Expression In MCF-7 Research Articles

Hydroxyacid Oxidase 1, a Glutamine Metabolism-Associated Protein, Predicts Poor Patient Outcome in Luminal Breast Cancer.

Breast cancer (BC), which remains the most prevalent malignancy among women, is characterised by significant heterogeneity across its molecular subtypes. Oestrogen receptor-positive (ER+) (luminal) BC represents approximately 75% of cases, and despite advancements in treatment there remains around a 40% recurrence rate. Cellular uptake of glutamine is conducted by solute carriers (SLCs), which are significantly associated with outcome in luminal BC. In this study, differential gene expression analysis was carried out using The Cancer Genome Atlas BC dataset. This identified hydroxyacid oxidase 1 (HAO1) as significantly overexpressed in luminal BC with a high expression of SLCs. Extended analysis in the METABRIC (n = 1980) and Breast Cancer Gene-Expression Miner (n = 4421) transcriptomic databases and the Nottingham (n = 952) BC tissue cohort showed a varied survival outcome for HAO1 expression at the genomic, transcriptomic, and proteomic levels. HAO1 copy number (CN) gain (p = 0.002) and high HAO1 protein expression (p = 0.019) were associated with poor prognosis in luminal BC, whereas high HAO1 mRNA expression correlated with better survival outcomes (p = 0.023) suggesting a complex regulatory mechanism affecting HAO1 at different biological levels. Importantly, in luminal BC patients treated with endocrine therapy, high protein expression of HAO1 predicted shorter distant-metastasis free survival (p = 0.042). The knockdown of SLC1A5 and SLC7A5 significantly reduced HAO1 expression in MCF-7 and ZR-751 BC cell lines. Protein analysis confirmed significant associations between HAO1 and SLC7A5 and SLC1A5, emphasising a potential role for the enzyme in glutamine metabolism and its potential as a therapeutic target. This study underscores the prognostic significance of HAO1 in luminal BC and its relationship with patient outcomes.

The effects of perfluorooctanoic acid on breast cancer metastasis depend on the phenotypes of the cancer cells: An in vivo study with zebrafish xenograft model

Per- and polyfluorinated substances (PFAS) have been associated with numerous human diseases. Recent in vitro studies have implicated the association of PFAS with an increased risk of breast cancer in humans. This study aimed to assess the toxic effects of PFAS during the development of human breast cancer using a zebrafish xenograft model. Perfluorooctanoic acid (PFOA) was used as a PFAS chemical of interest for this study. Two common breast cancer cell lines, MCF-7 and MDA-MB-231, were used to represent the diversity of breast cancer phenotypes. Human preadipocytes were co-implanted with the breast cancer cells into the zebrafish embryos to optimize the microenvironment for tumor cells in vivo. With this modified model, we evaluated the potential effects of the PFOA on the metastatic potential of the two types of breast cancer cells. The presence of human preadipocytes resulted in an enhancement to the metastasis progress of the two types of cells, including the promotion of cell in vivo migration and proliferation, and the increased expression levels of metastatic biomarkers. The enhancement of MCF-7 proliferation by preadipocytes was observed after 2 days post injection (dpi) while the increase of MDA-MB-231 proliferation was seen after 6 dpi. The breast cancer metastatic biomarkers, cadherin 1 (cdh1), and small breast epithelial mucin (sbem) genes demonstrated significant down- and upregulations respectively, by the co-injection of preadipocytes. In the optimized xenograft model, the PFOA consistently promoted cell proliferation and migration and altered the metastatic biomarker expression in MCF-7, which suggested a metastatic effect of PFOA on MCF-7. However, those effects were not consistently observed in MDA-MB-231. The presence of the preadipocytes in the xenograft model may provide a necessary microenvironment for the progress of tumor cells in zebrafish embryos. The finding suggested that the impacts of PFOA exposure on different phenotypes of breast cancers may differ.

HER4 Affects Sensitivity to Tamoxifen and Abemaciclib in Luminal Breast Cancer Cells and Restricts Tumor Growth in MCF-7-Based Humanized Tumor Mice.

The impact of the HER4 receptor on the growth and treatment of estrogen receptor-positive breast cancer is widely uncertain. Using CRISPR/Cas9 technology, we generated stable HER4 knockout variants derived from the HER4-positive MCF-7, T-47D, and ZR-75-1 breast cancer cell lines. We investigated tumor cell proliferation as well as the cellular and molecular mechanisms of tamoxifen, abemaciclib, AMG232, and NRG1 treatments as a function of HER4 in vitro. HER4 differentially affects the cellular response to tamoxifen and abemaciclib treatment. Most conspicuous is the increased sensitivity of MCF-7 in vitro upon HER4 knockout and the inhibition of cell proliferation by NRG1. Additionally, we assessed tumor growth and immunological effects as responses to tamoxifen and abemaciclib therapy in humanized tumor mice (HTM) based on MCF-7 HER4-wildtype and the corresponding HER4-knockout cells. Without any treatment, the enhanced MCF-7 tumor growth in HTM upon HER4 knockout suggests a tumor-suppressive effect of HER4 under preclinical but human-like conditions. This phenomenon is associated with an increased HER2 expression in MCF-7 in vivo. Independent of HER4, abemaciclib and tamoxifen treatment considerably inhibited tumor growth in these mice. However, abemaciclib-treated hormone receptor-positive breast cancer patients with tumor-associated mdm2 gene copy gains or pronounced HER4 expression showed a reduced event-free survival. Evidently, the presence of HER4 affects the efficacy of tamoxifen and abemaciclib treatment in different estrogen receptor-positive breast cancer cells, even to different extents, and is associated with unfavorable outcomes in abemaciclib-treated patients.

Abstract PO3-24-07: The role of Hepatitis A Virus Cellular Receptor 1 (HAVCR1) in breast cancer and the impact on penetration of blood brain barrier

Abstract Background: Hepatitis A Virus Cellular Receptor 1 (HAVCR1) is associated with the biological behaviour of several cancers, and has been shown to regulate junctional complexes. However, its role in breast cancer remains unexplored. This study aimed to investigate the effects of HAVCR1 on human breast cancer. Methods: We explored the UALCAN web portal, which is linked to the TCGA database, to determine the expression of HAVCR1 in breast cancer and the Kaplan-Meier plotter to evaluate the correlation with the survival of the patients. HAVCR1 siRNA was used to knock down the HAVCR1 expression in MCF-7 and MDA-MB-231 cell lines. In vitro cell growth, wounding healing, and electric cell impedance sensing (ECIS for assessing cellular proliferation, migration and adhesion) assays were used. Transepithelial resistance (TEER) and paracellular permeability (PCP) were assessed to determine changes in the barrier function of human breast cancer cells. The expression of HAVCR1 and prospective interactive binding molecules of HAVCR1 protein was assessed using quantitative polymerase chain reaction (qPCR). Cellular response to docetaxel was evaluated and shown (IC50). The invasiveness of breast cancer cells through the human cerebral microvessel endothelial cells (hCMEC/D3) cell layers was measured using an in vitro transwell cell invasion assay. Results: HAVCR1 gene expression levels were significantly lower (P< 0.001) in breast cancer tissue compared with normal tissues. Patients with high expression of HAVCR1 had a significantly better relapse-free survival than low expression (P< 0.001). There was no significant difference in overall survival (OS) between the HAVCR1 high and the low expression groups (P=0.22). However, in subgroup analysis, high HAVCR1 expression was associated with better OS in luminal A (P=0.04). Knockdown (KD) HAVCR1 in breast cancer cells influence cell function by enhanced adhesion, proliferation, and migration compared to wild type (WT) cell lines. It was noted that loss of HAVCR1 rendered both MCF-7 and MDA-MB-231 cells resistant to docetaxel (IC50 for WT and HAVCR1 KD respectively being 0.86nM vs 1.75nM for MCF-7 and 1.35nM vs 15.48nM for MDA-MB-231). HAVCR1 KD increase adhesion to hCMEC/D3 cells (53.14±8.23 vs ±99.37±8.97 for MCF-7, P< 0.001; 109.5±97.56 vs 147.1±96.63 for MDA-MB-231, P=0.228, WT and HAVCR1 KD). In the cerebral endothelial cell invasion assay, we found that HAVCR1 KD resulted in a significantly increased invasion of the breast cancer cells through the endothelium (19.27±8.74 vs 33.58±16.81 for MDA-MB-231, P=0.0114, WT and HAVCR1 KD). For the barrier function, the knockdown led to a decrease in TEER and an increase in PCP when compared with WT cells in both cell lines. HAVCR1 KD cells showed decreased barrier function, together with reduced expression of Claudin-1/8/10, occludin, MarvelD3, Tricellulin and ZO1. Conclusions: HAVCR1 is a potential biomarker for breast cancer prognosis. HAVCR1 influences the integrity of barrier function by influence the gene expression of tight junction in breast cancer, and influence the penetration to blood brain barrier. Together, it suggests that HAVCR1 may be a potential therapeutic target in breast cancer. Citation Format: Xiaoshan Cao, Binbin Cong, Wen Jiang, Tracey Martin. The role of Hepatitis A Virus Cellular Receptor 1 (HAVCR1) in breast cancer and the impact on penetration of blood brain barrier [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 PO3-24-07.

Long non‑coding RNA DANCR aggravates breast cancer through the miR‑34c/E2F1 feedback loop.

Emerging scientific evidence has suggested that the long non‑coding (lnc)RNA differentiation antagonizing non‑protein coding RNA (DANCR) serves a significant role in human tumorigenesis and cancer progression; however, the precise mechanism of its function in breast cancer remains to be fully understood. Therefore, the objective of the present study was to manipulate DANCR expression in MCF7 and MDA‑MB‑231 cells using lentiviral vectors to knock down or overexpress DANCR. This manipulation, alongside the analysis of bioinformatics data, was performed to investigate the potential mechanism underlying the role of DANCR in cancer. The mRNA and/or protein expression levels of DANCR, miR‑34c‑5p and E2F transcription factor 1 (E2F1) were assessed using reverse transcription‑quantitative PCR and western blotting, respectively. The interactions between these molecules were validated using chromatin immunoprecipitation and dual‑luciferase reporter assays. Additionally, fluorescence in situ hybridization was used to confirm the subcellular localization of DANCR. Cell proliferation, migration and invasion were determined using 5‑ethynyl‑2'‑deoxyuridine, wound healing and Transwell assays, respectively. The results of the present study demonstrated that DANCR had a regulatory role as a competing endogenous RNA and upregulated the expression of E2F1 by sequestering miR‑34c‑5p in breast cancer cells. Furthermore, E2F1 promoted DANCR transcription by binding to its promoter in breast cancer cells. Notably, the DANCR/miR‑34c‑5p/E2F1 feedback loop enhanced cell proliferation, migration and invasion in breast cancer cells. Thus, these findings suggested that targeting DANCR may potentially provide a promising future therapeutic strategy for breast cancer treatment.

Circ_0011385 increases cisplatin resistance in breast cancer via miR-615-5psuppression

Purpose: Circular RNAs (circRNAs) participate in chemo-resistance among different cancers, containing breast cancer (BC). Herein, this project aimed to investigate the role of circ_0011385 on cisplatin (DDP) resistance in BC cells.
 Methods: circ_0011385 and microRNA (miR)-615-5p content in DDP-resistant BC cells and their parent cells (MCF-7) was calculated by RT-qPCR. CCK-8 assessed inhibition rate, and further to calculate half inhibitory concentration (IC50). Clone formation and flow cytometry experiments detected clone numbers and apoptosis rate. Dual-luciferase reporter system validated targeting between circ_0011385 and miR-615-5p.
 Results: circ_0011385 was upregulated, whereas miR-615-5p was decreased in MCF-7/DDP cells. Silencing circ_0011385 or miR-615-5p overexpression enhanced BC cell sensitivity to DDP, repress proliferation, and expedite apoptosis rate. Circ_0011385 directly targeted miR-615-5p.
 CONCLUSION: Silencing circ_0011385 increases DDP sensitivity by targeting and up-regulating miR-615-5p expression in MCF-7/DDP.

Combination Treatment with EGFR Inhibitor and Doxorubicin Synergistically Inhibits Proliferation of MCF-7 Cells and MDA-MB-231 Triple-Negative Breast Cancer Cells In Vitro.

The role of the epidermal growth factor receptor (EGFR) in tumor progression and survival is often underplayed. Its expression and/or dysregulation is associated with disease advancement and poor patient outcome as well as drug resistance in breast cancer. EGFR is often overexpressed in breast cancer and particularly triple-negative breast cancer (TNBC), which currently lacks molecular targets. We examined the synergistic potential of an EGFR inhibitor (EGFRi) in combination with doxorubicin (Dox) in estrogen-positive (ER+) MCF-7 and MDA-MB-231 TNBC cell lines. The exposure of MDA-MB-231 and MCF-7 to EGFRi produced an IC50s of 6.03 µM and 3.96 µM, respectively. Dox induced MDA-MB-231 (IC50 9.67 µM) and MCF-7 (IC50 1.4 µM) cytotoxicity. Combinations of EGFRi-Dox significantly reduced the IC50 in MCF-7 (0.46 µM) and MBA-MB 231 (0.01 µM). Synergistic drug interactions in both cell lines were confirmed using the Bliss independence model. Pro-apoptotic Caspase-3/7 activation occurred in MCF-7 at 0.1-10 µM of EGFRi and Dox single treatments, whilst 1 μM Dox yielded a more potent effect on MDA-MB-231. EGFRi and Dox individually and in combination downregulated the EGFR gene expression in MCF-7 and MDA-MB-231 (p < 0.001). This study demonstrates EGFRi's potential for eliciting synergistic interactions with Dox, causing enhanced growth inhibition, apoptosis induction, and downregulation of EGFR in both cell lines.

EFFECT OF COWANIN ON CYCLIN D1 EXPRESSION IN MCF-7/HER2 BREAST CANCER CELLS

Objective: Cowanin, isolated from the stem bark of Asam kandis (Garcinia cowa Roxb.) has been known to have cytotoxic activity in MCF-7/HER2 breast cancer cells. Recent studies have reported that cowanin compounds can inhibit MCF-7/HER2 cell migration and the T47D cell cycle in the G0-G1 phase. This research aims to determine the effect of cowanin on the cyclin D1 protein expression in MCF-7/HER2 breast cancer cells.&#x0D; Methods: The treatment consisted of a negative control group and a group given cowanin at a concentration of IC50 value (10,51 µM). The expression of cyclin D1 protein was detected using the western blot method. Observations of protein area and density were carried out using ImageJ software. Data were analyzed using the independent T-test.&#x0D; Results: The research showed that cowanin compounds induced cell cycle arrest of MCF-7/HER2 breast cancer cells by reducing the expression of cyclin D1 protein (p&lt;0,05).&#x0D; Conclusion: The findings show that cowanin can significantly decrease the area and density of cyclin D1 protein

Dual effect of vitamin D3 on breast cancer-associated fibroblasts

BackgroundCancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Despite the well-known in vitro antitumoral effect of vitamin D3 (VD3), its impact on breast CAFs is almost unknown. In this study, we analyzed the ex vivo effects of calcitriol on CAFs isolated from breast cancer tissues.MethodsCAFs were cultured with 1 and 10 nM calcitriol and their phenotype; gene expression, protein expression, and secretion were assessed. Calcitriol-treated CAFs-conditioned media (CM) were used to analyze the effect of CAFs on the migration and protein expression of MCF-7 and MDA-MB-231 cells.ResultsTumor tissues from VD3-deficient patients exhibited lower levels of β-catenin and TGFβ1, along with higher levels of CYP24A1 compared to VD3-normal patients. In VD3-deficient patients, CAF infiltration was inversely associated with CYP24A1 levels and positively correlated with OPN levels. Calcitriol diminished CAFs’ viability, but this effect was weaker in premenopausal and VD3-normal patients. Calcitriol reduced mRNA expression of CCL2, MMP9, TNC, and increased PDPN, SPP1, and TIMP1. It also decreased the secretion of CCL2, TNC, and the activity of MMP-2, while increasing cellular levels of TIMP1 in CAFs from all patient groups. In nonmetastatic and postmenopausal patients, PDPN surface expression increased, and CAFs CM from these groups decreased MCF-7 cell migration after ex vivo calcitriol treatment. In premenopausal and VD3-deficient patients, calcitriol reduced IDO1 expression in CAFs. Calcitriol-treated CAFs CM from these patients decreased OPN expression in MCF-7 and/or MDA-MB-231 cells. However, in premenopausal patients, calcitriol-treated CAFs CM also decreased E-cadherin expression in both cell lines.ConclusionThe effects of calcitriol on breast CAFs, both at the gene and protein levels, are complex, reflecting the immunosuppressive or procancer properties of CAFs. The anticancer polarization of CAFs following ex vivo calcitriol treatment may result from decreased CCL2, TNC (gene and protein), MMP9, and MMP-2, while the opposite effect may result from increased PDPN, TIMP1 (gene and protein), and SPP1. Despite these multifaceted effects of calcitriol on molecule expression, CAFs’ CMs from nonmetastatic and postmenopausal patients treated ex vivo with calcitriol decreased the migration of MCF-7 cells.

The miR-221/222 regulates the ID1 gene expression through PTEN, c-JUN and ARF4 mediators to control cell proliferation

Objective: To explore the effect of miR-221/222 on cell proliferation and regulation of inhibitor of differentiation1 (ID1) expression concerning the value of miR-221/22 in cancer diagnosis, prognosis, or therapeutic use. Methods: Embryonic cell line (HEK293), breast cancer cell line (MCF7), and lymphoblast cell line (U937) were employed to investigate cell proliferation in the presence or absence of miR-221/222 using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The relation of miR-221/222 and expression of the ID1 gene was monitored via luciferase activity and real-time PCR. Quantitative PCR was used to detect the effect of miR-221/222 on the expression of cellular proteins, c-JUN, ARF4, and PTEN. Results: The miR-221/222 significantly increased the expression level of the ID1 gene in MCF7 and U937 cell lines but downregulation of the ID1 gene was revealed for transfected HEK293 cells. Level of the ID1 mRNA showed that miR-221/222 regulated the ID1 gene expression at the transcriptional level. Moreover, miR-221/222 enhanced the cellular proteins, ARF4 and c-JUN, and expression in MCF7 and U937, while inhibiting them in HEK293. These findings indicated that the effect of miR-221/222 on ARF4 and c-JUN expression in HEK293 may be mediated by different pathways from MCF7 and U937. Conclusions: Increasing levels of miR-221/222 expression are correlated with cell proliferation and the regulation of ID1 expression. ID1 regulation is a complex process and miR-221/222 may regulate its expression via c-JUN and ARF4. The value of miR-221/22 in cancer diagnosis, prognosis, or therapeutic use may be cancer type dependent.

Evaluation of Saudi Juniperus procera Extracts Cytotoxicity and Regulatory Mechanisms of Tumorigenesis Against Two Breast Cancer Cell Lines

Background Breast cancer poses a major threat to female health worldwide. One of the medicinal plants used in traditional Saudi medicine is the Juniperus procera (Arar) plant, which is used to cure various illnesses, including cancer. Objectives The cytotoxic properties of Saudi J. procera extracts and their regulatory mechanisms against tumorigenesis in breast cancer were investigated. Methods The antioxidant capacities of the fruit and leaf extracts were detected by the 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay and 2,2-diphenyl-1-1picrylhydrazyl (DPPH) assay. The cell viability of the extracts was determined by 3-(4,5-dimethylthiazoyl)-2,5-diphenyl-tetrazolium bromide assay. The cell migration was detected by a wound healing assay. The cell cycle and cell apoptosis were analyzed by flow cytometry. The reactive oxygen species (ROS) was evaluated using a DCFDA-cellular (the cell permeant reagent 2',7' –dichlorofluorescein diacetate) reactive oxygen species detection assay. The protein and gene expression levels were estimated by western blotting and Reverse transcription polymerase chain reaction (RT-PCR), respectively. Results Phytochemical analysis revealed a high content of phenolic and flavonoids in fruit and leaf extracts. Relative to leaf extract, the fruit extract showed higher antioxidant activities and exhibited more potent cytotoxic effects against two breast cancer cell lines: MCF-7 and MDA-MB-231. The fruit extract arrested the cell cycle of the tested cancer cell lines in S phase and G1 phase, and downregulated PI3K (phosphoinositide 3-kinase-protein kinase)/Akt (protein kinase B)/mTOR (Mammalian target of rapamycin) pathway. It also suppressed aromatase expression in MCF-7 but upregulated it in MDA-MB-231. Conclusion J procera fruit and leaf extracts have significant antitumor activities against MCF-7 and MDA-MB-231 breast cancer cell lines.

Crocin enhances the sensitivity to paclitaxel in human breast cancer cells by reducing BIRC5 expression.

Paclitaxel (PTX) is one of the first-line chemotherapeutic agents for treating breast cancer. However, PTX resistance remains a major hurdle in breast cancer therapy. Crocin, the main chemical constituent of saffron, shows anti-cancer activity against various types of cancer. However, the effect of crocin on the resistance of PTX in breast cancer is still unknown. CCK-8 and TUNEL assays were employed to detect cell viability and apoptosis, respectively. The targets of crocin were predicted using HERB database and the targets associated with breast cancer were acquired using GEPIA database. The Venn diagram was utilized to identify the common targets between crocin and breast cancer. Baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) expression was detected by qRT-PCR and western blot analysis. The correlation between BIRC5 expression and survival was analyzed by Kaplan-Meier plotter and PrognoScan databases. Our data suggested that crocin aggravated PTX-induced decrease of viability and increase of apoptosis in MCF-7 and MCF-7/PTX cells. BIRC5 was identified as the target of crocin against breast cancer. Crocin inhibited BIRC5 expression in MCF-7 and MCF-7/PTX cells. BIRC5 is overexpressed in breast cancer tissues, as well as PTX-sensitive and PTX-resistant breast cancer cells. BIRC5 expression is related to the poor survival of patients with breast cancer. Depletion of BIRC5 strengthened PTX-induced viability reduction and promotion of apoptosis in MCF-7 and MCF-7/PTX cells. Moreover, BIRC5 overexpression reversed the inhibitory effect of crocin on PTX resistance in breast cancer cells. In conclusion, crocin enhanced the sensitivity of PTX in breast cancer cells partially through inhibiting BIRC5 expression.

TFAP2C Activates CST1 Transcription to Facilitate Breast Cancer Progression and Suppress Ferroptosis.

Cystatin SN (CST1) appears to have pro-tumor effects in breast cancer (BC) and is involved in ferroptosis; however, there is no report on the regulation of ferroptosis by CST1 for BC development. The purpose of this study is to investigate the functions and mechanisms operated by CST1 in BC development and ferroptosis. Transcription Factor Activator Protein 2γ (TFAP2C) and CST1 levels in BC tissues and estrogen receptor (ER)+ cells were quantified by RT-qPCR and western blotting. After knocking down TFAP2C and CST1 expression in MCF7 and T47D cells, the proliferation, colony formation ability, apoptosis, and cell cycle were assessed. Ferroptosis was verified by detecting glutathione peroxidase 4 (GPX4) and 4-hydroxy-2-nonenal (4HNE) levels. The kits were used to test Fe2+, reactive oxygen species, malondialdehyde, and glutathione levels, and ultrastructure of mitochondria was observed through transmission electron microscope. Dual-luciferase reporter assay and chromatin immunoprecipitation test were carried out to investigate the interaction of TFAP2C and CST1. A transplanted tumor model was established to explore the function of TFAP2C in tumorigenesis by quantifying TFAP2C, CST1, Ki67, and GPX4 levels through western blotting and immunochemistry after silencing TFAP2C. TFAP2C and CST1 were predominantly expressed in BC cells. Silencing of TFAP2C or CST1 expression suppressed ER+ BC cell proliferation, promoted apoptosis and ferroptosis, and blocked cell cycle transition from G1 phase to S phase. TFAP2C knockdown in transplanted tumors inhibited tumor growth and GPX4 level. Upregulating CST1 nullified the anti-tumor effects of TFAP2C knockdown and TFAP2C promoted CST1 expression through transcription activation. TFAP2C activates CST1 transcription to facilitate BC development and block ferroptosis.

Self-assembly circular multi-DNA mediated rolling circle amplification for sensitive detection of PD-L1+ circulating tumor cells in the peripheral blood of cancer patients

It has been found that evaluating the status of programmed cell death ligand (PD-L1) in the circulating tumor cells (CTCs) was associated with improved efficacy to anti-PD-L1 inhibitors. However, high-quality detection of PD-L1 positive (PD-L1+) CTCs remains a huge challenge. In this study, we propose a short time, high efficiency and applicability method that fabricated by self-assembly circular multi-DNA (CMD) mediating rolling circle amplification (RCA) for highly sensitive and rapid analysis of PD-L1+ cells. Firstly, a short single-stranded circular DNA which could bind specifically with PD-L1 protein was designed. In addition, CMD were designed as self-assembly which could serve as a template in RCA process. Next, the exponential amplification of DNA was achieved by the rolling circle amplification and the fluorescence signal was detected by using the molecular beacons. The unique CMD mediated rolling circle amplification facilitated quantitative detection of PD-L1+ CTCs in the blood of different cancer patients. To visualize the results of CMD on the target cells, we used confocal microscope to assess PD-L1 expression in MCF-7 (breast cancer), OE33 (esophaguscancer) and HCC827 (lung cancer) cells. The results indicated that the CMD mediating RCA can perfectly combine with different kinds of cells, amplify the signal and the recovery rate was over 99 %. What’s more, we performed clinical tests in the blood of the corresponding patient (14 cases of breast cancer, 15 esophageal cancer and 11 lung cancer). We further quantified the number of CTCs in patient blood through establishing the standard curve and the limit of detection (LOD) was calculated to be 1 cell/mL. Moreover, the ring structure was designed to have replaceable sequence and to realize combining different CTCs surface markers (such as Her-2). This method has the advantages of high sensitivity, good expansibility, low cost and can be completed in a short time as well. Therefore, it is expected to develop a new CTCs typing detection kit and might thus provide reliable diagnostic results to provide individualized treatment strategies.

Sinensetin suppresses breast cancer cell progression via Wnt/β-catenin pathway inhibition.

Although there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer. Cell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 μM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on. Herein, our results showed that 120 μM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 μM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated β-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of β-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells. In summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/β-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

Hypoxia-mimetic by CoCl2 increases SLC7A5 expression in breast cancer cells in vitro

ObjectiveIncreased expression of the amino acid transporter solute Carrier Family 7 Member 5 (SLC7A5) has been observed in neoplastic cells during hypoxic conditions in vitro, indicating an adaptation for cell survival. To further explore this, we evaluated hypoxia-mimetic by CoCl2 as a model for hypoxia in breast cancer cell lines and the effect on SLC257A5 expression. Four different breast cancer cell lines (MCF7, T-47D, BT-474 and ZR-75-1) were exposed to 100 µM CoCl2 for 48 h. Subsequently, cell viability, gene- and protein expression analyses were performed.ResultsThe gene expression of VEGF, a marker of hypoxia, was significantly elevated in all four cell lines compared to the control (p < 0.0001), indicating that CoCl2 exposure generates a hypoxic response. Moreover, CoCl2 exposure significantly upregulated SLC7A5 gene expression in T-47D (p < 0.001), BT-474 (p < 0.0001) and ZR-75-1 (p < 0.0001) cells, as compared to vehicle control. Immunofluorescence staining showed increased SLC7A5 protein expression in MCF7, T-47D and BT-474 cells compared to vehicle control. This report suggests that hypoxia-mimetic by CoCl2 can be used as a simple model for inducing hypoxia in breast cancer cell lines and in fact influence SLC7A5 gene and protein expression in vitro.

Selected Fungicides as Potential EDC Estrogenic Micropollutants in the Environment.

An increasing level of pesticide exposition is being observed as a result of the consumption of large amounts of fruits, vegetables and grain products, which are key components of the vegetarian diet. Fungicides have been classified as endocrine-disrupting compounds, but their mechanisms of action have not yet been clarified. The effect of boscalid (B), cyprodinil (C) and iprodione (I) combined with Tamoxifen (T) and 17β-estradiol (E2) on cell viability, cell proliferation, reporter gene expression, ROS content, the cell membrane's function, cell morphology and antioxidant enzymes gene expression in MCF-7 and T47D-KBluc cell lines were investigated. The cell lines were chosen due to their response to 17β -estradiol. The selected fungicides are commonly used in Poland to protect crops against fungi. Our results revealed that the studied fungicides caused significant increases in cell viability and proliferation, and estrogenic activity was present in all studied compounds depending on their concentrations. Oxidative stress activated uncontrolled cancer cell proliferation by inducing ROS production and by inhibiting antioxidant defense. Our findings verify that the studied fungicides could possibly exhibit endocrine-disrupting properties and exposure should be avoided.

Effect of Dioscorea extract on Bax and Bcl-2 gene expression in MCF-7 and HFF cell lines

BackgroundIn cancer cells, the balance between proliferation and apoptosis is disturbed. There is a direct relationship between gene expression and the process of apoptosis. The two genes involved in apoptosis are Bax and Bcl-2, and it is now well established that some plant compounds can alter the expression of genes. The aim of this study is to determine the rate of change in the expression of these genes in the cell line MCF-7 treated with Dioscorea extract for 24, 48 and 72 h. For this purpose, the plant extract was prepared by Soxhlet method and diluted in different concentrations. MCF-7 and HFF cell lines were treated in three replicates with different concentrations of the extract at intervals of 24, 48, and 72 h. To evaluate the toxicity of the extract, the MTT assay was performed and the IC50 value was calculated. Both cell types were cultured at IC50 concentration with three treatments and three replicates. RNA extraction, cDNA synthesis and real-time PCR were then performed. Flow cytometry was performed to further confirm apoptosis.ResultsMTT results showed that 72 h treatment with Dioscorea extract in IC50 concentration had the greatest effect on the death of MCF-7 cancer cells, while the cells of the control cell line remained healthy. The results of the study of gene expression changes showed that when treated with the plant extract for 24 h, the increase in Bax gene expression and the decrease in Bcl-2 gene expression were not statistically significant. At 48-h treatment, the decrease in Bcl-2 expression was not statistically significant, whereas the increase in Bax expression, which was 2.1 times, was statistically significant. When treated with the plant extract for 72 h, Bax expression increased 2.72 times and Bcl-2 gene expression decreased 0.67 times. Flow cytometry showed that 72-h treatment with plant extract at a concentration of 438.35 µg/ml was the most effective treatment for MCF-7 cancer cell death.ConclusionsThe expression ratio of Bax gene to Bcl-2 is equal to 4.06, which indicates the induction of more apoptosis by treatment with plant extract.

Expression of polymeric immunoglobulin receptor (PIGR) and the effect of PIGR overexpression on breast cancer cells

Polymeric immunoglobulin receptor (PIGR) has a major role in mucosal immunity as a transporter of polymeric immunoglobulin across the epithelial cells. The aim of this study was to determine the effect of PIGR on cellular behaviours and chemo-sensitivity of MCF7 and MDA-MB468 breast cancer cell lines. Basal levels of PIGR mRNA and protein expression in MCF7 and MDA-MB468 cells were evaluated by real time quantitative polymerase chain reaction and Western blotting, respectively. MCF7/PIGR and MDA-MB468/PIGR stable cell lines, overexpressing the PIGR gene, were generated using a lentiviral vector with tetracycline dependent induction of expression. Cell viability, cell proliferation and chemo-sensitivity of PIGR transfected cells were evaluated and compared with un-transfected cells to determine the effect of PIGR overexpression on cell phenotype. The levels of PIGR mRNA and protein expression were significantly higher in MDA-MB468 cells than in MCF7 cells (380-fold, p < 0.0001). However, the differential expression of PIGR in these two cell lines did not lead to significant differences in chemosensitivity. Viral overexpression of PIGR was also not found to change any of the parameters measured in either cell line. PIGR per se did not affect cellular behaviours and chemosensitivity of these breast cancer cell lines.

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17β-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17β-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment.