Doxorubicin-resistant Breast Cancer Research Articles

SH003 overcomes drug resistance and immune checkpoints by inhibiting JAK-STAT3 signaling in MCF7/ADR cells

BackgroundBreast cancer has the most commonly diagnosed malignancy cancer worldwide in women and has a high mortality. Various anticancer drugs to treat breast cancer have been developed and tested but have failed because of drug resistance. ObjectivesThe efficacy of SH003 against doxorubicin-resistant MCF/ADR cells has not been evaluated. In this study, we aimed to examine whether SH003 could efficiently prevent the proliferation of MCF7/ADR cells. MethodsCell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by SH003 was measured by an annexin V-FITC/PI assay. Levels of p-STAT3, p-PD-L1, MDR and caspases were measured by western blot analysis. mRNA expression levels of MDR1, MRP1, -2, -3, -4, -5, -6, -9, BCRP and PD-L1 were measured by RT-PCR. Nuclear staining of STAT3 was measured by immunocytochemistry. The expression levels of VEGF, MMP-2 and MMP-9 were measured by ELISA. ResultsSH003 inhibited the proliferation of MCF7/ADR cells and induced their sub-G1 cell cycle arrest. SH003 also induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in MCF7/ADR cells. Furthermore, SH003 suppressed STAT3 transcriptional activity and, more importantly, reduced the cytokine levels of VEGF, MMP-2 and MMP-9 in MCF7/ADR cells. SH003 decreased the protein expression of PD-L1 and MDR1. Additionally, SH003 reduced the mRNA expression of PD-L1; MDR1; MRP1, -2, -3, -4, -5, -6, and -9; and BCRP. ConclusionOur results clearly demonstrate that SH003 inhibits cell growth and induces apoptosis by inhibiting STAT3 signaling. Additionally, SH003 decreased the levels of MDR1 and PD-L1 by inhibiting STAT3 signaling in MCF7/ADR breast cancer cells. These results support the possibility that SH003 could be useful as an herbal medicine to treat doxorubicin-resistant breast cancer.

Integrated Chromatin Accessibility and Transcriptome Landscapes of Doxorubicin-Resistant Breast Cancer Cells.

BackgroundDoxorubicin is one of the most effective chemotherapeutic drugs for breast cancer while its common drug resistance leads to poor patient prognosis and survival. Growing evidence indicate dynamically reorganized chromatin allows rapid access of the gene regulatory machinery to open genomic regions facilitating subsequent gene expression through direct transcription factor (TF) activation and regulatory element binding.MethodsTo better understand the regulatory network underlying doxorubicin resistance in breast cancer cells, we explored the systematic alterations of chromatin accessibility and gene expression by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) in combination with RNA sequencing, followed by integrative analysis to identify potential regulators and their targets associated with differentially accessible regions (DARs) in doxorubicin-resistant MCF7 (MCF7-DR) cells.ResultsA total of 3,963 differentially expressed genes (DEGs) related to doxorubicin resistance were identified, including dramatically up-regulated MT1E, GSTP1, LDHB, significantly down-regulated TFF1, UBB, DSCAM-AS1, and histone-modifying enzyme coding genes HDAC2, EZH2, PRMT5, etc. By integrating with transcriptomic datasets, we identified 18,228 DARs in MCF7-DR cells compared to control, which were positively correlated with their nearest DEGs (r = 0.6). There were 11,686 increased chromatin-accessible regions, which were enriched in up-regulated genes related to diverse KEGG pathways, such as the cell cycle, regulation of actin cytoskeleton, signaling pathways of MAPK, PI3K/Akt and Hippo, which play essential roles in regulating cell apoptosis, proliferation, metabolism, and inflammatory responses. The 6,542 decreased chromatin-accessible regions were identified for the declined doxorubicin-associated biological processes, for instance, endocrine and insulin resistance, central carbon metabolism, signaling pathways of TGF-beta and P53. Combining data from TCGA, analyses of the DAR sequences associated with the DNA-binding motifs of significantly enriched TF families including AP-1, TEAD and FOX, indicated that the loss-function of FOXA1 might play a critical role in doxorubicin-resistant breast cancer cells (DOX-R BCCs).ConclusionThese data exhibit the non-genetic landscape of chromatin accessibility and transcript levels in the DOX-R BCCs, and provide clear insights and resources for the detection of critical TFs and potential cis-regulatory elements-based putative therapeutic targets.

Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents.

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC50 values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

STAT5a Confers Doxorubicin Resistance to Breast Cancer by Regulating ABCB1.

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.

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.

Decrease of ABCB1 protein expression and increase of G1 phase arrest induced by oleanolic acid in human multidrug-resistant cancer cells.

Oleanolic acid (OA) is a natural compound that can be found in a number of edible and medicinal plants and confers diverse biological actions. However, the direct target of OA in human tumor cells remains poorly understood, preventing its application in clinical and health settings. A previous study revealed that overexpression of caveolin-1 in human leukemia HL-60 cells can increase its sensitivity to OA. The present study aimed to investigate the effects of OA on the doxorubicin-resistant human breast cancer MCF-7 cell line (MCF-7/DOX), harringtonine-resistant human leukemia HL-60 cells (HL-60/HAR) and their corresponding parental cell lines. Western blotting was performed to measure protein expression levels, whilst Cell Counting Kit-8 (CCK-8) assays, cell cycle analysis (by flow cytometry) and apoptosis assays (with Annexin V/PI staining) were used to assess drug sensitivity. CCK-8 assay results suggested that MCF-7/DOX cells, which overexpress the caveolin-1 protein, have similar OA susceptibility to their parent line. In addition, sensitivity of MCF-7/DOX cells to OA was not augmented by knocking down caveolin-1 using RNA interference. HL-60/HAR cells exhibited a four-fold increased sensitivity to OA compared with that in their parental HL-60 cells according to CCK-8 assay. Both of the resistant cell lines exhibited higher numbers of cells at G1 phase arrest compared with those in their parent lines, as measured via flow cytometry. Treatment of both MCF-7 cell lines with 100 µM OA for 48 h induced apoptosis, with increased effects observed in resistant cells. However, no PARP-1 or caspase-3 cleavage was observed, with some positive Annexin V staining found after HL-60/HAR cells were treated with OA, suggesting that cell death occurred via non-classical apoptosis or through other cell death pathways. It was found that OA was not a substrate of ATP-binding cassette subfamily B member 1 (ABCB1) in drug-resistant cells, as indicated by the accumulation of rhodamine 123 assessed using flow cytometry. However, protein expression of ABCB1 in both of the resistant cell lines was significantly decreased after treatment with OA in a concentration-dependent manner. Collectively, these results suggest that OA could reduce ABCB1 protein expression and induce G1 phase arrest in multidrug-resistant cancer cells. These findings highlight the potential of OA for cancer therapy.

The role of microRNAs on doxorubicin drug resistance in breast cancer.

Resistance to chemotherapeutic drugs is a serious challenge for effective therapy of cancers. Doxorubicin is a drug which is typically used for breast cancer treatment. Several mechanisms are involved in resistance to doxorubicin including overexpression of ATP-binding cassette (ABC) transporters, altering apoptosis, autophagy and cell cycle arrest. In this review, we focus on the potential effects of microRNAs on doxorubicin resistance in breast cancer. Literature review focusing on the 'microRNAs and doxorubicin drug resistance in breast cancer' was conducted comprehensively. The search was performed in PubMed, Scopus, Google and Google Scholar databases and reference lists of relevant articles were also included. MicroRNAs play essential role in resistance of breast cancer to doxorubicin by affecting several key cellular pathways, including overexpression of ABC transporters, altering apoptosis, autophagy and cell signaling pathways, cell cycle arrest, epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). Cancer treatment methods are moving from conventional therapies to targeted therapies such as using microRNAs. MiRNAs can act as regulatory molecules to overcome breast cancer doxorubicin resistance by controlling the expression levels of genes involved in different cellular pathways. Thus, exact elucidation of their role in different cellular processes can help overcome the breast cancer development and drug resistance.

CIP2A silencing alleviates doxorubicin resistance in MCF7/ADR cells through activating PP2A and autophagy

BackgroundCancerous inhibitor of protein phosphatase 2A (CIP2A) plays a critical role in the pathogenesis of various types of cancer. Here, we investigated whether manipulating CIP2A abundance could enhance the treatment effects of doxorubicin in MCF-7/ADR cells.MethodsCIP2A silencing was achieved by specific siRNAs. Proliferation of breast cancer cell line MCF-7/ADR under effective doxorubicin concentrations after CIP2A silencing was examined by MTT assay. Wound healing assay was performed to quantify cell migration and caspase-3/-7 activities were measured for assessing the extent of apoptosis.ResultsFirst, our data confirmed that MCF-7/ADR cell proliferation was suppressed by doxorubicin in a dose-dependent manner. Additionally, knocking down of CIP2A could further decrease MCF-7 cell proliferation and migration, even in the presence of doxorubicin. Mechanistically, we have found that CIP2A silencing promoted cell apoptosis relative to doxorubicin alone or vehicle control groups. Lastly, phosphatase2A (PP2A) activity was potentiated and the autophagy markers, LC3B and Beclin1, were upregulated after knocking down CIP2A.ConclusionOur findings support the potential benefits of using CIP2A inhibitor as a therapeutic agent to treat doxorubicin-resistant breast cancer.

Blockage of AMPK-ULK1 pathway mediated autophagy promotes cell apoptosis to increase doxorubicin sensitivity in breast cancer (BC) cells: an in vitro study

BackgroundActivation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro.MethodsCell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels.ResultsThe parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin.ConclusionsCollectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.

Ardeemins and citrinin dimer derivatives from Aspergillus terreus harbored in Pinellia ternate

One new alkaloid, 15b-β-hydroxy-16α-hydroxy-5-N-acetylardeemin (1), and one new dimeric derivative of citrinin, penicitrinol Q (5), together with three known alkaloids (2–4) and two citrinin dimers (6–7), were isolated from the solid culture of an endophytic Aspergillus terreus resided in Pinellia ternata. Their structures were established by detailed spectroscopic analysis, and the absolute configurations were determined by single crystal X‑ray diffraction and the comparison of ECD spectra. Penicitrinol Q (5) exhibited inhibitory effects against Staphylococcus aureus and Bacillus subtilis with MIC50 values of 4.3 and 6.2 μg/mL, respectively. When using a concentration of 30 μM, compound 2 showed 2.1-fold enhancement of doxorubicin susceptibility in treating doxorubicin-resistant human breast cancer cell line.

Abstract PS16-23: Elucidating the role of APC resulting in doxorubicin resistance in breast cancer

Abstract Chemoresistance is a leading cause of breast cancer related deaths. Therefore, understanding the molecularbasis for chemoresistance is essential for novel therapeutic advancement improving patient outcome. TheAdenomatous Polyposis Coli (APC) tumor suppressor is lost in up to 70% of sporadic breast cancer; however,little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated fromthe Apc Min/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we werethe first to show that APC loss decreased doxorubicin (DOX) induced apoptosis. Therefore, we investigatedthe mechanisms contributing to DOX resistance with APC loss to identify combination therapy options. DOX, acommonly used chemotherapeutic in breast cancer, inhibits topoisomerase IIa (Topo IIa), causing doublestranded breaks (DSBs), the most lethal form of DNA damage, and APC has been shown to affect Topo IIaactivity. DSB repair is mediated through HRR (homologous recombination repair) or NHEJ (nonhomologousend joining), which are regulated by the repair serine/threonine kinases: ataxia telangiectasia mutated (ATM)or ataxia telangiectasia and Rad3 related (ATR), but only NHEJ activates DNA-dependent protein kinase(DNA-PK). We hypothesized that APC loss prevents DOX-mediated apoptosis through alterations in HRR andNHEJ. To investigate the effect of APC loss on DNA damage repair pathways, we monitored damagerecognition pathways after 24-hour DOX treatment. The MMTV-PyMT;Apc Min/+ cells exhibited decreaseddamage as measured by γH2AX expression and comet assay. We also observed decreased ATMphosphorylation in MMTV-PyMT;Apc Min/+ following DOX treatment compared to control. Decreasedphosphorylation of Chk1 and Chk2 was also observed in DOX-treated MMTV-PyMT;Apc Min/+ cells. Using theATM inhibitor (KU55933) or the DNA-PK inhibitor (NU7441), we observed increased DOX-induced apoptosis inMMTV-PyMT;Apc Min/+ cells. These data suggest enhanced DNA repair in MMTV-PyMT;Apc Min/+ cells and will beconfirmed by measuring repair efficiency via reporter plasmids. In addition, knowing that APC affects activitiesby Topo IIa, we measured the amount of Topo IIa: DNA cleavage complexes following DOX treatment.Taken together, APC loss mediates DOX resistance via increasing DNA repair demonstrating the potential useof combination therapy to overcome chemoresistance. Citation Format: Jeni Prosperi, Casey Stefanski. Elucidating the role of APC resulting in doxorubicin resistance in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-23.

Honokiol antagonizes doxorubicin resistance in human breast cancer via miR-188-5p/FBXW7/c-Myc pathway.

Honokiol, a natural phenolic compound derived from Magnolia plants, is a promising anti-tumor compound that exerts a wide range of anti-cancer effects. Herein, we investigated the effect of honokiol on doxorubicin resistance in breast cancer. Doxorubicin-sensitive (MCF-7 and MDA-MB-231) and doxorubicin-resistant (MCF-7/ADR and MDA-MB-231/ADR) breast cancer cell lines were treated with doxorubicin in the absence or presence of honokiol; then, the following tests were performed: flow cytometry for cell apoptosis, WST-1 assay for cell viability, qPCR and western blot for the expression of miR-188-5p, FBXW7, and c-Myc. MiR-188-5p mimic, miR-188-5p inhibitor, siFBXW7, and c-Myc plasmids were transfected into cancer cells to evaluate whether miR-188-5p and FBXW7/c-Myc signaling are involved in the effect of honokiol on doxorubicin resistance in breast cancer. A dual luciferase reporter system was used to study the direct interaction between miR-188-5p and FBXW7. Honokiol sensitized doxorubicin-resistant breast cancer cells to doxorubicin-induced apoptosis. Mechanically, upregulation of miR-188-5p was associated with doxorubicin resistance, and honokiol enhanced doxorubicin sensitivity by downregulating miR-188-5p. FBXW7 was confirmed to be a direct target gene of miR-188-5p. FBXW7/c-Myc signaling was involved in the chemosensitization effect of honokiol. Honokiol induced apoptosis in MCF-7/ADR and MDA-MB-231/ADR cells. However, FBXW7 silencing or c-Myc transfection resulted in resistance to the honokiol-induced apoptotic effect. These findings suggest that downregulation of miR-188-5p by honokiol enhances doxorubicin sensitivity through FBXW7/c-Myc signaling in human breast cancer. Our study finds an important role of miR-188-5p in the development of doxorubicin resistance in breast cancer, and enriches our understanding of the mechanism of action of honokiol in cancer therapy.

Development of an engineered peptide antagonist against periostin to overcome doxorubicin resistance in breast cancer

BackgroundChemoresistance is one of the main problems in treatment of cancer. Periostin (PN) is a stromal protein which is mostly secreted from cancer associated fibroblasts in the tumor microenvironment and can promote cancer progression including cell survival, metastasis, and chemoresistance. The main objective of this study was to develop an anti-PN peptide from the bacteriophage library to overcome PN effects in breast cancer (BCA) cells.MethodsA twelve amino acids bacteriophage display library was used for biopanning against the PN active site. A selected clone was sequenced and analyzed for peptide primary structure. A peptide was synthesized and tested for the binding affinity to PN. PN effects including a proliferation, migration and a drug sensitivity test were performed using PN overexpression BCA cells or PN treatment and inhibited by an anti-PN peptide. An intracellular signaling mechanism of inhibition was studied by western blot analysis. Lastly, PN expressions in BCA patients were analyzed along with clinical data.ResultsThe results showed that a candidate anti-PN peptide was synthesized and showed affinity binding to PN. PN could increase proliferation and migration of BCA cells and these effects could be inhibited by an anti-PN peptide. There was significant resistance to doxorubicin in PN-overexpressed BCA cells and this effect could be reversed by an anti-PN peptide in associations with phosphorylation of AKT and expression of survivin. In BCA patients, serum PN showed a correlation with tissue PN expression but there was no significant correlation with clinical data.ConclusionsThis finding supports that anti-PN peptide is expected to be used in the development of peptide therapy to reduce PN-induced chemoresistance in BCA.

Bioactive Compounds of Raspberry Oil Emulsions Induced Oxidative Stress via Stimulating the Accumulation of Reactive Oxygen Species and NO in Cancer Cells.

There are growing interests in the complex combinations of natural compounds that may advance the therapy of cancer. Such combinations already exist in foods, and a good representative is seed oils. Two raspberry oils: cold pressed (ROCOP) and one extracted by supercritical CO2 (ROSCO2) were evaluated for their chemical characteristics and oil emulsions for cell suppression potential against colon adenocarcinoma (LoVo), doxorubicin-resistant colon adenocarcinoma (LoVo/DX), breast cancer (MCF7), doxorubicin-resistant breast cancer (MCF7/DX), and lung cancer (A549) cell lines. The cytotoxicity was also assessed on normal human dermal fibroblasts (NHDFs). With increasing concentration of raspberry oil emulsions (0.5–10%), increasing inhibition of cancer cell viability and proliferation in all of the lines was observed, with different degrees of potency between cancer types and oil tested. ROSCO2 strongly induced free radical production and DNA strand damage in LoVo and MCF7 cells especially doxorubicin-resistant lines. This suggests that ROSCO2 engages and effectively targets the vulnerabilities of the cancer cell. Generally, both ROSCO2 and ROCOP could be a nontoxic support in therapy of selected human cancers.

Fat mass and obesity-associated protein (FTO) mediates signal transducer and activator of transcription 3 (STAT3)-drived resistance of breast cancer to doxorubicin

ABSTRACT Excessive activation of signal transducer and activator of transcription 3 (STAT3) is implicated in breast cancer (BC) chemoresistance, but its underlying mechanism is not fully understood. There are STAT3 binding sites in fat mass and obesity-associated protein (FTO) promoter region, thus STAT3 may regulate the transcription of FTO. This study aimed to investigate the correlation between FTO and STAT3 in BC chemoresistance. Herein, FTO and STAT3 were highly expressed in doxorubicin-resistant BC (BC-DoxR) cells. CHIP assay verified the binding between STAT3 and FTO promoter in BC-DoxR cells. Dual luciferase reporter assay showed that FTO promoter activity was inhibited by S3I-201 (STAT3 inhibitor) but enhanced by epidermal growth factor (EGF, STAT3 activator) in BC-DoxR and BC cells. FTO mRNA and protein expression were suppressed by S3I-201 in BC-DoxR cells and EGF-stimulated BC cells. Notably, FTO regulated total N6-methyladenosine (m6A) levels in BC-DoxR and BC cells but could not affect STAT3 mRNA expression, indicating that FTO was not involved in the m6A modification of STAT3. However, FTO could activate STAT3 signaling in BC-DoxR and BC cells. Besides, FTO knockdown inhibited the doxorubicin resistance of BC-DoxR cells, while FTO overexpression enhanced the doxorubicin resistance and weakened the doxorubicin sensitivity of BC cells. Moreover, decreased doxorubicin resistance by STAT3 knockdown was abolished by FTO overexpression and decreased doxorubicin sensitivity by STAT3 overexpression was reversed by FTO knockdown, indicating that FTO was implicated in STAT3-mediated doxorubicin resistance and impairment of doxorubicin sensitivity of BC cells. Altogether, our findings provide a mechanism underlying BC doxorubicin resistance.

MiR-34a Reverses Doxorubicin Resistance in Breast Cancer

Chemotherapy is effectively used for treating breast cancer, but the problem of tumor resistance to chemotherapy drugs has been plaguing scientists. Our study investigated miR-34a?s effect on the sensitivity of drug-resistant strains to chemotherapeutic drugs using doxorubicin-resistant strains of breast cancer cells. Cell survival rate was detected by MTT assay. The doxorubicin-resistant strain rMCF-7 was obtained. The cell scratching method and CCK-8 method were used to detect cell migration and proliferation.Western Blot was performed for measuring SIRT1, p-AKT, AKT, p-mTOR and mTOR level. miR-34a significantly reduced the survival rate o doxorubicin-resistant breast cancer cell line rMCF-7 and significantly enhanced doxorubicin?s effect on inhibiting cell proliferation and cell migration. Compared with the doxorubicin group alone, miR-34a and doxorubicin combination group significantly downregulated SIRT1, p-AKT/AKT and p-mTOR/mTOR related proteins in rMCF-7 cells. miR-34a can reverse the resistance of doxorubicin in breast cancer in vitro and the mechanism may be through inhibition of SIRT and AKT signaling.

LncRNA MALAT1 promotes breast cancer progression and doxorubicin resistance via regulating miR-570–3p

BackgroundMetastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that regulates disease progression in various types of cancers. The aim of this study was to explore the role of MALAT1 in breast cancer (BC) progression and doxorubicin resistance. MethodsReal-time polymerase chain reaction (RT-PCR) was used to determine the expression of MALAT1 in BC tissues and cells; MTT and Transwell assay were used to detect the proliferation, migration and invasion of BC cells, respectively; drug resistance test was performed to assess the sensitivity of BC cells to doxorubicin; dual-luciferase reporter gene assay was conducted to verify the interaction between MALAT1 and miR-570–3p. ResultsMALAT1 was highly expressed in BC tissues compared with normal tissues adjacent to cancer as well as in BC cells. In addition, inhibition the expression of MALAT1 could significantly suppress the proliferation, migration and invasion of BC cells. Meanwhile, down-regulation of MALAT1 sensitized BC cells to doxorubicin. Moreover, bioinformatics analysis suggested that miR-570–3p was the potential downstream target of MALAT1. Dual-luciferase reporter gene assay confirmed that MALAT1 could directly target miR-570–3p. Additionally, miR-570–3p was lowly expressed in BC tissues and cells. Up-regulation of miR-570–3p not only significantly inhibited the proliferation, metastasis, and invasion of BC cells, but also increased the sensitivity of BC cells to doxorubicin. ConclusionMALAT1 functions as a novel oncogenic lncRNA in regulating the progression and doxorubicin resistance of BC by targeting miR-570–3p.

Reducing Doxorubicin resistance in breast cancer by liposomal FOXM1 aptamer: In vitro and in vivo

AimsDrug resistance is one of the main obstacles in cancer chemotherapy. The forkhead box M1 (FOXM1) is a transcription factor and its overexpression in breast cancer is related to resistance to chemotherapy. In this study, we prepare liposomal FOXM1 aptamer (Lip-FOXM1apt) and evaluate its effects on Doxorubicin (Dox) resistance in vitro and in vivo. Main methodsMTT assay, cell association, cellular uptake, Annexin V-FITC/PI dual staining assay were investigated in MDA-MB-231, MCF-7, 4T1. In vivo studies were performed in 4T1 tumor-bearing BALB/c mice. Key findingsWe found that the combination therapy of Dox and Lip-FOXM1apt significantly increases both Dox cytotoxicity on cancer cells as well as Dox-induced apoptosis. Administering Lip-FOXM1apt remarkably improved the anti-tumor efficacy of Dox in mice model that was strikingly more effective than Dox monotherapy. SignificanceTaken together, this study provides a new strategy to overcome Dox resistance and merits further investigation.

Overcoming anticancer resistance by photodynamic therapy-related efflux pump deactivation and ultrasound-mediated improved drug delivery efficiency

One of the major obstacles to successful chemotherapy is multi-drug resistance (MDR). A multi-drug resistant cancerous cell abnormally overexpresses membrane transporters that pump anticancer drugs out of the cell, resulting in low anticancer drug delivery efficiency. To overcome the limitation, many attempts have been performed to inhibit the abilities of efflux receptors chemically or genetically or to increase the delivery efficiency of anticancer drugs. However, the results have not yet been satisfactory. In this study, we developed nanoparticle-microbubble complexes (DOX-NPs/Ce6-MBs) by conjugating doxorubicin loaded human serum albumin nanoparticles (DOX-NPs) onto the surface of Chlorin e6 encapsulated microbubbles (Ce6-MBs) in order to maximize anticancer efficiency by overcoming MDR. Under the ultrasound irradiation, DOX-NPs and Ce6 encapsulating self-assembled liposomes or micelles were effectively delivered into the cells due to the sonoporation effect caused by the microbubble cavitation. At the same time, reactive oxygen (ROS) generated from intracellularly delivered Ce6 by laser irradiation arrested the activity of ABCG2 efflux receptor overexpressed in doxorubicin-resistant breast cancer cells (MCF-7/ADR), resulting in increased the chemotherapy efficacy. In addition, the total number of side population cells that exhibit the properties of cancer stem-like cells were also reduced by the combination of photodynamic therapy and chemotherapy. In conclusion, DOX-NPs/Ce6-MBs will provide a platform for simultaneously overcoming MDR and increasing drug delivery and therefore, treatment efficiency, under ultrasound irradiation.

Identification of CCN1 transferred by extracellular vesicles derived from breast cancer cells responsible for doxorubicin-resistance.

e12623 Background: The anthracycline drug, doxorubicin, is one of the most frequently used chemotherapeutic drugs for breast cancer. However, not all patients respond to doxorubicin-containing combination chemotherapy due to chemo-resistance (natural or acquired). Preceding researches have shown that extracellular vesicles (EVs) can be readily harvested from peripheral blood for further biological analysis and thus perform an attractive source of tumor derived materials for identification of chemo-resistance factors. This study aims to provide a theoretical basis to use CCN1-containing EVs as new biomarkers and therapeutic targets for doxorubicin-resistance in breast cancer. Methods: A strategy combining RNA-sequencing technique and bioinformatic analysis was used to determine the level of differential expressed mRNAs in EVs samples from cellular supernatant (n = 4) and blood serum (n = 8) from chemo-resistant and chemo-sensitive patients. Serial gene expression analysis in patients (n = 203) who treated with neoadjuvant (GSE32603) were also employed to identified drug-resistance-related proteins. The expression of CCN1 was detected in doxorubicin-resistant cell lines (MCF-7/DOX),MCF-7 and their EVs by RT-qPCR and western blot. Then, we used cell apoptosis and immunofluorescence stain to confirm the ability of CCN1-containing EVs in conferring drug resistance. Immunohistochemistry stain and RT-qPCR were used to prove the expression level of CCN1 in tumor tissues from breast cancer patients (n = 80) and in serum EVs from patients treated with the whole neoadjuvant chemotherapy. Results: We combined the differentially expressed mRNAs from three datasets and found identified CCN1 as a drug-resistance-related factor in breast cancer. The expression level of CCN1 is much higher in MCF-7/DOX cells and their EVs compared with MCF-7 cells and their EVs. The addition of CCN1-containing EVs has been found significantly increase the resistance to doxorubicin in sensitive cells. Moreover, a higher expression level of CCN1 was detected in the progressive disease (PD)/stable disease (SD) group than in the partial response (PR)/complete response (CR) group both in tissues and serum EVs. Conclusions: All These findings demonstrate that CCN1-containing EVs appear to be essential in conferring resistance to doxorubicin. More importantly, we exhibit the potential applications of CCN1-containing EVs for predicting the efficiency and outcome of patients treated with doxorubicin-based chemotherapy.