miR-503-5p induces doxorubicin resistance in triple-negative breast cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 1083-1083
Author(s):  
Iris Garrido-Cano ◽  
Anna Adam-Artigues ◽  
Ana Lameirinhas ◽  
Birlipta Pattanayak ◽  
Eduardo Tormo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Doxorubicin Resistance ◽  
Tnbc Cell Line ◽  
Gain Loss ◽  
Resistance To Doxorubicin

1083 Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype comprising approximately 15% of BC. Conventional cytotoxic chemotherapies continue to be the mainstay for treatment of this BC, which lacks targetable markers. In this context, microRNAs have been described to have an important role. The aim of this work was to elucidate the function of miR-503-5p in doxorubicin resistance in TNBC. Methods: miR-503-5p expression was evaluated in the TNBC cell line with acquired resistance to doxorubicin (MDA-MB-231R) and its parental cell line (MDA-MB-231), by qRT-PCR. Studies of gain/loss of function of miR-503-5p were carried out in MDA-MB-231 and MDA-MB-231R cells by transient transfection of mimics and inhibitors. Cells were treated with doxorubicin, and viability was measured by flow cytometry and MTT assay. The role of miR-503-5p was also evaluated in vivo by Chicken Chorioallantoic Membrane (CAM) assay. MDA-MB-231 cells transfected with miR-503-5p mimic or scramble miRNA were inoculated onto the CAM of fertilized chicken eggs. After 48 hours, tumours were treated with doxorubicin or supplemented media for 48 hours and tumour growth was measured. miR-503-5p expression was quantified by qRT-PCR in a retrospective cohort of 74 TNBC patients treated with anthracycline + taxane regimens. Overall survival analysis for miR-503-5p in TNBC patients from METABRIC dataset was evaluated by the KM plotter online tool. Results: miR-503-5p was significantly upregulated in the resistant MDA-MB-231R TNBC cell line when compared to its parental cell line MDA-MB-231 (̃3.5-fold; p< 0.0001). Then, gain/loss function assays showed that upregulation of miR-503-5p in MDA-MB-231 cells increased resistance to doxorubicin ( p< 0.0001) and its downregulation in MDA-MB-231R cells had the opposite effect ( p< 0.0001). Moreover, the role of miR-503-5p was also confirmed in the CAM assay in vivo model, where miR-503-5p overexpression inhibited the effect of doxorubicin. In our cohort of patients, miR-503-5p expression levels in core biopsies sampled before preoperative chemotherapy were associated with residual cancer burden (RCB). miR-503-5p expression was significantly higher in patients with poor response to chemotherapy (RCB II and III; median, 95% CI: 0.00055, 0.00024 - 0.00136) than in patients with good response (RCB 0 and I; median, 95% CI: 0.00018, 0.00011 - 0.00034; p = 0.036). Moreover, we confirmed that TNBC patients with high expression of miR-503-5p had worse overall survival than patients with low expression ( p= 0.016). Conclusions: We identified miR-503-5p as a modulator of doxorubicin resistance in TNBC. Our in vitro findings are supported by the clinical data of TNBC patients and in vivo assays. Hence, the inhibition of miR-503-5p may be a promising strategy to improve chemotherapeutic efficacy. Moreover, the expression levels of miR-503-5p may be used as a biomarker for therapy response in TNBC.

scholarly journals EGCG-Derivative G28 Shows High Efficacy Inhibiting the Mammosphere-Forming Capacity of Sensitive and Resistant TNBC Models

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1027 ◽  
Author(s):  
Ariadna Giró-Perafita ◽  
Marc Rabionet ◽  
Marta Planas ◽  
Lidia Feliu ◽  
Joaquim Ciurana ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Fatty Acid Synthase ◽  
Epithelial Mesenchymal Transition ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Enzyme Activity Assay ◽  
Mesenchymal Transition ◽  
Tnbc Cell Line ◽  
Synthetic Derivatives

Recent studies showed that Fatty Acid Synthase (FASN), a lipogenic enzyme overexpressed in several carcinomas, plays an important role in drug resistance. Furthermore, the enrichment of Breast Cancer Stem Cell (BCSC) features has been found in breast tumors that progressed after chemotherapy. Hence, we used the triple negative breast cancer (TNBC) cell line MDA-MB-231 (231) to evaluate the FASN and BCSC population role in resistance acquisition to chemotherapy. For this reason, parental cell line (231) and its derivatives resistant to doxorubicin (231DXR) and paclitaxel (231PTR) were used. The Mammosphere-Forming Assay and aldehyde dehydrogenase (ALDH) enzyme activity assay showed an increase in BCSCs in the doxorubicin-resistant model. Moreover, the expression of some transcription factors involved in epithelial-mesenchymal transition (EMT), a process that confers BCSC characteristics, was upregulated after chemotherapy treatment. FASN inhibitors C75, (−)-Epigallocatechin 3-gallate (EGCG), and its synthetic derivatives G28, G56 and G37 were used to evaluate the effect of FASN inhibition on the BCSC-enriched population in our cell lines. G28 showed a noticeable antiproliferative effect in adherent conditions and, interestingly, a high mammosphere-forming inhibition capacity in all cell models. Our preliminary results highlight the importance of studying FASN inhibitors for the treatment of TNBC patients, especially those who progress after chemotherapy.

The Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine Treatment in Resistant 2D and 3D Model Triple Negative Breast Cancer Cell Line: ABCG2 Expression Data

2021 ◽  
Vol 21 ◽  
Author(s):  
Fatma Kubra Ata ◽  
Serap Yalcin
Keyword(s):  
Breast Cancer ◽  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profile ◽  
Three Dimensional ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Xtt Assay ◽  
Gene And Protein Expression

Background: Chemotherapeutics have been commonly used in cancer treatment. Objective: In this study, the effects of Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine have been evaluated on two-dimensional (2D) (sensitive and resistance) cell lines and three dimensional (3D) spheroid structure of MDA-MB-231. The 2D cell culture lacks a natural tissue-like structural so, using 3D cell culture has an important role in the development of effective drug testing models. Furthermore, we analyzed the ATP Binding Cassette Subfamily G Member 2 (ABCG2) gene and protein expression profile in this study. We aimed to establish a 3D breast cancer model that can mimic the in vivo 3D breast cancer microenvironment. Methods: The 3D spheroid structures were multiplied (globally) using the three-dimensional hanging drop method. The cultures of the parental cell line MDA-MB-231 served as the controls. After adding the drugs in different amounts we observed a clear and well-differentiated spheroid formation for 24 h. The viability and proliferation capacity of 2D (sensitive and resistant) cell lines and 3D spheroid cell treatment were assessed by the XTT assay. Results: Cisplatin, Irinotecan, 5-Fu, and Gemcitabine-resistant MDA-MB-231 cells were observed to begin to disintegrate in a three-dimensional clustered structure at 24 hours. Additionally, RT-PCR and protein assay showed overexpression of ABCG2 when compared to the parental cell line. Moreover, MDA-MB-231 cells grown in 3D showed decreased sensitivity to chemotherapeutics treatment. Conclusion: More resistance to chemotherapeutics and altered gene expression profile was shown in 3D cell cultures when compared with the 2D cells. These results might play an important role to evaluate the efficacy of anticancer drugs, explore mechanisms of MDR in the 3D spheroid forms.

Effective treatment of triple-negative breast cancer with targeted cytotoxic somatostatin analogue AN-162 (AEZS-124)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 619-619
Author(s):  
S. Seitz ◽  
A. V. Schally ◽  
S. Gluck ◽  
F. Rick ◽  
L. Szalontay ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Fluorescence Microscopy ◽  
Tumor Growth ◽  
Cell Line ◽  
Triple Negative ◽  
Somatostatin Analogue ◽  
Tnbc Cell ◽  
Tnbc Cell Line ◽  
Fluorescence Assays

619 Background: Triple negative breast cancers (TNBC) represent a distinct subtype of breast cancer being negative to ER, PR and HER2 and are associated with poor prognosis. Limited systemic treatment options exist for TNBC. TNBC cells express somatostatin receptors (SSTR). Therefore, to investigate preclinical characteristics of TNBC we used a novel targeted cytotoxic somatostatin analogue AN-162 containing doxorubicin (DOX) which binds to the subtypes 2, 3 and 5 of SSTR. Methods: The expression of SSTR in HCC 1806 human TNBC cell line was detected by RT-PCR. Cytotoxic effect of AN-162 in vitro was visualized by ethidium bromide staining fluorescence microscopy. Internalization of AN-162 into HCC 1806 cells was tested by 125Iodide-labeled AN-162 uptake assays and the presence of DOX in the nucleus was measured by fluorescence assays after separating the nucleus from the cytoplasm. For in vivo experiments, HCC 1806 TNBC cells were xenografted subcutaneously into nude mice which were then randomized into four groups receiving AN-162, DOX, an unconjugated mixture of DOX and somatostatin analogue RC-160 at the same equimolar dose of 2.5 μmol/kg (1.45 mg/kg Dox equivalent) i.v. (q7d 4x) and vehicle solution control. Results: HCC 1806 TNBC cell line was positive for the expression of all five SSTR receptor subtypes. Ethidum bromide staining of cells treated with 2.5 μM of AN-162 for 30 min demonstrated cell death after 24h by fluorescence microscopy. Uptake assays with AN-162 showed specific internalization of AN-162 into the cells mediated through the sstrs. After treatment of the cells with 2.5 μM AN-162 for 10 or 30 min, DOX could be detected in the nucleus by fluorescence assays. In vivo, AN-162 significantly (p<0.05) inhibited tumor growth of HCC 1806 xenografts compared to Control, DOX and the unconjugated mixture of DOX+RC-160 from day 14 and the inhibition remained significant until the end of the study on day 35. Conclusions: Our results indicate that treatment with targeted cytotoxic somatostatin analogue AN-162 produces a greater inhibition of tumor growth than DOX alone in somatostatin receptor positive TNBC. Our findings support the concept of targeted chemotherapy based on cytotoxic peptide analogues for the treatment of breast cancer and other cancers. No significant financial relationships to disclose.

scholarly journals Human drug efflux transporter ABCC5 confers acquired resistance to pemetrexed in breast cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jihui Chen ◽  
Zhipeng Wang ◽  
Shouhong Gao ◽  
Kejin Wu ◽  
Fang Bai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Abc Transporters ◽  
Breast Cancer Cells ◽  
Breast Cancer Treatment ◽  
Control Group ◽  
Mcf 7

Abstract Aim Pemetrexed, a new generation antifolate drug, has been approved for the treatment of locally advanced or metastatic breast cancer. However, factors affecting its efficacy and resistance have not been fully elucidated yet. ATP-binding cassette (ABC) transporters are predictors of prognosis as well as of adverse effects of several xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and can contribute to the optimization of breast cancer treatment regimen. Methods First, we measured the expression levels of ABC transporter family members in cell lines. Subsequently, we assessed the potential role of ABC transporters in conferring resistance to pemetrexed in primary breast cancer cells isolated from 34 breast cancer patients and the role of ABCC5 in mediating pemetrexed transport and apoptotic pathways in MCF-7 cells. Finally, the influence of ABCC5 expression on the therapeutic effect of pemetrexed was evaluated in an in vivo xenograft mouse model of breast cancer. Results The expression levels of ABCC2, ABCC4, ABCC5, and ABCG2 significantly increased in the pan-resistant cell line, and the ABCC5 level in the MCF-7-ADR cell line was 5.21 times higher than that in the control group. ABCC5 expression was inversely correlated with pemetrexed sensitivity (IC50, r = 0.741; p < 0.001) in breast cancer cells derived from 34 patients. Furthermore, we found that the expression level of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cells, with IC50 values of 0.06 and 0.20 μg/mL in ABCC5 knockout and over-expression cells, respectively. In the in vivo study, we observed that ABCC5 affected the sensitivity of pemetrexed in breast tumor-bearing mice, and the tumor volume was much larger in the ABCC5-overexpressing group than in the control group when compared with their own initial volumes (2.7-fold vs. 1.3-fold). Conclusions Our results indicated that ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and it may serve as a target or biomarker for the optimization of pemetrexed regimen in breast cancer treatment.

A Molecular Mechanism of Tumor Mediated Suppression of Dendritic Cell Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1735-1735
Author(s):  
Matthew R Farren ◽  
Louise Carlson ◽  
Haley Spangler ◽  
Kelvin P Lee
Keyword(s):  
T Cell ◽  
Dendritic Cell ◽  
Immune Responses ◽  
Cell Line ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Protein Levels ◽  
Stat3 Signaling ◽  
Myeloid Progenitor

Abstract Abstract 1735 The immune system plays a key role in preventing and controlling tumor growth. Cancer frequently induces a state of immune suppression in patients mediated, in part, through inhibition of dendritic cell (DC) differentiation. This results in the accumulation of actively immunosuppressive myeloid derived suppressor cells (MDSCs) and a loss of DCs (critical in the induction of T cell mediated immune responses), thus compromising the ability to initiate anti-tumor immune responses. This is mediated by tumor derived factors (TDFs)(e.g. VEGF) that inhibit DC differentiation by driving STAT3 hyperactivation. The downstream target(s) of this STAT3 signaling that inhibits DC differentiation, however, has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor to DC differentiation and demonstrated that PKC βII inhibition (signaling or expression) prevents DC differentiation. We've also found that PKC βII positively regulates its own expression and that, under certain circumstances, the PKCβ promoter is negatively regulated. These observations lead us to hypothesize that TDF activation of Stat3 inhibits DC differentiation by down regulating PKC βII expression. We've previously shown that culture in tumor conditioned media (TCM) decreased PKC βII protein levels and significantly reduced PKC βII mRNA transcript levels in KG1, a myeloid progenitor-like cell line long used to model DC differentiation. We've also previously seen that decreased PKC βII expression following culture in TCM significantly impaired DC differentiation, compared to cells grown in the absence of tumor conditions; however, the mechanism by which Stat3 signaling down regulated PKC βII expression remained unclear. We now show that culture in TCM reduced PKCβ promoter driven transcription 7-fold, compared to cells grown in normal media (p<0.01). Given the previously described importance of Stat3 hyperactivation in tumor-mediated suppression of DC differentiation, and since PKC βII down regulation appears to occur at the promoter level, we examined the role of Stat3 in regulating PKC βII expression. Culture in TCM rapidly (<5 min.) induced Stat3 phosphorylation, an indication of activation. By chromatin immunoprecipitation, we found that TCM treatment induces direct interaction between Stat3 and the PKCβ promoter, suggesting that Stat3 signaling may act to decrease PKC βII expression. To directly test the role of Stat3 signaling in regulation of PKC βII expression, we generated a series of clones stably expressing wild type (WT) or constitutive active (CA) Stat3 constructs in K562, a second DC progenitor-like cell line. We've previously seen that clones stably expressing the CA-Stat3 construct have decreased PKC βII protein levels and significantly decreased PKC βII mRNA levels, compared to the parental cell line and WT-Stat3 clones. We now show that this decrease in PKC βII expression was dependent on constitutive Stat3 signaling, as pharmacologic Stat3 inhibition restored PKC βII expression to levels seen in the parental cell line. Consistent with the proposed model and our previous work, decreased PKC βII expression in clones expressing CA-Stat3 resulted in significantly inhibited phorbol ester driven DC differentiation (p<0.05)(as measured by allogenic T cell proliferation, a key measure of DC differentiation). Interestingly, we've also found that PKC βII antagonizes Stat3 signaling: myeloid progenitor-like cells (KG1a) overexpressing PKC βII do not exhibit Stat3 activation in response to TCM: PKC βII overexpression or activation led to down regulation of the receptors for G-CSF, IL-6, and VEGF, TDFs demonstrated to inhibit myeloid progenitor to DC differentiation. These findings suggest a novel mechanism by which PKC βII negatively regulates the potential of a cell to respond to (tumor derived) inflammatory cytokines. This work demonstrates that tumor driven Stat3 hyperactivation down regulates PKCβ promoter activity, resulting in decreased PKC βII protein levels. In agreement with our previous work, this decrease in PKC βII expression impairs a cell's potential to undergo DC differentiation. Additionally, this work suggests that PKC βII signaling impairs a cell's potential to signal via Stat3 in response to TDFs, perhaps providing an avenue by which to block or reverse tumor mediated suppression of DC differentiation in cancer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Drug Efflux Transporter ABCC5 Confers Acquired Resistance to Pemetrexed in Breast Cancer

2021 ◽  
Author(s):  
Jihui Chen ◽  
Zhipeng Wang ◽  
Shouhong Gao ◽  
Kejin Wu ◽  
Fang Bai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Abc Transporters ◽  
Control Group ◽  
Apoptosis Pathway ◽  
Over Expression ◽  
Mcf 7

Abstract AimPemetrexed, a new generation antifolate drug, is approved for the treatment for locally advanced or metastatic breast cancer, but factors affecting the efficacy and resistance of it have yet to be fully explicit. ATP-binding cassette (ABC) transporters have been reported as prognostic and adverse effects predictors of many xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and may contribute to treatment regimen optimization for breast cancer.MethodsFirstly, the expression of ABC transporters family members was measured in cell lines, thereafter examined the potential role of ABC transporter in conferring resistance to pemetrexed in primary cancer cell lines isolated from 34 breast cancer patients, and then the role of ABCC5 in mediating transport of pemetrexed and apoptosis pathway in MCF-7 cell line was assessed. Finally, the functions of ABCC5 on therapeutic effect of pemetrexed was evaluated in breast cancer bearing mice.ResultsThe expressions of ABCC2, ABCC4, ABCC5 and ABCG2 were significantly increased in pan-resistance cell line, and the ABCC5, the most obvious one, was 5.21 times higher than that of the control group. The expression of ABCC5 was inversely correlated with sensitivity (IC50) of pemetrexed (r = 0.741; p<0.001) in breast cancer cells from 34 patients. Furthermore, we found that the expression of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cell line, and the IC50 were 0.06 μg/ml and 0.20 μg/ml in ABCC5 knock-down and over-expression cells, respectively. In in vivo study, we found ABCC5 affected the sensitivity of pemetrexed in breast cancer bearing mice, and the tumor volume was much larger in ABCC5 over-expression group than that in control group (2.7 folds vs 1.3 folds).ConclusionsOur results indicated ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and may be a biomarker for regimen optimization of pemetrexed in breast cancer treatment.

scholarly journals STAT5a Confers Doxorubicin Resistance to Breast Cancer by Regulating ABCB1

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhaoqing Li ◽  
Cong Chen ◽  
Lini Chen ◽  
Dengdi Hu ◽  
Xiqian Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Resistant Cell ◽  
Vital Role ◽  
Cancer Targeting ◽  
Doxorubicin Resistance ◽  
Daunting Challenge

Chemoresistance is a daunting challenge to the prognosis of patients with breast cancer. Signal transducer and activator of transcription (STAT) 5a plays vital roles in the development of various cancers, but its function in breast cancer is controversial, and its role in chemoresistance in breast cancer remains unexplored. Here we identified STAT5a as a chemoresistance inducer that regulates the expression of ABCB1 in breast cancer and can be targeted by pimozide, an FDA-approved psychotropic drug. First, we found that STAT5a and ABCB1 were expressed at higher levels in doxorubicin-resistant cell lines and chemoresistant patients, and their expression was positively correlated. Then, we confirmed the essential roles of STAT5a and ABCB1 in doxorubicin resistance in breast cancer cells and the regulation of ABCB1 transcription by STAT5a. Subsequently, the efficacy of pimozide in inhibiting STAT5a and sensitizing doxorubicin-resistant breast cancer cells was tested. Finally, we verified the role of STAT5a in doxorubicin resistance in breast cancer and the efficacy of pimozide in reversing this resistance in vivo. Our study demonstrated the vital role of STAT5a in doxorubicin resistance in breast cancer. Targeting STAT5a might be a promising strategy for treating doxorubicin-resistant breast cancer. Moreover, repurposing pimozide for doxorubicin resensitization is attractive due to the safety profile of pimozide.

scholarly journals Role and mechanism of miR-130a-3p in the chemosensitivity of retinoblastoma cells to vincristine

2021 ◽  
Author(s):  
Xiulan Lu ◽  
Huifang Tu ◽  
Dongrun Tang ◽  
Xiaoming Huang ◽  
Fengyuan Sun
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Resistance Index ◽  
Parental Cell ◽  
Luciferase Assay ◽  
Parental Cell Line ◽  
Pax6 Expression ◽  
Retinoblastoma Cells ◽  
Primary Obstacle

Abstract Purpose Chemoresistance remains the primary obstacle threatening the prognosis of retinoblastoma (RB). microRNAs (miRNAs) are acknowledged as critical regulator of drug resistance. This study explored the molecular mechanism of miR-130a-3p affecting the chemosensitivity of RB to vincristine (VCR). Methods miR-130a-3p expression of VCR-sensitive and VCR-resistant RB tissues was detected using RT-qPCR. VCR-resistant RB cell line Y79/VCR was induced. miR-130a-3p expression of Y79/VCR cell line and its corresponding parental cell line was detected. Y79/VCR cells were subjected to miR-130a-3p overexpression treatment. The cell proliferation was measured using MTT assay, and the IC50 value and drug resistance index were examined using CCK-8 assay. The targeting relationship between miR-130a-3p and PAX6 was predicted through bioinformatics analysis and verified using dual-luciferase assay. Functional rescue experiments were conducted to confirm the role of PAX6 in chemosensitivity of RB cells. The effect of miR-130a-3p on tumorigenesis and VCR sensitivity was observed in vivo. Results miR-130a-3p was downregulated in VCR-resistant RB tissues and cells. Overexpression of miR-130a-3p repressed the proliferation of VCR-resistant RB cells and enhanced chemosensitivity. miR-130a-3p targeted PAX6 expression. Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of RB. Overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo. Conclusion miR-130a-3p enhanced the chemosensitivity of RB cells to VCR by targeting PAX6 expression.

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