Accumulation Of Doxorubicin In Cells Research Articles

ZIF-8-mediated block copolymer micelles and its application in drug delivery

Block copolymer micelles have been widely used in drug delivery. Great efforts have been made to optimize the function of block copolymer micelles and further enhance their bioavailability. We propose a means of embedding ZIF-8 in block copolymer micelles to regulate its stability for improving the drug release kinetics. The block copolymer poly (2-(diisopropyl amino-ethyl methacrylate)-block-poly (vinylimidazole-co-poly (ethylene glycol) methyl methacrylate) (PDPA18-b-P(Vim18-co-P(EG)5MA21) (PDPV) bearing imidazole moiety was synthesized through reversible addition-cracking chain transfer (RAFT) polymerization. PDPV can self-assemble into micelles in aqueous solution and then be loaded with doxorubicin (DOX) to obtain DOX@PDPV, followed by Zn coordination to anchor ZIF-8 to the PVim moiety in DOX@PDPV to obtain the final drug delivery system DOX@PDPV@ZIF-8 hybrids. The DOX@PDPV@ZIF-8 hybrids can avoid the influence of the critical micelle concentration (CMC) on it, which was further prevent the premature leakage of DOX under physiological conditions. Furthermore, the pH-responsive properties of PDPA and ZIF-8 facilitated enabled the effective release of DOX in weakly acidic tumor environments. In vitro studies also confirmed that DOX@PDPV@ZIF-8 improved DOX accumulation in cells, leading to the inhibition of cancer cell growth. Therefore, the integrated design provided a promising strategy to regulate the stability of block copolymer micelles, improve drug release kinetics, and ultimately inhibit the growth of cancer cell.

Abstract 4556: USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells

Abstract Background: Resistance to chemotherapy remains an obstacle for triple-negative breast cancer (TNBC) patients. The core components of the ubiquitin-proteasome system have been demonstrated to regulate chemoresistance of TNBC. As a novel deubiquitinase (DUB), ubiquitin-specific peptidase 51 (USP51) plays a pivotal role in chemotherapeutic resistance in multiple malignancies. Herein, we sought to better understand how USP51 performs a cell-intrinsic role in mediating chemotherapeutic resistance in TNBC. Methods: Western blotting, RNA-sequencing and CCK8 assays were used to identify the DUBs for chemoresistance of breast cancer. Coimmunoprecipitation, GST-pulldown, protein deubiquitination and immunohistochemistry assays were then used to determine the biological phenotypes of ectopic USP51 in TNBC chemoresistance and associated signaling pathways in several different cell lines, mouse models and patient tissue samples. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC)-based deubiquitinase activity, cellular thermal shift and surface plasmon resonance (SPR) analyses were performed to investigate the activity of USP51 inhibitors. Results: To identify the critical DUBs involved in breast cancer chemoresistance, we established doxorubicin-resistant Cal51 and MDA-MB-231 TNBC cell lines by multiple dose exposure. We found ectopic USP51 in doxorubicin-resistant cells compared with their parental cells. Moreover, USP51-interfered chemoresistant tumor cells exhibited impaired cell viability as well as increased DNA damage and cell apoptosis upon treatment with doxorubicin. On the contrary, overexpression of USP51 performed the opposite effects to enhance cell viability but decrease DNA damage and apoptosis in response to doxorubicin; however, these outcomes were not shown for its catalytically inactive mutant USP51C372S. At the molecular level, GRP78 was identified as a bona fide substrate of USP51. Importantly, knockdown USP51 increased doxorubicin-induced DNA damage and apoptosis, which was rescued by overexpression of GRP78 in vitro and in vivo. In addition, the activity of ABCB1, the main efflux pump of doxorubicin, was enhanced by GRP78. Consistently, the expression of USP51, GRP78 and ABCB1 were correlated with chemoresistant phenotypes in TNBC patients. Of note, we also explored a small molecular inhibitor of USP51, WCY-4-1, which conferred chemosensitization in TNBC. Conclusion: These findings collectively indicated that ectopic USP51 enhances the activity of ABCB1 by deubiquitinating and stabilizing GRP78, which leads to decreased accumulation of doxorubicin as well as decreased DNA damage and cell apoptosis in TNBC. Our results also revealed that specific inhibition of USP51 significantly impairs doxorubicin-resistance in TNBC. Citation Format: Yang Ou, Kun Zhang, Qiuying Shuai, Chenyang Wang, Huayu Hu, Lixia Cao, Chunchun Qi, Min Guo, Zhaoxian Li, Jie Shi, Yuxin Liu, Siyu Zuo, Xiao Chen, Yanjing Wang, Mengdan Feng, Hang Wang, Yi Shi, Guang Yang, Shuang Yang. USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4556.

Exploring doxorubicin transport in 2D and 3D models of MDA-MB-231 sublines: impact of hypoxia and cellular heterogeneity on doxorubicin accumulation in cells.

Triple-negative breast cancer (TNBC) treatment is challenging due to its aggressive nature and heterogeneity of this type of cancer, characterized by various subtypes and intratumoral diversity. Doxorubicin (DOX) plays a crucial role in TNBC chemotherapy reducing the tumor size and improving patient survival. However, decreased drug uptake and increased resistance in specific cell subpopulations reduce the effectiveness of the treatment. This study explored the differences in DOX transport in MDA-MB-231 phenotypic sublines in cell monolayer (2D model) and cell spheroids (3D cultures). Cell spheroids were formed using magnetic 3D Bioprinting method. DOX transport into cells and spheroids was evaluated using fluorescence microscopy after different incubation durations with DOX in normoxia and hypoxia. In hypoxia, DOX transport into cells was 2.5 to 5-fold lower than in normoxia. The subline F5 monolayer-cultured cells exhibited the highest DOX uptake, while subline H2 cells showed the lowest uptake in normoxia and hypoxia. In 3D cultures, DOX transport was up to 2-fold lower in spheroids formed from subline H2 cells. Spheroids from subline D8 and MDA-MB-231 parent cells had the highest DOX uptake. A correlation was observed between the characteristics of the cells and their resistance to anticancer drugs. The results indicate that different cancer cell subpopulations in tumours due to differences in drug uptake could significantly impact treatment efficacy.

Gamma-tocotrienol reverses multidrug resistance of breast cancer cells with a mechanism distinct from that of atorvastatin

In addition to its antioxidant properties, γ-tocotrienol also has the ability to inhibit HMG-CoA reductase, which is the key enzyme in the mevalonate pathway for cholesterol biosynthesis. Statins, the competitive inhibitors of HMG-CoA reductase, display potent anticancer activity and reversal ability of multidrug resistance in a variety of tumor cells, which is believed to be due to their inhibition of HMG-CoA reductase. Here, we determined the role of the mevalonate pathway in γ-tocotrienol-mediated reversal of multidrug resistance in cancer cells. We found both γ-tocotrienol and atorvastatin effectively reversed multidrug resistance of MCF-7/Adr and markedly inhibited the intracellular levels of FPP and GGPP. Exogenous addition of mevalonate or FPP and GGPP almost completely prevented the reversal ability of atorvastatin but only partly attenuated the reversal effect of γ-tocotrienol on doxorubicin resistance. In addition, γ-tocotrienol actively inhibited the expression of P-gp and increased the accumulation of doxorubicin in cells, which led to the enhanced G2/M arrest and cell apoptosis. Taken together, γ-tocotrienol reversed the multidrug resistance of MCF-7/Adr with a mechanism distinct from that of atorvastatin. Instead of the mevalonate pathway, the inhibition of P-gp expression is a potential mechanism by which γ-tocotrienol reverses multidrug resistance in MCF-7/Adr.

Theranostic, pH-Responsive, Doxorubicin-Loaded Nanoparticles Inducing Active Targeting and Apoptosis for Advanced Gastric Cancer.

This study developed a kind of magnetic-polymer nanocarrier with folate receptor-targeting and pH-sensitive multifunctionalities to carry doxorubicin (DOX) for treatment of advanced gastric cancer (AGC). Folate-conjugated, pH-sensitive, amphiphilic poly(β-aminoester) self-assembled with hydrophobic oleic acid-modified iron oxide nanoparticles, and the resulting hydrophobic interaction area is a reservoir for lipophilic DOX (F-P-DOX). Confocal microscopy illustrated that F-P-DOX treatment could keep higher DOX accumulation in cells than P-DOX (without folate conjugation), and therefore get a higher efficiency of DOX internalization at pH 6.5 than at pH 7.4. Electron microscope characterization and real-time polymerase chain reaction revealed cell apoptosis promoted by F-P-DOX. The better efficacy of F-P-DOX on GC than free DOX and P-DOX was determined by MTT assay and xenograft model. Moreover, the accumulation of F-P-DOX in the tumor site was detected by magnetic resonance imaging (MRI). All those observations suggest F-P-DOX could be a promising theranostic candidate for AGC treatment.

Characterization of dihydroartemisinin-resistant colon carcinoma HCT116/R cell line

Dihydroartemisinin (DHA) is an important artemisinin derivative and presents profound anti-tumor potential. A DHA-resistant cell line named HCT116/R derived from colon carcinoma cell line HCT116 was established in our previous study. Herein, we found that HCT116/R cells were much more resistant to DHA- or artesunate-induced proliferation inhibition and more tolerant to DHA-induced cell cycle arrest and apoptosis compared with those of the parent HCT116 cells. The protein levels of P-glycoprotein and MDR-associated protein 1 and the accumulation of doxorubicin in cells were similar in both cell lines. Moreover, HCT116/R cells were still sensitive to camptothecin- and doxorubicin-induced cell growth inhibition. To further explore the characterization of HCT116/R cell line, a proteomic study employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Eight different expressed proteins between the two cell lines were identified including some heat shock proteins, annexins, etc. This study not only indicates that exposure to DHA may not induce a tumor multi-drug-resistant phenotype but also affords new clues for the further investigation of the anti-cancer mechanisms of DHA and other artemisinin derivatives.

Ionophoric activity of pluronic block copolymers.

Pluronic block copolymers (triblock copolymers of poly(ethylene oxide) and poly(propylene oxide)) exhibit a chemosensitizing effect on multidrug resistant cell lines. Changes in membrane permeability are hypothesized to be responsible because inhibition of drug transport mediated by both the multidrug-resistance-associated protein and the P-glycoprotein drug efflux system has been observed. To test this hypothesis, we now have studied the ion conductivity mediated by Pluronic L61. Besides a detergent-like action, the copolymer was able to form regular channels and to exhibit carrier activity. Long living ion channels were formed by polymer oligomerization. Aggregate equilibrium was shifted toward L61 monomers and dimers, which operated as mobile carriers. Copolymer-induced membrane permeability for potassium ions (1 M KCl) was less than 10(-8) cm s(-1), whereas the permeability for uncharged doxorubicin molecules (1 mM) was equal to 5 x 10(-4) cm s(-1). The results are consistent with reports about an increased doxorubicin accumulation in cells (Venne, Li, S., Mandeville, R., Kabanov, A., and Alakhov, V. Y. (1996) Cancer Res. 56, 3626-3629). However, the increased permeability contrasts with the polymer-mediated decrease of drug efflux from cells. Preferential polymer binding to membrane proteins may mask the unspecific effect of L61 observed on lipid bilayers.

Ketotifen reverses MDR1-mediated multidrug resistance in human breast cancer cells in vitro and alleviates cardiotoxicity induced by doxorubicin in vivo.

To investigate the effect of the antihistamine ketotifen on multidrug resistance in human breast cancer cells and doxorubicin toxicity in mice. Clonogenicity assays were used to test the effect of ketotifen on human multidrug resistant breast cancer cell lines exposed to chemotherapeutic agents. Flow cytometry was used to measure accumulation of doxorubicin in cells. Fluorimetry was used to measure accumulation of doxorubicin in cardiac tissues. Histological analysis and toxicity studies in mice were used to test the effect of ketotifen on doxorubicin-induced toxicity. Ketotifen was found to restore the sensitivity of P-glycoprotein-overexpressing multidrug-resistant MCF-7/adr cells to doxorubicin, mitoxantrone, VP-16 and vinblastine, but not to methotrexate or camptothecin. Ketotifen, however, was unable to restore sensitivity of BCRP-overexpressing MCF-7/mx cells or MRP-overexpressing MCF-7/vp cells to mitoxantrone or VP-16, respectively. In vivo, pretreatment of mice with ketotifen caused an increased accumulation of doxorubicin in cardiac tissue, consistent with a block in drug clearance. However, unlike verapamil, ketotifen pretreatment did not enhance doxorubicin toxicity but in fact provided protection, both at the level of cardiac tissue damage and in terms of survival. Taken together, these observations show that ketotifen is unique in its ability both to reverse multidrug resistance due to P-glycoprotein overexpression and to provide cardioprotection to doxorubicin.

Flow cytometric analysis of doxorubicin accumulation in cells from human and rodent cell lines.

The correlation between intracellular accumulation of doxorubicin (DOX) and drug cytotoxicity was studied in cells from seven cell lines of rodent and human origin. We used three Chinese hamster ovary cell lines (AuxB1, CHRC5, and UV20), two murine tumor cell lines (KHT-LP1 and EMT6/Ro), and two human tumor cell lines (MGH-U1 and DLD-1). Intracellular DOX was measured by its fluorescence intensity with flow cytometry, and drug cytotoxicity was quantified with clonogenic assays. When data for all of the cell lines were combined, cell killing was correlated with the intracellular concentration of DOX (r = -.88). For the cell line AuxB1 and its DOX-resistant subline CHRC5, in which cells express high levels of P-glycoprotein, the relationship between cell survival and the intracellular concentration of DOX was stronger than that for the other cell lines. These results suggest that differences in intracellular accumulation of DOX account for much of the heterogeneity in response to the drug in cells from different cell lines, although additional mechanisms also contribute to variation in drug sensitivity. Flow cytometric analysis of intracellular DOX fluorescence is a simple assay that should be tested in cells from human tumors as a possible predictor of tumor response. For a given cell line, this technique also provides a rapid way to monitor the development of drug resistance after multiple courses of chemotherapy.