Accumulation Of Doxorubicin Research Articles

Potentiation of growth suppression and modulation of multidrug resistance by gamma and beta interferons in MDA-MB-231 breast cancer cell line.

Multi-drug resistance (MDR) might be acquired by the cancer cells during chemotherapy, and ATP-binding cassette (ABC) transporters play a significant role in MDR. Interferon-γ (IFN-γ) and IFN-β can inhibit cancer cell proliferation; however, the effects and mechanism of these cytokines on the growth and MDR are still unclear. To investigate the effects of IFN-γ and IFN-β, alone or in combination, on viability, resistance, and the expression of ABC transporters of the MDA-MB-231 breast cancer cell line. Using the MDA-MB-231 cell line, we assessed the effects of 20, 100, and 500 IU/ml of IFN-γ and IFN-β, alone or in combination, on cell viability by methyl thiazolyl tetrazolium(MTT) assay; and then we performed the Uptake and Efflux experiment to evaluate the effect of these IFNs on the cell resistance. Then, using quantitative real-time PCR, we evaluated changes in the expression of ABCB1, ABCC1, and ABCG2 mRNA levels. We discovered that IFN-γ and IFN-β might both reduce viability, either alone or in combination. The combination of IFNs also displayed synergistic responses, particularly when utilizing equivalent dosages of 500 or 100 IU/ml. The combination of IFN-γ and IFN-β resulted in a significant increase in Doxorubicin accumulation and down-regulation of the ABCC1 gene at the mRNA level. Our study suggested that equal doses of IFN-γ and IFN-β in combination might result in potentiated responses against cancer, especially, along with chemotherapy agents.

Reactive Oxygen Species-Regulated Conjugates Based on Poly(jasmine) Lactone for Simultaneous Delivery of Doxorubicin and Docetaxel.

In cancer therapy, it is essential to selectively release cytotoxic agents into the tumor to prevent the adverse effects associated with anticancer drugs. Thus, in this study, a stimuli-sensitive polymer-drug conjugate was synthesized for selective drug release. Doxorubicin (DOX) and docetaxel (DTX) were conjugated onto novel poly(jasmine lactone) based copolymer via a thioketal (TK) linker. In addition, a photosensitizer (chlorin e6) was attached to the polymer, which served as a reactive oxygen species generator to cleave the TK linker. The conjugate is readily self-assembled into micelles less than 100 nm in size. Micelles demonstrate a notable increase in their ability to cause cell death when exposed to near-infrared (NIR) light on MDA-MB-231 breast cancer cells. The increase in cytotoxicity is higher than that observed with the combination of free DOX and DTX. The accumulation of DOX in the nucleus after release from the micelles (laser irradiation) was also confirmed by confocal microscopy. In the absence of light, micelles did not show any toxicity while the free drugs were found toxic irrespective of the light exposure. The obtained results suggest the targeted drug delivery potential of micelles regulated by the external stimuli, i.e., NIR light.

Simultaneous therapeutic and diagnostic applications of magnetic PLGA nanoparticles loaded with doxorubicin in rabbit.

In this study, DOX (Doxorubicin) and Fe3O4 magnetic nanocrystals (SPIONs (Superparamagnetic iron oxide nanocrystals)) were encapsulated in the PLGA-PEG: poly(lactide-co-glycolide)-b-poly(ethylene glycol) nanoparticles for theranostic purposes. The final prepared formulation which is called NPs (Nanoparticles) exhibited a particle size with a mean diameter of ~ 209nm and a sufficient saturation magnetization value of 1.65 emu/g. The NPs showed faster DOX release at pH 5.5 compared to pH 7.4. Also, the cytotoxicity effect of NPs increased compared to Free-DOX alone in C6 glioma cancer cells. For in vivo investigations, the 2.2 Kg rabbits were injected with NPs formulations via a central articular anterior vein in their ears. Furthermore, the images of rabbit organs were depicted via MR (Magnetic resonance) and fluorescent imaging techniques. A negative contrast (dark signal) was observed in T2 (Relaxation Time) weighted MR images of IV (Intravenously)-injected rabbits with NPs compared to the control ones. The organ's florescent images of NPs-injected rabbits showed a high density of red color related to the accumulation of DOX in liver and kidney organs. These data showed that the NPs have no cytotoxicity effect on the heart. Also, the results of histopathological tests of different organs showed that the groups receiving NPs and Free-DOX were almost similar and no significant difference was seen, except for the cardiac tissue in which the pathological effects of NPs were significantly less than the Free-DOX. Additionally, pharmacokinetic studies were also conducted at the sera and whole bloods of IV-injected rabbits with NPs and Free-DOX. The pharmacokinetic parameters showed that NPs could enhance the DOX retention in the serum compared to the Free-DOX. Altogether, we aimed to produce a powerful delivery nanosystem for its potential in dual therapeutic and diagnostic applications which are called theranostic agents.

Enhanced chemotherapy response in hepatocellular carcinoma: synergistic effects of miR-122 and doxorubicin co-delivery system inducing apoptosis and DNA damage

BackgroundCancer cells can resist chemotherapy through various mechanisms, diminishing treatment outcomes. Research had indicated that combining miR-122 with doxorubicin (DOX) can improve hepatocellular carcinoma (HCC) therapy.ResultsTo explore this, we created a one-pot co-delivery system, Fe-miR-122/DOX, by coordinating miR-122, DOX, and FeII ions into nanoparticles. These nanoparticles display uniform particle sizes, well-defined morphology, and exceptional colloidal stability in 10% FBS and 20% FBS solution over 24 h. When the ratio of DOX to miR-122 was set at 20:1, the loading efficiency of both drugs reached 54.7% and 55.5%, respectively. Cell experiments confirmed that Fe-miR-122/DOX efficiently delivers both miR-122 and DOX, enabling cytoplasmic delivery through lysosomal escape, facilitated by the positive charge of the nanoparticles. Functionally, miR-122 increases intracellular accumulation of DOX by downregulating P-glycoprotein (P-gp) expression, and it promotes apoptosis by downregulating B-cell lymphoma 2 (Bcl-2), which leads to the upregulation of Caspase-3. Additionally, Fe-miR-122/DOX disrupts cIAPs-mediated anti-apoptotic signals, downregulates PARP-1 expression, hinders DNA repair, promotes DNA fragmentation, enhances caspase-3 expression, and triggers programmed cell death, synergistically enhancing its antitumor efficacy. This synergistic mechanism disrupts DNA repair, amplifying DNA damage and apoptosis. Our cytotoxicity and apoptosis assays (with a HepG2 cell apoptosis rate of 85.98%) demonstrated the potent antitumor capability of Fe-miR-122/DOX.ConclusionsThis innovative system has demonstrated good biocompatibility and has the potential to transform HCC therapy. Future research could focus on optimizing the co-delivery system and assessing its efficacy in clinical trials.

Saikosaponin D potentiates the antineoplastic effects of doxorubicin in drug-resistant breast cancer through perturbing NQO1-mediated intracellular redox balance

BackgroundDrug resistance to doxorubicin (DOX) significantly limits its therapeutic efficacy in breast cancer (BC) patients. Saikosaponin D (SSD), a triterpene saponin derived from the traditional herb Radix Bupleuri, has shown promise as a chemotherapeutic sensitizer in preclinical studies due to its notable antitumor activity. However, the role and mechanism of SSD in DOX-resistant BC cells remain largely unexplored. PurposeThis study aimed to investigate the chemosensitizing effect of SSD on DOX-resistant BC and the underlying molecular mechanisms both in vitro and in vivo. MethodsIn vitro assays, including cell viability, clone formation, three-dimensional tumor spheroid growth, and apoptosis analysis, were conducted to evaluate the synergistic effect of SSD and DOX on resistant BC cells. Reactive oxygen species (ROS), GSH/GSSG, NADPH/NADP+, and NADH/NAD+ detections were employed to assess the impact of SSD on cellular redox homeostasis. Western blotting, cell cycle distribution assay, and DOX uptake assay were performed to further elucidate the possible antineoplastic mechanism of SSD. Finally, a subcutaneous MCF7/DOX cell xenografted model in nude mice was established to identify the in vivo anticarcinogenic effect of SSD combined with DOX. ResultsSSD significantly inhibited cell viability, proliferation, and clone formation, enhancing DOX's anticancer efficacy in vitro and in vivo. Mechanistically, SSD reduced STAT1, NQO1, and PGC-1α protein levels, leading to cellular redox imbalance, excessive ROS generation, and depletion of GSH, NADPH, and NADH. SSD induced DNA damage by disrupting redox homeostasis, resulting in G0/G1 phase cell cycle arrest. Additionally, SSD increased DOX accumulation in BC cells via inhibiting P-gp protein expression and efflux activity. ConclusionWe demonstrated for the first time that SSD enhances the sensitivity of chemoresistant BC cells to DOX by disrupting cellular redox homeostasis through inactivation of the STAT1/NQO1/PGC-1α signaling pathway. This study provides evidence for SSD as an adjuvant agent in drug-resistant BC treatment.

Abstract Tu140: Evaluation of the effects of mitoquinone on doxorubicin-induced acute cardiac damage

Background: Doxorubicin (DOX)-induced cardiotoxicity remains a high priority to overcome due to irreversibility and lack of abundant preventions. DOX damages the heart tissue principally by overproduction of mitochondrial superoxide and intercalation with the topoisomerase II in the nucleus. Ultimately, cardiac cells become apoptotic, and heart function declines. Our lab has shown that Mitoquinone (MQ), a mitochondria-targeted antioxidant, improves H9c2 cell viability in the presence of DOX. We hypothesize that MQ would counteract DOX to preserve cardiac cells and function in isolated rat hearts, possibly by reducing mitochondrial superoxide production and intracellular accumulation. Methods: H9c2 cells (passages < 18) were incubated with DOX (40 µM) with or without MQ (0.005 μM - 5 μM) for 24 hours before measuring mitochondrial superoxide anion levels and DOX intracellular accumulation. Furthermore, heart rate (HR) and left ventricular end-systolic pressure (LVESP) were recorded before and after a 60-minute infusion of DOX or DOX with MQ in isolated rat hearts. TUNEL staining was performed on heart tissue slides to assess apoptosis. All the data was expressed as a ratio between treatment and DOX alone or between final to initial values. Statistical significance was evaluated using ANOVA with Student Newman Keuls post-hoc analysis. Results: Hearts perfused with DOX (25 µM, n = 5) displayed lower final LVESP with 39 ± 5% of the initial baseline. By contrast, hearts treated with 0.25 µM MQ (n = 4) or 1 µM MQ (n = 5) manifested exhibited a higher final LVESP to 44 ± 8% and 90 ± 6% of initial baselines, respectively. Additionally, MQ administration significantly reduced cardiac cell apoptosis to 8 ± 1% (0.25 µM, n = 2) and 6 ± 1% (1 µM, n = 5, p < 0.05) when compared to DOX (88 ± 2%, n = 4). In contrast to DOX, MQ (1 µM, n = 4) showed 27 ± 9% (n = 4) reduction in mitochondrial superoxide levels and 54 ± 6% (n = 6, p < 0.05) less intracellular DOX deposition in H9c2 cells. Conclusion: This study suggests acute DOX administration to the heart severely compromises cardiac systolic function with higher induction of cell apoptosis. MQ exerts cardio-protection with higher systolic function and cell viability by attenuating mitochondrial superoxide levels and intracellular DOX accumulation.

Kupffer cells determine intrahepatic traffic of PEGylated liposomal doxorubicin

Intrahepatic accumulation dominates organ distribution for most nanomedicines. However, obscure intrahepatic fate largely hampers regulation on their in vivo performance. Herein, PEGylated liposomal doxorubicin is exploited to clarify the intrahepatic fate of both liposomes and the payload in male mice. Kupffer cells initiate and dominate intrahepatic capture of liposomal doxorubicin, following to deliver released doxorubicin to hepatocytes with zonated distribution along the lobule porto-central axis. Increasing Kupffer cells capture promotes doxorubicin accumulation in hepatocytes, revealing the Kupffer cells capture-payload release-hepatocytes accumulation scheme. In contrast, free doxorubicin is overlooked by Kupffer cells, instead quickly distributing into hepatocytes by directly crossing fenestrated liver sinusoid endothelium. Compared to free doxorubicin, liposomal doxorubicin exhibits sustained metabolism/excretion due to the extra capture-release process. This work unveils the pivotal role of Kupffer cells in intrahepatic traffic of PEGylated liposomal therapeutics, and quantitively describes the intrahepatic transport/distribution/elimination process, providing crucial information for guiding further development of nanomedicines.

The Role of PTEN in Chemoresistance Mediated by the HIF-1α/YY1 Axis in Pediatric Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Current chemotherapy treatment regimens have improved survival rates to approximately 80%; however, resistance development remains the primary cause of treatment failure, affecting around 20% of cases. Some studies indicate that loss of the phosphatase and tensin homolog (PTEN) leads to deregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, increasing the expression of proteins involved in chemoresistance. PTEN loss results in deregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces hypoxia-inducible factor 1-alpha (HIF-1α) expression in various cancers. Additionally, it triggers upregulation of the Yin Yang 1 (YY1) transcription factor, leading to chemoresistance mediated by glycoprotein p-170 (Gp-170). The aim of this study was to investigate the role of the PTEN/NF-κB axis in YY1 regulation via HIF-1α and its involvement in ALL. A PTEN inhibitor was administered in RS4;11 cells, followed by the evaluation of PTEN, NF-κB, HIF-1α, YY1, and Gp-170 expression, along with chemoresistance assessment. PTEN, HIF-1α, and YY1 expression levels were assessed in the peripheral blood mononuclear cells (PBMC) from pediatric ALL patients. The results reveal that the inhibition of PTEN activity significantly increases the expression of pAkt and NF-κB, which is consistent with the increase in the expression of HIF-1α and YY1 in RS4;11 cells. In turn, this inhibition increases the expression of the glycoprotein Gp-170, affecting doxorubicin accumulation in the cells treated with the inhibitor. Samples from pediatric ALL patients exhibit PTEN expression and higher HIF-1α and YY1 expression compared to controls. PTEN/Akt/NF-κB axis plays a critical role in the regulation of YY1 through HIF-1α, and this mechanism contributes to Gp-170-mediated chemoresistance in pediatric ALL.

Dual Inhibitors of P-gp and Carbonic Anhydrase XII (hCA XII) against Tumor Multidrug Resistance with Piperazine Scaffold.

A new series of piperazine derivatives were synthesized and studied with the aim of obtaining dual inhibitors of P-glycoprotein (P-gp) and carbonic anhydrase XII (hCA XII) to synergistically overcome the P-gp-mediated multidrug resistance (MDR) in cancer cells expressing the two proteins, P-gp and hCA XII. Indeed, these hybrid compounds contain both P-gp and hCA XII binding groups on the two nitrogen atoms of the heterocyclic ring. All compounds showed good inhibitory activity on each protein (P-gp and hCA XII) studied individually, and many of them showed a synergistic effect in the resistant HT29/DOX and A549/DOX cell lines which overexpress both the target proteins. In particular, compound 33 displayed the best activity by enhancing the cytotoxicity and intracellular accumulation of doxorubicin in HT29/DOX and A549/DOX cells, thus resulting as promising P-gp-mediated MDR reverser with a synergistic mechanism. Furthermore, compounds 13, 27 and 32 induced collateral sensitivity (CS) in MDR cells, as they were more cytotoxic in resistant cells than in the sensitive ones; their CS mechanisms were extensively investigated.

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.

Brain-targeting biomimetic disguised manganese dioxide nanoparticles via hybridization of tumor cell membrane and bacteria vesicles for synergistic chemotherapy/chemodynamic therapy of glioma

Glioma is a prevalent brain malignancy associated with poor prognosis. Although chemotherapy serves as the primary treatment for brain tumors, its effectiveness is hindered by the limited ability of drugs to traverse the blood–brain barrier (BBB) and the development of drug resistance linked to tumor hypoxia. Herein, we report the creation of hybrid camouflaged multifunctional nanovesicles comprising membranes of tumor C6 cells (mT) and bacterial outer membrane vesicles (OMVs) and co-loaded with manganese dioxide nanoparticles (MnO2 NPs) and doxorubicin (DOX) to synergistically enhance the chemotherapy/chemodynamic therapy (CDT) of glioma. Owing to OMV-mediated BBB penetration and mT-inherited tumor-homing properties, MnO2-DOX@mT/OMVs can penetrate the BBB and enhance the tumor cell-specific uptake of DOX via “proton sponge effect”-mediated lysosomal escape. This enhances the apoptotic effect induced by DOX and minimizing DOX-associated cardiotoxicity by facilitating the accumulation of DOX at the tumor site. Furthermore, the MnO2 NPs in MnO2-DOX@mT/OMVs can generate potent CDT by accelerating the Fenton-like reaction with DOX-generated H2O2 and achieving glutathione (GSH)-depletion-induced glutathione peroxidase 4 (GPX4) inactivation. These results showed that MnO2-DOX@mT/OMVs, designed for brain tumor targeting, significantly inhibited tumor growth and exhibited favorable biological safety. This innovative approach offers the augmentation of anticancer treatment efficacy via a potential combination of chemotherapy and CDT.

Enhanced Cellular Doxorubicin Uptake via Delayed Exposure Following Nanosecond Pulsed Electric Field Treatment: An In Vitro Study.

The combination of nanosecond Pulsed Electric Field (nsPEF) with pharmaceuticals is a pioneering therapeutic method capable of enhancing drug uptake efficacy in cells. Utilizing nsPEFs configured at 400 pulses, an electric field strength of 15 kV/cm, a pulse duration of 100 ns, and a repetition rate of 10 pulses per second (PPS), we combined the nsPEF with a low dose of doxorubicin (DOX) at 0.5 μM. Upon verifying that cells could continuously internalize DOX from the surrounding medium within 1 h post nsPEF exposure, we set the DOX exposure period to 10 min and contrasted the outcomes of varying sequences of DOX and nsPEF administration: pulsing followed by DOX, DOX followed by pulsing, and DOX applied 40 min after pulsing. Flow cytometry, CCK-8 assays, and transmission electron microscopy (TEM) were employed to examine intracellular DOX accumulation, cell viability, apoptosis, cell cycle, and ultrastructural transformations. Our findings demonstrate that exposing cells to DOX 40 min subsequent to nsPEF treatment can effectively elevate intracellular DOX levels, decrease cell viability, and inhibit the cell cycle. This research work presents a novel approach to enhance DOX uptake efficiency with moderate conditions of both DOX and nsPEF.

RNA editing enzyme ADAR2 regulates P-glycoprotein expression in murine breast cancer cells through the circRNA-miRNA pathway

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.

Implications of chronic moderate protein-deficiency malnutrition on doxorubicin pharmacokinetics and cardiotoxicity in early post-weaning stage

BackgroundMalnutrition is a common problem in developing countries, and the impact of severe malnutrition on optimal treatment outcomes of chemotherapy in pediatric cancer patients is well documented. However, despite being a more prevalent and distinct entity, moderate malnutrition is until now unexplored for its effects on treatment outcomes. AimsIn this study we aimed to investigate the molecular basis of altered pharmacokinetics and cardiotoxicity of doxorubicin observed in early-life chronic moderate protein deficiency malnutrition. Materials and methodsWe developed an animal model of early-life moderate protein-deficiency malnutrition and validated it using clinical samples. This model was used to study pharmacokinetic and toxicity changes and was further utilized to study the molecular changes in liver and heart to get mechanistic insights. Key findingsHere we show that moderate protein-deficiency malnutrition in weanling rats causes changes in drug disposition in the liver by modification of hepatic ABCC3 and MRP2 transporters through the TNFα signalling axis. Furthermore, malnourished rats in repeat-dose doxorubicin toxicity study showed higher toxicity and mortality. A higher accumulation of doxorubicin in the heart was observed which was associated with alterations in cardiac metabolic pathways and increased cardiotoxicity. SignificanceOur findings indicate that moderate malnutrition causes increased susceptibility towards toxic side effects of chemotherapy. These results may necessitate further investigations and new guidelines on the dosing of chemotherapy in moderately malnourished pediatric cancer patients.

Differential synergistic effects of palbociclib and doxorubicin on doxorubicin-resistant cancer cells with diverse tumor origins

Palbociclib is a dual inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). Palbociclib has frequently been studied in breast cancer cells and has also been linked to function of P-glycoprotein (P-gp), main protein responsible for cancer drug resistance. However, the effect of Palbociclib on cancer drug resistance and specifically doxorubicin-resistant cells overexpressing P-gp have limitedly been studied in the literature. Here, we aimed to decipher the possible synergistic effects of Palbociclib and Doxorubicin combination treatment in doxorubicin-resistant not only breast cancer, which has restrictedly been studied previously, but leukemia and cervical cancer cell lines in the presence of sensitive counterparts to totally explore the mechanistic properties of the Palbociclib in cancer drug resistance. Our results underlined that Palbociclib differentially displayed synergistic effect with doxorubicin in a cell type-specific manner and increased the efficacy of Doxorubicin in Doxorubicin-resistant cells. As a monotherapy, palbociclib has been shown to decrease the expression of MDR-1 in doxorubicin-resistant cells, and when used in combination with doxorubicin, it has been shown to increase the accumulation of doxorubicin in the cell and consequently induce apoptosis. This is the first report that proposes the Palbociclib as a candidate for combination therapy to limit the Doxorubicin resistance in different cancer origins in clinics.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Phenylspirodrimane with Moderate Reversal Effect of Multidrug Resistance Isolated from the Deep-Sea Fungus Stachybotrys sp. 3A00409.

Two new phenylspirodrimanes, stachybotrins K and L (1 and 2), together with eight known analogues (3-10), were isolated from deep-sea-derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Absolute configurations of new compounds were determined through a comparison of their circular dichroism (CD) spectra with other reported compounds. The possible reversal effects of all compounds were assayed in the resistant cancer cell lines. Stachybotrysin B (8) can reverse multidrug resistance (MDR) in ABCB1-overexpression cells (KBv200, Hela/VCR) at the non-cytotoxic concentration. Doxorubicin accumulation assay and molecular-docking analysis reveal that the mechanism of its reversal MDR effect may be related to the increase in the intracellular concentration of substrate anticancer drugs.

PEGylated graphene oxide-mediated stimulation of vascular endothelial cells and responsive release of PD-1/PD-L1 inhibitor for efficient chemo-immunotherapy against cancer

Immune checkpoint blockade (ICB) has emerged as a promising immunotherapeutic modality against cancer in the clinic. However, only 10–30% of patients respond to ICB, primarily due to poor immunogenicity and insufficient T cell infiltration in solid tumors. Herein, we presented an approach for high-performance cancer treatment using the programmed cell death protein-1 and programmed cell death ligand-1 (PD-1/PD-L1) inhibitor (BMS-202)-loaded PEGylated graphene oxide (GPi). On the one hand, GPi dissociated tight junctions of vascular endothelial cells (VECs) in tumor, thus promoting the extravasation and intratumoral accumulation of liposomal doxorubicin (LipDox), which then effectively induced immunogenic cell death of tumor cells. On the other hand, GPi also stimulated VECs to upregulate the expression of cell-cell interaction molecules, such as intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1, which facilitated the infiltration of T cells in tumor. Beyond acting as a stimulator of VECs, GPi could exert responsive release of BMS-202 under the acidic tumor microenvironment and blockade PD-1/PD-L1 axis in tumors. Finally, the alternating administration of GPi and LipDox effectively inhibited tumor growth in a 4T1 tumor model, providing a novel treatment mode for chemo-immunotherapy.

Abstract 4762: Tumor treating fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin in vitro and in vivo

Abstract Background: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. While the mechanism of action of TTFields has been associated with an antimitotic effect and impaired DNA damage repair mechanisms, the application of TTFields has also been shown to increase the membrane permeability of glioblastoma cells. The current study examined whether increased cell membrane permeability is relevant in other tumor types and may be leveraged to facilitate uptake of the anticancer agent doxorubicin (DOX). Methods: Breast mammary carcinoma (4T1), breast adenocarcinoma (MCF-7), uterine sarcoma (MES-SA), lung fibroblasts (MRC-5), and brain endothelial (HBMVEC) cells were treated with TTFields using the inovitroTM system. Intracellular 7-aminoactinomycin D (7-AAD) accumulation and cell count were measured using flow cytometry to determine membrane permeability and cytotoxicity, respectively. TTFields (300 kHz) together with DOX were applied to 4T1 cells DOX-sensitive (4T1-S) and DOX-resistant (4T1-R, generated by repeated DOX exposure of the 4T1-S cells), followed by flow cytometry analysis of DOX accumulation (24 h treatment) and cell survival using cell count (72 h treatment). For proof of concept, mice were orthotopically inoculated with 4T1-S cells, followed by TTFields treatment for 72 h and injection of DOX (5 mg/kg) 24 h before ending TTFields treatment. DOX fluorescence was measured by flow cytometry in single-cell tumor suspension and whole tumor in vivo imaging system (IVIS). For the efficacy studies, mice were orthotopically inoculated with 4T1-S or 4T1-R cells. Tumors were allowed to grow, and then the mice were treated with TTFields (300 kHz) or sham-heat continuously for 8 days, with DOX (1 mg/kg for 4T1-S cells; 5 mg/kg for 4T1-R cells) or vehicle 2- and 6-days following treatment initiation. Tumor volumes were measured using caliper. Results: TTFields increased 7-AAD accumulation in cancer but not normal cells. Maximal TTFields-induced cellular permeability was identified at 300 kHz for 4T1 cells, whereas TTFields-induced cytotoxicity was the highest at 150 kHz. The application of TTFields allowed DOX accumulation to the same extent in both DOX-resistant and DOX-sensitive cells and sensitized both cell types to DOX cytotoxicity. A 2- to 3-fold higher DOX accumulation was observed in tumors isolated from mice treated with TTFields relative to control mice. Significantly lower tumor volume was observed in mice treated with TTFields and DOX relative to control or mice treated with DOX alone. Conclusions: TTFields increased cancer cell permeability, resulting in enhanced intracellular accumulation of DOX. Concomitant treatment with TTFields and DOX can improve treatment efficacy in DOX-resistant tumors and sensitize DOX-sensitive tumors to lower DOX concentrations. Citation Format: Bella Koltun, Tali Voloshin, Tal Kan, Cfir David, Lilach Koren, Yaara Porat, Alexandra Volodin, Noa Kaynan, Anat Klein-Goldberg, Rom Paz, Boris Brant, Yiftah Barsheshet, Efrat Zemer-Tov, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin in vitro and in vivo [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 4762.

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.