role in supporting breast cancer cell growth and metastasis. SDHA was upregulated in breast cancer and associated with poor prognosis. Our findings also suggest that SDHA plays a crucial role in promoting breast cancer cell growth and migration, indicating its therapeutic potential. Targeting SDHA could provide a novel strategy for breast cancer treatment, particularly in overcoming chemoresistance and inhibiting tumor progression.

HER2-positive breast cancer has an aggressive tumour progression among breast cancers characterized by the overexpression of HER2. Trastuzumab is an FDA-approved drug and has significantly improved outcomes for patients; however, drug resistance remains a major challenge. Tumour heterogeneity, describing genetic, epigenetic, and phenotypic differences within and between tumours, complicates tumour treatment and contributes to drug resistance. Understanding the mechanisms underlying Trastuzumab resistance, such as tumour heterogeneity, is crucial for developing new and effective therapeutic strategies. This study investigates the role of ITGB3 heterogeneity in Trastuzumab resistance, focusing on its impact on TGF-β signalling and migration marker response. It also evaluates the potential of combining Trastuzumab with the integrin β3 inhibitor cilengitide to overcome resistance associated with ITGB3 levels. Trastuzumab-resistant HER2-positive HCC1954 and SKBR3 breast cancer cell lines were generated and analysed for ITGB3 expression heterogeneity. The impact of ITGB3 on TGF-β-responsive genes (WWP1, CARM1, RASGRP1, THBS1, KCTD5, SGCA, EIF3S6, MCAM, FXR2, MTMR3, SOCS3, SLC2A4RG, MMP2, MMP9, and HSP47) and cell migration (Col4a1, fibronectin, ICAM1, Timp2, and vimentin) was analysed using luciferase reporter assays and real-time PCR. The effects of combined treatment with Trastuzumab and cilengitide were also evaluated via wound closure assay. ITGB3 expression varied significantly among resistant clones, correlating with increased expression of TGF-β-responsive genes and enhanced migration markers. Combined treatment with Trastuzumab and cilengitide significantly reduced TGF-β signalling and migration-related gene expression, particularly in high ITGB3-expressing cells. ITGB3 plays a critical role in Trastuzumab resistance through the modulation of TGF-β signalling, migration, and contributing to tumour heterogeneity. Targeting ITGβ3, alone or in combination with cilengitide, offers a promising strategy to resensitize resistant HER2-positive breast cancer cells to Trastuzumab. These findings provide valuable insights into the mechanisms of Trastuzumab resistance and suggest potential therapeutic avenues for improving patient outcomes.

Introduction: Earlier, we documented that a combination of vitamins A and D3 synergistically inhibited the growth of MCF-7, T48:A18 and SKBR3 breast cancer cells with the best activity seen in the ER+ cell line MCF-7. Transcriptomic analysis of treated MCF-7 cells also showed that the combination significantly upregulated the apoptosis and unfolded protein response canonical pathways, and reduced estrogen signaling. Objective: This study aimed to explore the impact of increasing vitamin A and D3 dose combinations over time in a postmenopausal model of breast cancer using ovariectomized athymic female mice bearing MCF-7 xenografts and further analyze mechanisms of action in MCF-7 cells using RNA-seq analysis. Methods: MCF-7 breast cancer cells were grown in culture for the xenograft experiments. Athymic female mice were injected with MCF-7 cells (1 x 10^6 in 100 µl of 50% Matrigel mixed with sterile PBS) via subcutaneous injection. Once the tumors reached an average volume of 100 mm³, the mice were randomly divided into four groups and treated with different vitamin A and D3 combinations. Tumor sizes and mouse body weights were monitored on a biweekly basis. After the treatment period, the mice were euthanized, and the tumors were surgically removed and measured. RNA-seq data from the treated MCF-7 cells were then further evaluated using IPA. Results: As compared with controls, treatment with vitamin A (25,000 IU) and vitamin D (10,000 IU) led to a significant reduction in tumor volume &gt;70%, (p &lt; 0.05-0.01) in OVX athymic mice with MCF-7 xenografts as determined by a two-tailed Student T test. Over the treatment period, the tumor volume in mice treated with vitamin A (10,000 IU) and vitamin D (5,000 IU) or vitamin A (25,000 IU) and vitamin D (5,000 IU) also trended downward and was statistically significant using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (p&lt;0.05 and p&lt;0.0001, respectively) but was not significant using a two-tailed Student T test. In cultured MCF-7 cells, Ingenuity Pathway Analysis of mRNA-seq data showed that the vitamin A and D combination significantly altered the expression of 101 genes out of 864 in the molecular mechanisms in cancer canonical pathway, downregulating gene expression in the integrin/P13K/Akt/mTOR pathway. Conclusions: The findings showed that the combination of vitamins A and D3 effectively reduced tumor burden in a postmenopausal MCF-7 xenograft mouse model, with effects that were both dose-dependent and time-dependent. The combination also significantly altered the expression of genes in the molecular mechanisms of cancer canonical pathway in cultured MCF-7 cells. These preclinical data support the use of vitamins A and D3 in the management of estrogen-dependent breast cancers, with the caveat that higher doses and longer treatment periods may be needed to observe anti-tumor effects. Keywords: Apoptosis, autophagy, breast cancer, cell cycle, integrin, postmenopausal, P13K, tumor load, xenograft

Gold nanoparticles (AuNPs) have shown promise for applications in the diagnosis and treatment of different diseases, including cancer. Understanding the effect of AuNPs on metabolic reprogramming in cancer cells at the single cell level is of high importance for improving the efficacy and safety. Fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide (phosphate) hydrogen (NAD(P)H) as a main metabolic cofactor and an indicator of metabolic reprogramming in cancer cells enables real-time monitoring of cancer cell metabolism in response to different treatments, including AuNPs. However, NPs such as AuNPs can be a potential source of signals themselves, which provides opportunities to measure the NP internalization, but it is also important to minimize confounding effects on metabolic measurements. In this study, we detected inherent photoluminescence (PL) from the AuNPs in treated prostate cancer cells (PC-3 cell line) as well as in solution at the NAD(P)H emission wavelength. We developed an analysis approach to minimize the confounding effect of the AuNPs' PL on metabolic measurements. On the other hand, we assessed the reliability of the intracellular AuNPs' PL as an estimator of AuNP uptake. To assess if intracellular AuNPs' PL may be dependent on the exposed cell type, we performed NAD(P)H FLIM imaging of AuNP-exposed SKBR-3 breast cancer cells, where we observed a similar AuNP PL but at a much lower level compared to PC-3 cells. We proposed that this difference can be attributed to the different levels of AuNP uptake or varying intracellular microenvironments.

Tissue specimen quality is becoming increasingly important for basic research and routine clinical results. Warm ischemia time (WIT) affects human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) scores. However, the role of WIT on HER2 modulation remains unclear. We hypothesized that the WIT-mediated increase in HER2 IHC scores was caused by hypoxia. Therefore, this study aimed to determine the mechanism by which WIT mediates the increase in HER2. HER2 mRNA expression was measured in 4T1, SKBR3, and HCC1954 breast cancer cell lines using real-time PCR following hypoxia exposure. The membrane proteins were isolated and extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) or evaluated through non-permeabilized immunofluorescent analysis. Hypoxic conditions up-regulated GLUT1 mRNA expression but not HER2 expression. The HER2 membrane protein fraction increased in response to hypoxic conditions. Nonpermeabilized immunofluorescence analysis showed that membrane-bound HER2 was also promoted under hypoxic conditions. HER2 is not regulated at the mRNA level; however, the level of membrane-bound HER2 increases in response to hypoxia.

Among the known breast cancers, the subtype with HER2 receptors-overexpressing cells is associated with a poor prognosis. The adopted monoclonal antibody Trastuzumab has improved clinical outcomes, but it is associated with drug resistance and relatively high costs. The present work adopted the peptide solid-phase synthesis method to synthesise branched poly(ε-lysine) peptide dendrons with 8 branching arms integrating, at their carboxy terminal molecular root, either an arginine or the HER2 receptor-binding sequence LSYCCK or the scramble sequence CSCLYK. These dendrons were synthesised in quantities higher than 100 mg/batch and with a purity exceeding 95%. When tested with two types of breast cancer cells, the dendrons led to levels of inhibition in the HER2 receptor-overexpressing breast cancer cells (SKBR3) comparable to Trastuzumab and higher than breast cancer cells with low receptor expression (MDA-MB-231) where inhibition was more moderate. Noticeably, the presence of the amino acid sequence LSYCCK at the dendron molecular root did not appear to produce any additional inhibitory effect. This was demonstrated also when the scramble sequence CSCLYK was integrated into the dendron and by the lack of any antiproliferative effect by the control linear target sequence. The specific inhibitory effect on proliferation was finally proven by the absence of cytotoxicity and normal expression of the cell migration marker N-Cadherin. Therefore, the present study shows the potential of poly(ε-lysine) dendrons as a cost-effective alternative to Trastuzumab in the treatment of HER2-positive breast cancer.