Doxorubicin-resistant Breast Cancer Research Articles

Effectiveness of Pheophorbide-a mediated Photodynamic Therapy against Doxorubicin Resistant Breast Cancer

SignificanceEmploying mechanistically distinct therapies like Photodynamic Therapy (PDT) can be a promising strategy to overcome the multidrug- resistance in breast cancer resulting in the killing of chemotherapeutic drug-resistant cancer cells. ApproachIn this study, Pheophorbide a (Pheo-a), is being used as a potential photosensitizer to induce cell death in Doxorubicin (DOX) resistant breast cancer MCF-7 cells. Efficacy of Pheo-a mediated PDT against MCF-7/DOX cancer cells was evaluated by MTT proliferation assay and analyzing cellular morphological changes, live dead cell death assay. ResultsAll experiments were performed thrice in triplicate, and the results obtained were analysed for statistical significance. Results showed that the Pheo-a can induce photodynamic cell death in resistant cancer cells treated with a 2.5 μM concentration of Pheo-a followed by irradiation with a 660 nm red laser at a light dose of 10 J/cm2. ConclusionsThus, the results from this study suggest the utilization of chlorophyll-based PSs as a rational therapeutic intervention for the eradication of chemo drug-resistance in cancers.

LINC00460-FUS-MYC feedback loop drives breast cancer metastasis and doxorubicin resistance.

Therapeutic resistance and metastasis largely contribute to mortality from breast cancer and therefore understanding the underlying mechanisms of such remains an urgent challenge. By cross-analysis of TCGA and GEO databases, LINC00460 was identified as an oncogenic long non-coding RNA, highly expressed in Doxorubicin resistant breast cancer. LINC00460 was further demonstrated to promote stem cell-like and epithelial-mesenchymal transition (EMT) characteristics in breast cancer cells. LINC00460 interacts with FUS protein with consequent enhanced stabilization, which further promotes MYC mRNA maturation. LINC00460 expression was transcriptionally enhanced by c-MYC protein, forming a positive feedback loop to promote metastasis and Doxorubicin resistance. LINC00460 depletion in Doxorubicin-resistant breast cancer cells restored sensitivity to Doxorubicin and increased the efficacy of c-MYC inhibitor therapy. Collectively, these findings implicate LINC00460 as a promising prognostic biomarker and potential therapeutic target to overcome Doxorubicin resistance in breast cancer.

Role of glutaminyl-peptide cyclotransferase in breast cancer doxorubicin sensitivity

ABSTRACT Doxorubicin (DOX) is one of the most effective and widely used chemotherapeutic drugs. However, DOX resistance is a critical risk problem for breast cancer treatment. Previous studies have demonstrated that metadherin (MTDH) involves in DOX resistance in breast cancer, but the exact mechanism remains unclear. In this study, we found that glutaminyl-peptide cyclotransferase (QPCT) was a MTDH DOX resistance-related downstream gene in breast cancer. Elevated expression of QPCT was found in the GEPIA database, breast cancer tissue, and breast cancer cells. Clinical data showed that QPCT expression was positively associated with poor prognosis in DOX-treated patients. Overexpression of QPCT could promote the proliferation, invasion and migration, and reduce DOX sensitivity in MCF-7 and MDA-MB-231 cells. Mechanistically, MTDH positively regulates the expressions of NF-κB (p65) and QPCT, and NF-κB (p65) directly regulates the expression of QPCT. Therefore, MTDH/NF-κB (p65)/QPCT signal axis was proposed. Collectively, our findings delineate the mechanism by which the MTDH/NF-κB (p65) axis regulate QPCT signaling and suggest that this complex may play an essential role in breast cancer progression and affect DOX sensitivity.

Allicin Overcomes Doxorubicin Resistance of Breast Cancer Cells by Targeting the Nrf2 Pathway.

Breast cancer (BC) is a lethal disorder that threatens the life safety of the majority of females globally, with rising morbidity and mortality year by year. Doxorubicin is a cytotoxic anthracycline antibiotic that is widely used as one of the first-line chemotherapy agents for patients with BC. However, the efficacy of doxorubicin in the clinic is largely limited by its serious side effects and acquired drug resistance. Allicin (diallyl thiosulfinate), as the major component and key active compound present in freshly crushed garlic, has shown potential effects in suppressing chemotherapy resistance in various cancers. Our research aimed to explore the relationship between allicin and doxorubicin resistance in BC. To generate doxorubicin-resistant BC cell lines (MCF-7/DOX and MDA-MB-231/DOX), doxorubicin-sensitive parental cell lines MCF-7 and MDA-MB-231 were continuously exposed to stepwise increased concentrations of doxorubicin over a period of 6 months. CCK-8, colony formation, flow cytometry, RT-qPCR, and western blotting assays were performed to investigate the effects of allicin and/or doxorubicin treatment on the viability, proliferation and apoptosis and the expression of Nrf2, HO-1, phosphate AKT and AKT in doxorubicin-resistant BC cells. Our results showed that combined treatment of allicin with doxorubicin exhibited better effects on inhibiting the proliferation and enhancing the apoptosis of doxorubicin-resistant BC cells than treatment with allicin or doxorubicin alone. Mechanistically, allicin suppressed the levels of Nrf2, HO-1, and phosphate AKT in doxorubicin-resistant BC cells. Collectively, allicin improves the doxorubicin sensitivity of BC cells by inactivating the Nrf2/HO-1 signaling pathway.

METTL3/IGF2BP3-regulated m6A modification of HYOU1 confers doxorubicin resistance in breast cancer

Chemoresistance is a main reason for therapeutic failure and poor prognosis for breast cancer (BC) patients, especially for triple-negative BC patients. How the molecular mechanisms underlying the chemoresistance to doxorubicin (Dox) in BC is not well understood. Here, we revealed that METTL3/IGF2BP3-regulated m6A modification of HYOU1 increased Dox resistance in BC cells. CCK-8 and Annexin V-FITC/PI staining assays were employed to measure viability and cell death. Western blotting and qRT–PCR assays were applied to assay the expression of genes. Knockdown and rescue experiments were used to assay the role of METTL3, IGF2BP3 and HYOU1 in regulating BC cell responses to Dox. RIP, MeRIP and dual-luciferase activity assays were applied to examine the function of METTL3/IGF2BP3 in the m6A modification of HYOU1 mRNA. It was found that global mRNA m6A methylation levels were upregulated in Dox-resistant BC cell lines. The methyltransferase METTL3 was upregulated in Dox-resistant BC cell lines, and downregulation of METTL3 could overcome this resistance. Furthermore, HYOU1 was identified as a downstream target of METTL3-mediated m6A modification. Downregulation of HYOU1 could overcome Dox resistance, while forced expression of HYOU1 resulted in Dox resistance in BC cells. METTL3 cooperated with IGF2BP3 to modulate the m6A modification of HYOU1 mRNA and increase its stability. Collectively, our findings unveiled the key roles of the METTL3/IGF2BP3/HYOU1 axis in modulating Dox sensitivity in BC cells; thus, targeting this axis might be a potential strategy to increase Dox efficacy in the treatment of BC.

A (Traditional Chinese Medicine) TCM-Inspired Doxorubicin Resistance Reversing Strategy: Preparation, Characterization, and Application of a Co-loaded pH-Sensitive Liposome.

In this study, nano-strategy for combined medication of active compounds from traditional Chinese medicine herbs was proposed to achieve the synergistic effects of inhibiting the doxorubicin (DOX) resistance, reducing the cardio-toxicity, and improving the treatment efficacy simultaneously. Dihydroartemisinin (DHA) and tetrandrine (TET) were co-delivered for the first time to treat DOX resistance of breast cancer with multi-pathway mechanism. Tumor micro-environment sensitivity prescription was adopted to enhance the reversal effect of DOX resistance nearly 50 times (resistance index, RI was 46.70) and uptake ability. The DHA-TET pH-sensitive liposomes (DHA-TET pH-sensitive LPs) had a good spherical structure and a uniform dispersion structure with particle size, polydispersity index (PDI), and zeta potential of 112.20 ± 4.80 nm, 0.20 ± 0.02, and - 8.63 ± 0.74 Mv, and was stable until 14 days under the storage environment of 4°C and for 6 months at room temperature environment. With the DOX resistance reversing ability increased, the inhibition effect of DHA-TET pH-sensitive LPs on both MCF-7/ADR cells and MCF-7 cells was significantly enhanced; meanwhile, the toxicity on cardiac cell (H9c2) was lowered. Ferroptosis induced by the DHA was investigated showing that the intracellular reactive oxygen species (ROS) and lipid peroxidation were increased to promote the synergistic effect through the due-loaded nano-carrier, providing a promising alternative for future clinical application.

Chemical Analysis of Ethanolic Extract of Ripe Fruit, N-Hexane Fraction of Half-Ripe Fruit, and Water Fraction of Half-Ripe Fruit of Sesoot (Garcinia Picrorrhiza Miq.) with Liquid Chromatography-Mass Spectrometry (Lc-Ms)

Garcinia is a plant belonging to the family of Guttiferae or Cluciaceae and has more than 180 famous species. Most of Garcinia species contains many prenelated xanthones, biflavonoids, and benzophenones. Using Liquid Chromatography - Mass Spectrometry (LC-MS), the development of electrospray ionization (ESI) could be detected and could be applied to a wide range of molecular biology such as xanthones, biflavonoids, and benzophenones and their derivatives. This method with a high speed of scanning at once could be measured a high degree of multiplexing and many components can be measured with a single analysis. These results were separated and analyzed using literature software of Dictionary of Natural Product 2006. The ethanolic extract of ripe fruit (IC), the n-hexane fraction of half ripe (IIB), and the water fraction of half ripe fruit (IVB) of sesoot (G. picrorrhiza Miq.) showed that the major compounds of them were xanthones, benzophenones with their derivatives, furanone and pyranone which were proven as cytotoxic to the brine shrimp nauplii using Brine Shrimp Lethality Test (BSLT) and to the doxorubicin-resistant breast cancer cell line of MCF-7, the doxorubicin-sensitive breast cancer cell line of T47D.

Jatrophone: a cytotoxic macrocylic diterpene targeting PI3K/AKT/NF-κB pathway, inducing apoptosis and autophagy in resistant breast cancer cells

BackgroundBreast cancer is a prevalent malignant tumor that affects women worldwide. The primary challenge in treating breast cancer is combating drug resistance, which contributes to relapse and metastasis. Jatrophone is a unique macrocyclic jatrophane diterpene found in various Jatropha and Euphorbia species. It possesses diverse biological and pharmacological activities, including anticancer activity. However, it is unclear whether jatrophone can overcome drug resistance in breast cancer.MethodsThis study includes the investigation of the cytotoxicity of jatrophone on doxorubicin-resistant breast cancer cells (MCF-7ADR) and the underlying molecular mechanisms. The effects of jatrophone on cell viability were determined using the sulforhodamine B (SRB) assay, while flow cytometry was used to evaluate cell cycle progression, apoptosis, and autophagy. A scratch assay was conducted to observe cell migration, and western blotting was used to measure downstream protein levels (PI3K, AKT, and NF-κB). Unpaired Student’s t-tests were used for comparison between the two groups and the results were analyzed by one-way ANOVA with Tukey- Kremer post hoc test.ResultsIt was shown that jatrophone exhibited potent cytotoxic activity on MCF-7ADR cells in a dose-dependent manner, with an IC50 value of 1.8 µM. It also significantly induced cell cycle S and G/M phase arrest. Interestingly, jatrophone induced both early and late apoptotic cell death, as well as autophagic cell death, with negligible necrosis. Furthermore, jatrophone treatment diminished the migration of MCF-7ADR cells. At the molecular level, jatrophone treatment significantly down-regulated the expression levels of PI3K, AKT, and NF-κB. β.ConclusionsThe results of the study suggest that jatrophone decreases the proliferation of MCF-7/ADR cells at a low micromolar concentration; induces cell cycle arrest; promotes apoptotic, and autophagic cell death; inhibits migration and EMT; and works on resistance by a mechanism involving the inhibition of the PI3K/Akt/ NF-κB pathway. These findings provide evidence of the potential of jatrophone to be a promising lead compound for targeting doxorubicin-resistant breast cancer cells and could be further investigated for its clinical application as a chemotherapy adjuvant.

Breviscapine reverses doxorubicin resistance in breast cancer and its related mechanisms.

Based on the effect of breviscapine (BRE) on reversing drug resistance of human breast cancer cell line MCF-7/doxorubicin (Dox), the mechanism was preliminarily explored. The methyl thiazolyl tetrazolium (MTT) method was introduced to detect inhibitory effect of Dox alone or in combination with BRE on MCF-7 (M) and MCF-7/Dox (MD) cells, and the inhibitory concentration (IC50 ) was obtained. Cell apoptosis and Dox concentration was assessed by flow cytometry. The drug resistance multiple and reversal fold were calculated. Western blot was performed to evaluate the expression of Bcl-2, Bax, EGFR, p-EGFR, P-gp, caspase-3, and cleaved-caspase-3 protein in cells. The efflux of Rho-123 was measured by flow cytometry and fluorescence microscopy. The IC50 of Dox on MD and M cells was 16.67 and 0.71 μg/mL, respectively, with a drug resistance ratio of 23.48 times. The IC50 of Dox combined with BRE on MD cells was 5.62 μg/mL, with a reversal ratio of 2.97 times. BRE greatly enhanced Dox-induced apoptosis of MD cells. Bax and cleaved-caspase-3 (proapoptotic protein) expression were obviously increased, while Bcl-2 (antiapoptotic protein) expression was significantly decreased after BRE treatment. BRE inhibited EGFR activation and P-gp expression. BRE increased the intracellular accumulation of Dox in MD cells by P-gp. BRE could increase the MD sensitivity to Dox via increasing Bax and cleaved-caspase-3 expression and inhibiting Bcl-2 expression, thereby promoting cell apoptosis. BRE reversed Dox resistance of MD cells by increasing Dox intracellular accumulation through inhibiting P-gp expression via EGFR.

Combined doxorubicin and arctigenin treatment induce cell cycle arrest-associated cell death by promoting doxorubicin uptake in doxorubicin-resistant breast cancer cells.

Chemotherapy failure is often caused by drug resistance, for which no effective treatment strategy has been established. Many studies have been undertaken with the aim of overcoming drug resistance using natural products. Arctigenin (ATG), a natural product, has been investigated for its anti-cancer effects in HER2-overexpressing, ER-positive, and triple-negative breast cancer cells. We investigated the efficacy of ATG against self-established doxorubicin (DOX)-resistant breast cancer cells (MCF-DR and MDA-DR cells) derived from MCF-7 and MDA-MB-231 cells, respectively. ATG was found to increase DOX intracellular levels by downregulating multidrug Resistance 1 (MDR1) mRNA expression in DOX-resistant cells. In addition, combined treatment with DOX and ATG (DOX/ATG) reduced the viability of and colony formation by DOX-resistant cells. DOX/ATG also significantly induced G2/M cell cycle arrest by suppressing the Cyclin D1/CDK4/RB pathways and suppressed the expressions of MDR1 and Cyclin D1 by inhibiting the Mitogen-activated protein kinase (MAPK)/Activating protein-1 (AP-1) signaling pathways. Furthermore, DOX/ATG induced DNA damage and attenuated the expressions of RAD51 and Ku80. However, PARP1 (Poly [ADP-ribose] polymerase1) cleavage and AIF (Apoptosis-inducing factor) induced apoptosis did not occur despite DNA damage-induced cell death. Rather, flow cytometry showed that DOX/ATG caused necrosis. In summary, DOX/ATG increased intracellular DOX levels by inhibiting MDR1 and inducing G2/M arrest by inhibiting the Cyclin D1/CDK4/RB pathways and causing necrosis by damaging DNA. Our results suggest that ATG might be used as an adjuvant to enhance the efficacy of DOX in DOX-resistant breast cancer.

Doxorubicin resistance in breast cancer is mediated via the activation of FABP5/PPARγ and CaMKII signaling pathway.

Breast cancer is the most prevalent malignancy among women. Doxorubicin (Dox) resistance was one of the major obstacles to improving the clinical outcome of breast cancer patients. The purpose of this study was to investigate the relationship between the FABP signaling pathway and Dox resistance in breast cancer. The resistance property of MCF-7/ADR cells was evaluated employing CCK-8, Western blot (WB), and confocal microscopy techniques. The glycolipid metabolic properties of MCF-7 and MCF-7/ADR cells were identified using transmission electron microscopy, PAS, and Oil Red O staining. FABP5 and CaMKII expression levels were assessed through GEO and WB approaches. The intracellular calcium level was determined by flow cytometry. Clinical breast cancer patient's tumor tissues were evaluated by immunohistochemistry to determine FABP5 and p-CaMKII protein expression. In the presence or absence of FABP5 siRNA or the FABP5-specific inhibitor SBFI-26, Dox resistance was investigated utilizing CCK-8, WB, and colony formation methods, and intracellular calcium level was examined. The binding ability of Dox was explored by molecular docking analysis. The results indicated that the MCF-7/ADR cells we employed were Dox-resistant MCF-7 cells. FABP5 expression was considerably elevated in MCF-7/ADR cells compared to parent MCF-7 cells. FABP5 and p-CaMKII expression were increased in resistant patients than in sensitive individuals. Inhibition of the protein expression of FABP5 by siRNA or inhibitor increased Dox sensitivity in MCF-7/ADR cells and lowered intracellular calcium, PPARγ, and autophagy. Molecular docking results showed that FABP5 binds more powerfully to Dox than the known drug resistance-associated protein P-GP. In summary, the PPARγ and CaMKII axis mediated by FABP5 plays a crucial role in breast cancer chemoresistance. FABP5 is a potentially targetable protein and therapeutic biomarker for the treatment of Dox resistance in breast cancer.

Reversal Effect of Saikosaponin A and Saikosaponin B on Doxorubicin-resistant Breast Cancer Cells and its Mechanism

Background One of the key factors limiting the effectiveness of chemotherapy treatment for malignancies is multidrug resistance (MDR). The MDR phenotype is related to P-glycoprotein (P-gp) expression and function. The main triterpenoid saponins generated from Bupleurum chinense DC (BCDC), saikosaponin A (SSa), has been found to have anti-tumor potential. Saikosaponin B (SSb) has the potential for utility in combination with anticancer drugs as the secondary saikosaponins. Objective In this study, we looked into the impact of SSa and SSb on doxorubicin (Dox)-resistant breast cancer cells and its underlying mechanisms. Materials and Methods Dox-resistant breast cancer cells (MCF-7ADR) and MCF-7 cells were used in the study. The experimental cells were divided into a different concentration SSa administration group, a different concentration SSb administration group, and a control group, and the related biochemical parameters of MCF-7 and MCF-7ADR cells were detected. Results We discovered that SSa and SSb both suppressed MCF-7 and MCF-7ADR cell proliferation in a dose-dependent manner. Additionally, SSa at 2.5 and 5.0 µg/mL and SSb at 3.0 and 7.0 µg/mL could significantly enhance the cytotoxicity of Dox and reverse MDR in MCF-7ADR cells. The combination of Dox and SSa or SSb induced obvious synergistic effects. SSa and SSb could increase the sensitivity of MCF-7ADR cells to Dox by decreasing P-gp expression, increasing intracellular accumulation, and delaying the drug efflux of rhodamine 123 (Rh123, a P-gp substrate). Additionally, SSa and SSb both induced G1-phase arrest in MCF-7ADR cells in the presence of Dox. Conclusion According to the study, SSa and SSb may be novel MDR reversal medicines for breast cancer chemotherapy and have significant therapeutic significance for MDR during tumor therapy.

METTL3-mediated m6A modification of lnc KCNQ1OT1 promotes doxorubicin resistance in breast cancer by regulating miR-103a-3p/MDR1 axis

ABSTRACT Doxorubicin (DOX) resistance in breast cancer (BC) poses a huge challenge for the therapeutic effect on BC. Lnc KCNQ1OT1 play crucial roles in chemotherapy resistance. However, the role and mechanism of lnc KCNQ1OT1 in DOX resistance BC have not been investigated, which merits further exploration. Based on MCF‐7 and MDA-MB-231 cells, MCF‐7/DOX and MDA-MB-231/DOX cells were established using gradient concentrations of DOX. IC50 values and cell viability were determined using MTT. Cell proliferation was investigated by colony formation. Flow cytometry was performed to examine cell apoptosis and cell cycle. Gene expression was examined using qRT-PCR and western blot. The interactions among METTL3, lnc KCNQ1OT1, miR-103a-3p, and MDR1 were validated with MeRIP-qPCR, RIP, and dual-luciferase reporter gene assays. The results showed that Lnc KCNQ1OT1 was highly expressed in DOX-resistant BC cells, and lnc KCNQ1OT1 depletion could enhance DOX sensitivity in BC cells and DOX-resistant BC cells. Besides, lnc KCNQ1OT1 was modulated by MELLT3 in the manner of m6A modification. MiR-103a-3p could interact with lnc KCNQ1OT1 and MDR1. Overexpression of MDR1 abolished the impacts of lnc KCNQ1OT1 depletion on DOX resistance in BC. In conclusion, our results unveiled that in BC cells and DOX-resistant BC cells, lnc KCNQ1OT1 could be mediated by METTL3 through m6A modification to elevate and stabilize its expression, further inhibitingmiR-103a-3p/MDR1 axis to promote DOX resistance, which might provide novel thought to overcome DOX resistance in BC.

Caffeic acid phenethyl ester reverses doxorubicin resistance in breast cancer cells via lipid metabolism regulation at least partly by suppressing the Akt/mTOR/SREBP1 pathway.

Chemotherapy is one of the common treatment methods for breast cancer, but chemoresistance is a severe challenge. Caffeic acid phenethyl ester (CAPE) is an active ingredient of propolis extract and has been shown to have a variety of beneficial effects, and its potential as a treatment for breast cancer is worth exploring. The effects of CAPE on doxorubicin (DOX) resistance were determined by cell counting kit-8 (CCK-8) assay, colony-formation assay, and flow cytometry. Oil Red O staining and the detection of free fatty acids, triglycerides, phospholipids, and cholesterol were performed to assess the status of lipid metabolism. Quantitative polymerase chain reaction (qPCR) and western blotting were applied to investigate the molecules involved in lipid metabolism and the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/sterol regulatory element binding protein 1 (SREBP1) pathway. CAPE treatment reversed DOX resistance in breast cancer cells and suppressed their lipid metabolism. In addition, CAPE combined with DOX remarkably suppressed SREBP1 expression in part by inhibiting Akt/mTOR pathway activation. Furthermore, by inhibiting lipid metabolism, partly via the Akt/mTOR/SREBP1 pathway, CAPE ultimately reversed DOX resistance in breast cancer. Our results suggest that CAPE treatment reversed DOX resistance in breast cancer cells, at least in part by inhibiting Akt/mTOR/SREBP1 pathway-mediated lipid metabolism, indicating that CAPE may be an effective substance to assist in the treatment of breast cancer.

Isorhamnetin induces cell cycle arrest and apoptosis by triggering DNA damage and regulating the AMPK/mTOR/p70S6K signaling pathway in doxorubicin-resistant breast cancer

BackgroundAcquired resistance to doxorubicin (DOX) inevitably limits its clinical use against breast cancer (BC). Isorhamnetin (IS), a native flavonoid which extensively available in vegetables, fruits, and phytomedicine, has been deemed to the probable cancer chemopreventive agent in preceding explorations since it exhibits satisfied antitumor activity. So far, the strategy for alleviating DOX resistance by using IS as a sensitizer against resistant BC has not yet been covered. PurposeTo investigate the effect of IS on potentiating the chemoreceptivity of drug-resistant BC cells to DOX in vitro and in vivo and elucidate the possible molecular mechanisms. MethodsMTS assays, colony formation assays, three-dimensional (3D) tumor spheroid model, and migration assay were deployed to verify the inhibiting action of IS in the presence or absence of DOX on resistant BC cells in vitro. Apoptosis, cell cycle regulation, and endocellular reactive oxygen species (ROS) were determined by flow cytometry. Protein levels were monitored by western blotting. Nuclear staining and EdU proliferation were photographed with a confocal laser scanning microscope. The effects of the IS and DOX combination on the tumorigenesis in the xenograft experiments were evaluated for further confirming the in vitro cytotoxicity. ResultsIS significantly inhibited cell proliferation and migration and enhanced the antitumor competence of DOX against resistant BC cells both in vitro and in vivo. Adjuvant IS (50 μM) effectively enhanced the proapoptotic impacts of DOX in resistant BC cells (35.38 ± 3.18%, vs. 5.83 ± 0.68% in the DOX group) by suppressing the expression of bcl 2 in addition to enhancing cleaved caspase 3, ultimately leading to DNA condensation and fragmentation. IS (20, 30, and 50 μM) treatments induced significant increases in the G2/M populations (41.60 ± 1.28%, 44.60 ± 1.14%, and 50.64 ± 0.67%, vs. 35.84 ± 1.56% in the untreated control in MCF7/ADR cells, p < 0.01) via regulating CDK1/Cyclin B1 complex expression, subsequently triggering the inhibition of BC proliferation. In addition, IS (10, 20, 30, and 50 μM) stimulated the production of interstitial ROS in MCF7/ADR cells, by 3.99-, 4.20-, 6.29-, and 6.78-fold, respectively, versus the untreated group (p < 0.001), which were involved in DNA damage and AMPK-caused intercept of the mTOR/p70S6K signaling. ConclusionOur study suggested the anti-breast cancer actions of IS as a DOX sensitizer and expounded the underlying molecular mechanisms, showing that IS could be deemed to a capable alternative for resistant BC cure.

Cytotoxic and chemomodulatory effects of Phyllanthus niruri in MCF-7 and MCF-7ADR breast cancer cells

The members of the genus Phyllanthus have long been used in the treatment of a broad spectrum of diseases. They exhibited antiproliferative activity against various human cancer cell lines. Breast cancer is the most diagnosed cancer and a leading cause of cancer death among women. Doxorubicin (DOX) is an anticancer agent used to treat breast cancer despite its significant cardiotoxicity along with resistance development. Therefore, this study was designed to assess the potential cytotoxicity of P. niruri extracts (and fractions) alone and in combination with DOX against naïve (MCF-7) and doxorubicin-resistant breast cancer cell lines (MCF-7ADR). The methylene chloride fraction (CH2Cl2) showed the most cytotoxic activity among all tested fractions. Interestingly, the CH2Cl2-fraction was more cytotoxic against MCF-7ADR than MCF-7 at 100 µg/mL. At sub-cytotoxic concentrations, this fraction enhanced the cytotoxic effect of DOX against the both cell lines under investigation (IC50 values of 0.054 µg/mL and 0.14 µg/mL vs. 0.2 µg/mL for DOX alone against MCF-7) and (1.2 µg/mL and 0.23 µg/mL vs. 9.9 µg/mL for DOX alone against MCF-7ADR), respectively. Further, TLC fractionation showed that B2 subfraction in equitoxic combination with DOX exerted a powerful synergism (IC50 values of 0.03 µg/mL vs. 9.9 µg/mL for DOX alone) within MCF-7ADR. Untargeted metabolite profiling of the crude methanolic extract (MeOH) and CH2Cl2 fraction exhibiting potential cytotoxicity was conducted using liquid chromatography diode array detector-quadrupole time-of-flight mass spectrometry (LC-DAD-QTOF). Further studies are needed to separate the active compounds from the CH2Cl2 fraction and elucidate their mechanism(s) of action.

Exosomal transfer of miR-181b-5p confers senescence-mediated doxorubicin resistance via modulating BCLAF1 in breast cancer.

Doxorubicin resistance represents a major clinical challenge for treating patients with advanced breast cancer (BC). Exosomes, exchanging genetic cargo between heterogeneous populations of tumour cells, have been proposed to mediate drug resistance and cancer progression in other cancer types. However, their specific role in mediating doxorubicin resistance in BC remains unclear. Here, we demonstrate the important role of exosomal miR-181b-5p (exo-miR-181b-5p) in mediating doxorubicin resistance. Small-RNA sequencing and bioinformatic analyses were used to screen miRNAs mediating doxorubicin resistance in BC, which were further verified by RT-qPCR. SA-β-gal staining assays allowed us to measure cellular senescence. Exosomes from patients' serum before and after neoadjuvant chemotherapy were isolated for exo-miR-181b-5p quantification. Doxorubicin-resistant BC cell lines exhibited upregulated exosomal miR-181b-5p. Addition of exo-miR-181b-5p actively fused with recipient cells and transferred a drug-resistant phenotype. Overexpression of miR-181b-5p downregulated p53/p21 levels and inhibited doxorubicin-induced G1 arrest and senescence by suppressing BCLAF1 expression in vitro. Further, in vivo experiments showed treatment of exo-miR-181b-5p inhibitors exhibited superior tumour control and reversed the doxorubicin-resistance phenotype, accompanied with increased tumoral BCLAF1. Our data suggests exo-miR-181b-5p as a prognostic biomarker and a therapeutic potential for exo-miR-181b-5p inhibitors in the treatment of doxorubicin-resistant BC patients.

Cancer Cells and Platelets Have a Synergistic Role on the Acceleration of Clot Formation and Fibrin Polymerization. a Thromboelastometric Study

Introduction The histological type and the aggressiveness of cancer cells as well as the acquisition of resistance to chemotherapy are significant predictors for the high risk of cancer associated thrombosis (CAT). It is well established that cancer cells, by expressing tissue factor (TF) trigger thrombin generation. Cancer cell derived extracellular vesicles (CaCa-dEV) bearing TF, in the microenvironment, further enhance thrombin generation. The impact of cancer cells on clot formation kinetics and the qualitative characteristics of the clot has not been evaluated yet. Aim The present study aimed to analyze the variables which influence the kinetics and the qualitative characteristics of the clot after triggering thrombin generation by cells from pancreatic cancer (BXPC3), breast cancer (MCF7 and MCF7R10) and multiple myeloma (RPMI-8226 and U266) - associated with different risk of CAT. The impact of the resistance to chemotherapy on the clot characteristics was also studied. Methods Cancer cell lines (BXPC3, MCF7, MCF7R10, RPMI-8226 and U266) and primary umbilical vein endothelial cells (HUVEC) for the control experiments were used according to standardized and validated experimental procedure. Doxorubicin resistant breast cancer cells (MCF7R10) were obtained by treating native breast cancer cells (MCF7) by increasing concentrations of Doxorubicin from 5 nM to 10 µM.. Thromboelastometry, using the ROTEM® delta device (from Warfen Paris, France), was used for the study of clot formation and firmness in whole blood (WB), platelet rich and platelet poor plasma (PRP and PPP respectively) in the presence of cancer cells or HUVEC (control). A suspension of 200 μL with 105 cells was placed in each sterilized cup. Following an incubation of 4 hour, culture media was removed and 330 μL of WB, PRP or PPP were added followed by addition of 20 µM of CaCl2 (0.2 M). Clotting time (CT), α-angle and maximum clot firmness (MCF) were analyzed. Results In PPP, pancreatic cancer cells BXPC3 as well as chemotherapy sensitive and resistant breast cancer cells (MCF7 and MCF7R10), significantly reduced the CT. MCF7R10 cells significantly reduced the CT as compared to MCF7. In contrast, myeloma cells (RPMI8226 and U266), did not significantly affect the CT. All studied cancer cell lines significantly increased the α-angle as compared to HUVEC, with the most potent effect from BXPC3 cells. The studied cancer cell lines did not significantly affect MCF. In PRP, BXPC3 cells significantly reduced the CT whereas MCF7 and MCF7R10 did not significantly change the CT, and myeloma cells significantly prolonged the CT as compared to HUVEC. BXPC3 cells significantly increased the α-angle as compared to the HUVEC, whereas the other studied cancer cell lines did not have any significant effect on this parameter. BXPC3, MCF7 and MCF7R10 cells did not significantly affect the MCF whereas the myeloma cells significantly decreased the MCF as compared to HUVEC. In WB, BXPC3 and MCF7 cells significantly decreased the CT whereas MCF7R10 did not show any significant effect and the myeloma cells significantly prolonged it. The α-angle significantly increased in the presence of BXPC3 cells whereas the other cell lines did not show any significant effect on this parameter. The presence of cancer cells resulted in significant decrease of the MCF as compared to the control (HUVEC), with the most potent effect from myeloma. In the control experiment (HUVEC), the comparison of the thromboelastometric profile of the three experimental systems (PPP, PRP and WB) showed that platelets (PRP) had a substantial impact on CT, α-angle and MCF whereas the erythrocytes (in WB) did not further modify these parameters. The same pattern of the role of platelets was observed in the presence of the studied cancer cell lines. Discussion The present study provides experimental evidence documenting that cancer cells impact clot formation kinetics without affecting clot firmness. The impact of cancer cells on clot formation kinetics is determined principally by the histological type. The pancreatic adenocarcinoma cells have a more pronounced effect on clot formation kinetics as compared to the breast cancer cells (sensitive or resistant to Doxorubicine). In contrast, multiple myeloma cells delay fibrin formation and polymerization and decrease the clot firmness. Platelets have a synergistic effect with cancer cells on the acceleration of the clot formation kinetics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhanced antitumor effect of doxorubicin through active-targeted nanoparticles in doxorubicin-resistant triple-negative breast cancer

Breast cancer is the most common cancer in women worldwide, and the incidence and mortality rates have been increasing. Chemotherapy remains the main treatment strategy for triple-negative breast cancer (TNBC). However, multidrug resistance often affects the efficiency of chemotherapy and is the main reason for the failure of current clinical cancer treatments in patients with TNBC. Therefore, we developed a nanocarrier with biodegradable polymer materials and encapsulated a clinical chemotherapeutic drug to form an active targeting nanoparticle drug delivery system. In this study, the biopolymers fucoidan (FD) and arginine-gelatin (Arg-GL) were used to synthesize nanoparticles through electrostatic forces and to encapsulate the chemotherapeutic drug doxorubicin (Dox). Our study demonstrated that the Dox-loaded FD/Arg-GL nanoparticles exhibited high drug loading capacity and pH-responsive characteristics. The results of in vitro experiments showed that the nanoparticles actively targeted P-selectin expressed by cancer cells, thereby enhancing the anticancer effect and inhibiting the migration and invasion of Dox-resistant TNBC cells. Moreover, in vivo studies further confirmed the antitumor activity of the nanoparticles, as they enabled the drug to accumulate at the tumor site. In conclusion, Dox-loaded FD/Arg-GL nanoparticles offer a potential therapeutic strategy for drug-resistant TNBC.

CircRNA-CREIT inhibits stress granule assembly and overcomes doxorubicin resistance in TNBC by destabilizing PKR

BackgroundCircular RNAs (circRNAs) represent a novel type of regulatory RNA characterized by high evolutionary conservation and stability. CircRNAs are expected to be potential diagnostic biomarkers and therapeutic targets for a variety of malignancies. However, the regulatory functions and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) are largely unknown.MethodsBy using RNA high-throughput sequencing technology, qRT-PCR and in situ hybridization assays, we screened dysregulated circRNAs in breast cancer and TNBC tissues. Then in vitro assays, animal models and patient-derived organoids (PDOs) were utilized to explore the roles of the candidate circRNA in TNBC. To investigate the underlying mechanisms, RNA pull-down, RNA immunoprecipitation (RIP), co immunoprecipitation (co-IP) and Western blotting assays were carried out.ResultsIn this study, we demonstrated that circRNA-CREIT was aberrantly downregulated in doxorubicin resistant triple-negative breast cancer (TNBC) cells and associated with a poor prognosis. The RNA binding protein DHX9 was responsible for the reduction in circRNA-CREIT by interacting with the flanking inverted repeat Alu (IRAlu) sequences and inhibiting back-splicing. By utilizing in vitro assays, animal models and patient-derived organoids, we revealed that circRNA-CREIT overexpression significantly enhanced the doxorubicin sensitivity of TNBC cells. Mechanistically, circRNA-CREIT acted as a scaffold to facilitate the interaction between PKR and the E3 ligase HACE1 and promoted proteasomal degradation of PKR protein via K48-linked polyubiquitylation. A reduced PKR/eIF2α signaling axis was identified as a critical downstream effector of circRNA-CREIT, which attenuated the assembly of stress granules (SGs) to activate the RACK1/MTK1 apoptosis signaling pathway. Further investigations revealed that a combination of the SG inhibitor ISRIB and doxorubicin synergistically inhibited TNBC tumor growth. Besides, circRNA-CREIT could be packaged into exosomes and disseminate doxorubicin sensitivity among TNBC cells.ConclusionsOur study demonstrated that targeting circRNA-CREIT and SGs could serve as promising therapeutic strategies against TNBC chemoresistance.