DOX-resistant Cells Research Articles

Apigenin alleviates resistance to doxorubicin in breast cancer cells by acting on the JAK/STAT signaling pathway.

Breast cancer is the most frequent cancer among women. Despite the effectiveness of Doxorubicin (DOX) as a chemotherapeutic for the treatment of breast cancer, the therapy-resistance remains unsolvable. Apigenin is a natural dietary flavonoid with potential anticancer activities. Our study's intention was to evaluate the effect of Apigenin on DOX resistance in MCF-7 cells. DOX-resistant MCF-7 cell line (MCF-7R) was developed by treating MCF-7 cells with increasing concentrations of DOX (0-100 µM). The viability of cell lines was assayed using MTT method. Quantitative polymerase chain reaction method was performed to measure multidrug-resistance 1 (MDR1) gene expression level. The expression of MDR1, Janus kinase 2 (JAK2) and Signal transducer and activator of transcription 3 (STAT3) proteins were determined by western blotting. MCF-7R cell line showed resistance to DOX in comparison to MCF-7 cells. Apigenin had a significant effect on the reduction of viability of both MCF-7 and MCF-7R cell lines. However, DOX-resistance in the MCF-7 cell line was considerably decreased due to the co-treatment of MCF-7R cells with Apigenin. This natural compound also downregulated the expression of MDR1 at mRNA and protein levels both in resistant and non-resistant cells. Apigenin significantly prohibited the phosphorylation and activation of JAK2 and STAT3 proteins both in MCF-7 and MCF-7R cell lines. The present results suggested, for the first time, Apigenin as an ideal therapeutic for ameliorating DOX resistance in breast cancer. These data also proposed a novel mechanism for the anti-resistance activity of Apigenin by regulating the JAK2/STAT3/MDR1 axis.

Reversing multi-drug resistance by polymeric metformin to enhance antitumor efficacy of chemotherapy

Multi-drug resistance (MDR) in breast cancer poses a great threat to chemotherapy. The expression and function of the ATP binding cassette (ABC) transporter are the major cause of MDR. Herein, a linear polyethylene glycol (PEI) conjugated with dicyandiamide, which called polymeric metformin (PolyMet), was successfully synthesized as a simple and biocompatible polymer of metformin. PolyMet showed the potential to reverse MDR by inhibiting the efflux of the substrate of ATP-binding cassette (ABC) transporter from DOX resistant MCF-7 cells (MCF-7/DOX). To test its MDR reversing effect, PolyMet was combined with DOX to treat mice carrying MCF-7/DOX xenografts. In order to decrease the toxicities of DOX and delivery PolyMet and DOX to tumor at the same time, PolyMet was complexed with poly-γ-glutamic acid-doxorubicin (PGA-DOX) electrostatically at the optimal ratio of 2:3, which were further coated with lipid membrane to form lipid/PolyMet-(PGA-DOX) nanoparticles (LPPD). The particle size of LPPD was 165.8 nm, and the zeta potential was +36.5 mV. LPPD exhibited favorable cytotoxicity and cellular uptake in MCF-7/DOX. Meanwhile, the bioluminescence imaging and immunohistochemical analysis indicated that LPPD effectively conquered DOX-associated MDR by blocking ABC transporters (ABCB1 and ABCC1) via PolyMet. Remarkably, LPPD significantly inhibited the tumor growth and lowered the systemic toxicity in a murine MCF-7/DOX tumor model. This is the first time to reveal that PolyMet can enhance the anti-tumor efficacy of DOX by dampening ABC transporters and activating the AMPK/mTOR pathway, which is a promising strategy for drug-resistant breast cancer therapy.

Abstract 1063: Combination strategies to overcome doxorubicin induced senescence in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor-2 over-expression. Compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence. While immunotherapy, PARP inhibitors, and sacituzumab govitecan have shown benefit in a subset of TNBC patients, chemotherapy with doxorubicin remains a mainstay of treatment. Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells are able to secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. It has been suggested that senescence is a potential mechanism of resistance in doxorubicin (dox) treated cells. Transitioning senescence mediated resistance to apoptosis is crucial to the treatment of TNBC. The objective of this study was to evaluate the combination BCL-2 and HDAC inhibitors with dox to overcome resistance and induce apoptosis. Experimental Procedures: TNBC cell lines resistant and sensitive to doxorubicin were identified using a CellTiter-Glo Viability Assay and resistant cell lines were selected for further analysis. To assess proliferation, dox resistant TNBC cell lines were plated in 96-well plates. Cells were exposed to vehicle control, dox, BCL-2 inhibitor, HDAC inhibitor or the combination of dox and BCL-2 inhibitor or HDAC inhibitor for 72 hours. Cellular proliferation was assessed using the BioSpa live cell analysis system. Apoptosis at 24 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and as single agents. Immunoblotting was performed to evaluate the downstream effects of apoptosis and senescence of drugs as single agents and in combination. Results: The addition of a BCL-2 inhibitor and HDAC inhibitor to doxorubicin resulted in decreased proliferation in resistant TNBC cell lines compared to single agents and vehicle control by live cell microscopy. Furthermore, the combination of dox and a BCL-2 inhibitor resulted in increased apoptosis when compared to single agents and control using Annexin V staining. The cyclin dependent kinase inhibitors p21 and p16 were over-expressed in cells exposed to combination treatment. Apoptotic proteins BAD, PUMA, cleaved PARP and the anti-apoptotic protein BCL-2 were upregulated in cells treated with a BCL-2 inhibitor with or without dox. The pro-mitotic protein cyclin-B1 was downregulated in resistant cells treated with single agent BCL-2 inhibitor and in combination with dox. Additional mechanistic studies are ongoing. Conclusion: The combination of doxorubicin with BCL-2 and HDAC inhibitors resulted in decreased cellular proliferation and increased apoptosis. Potential senolytic drugs used with dox represent an exciting potential to overcome dox resistance and warrant further investigation. Citation Format: Anna R. Schreiber, Stephen Smoots, Betelehem W. Yacob, Adrian TA Dominguez, Cecilia Levandowski, Jennifer R. Diamond, Todd M. Pitts. Combination strategies to overcome doxorubicin induced senescence in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1063.

Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 136.

The significance of interferon gamma inducible protein 16 (IFI16) expression in drug resistant ovarian cancer cell lines.

Inherent or developed during treatment drug resistance is the main reason for the low effectiveness of chemotherapy in ovarian cancer. IFI16 is a cytoplasmic/nuclear protein involved in response to virus's infection and cell cycle arrest associated with the cellular senescence. Here we performed a detailed IFI16 expression analysis in ovarian cancer cell lines sensitive (A2780) and resistant to doxorubicin (DOX) (A2780DR1 and A2780DR2) and paclitaxel (PAC) (A2780PR1). IFI16 mRNA level, protein level in the nuclear and cytoplasmic fraction (Western blot analysis), the protein expression in cancer cells and nuclei (immunofluorescence analysis) and cancer patient lesions (immunohistochemistry) were performed in this study. We observed upregulation of IFI16 expression in drug resistant cell lines with dominant cytoplasmic localization in DOX-resistant cell lines and nuclear one in the PAC-resistant cell line. The most abundantly overexpressed isoforms of IFI16 were IFI16A and IFI16C. Finally, an analysis of a histological type of ovarian cancer (immunohistochemistry) showed expression in serous ovarian cancer. Expression of IFI16 in drug-resistant cell lines suggests its role in drug resistance development in ovarian cancer. Expression in serous ovarian cancer suggests its role in the pathogenesis of this histological type.

PH and Redox-Dual Sensitive Chitosan Nanoparticles Having Methyl Ester and Disulfide Linkages for Drug Targeting against Cholangiocarcinoma Cells.

The aim of this study is to prepare pH- and redox-sensitive nanoparticles for doxorubicin (DOX) delivery against DOX-resistant HuCC-T1 human cholangiocarcinoma (CCA) cells. For this purpose, L-histidine methyl ester (HIS) was attached to chitosan oligosaccharide (COS) via dithiodipropionic acid (abbreviated as ChitoHISss). DOX-incorporated nanoparticles of ChitoHISss conjugates were fabricated by a dialysis procedure. DOX-resistant HuCC-T1 cells were prepared by repetitive exposure of HuCC-T1 cells to DOX. ChitoHISss nanoparticles showed spherical morphology with a small diameter of less than 200 nm. The acid pH and glutathione (GSH) addition induced changes in the size distribution pattern of ChitoHISss nanoparticles from a narrow/monomodal distribution pattern to a wide/multimodal pattern and increased the fluorescence intensity of the nanoparticle solution. These results indicate that a physicochemical transition of nanoparticles can occur in an acidic pH or redox state. The more acidic the pH or the higher the GSH concentration the higher the drug release rate was, indicating that an acidic environment or higher redox states accelerated drug release from ChitoHISss nanoparticles. Whereas free DOX showed decreased anticancer activity at DOX-resistant HuCC-T1 cells, DOX-incorporated ChitoHISss nanoparticles showed dose-dependent anticancer activity. Intracellular delivery of DOX-incorporated ChitoHISss nanoparticles was relatively increased at an acidic pH and in the presence of GSH, indicating that DOX-incorporated ChitoHISss nanoparticles have superior acidic pH- and redox-sensitive behavior. In an in vivo tumor xenograft model, DOX-incorporated ChitoHISss nanoparticles were specifically delivered to tumor tissues and then efficiently inhibited tumor growth. We suggest that ChitoHISss nanoparticles are a promising candidate for treatment of CCA.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Doxorubicin and Topotecan Resistant Ovarian Cancer Cell Lines.

Epithelial ovarian cancer has the highest mortality among all gynecological malignancies. The main reasons for high mortality are late diagnosis and development of resistance to chemotherapy. Resistance to chemotherapeutic drugs can result from altered expression of drug-resistance genes regulated by miRNA. The main goal of our study was to detect differences in miRNA expression levels in two doxorubicin (DOX)- and two topotecan (TOP)-resistant variants of the A2780 drug-sensitive ovarian cancer cell line by miRNA microarray. The next aim was to recognize miRNAs as factors responsible for the regulation of drug-resistance genes. We observed altered expression of 28 miRNA that may be related to drug resistance. The upregulation of miR-125b-5p and miR-935 and downregulation of miR-218-5p was observed in both DOX-resistant cell lines. In both TOP-resistant cell lines, we noted the overexpression of miR-99a-5p, miR-100-5p, miR-125b-5p, and miR-125b-2-3p and decreased expression of miR-551b-3p, miR-551b-5p, and miR-383-5p. Analysis of the targets suggested that expression of important drug-resistant genes such as the collagen type I alpha 2 chain (COL1A2), protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase—EPHA7, Roundabout Guidance Receptor 2 (ROBO2), myristoylated alanine-rich C-kinase substrate (MARCK), and the ATP-binding cassette subfamily G member 2 (ABCG2) can be regulated by miRNA.

Doxorubicin-NO Releaser Molecular Hybrid Activatable by Green Light to Overcome Resistance in Breast Cancer Cells.

The biological activity of a molecular hybrid (DXNO-GR) joining doxorubicin (DOX) and an N-nitroso moiety releasing nitric oxide (NO) under irradiation with the biocompatible green light has been investigated against DOX-sensitive (MCF7) and -resistant (MDA-MB-231) breast cancer cells in vitro. DXNO-GR shows significantly higher cellular internalization than DOX in both cell lines and, in contrast to DOX, does not experience cell efflux in MDR overexpressing MDA-MB-231 cells. The higher cellular internalization of the DXNO-GR hybrid seems to be mediated by bovine serum albumin (BSA) as a suitable carrier among serum proteins, according to the high binding constant measured for DXNO-GR, which is more than one order of magnitude larger than that reported for DOX. Despite the higher cellular accumulation, DXNO-GR is not toxic in the dark but induces remarkable cell death following photoactivation with green light. This lack of dark toxicity is strictly related to the different cellular compartmentalization of the molecular hybrid that, different from DOX, does not localize in the nucleus but is mainly confined in the Golgi apparatus and endoplasmic reticulum and therefore does not act as a DNA intercalator. The photochemical properties of the hybrid are not affected by binding to BSA as demonstrated by the direct detection of NO photorelease, suggesting that the reduction of cell viability observed under light irradiation is a combined effect of DOX phototoxicity and NO release which, ultimately, inhibits MDR1 efflux pump in DOX-resistant cells.

Lemon-Derived Extracellular Vesicles Nanodrugs Enable to Efficiently Overcome Cancer Multidrug Resistance by Endocytosis-Triggered Energy Dissipation and Energy Production Reduction.

Multidrug resistance remains a great challenge for cancer chemotherapy. Herein, a biomimetic drug delivery system based on lemon‐derived extracellular vesicles (EVs) nanodrugs (marked with heparin‐cRGD‐EVs‐doxorubicin (HRED)) is demonstrated, achieving highly efficient overcoming cancer multidrug resistance. The HRED is fabricated by modifying functional heparin‐cRGD (HR) onto the surface of EVs and then by loading with doxorubicin (DOX). The obtained HRED enable to effectively enter DOX‐resistant cancer cells by caveolin‐mediated endocytosis (main), macropinocytosis (secondary), and clathrin‐mediated endocytosis (last), exhibiting excellent cellular uptake capacity. The diversified endocytosis capacity of HRED can efficiently dissipate intracellular energy and meanwhile trigger downstream production reduction of adenosine triphosphate (ATP), leading to a significant reduction of drug efflux. Consequently, they show excellent anti‐proliferation capacities to DOX‐resistant ovarian cancer, ensuring the efficiently overcoming ovarian cancer multidrug resistance in vivo. The authors believe this strategy provides a new strategy by endocytosis triggered‐energy dissipation and ATP production reduction to design drug delivery system for overcoming cancer multidrug resistance.

Phytic acid-modified CeO2 as Ca2+ inhibitor for a security reversal of tumor drug resistance.

Ca2+ plays critical roles in the development of diseases, whereas existing various Ca regulation methods have been greatly restricted in their clinical applications due to their high toxicity and inefficiency. To solve this issue, with the help of Ca overexpressed tumor drug resistance model, the phytic acid (PA)-modified CeO2 nano-inhibitors have been rationally designed as an unprecedentedly safe and efficient Ca2+ inhibitor to successfully reverse tumor drug resistance through Ca2+ negative regulation strategy. Using doxorubicin (Dox) as a model chemotherapeutic drug, the Ca2+ nano-inhibitors efficiently deprived intracellular excessive free Ca2+, suppressed P-glycoprotein (P-gp) expression and significantly enhanced intracellular drug accumulation in Dox-resistant tumor cells. This Ca2+ negative regulation strategy improved the intratumoral Dox concentration by a factor of 12.4 and nearly eradicated tumors without obvious adverse effects. Besides, nanocerias as pH-regulated nanozyme greatly alleviated the adverse effects of chemotherapeutic drug on normal cells/organs and substantially improved survivals of mice. We anticipate that this safe and effective Ca2+ negative regulation strategy has potentials to conquer the pitfalls of traditional Ca inhibitors, improve therapeutic efficacy of common chemotherapeutic drugs and serves as a facile and effective treatment platform of other Ca2+ associated diseases.Electronic Supplementary MaterialSupplementary material (further details of the XRD pattern of CeO2, TEM images, XPS spectra, cellular uptake study, cytotoxicity data, apoptosis study, biodistribution, and biosecurity of nanocerias in vivo, etc.) is available in the online version of this article at 10.1007/s12274-022-4069-0.

Inhibition of ABCC9 by zinc oxide nanoparticles induces ferroptosis and inhibits progression, attenuates doxorubicin resistance in breast cancer

BackgroundZinc oxide nanoparticles (ZONs) are a type of nanomaterial that has presented anti-cancer properties in breast cancer (BC). However, the function of ABCC9 in BC and its correlation with ZONs are still elusive.MethodsHere, we identified the crucial role of ABCC9 in modulating ferroptosis and doxorubicin (Dox) resistance in BC and the targeted function of ZONs to ABCC9.ResultsThe silencing of ABCC9 significantly repressed the viability of BC cells. The knockdown of ABCC9 decreased the numbers of Edu-positive BC cells. Conversely, BC cell apoptosis was increased by the inhibition of ABCC9. Besides, the silencing of ABCC9 reduced the capability of migration and invasion of BC cells. Significantly, tumorigenicity analysis demonstrated that the tumor growth of BC cells was suppressed by the depletion of ABCC9 in the xenograft model of nude mice. Moreover, the treatment of ferroptosis activator erastin repressed cell viability of BC cells and ABCC9 overexpression rescued the repression. Similarly, the numbers of Edu-positive BC cells were inhibited by erastin and the overexpression of ABCC9 reversed the inhibitory effect of erastin. The levels of GSH were decreased and MDA, lipid ROS, and iron levels were increased by the treatment of erastin, while the ABCC9 overexpression could reverse these results in BC cells. Consistently, erastin suppressed the expression of ferroptosis inhibitory factors, including GPX4 and SLC7A11, in BC cells and the overexpression of ABCC9 rescued the expression. The IC50 value of Dox was reduced by the knockdown of ABCC9 in Dox-resistant BC cells (BC/Dox). The numbers of Edu-positive BC/Dox cells were attenuated by the depletion of ABCC9. Meanwhile, the apoptosis of BC/Dox cells was stimulated by the silencing of ABCC9. Furthermore, the treatment of ZONs attenuated Dox resistance of BC cells. ZONs remarkably repressed the expression of ABCC9 in BC/Dox cells. ZONs inhibited the cell viability of BC/Dox cells and the overexpression of ABCC9 reversed the repression. Moreover, the treatment of ZONs reduced GSH levels and enhanced MDA, lipid ROS, and iron levels in erastin-stimulated BC/Dox cells.ConclusionsIn conclusion, we discovered that the inhibition of ABCC9 by zinc oxide nanoparticles induces ferroptosis and attenuates Dox resistance in BC.

(WR)8WKβA]-Doxorubicin Conjugate: A Delivery System to Overcome Multi-Drug Resistance against Doxorubicin.

Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKβA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKβA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 μM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 μM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKβA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 μM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKβA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

Has_circ_0010220 regulates the miR-574-3p/IL-6 axis to increase doxorubicin resistance in osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy. It has an aggressive nature and produces drug resistance in diseased patients, which in turn causes obstacles in treating cancer with chemotherapy. The objective of our investigation was to analyze the function and hsa_circ_0010220 mechanism in doxorubicin (DOX) resistance to OS. The hsa_circ_0010220, IL-6, and miR-574-3p levels in OS diseased tissues and cell resistance towards DOX drug were elucidated by qRT-PCR and Elisa assay. The DOX half-inhibitory concentration (IC50) was quantified by Cell Counting Kit-8. For this study, we used RNA pull-down, RNA immunoprecipitation, and a dual-luciferase reporter experiment to identify the proteins that interacted with has_circ_0010220, IL-6, and miR-574-3p in OS cells that have developed resistance towards DOX. The results indicated upregulated Hsa_circ_0010220 and IL-6 expression, However, DOX-resistant OS tissues and cells showed a downregulation of miR-574-3p. Reducing DOX resistance in vitro was achieved by silencing Has_circ_0010220. Further, by sponging miR-574-3p, increasing has_circ_0010220 boosted DOX resistance. However, miR-574-3p bound to IL-6 and inhibited DOX resistance. Additionally, it was discovered that hsa_circ_0010220 sponged miR-574-3p for upregulating IL-6 expression. Hsa_circ_0010220 encouraged OS resistance to DOX by miR-574-3p/IL-6 axis regulation, suggesting its potency as a promising biomarker for treating OS.

Exosomal transfer of circ_0006174 contributes to the chemoresistance of doxorubicin in colorectal cancer by depending on the miR-1205/CCND2 axis.

Exosomes are the mediators of intercellular signal transduction, and they have been involved in the carcinogenesis and chemoresistance of tumor cells. Herein, we intended to investigate whether circular RNA (circRNA) circ_0006174 can regulate chemoresistance of doxorubicin (DOX) in colorectal cancer via exosomes. Forty-one pairs of normal and CRC (DOX sensitive, n = 16; DOX resistant, n = 25) samples were collected. The resistant cell lines (LoVo/DOX and HCT116/DOX) were constructed by exposure of parental cell lines (LoVo and HCT116) to DOX. The detection of circ_0006174, microRNA-1205 (miR-1205), and cyclin D2 (CCND2) was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8(CCK-8) was applied for determining the half of inhibitory concentration (IC50) of DOX and cell proliferation. The migration and invasion capacities were analyzed via transwell assay. Exosomes were extracted using ultracentrifugation. Protein levels were determined using western blot. Dual-luciferase reporter assay was used for affirming target interaction. In vivo experiment was performed by establishing xenograft models in mice. Circ_0006174 level was upregulated in DOX-resistant CRC tissues and cells. The downregulation of circ_0006174 inhibited DOX resistance, cell proliferation, migration, and invasion in DOX-resistant CRC cells. Interestingly, the abundant circ_0006174 was enriched in exosomes derived from DOX-resistant CRC cells. Furthermore, circ_0006174 could enhance DOX resistance via the exosomal intercellular transfer. Moreover, we validated the target relation of circ_0006174/miR-1205 or miR-1205/CCND2. The effect of exosomal circ_0006174 on DOX resistance was achieved by upregulating the miR-1205-mediated CCND2. In vivo, knockdown of circ_0006174 also enhanced tumor sensitivity to DOX by targeting miR-1205/CCND2 axis. Altogether, these findings unraveled that circ_0006174-enriched exosomes elevated DOX chemoresistance in CRC by the miR-1205/CCND2 axis. The exosomal circ_0006174 can be used as an available biomarker for the diagnosis of chemoresistance in CRC.

HMGB1 Promotes Resistance to Doxorubicin in Human Hepatocellular Carcinoma Cells by Inducing Autophagy via the AMPK/mTOR Signaling Pathway.

Chemoresistance remains as a major hindrance in the treatment of hepatocellular carcinoma (HCC). High mobility group box protein 1 (HMGB1) enhances autophagic flux and protects tumor cells from apoptosis, which results in acquired drug resistance. However, the exact mechanisms underlying HMGB1-modulated autophagy in HCC chemoresistance remain to be defined. In the present study, we found that administration of doxorubicin (DOX) significantly promoted HMGB1 expression and induced HMGB1 cytoplasmic translocation in human HCC cell lines BEL7402 and SMMC7721, which enhanced autophagy that contributes to protecting HCC cells from apoptosis and increasing drug resistance. Moreover, we observed HMGB1 translocation and elevation of autophagy in DOX-resistant BEL7402 and SMMC7721 cells. Additionally, inhibition of HMGB1 and autophagy increased the sensitivities of BEL-7402 and SMMC-7721 cells to DOX and re-sensitized their DOX-resistant cells. Subsequently, we confirmed with HMGB1 regulated autophagy by activating the 5ʹ adenosine monophosphate-activated protein kinase (AMPK)/mTOR pathway. In summary, our results indicate that HMGB1 promotes acquired DOX resistance in DOX-treated BEL7402 and SMMC7721 cells by enhancing autophagy through the AMPK/mTOR signaling pathway. These findings provide the proof-of-concept that HMGB1 inhibitors might be an important targeted treatment strategy for HCC.

TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

BackgroundChemoresistance is a critical risk problem for breast cancer treatment. However, mechanisms by which chemoresistance arises remains to be elucidated. The expression of T-box transcription factor 15 (TBX-15) was found downregulated in some cancer tissues. However, role and mechanism of TBX15 in breast cancer chemoresistance is unknown. Here we aimed to identify the effects and mechanisms of TBX15 in doxorubicin resistance in breast cancer.MethodsAs measures of Drug sensitivity analysis, MTT and IC50 assays were used in DOX-resistant breast cancer cells. ECAR and OCR assays were used to analyze the glycolysis level, while Immunoblotting and Immunofluorescence assays were used to analyze the autophagy levels in vitro. By using online prediction software, luciferase reporter assays, co-Immunoprecipitation, Western blotting analysis and experimental animals models, we further elucidated the mechanisms.ResultsWe found TBX15 expression levels were decreased in Doxorubicin (DOX)-resistant breast cancer cells. Overexpression of TBX15 reversed the DOX resistance by inducing microRNA-152 (miR-152) expression. We found that KIF2C levels were highly expressed in DOX-resistant breast cancer tissues and cells, and KIF2C was a potential target of miR-152. TBX15 and miR-152 overexpression suppressed autophagy and glycolysis in breast cancer cells, while KIF2C overexpression reversed the process. Overexpression of KIF2C increased DOX resistance in cancer cells. Furthermore, KIF2C directly binds with PKM2 for inducing the DOX resistance. KIF2C can prevent the ubiquitination of PKM2 and increase its protein stability. In addition, we further identified that Domain-2 of KIF2C played a major role in the binding with PKM2 and preventing PKM2 ubiquitination, which enhanced DOX resistance by promoting autophagy and glycolysis.ConclusionsOur data identify a new mechanism by which TBX15 abolishes DOX chemoresistance in breast cancer, and suggest that TBX15/miR-152/KIF2C axis is a novel signaling pathway for mediating DOX resistance in breast cancer through regulating PKM2 ubiquitination and decreasing PKM2 stability. This finding suggests new therapeutic target and/or novel strategy development for cancer treatment to overcome drug resistance in the future.

ERRα contributes to HDAC6-induced chemoresistance of osteosarcoma cells.

Chemotherapy resistance is an important problem for clinical therapy of osteosarcoma (OS). The potential effects of histone deacetylases (HDACs) on OS chemoresistance are studied. The expression of HDACs in OS cells resistance to doxorubicin (Dox) and cisplatin (CDDP) is checked. Among 11 members of HDACs, levels of HDAC6 are significantly upregulated in OS cells resistance to Dox and CDDP. Inhibition of HDAC6 via its specific inhibitor ACY1215 restores chemosensitivity of OS-resistant cells. Further, HDAC6 directly binds with estrogen-related receptors alpha (ERRα) to regulate its acetylation and protein stability. Inhibition of ERRα further strengthens ACY1215-increased chemosensitivity of OS-resistant cells. Mechanistically, K129 acetylation is the key residue for HDAC6-regulated protein levels of ERRα. Collectively, we find that ERRα contributes to HDAC6-induced chemoresistance of OS cells. Inhibition of HDAC6/ERRα axis might be a potential approach to overcome chemoresistance and improve therapy efficiency for OS treatment. 1. HDAC6 was significantly upregulated in Dox and CDDP resistant OS cells; 2. Inhibition of HDAC6 can restore chemosensitivity of OS cells; 3. HDAC6 binds with ERRα at K129 to decrease its acetylation and increase protein stability; 4. ERRα contributes to HDAC6-induced chemoresistance of OS cells.

Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C–S–S-Dox conjugate, where S–S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C–S–S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C–S–S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 μM after 72 h of incubation, which was comparable to Dox (5 μM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5–7%) and heart cells (H9C2, <9%) at concentrations of 5–10 μM (compared to free Dox at 5 μM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R5K]W7A-Dox (5 μM) in H9C2 cells. In comparison, Dox (5 μM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R5K]W7A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC50 of 2.3 and 4.3 μM, respectively) compared to free Dox (LC50 of 36–41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R5K]W7A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R5K]W7A-Dox.

Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells.

Since its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMPO.

A pore-forming protein-induced surface-enhanced Raman spectroscopic strategy for dynamic tracing of cell membrane repair

SummaryThe plasma membrane repair holds significance for maintaining cell survival and homeostasis. To achieve the sensitive visualization of membrane repair process for revealing its mechanism, this work designs a perforation-induced surface-enhanced Raman spectroscopy (SERS) strategy by conjugating Raman reporter (4-mercaptobenzoic acid) loaded gold nanostars with pore-forming protein streptolysin O (SLO) to induce the SERS signal on living cells. The SERS signal obviously decreases with the initiation of membrane repair and the degradation of SLO pores due to the departure of gold-nanostar-conjugated SLO. Thus, the designed strategy can dynamically visualize the complete cell membrane repair and provide a sensitive method to demonstrate the SLO endocytosis- and exocytosis-mediated repairing mechanism. Using DOX-resistant MCF-7 cells as a model, a timely repair-blocking technology for promoting the highly efficient treatment of drug-resistant cancer cells is also proposed. This work opens an avenue for probing the plasma membrane repairing mechanisms and designing the precision therapeutic schedule.