Multidrug-resistant Cells Research Articles

Cannabis sativa mediated palladium nanoparticles as an effective nanodrug against multi-drug resistant bacteria and A549 lung cancer cells

The present paper reports a biogenic synthesis of palladium nanoparticles (PdNPs) involving Cannabis sativa leaf extract. The physicochemical characterisations of biogenic synthesised PdNPs were carried out to determine size, shape, morphology, atomic composition, and crystal structure via different techniques involving Ultraviolet–visible (UV–vis), Fourier transform infrared (FT-IR), Field emission scanning electron microscopy (FE-SEM), Transmission electron microscopy (TEM), and X-ray diffraction (XRD). The standard particle size of the synthesized PdNPs was found to be 3.49 ± 0.5 nm from the TEM analysis, while XRD analysis showed a crystal size of 4.6 nm with a face-centred-cubic (fcc) structure. FT-IR study of the green synthesis confirmed the presence of phytochemicals which act as reducing and stabilising agents. The synthesised biogenic PdNPs were subjected to antimicrobial analysis for isolated strains of multi-drug resistant (MDR) Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Salmonella abony where minimum inhibitory concentration (MIC) was found in the range of 52.04–68.3 μg/mL. Moreover, for the cytotoxic effects of biogenic PdNPs, A549 lung cell lines were used, and the half-maximal inhibitory concentration (IC50) value was evaluated as 22.17 µg/mL. The present investigation suggests the possible role of C. sativa extract capped PdNPs in the clinical management of lung cancer cells and MDR pathogens.

Pentagalloyl Glucose-Targeted Inhibition of P-Glycoprotein and Re-Sensitization of Multidrug-Resistant Leukemic Cells (K562/ADR) to Doxorubicin: In Silico and Functional Studies.

Combining phytochemicals with chemotherapeutic drugs has demonstrated the potential to surmount drug resistance. In this paper, we explore the efficacy of pentagalloyl glucose (PGG) in modulating P-gp and reversing multidrug resistance (MDR) in drug-resistant leukemic cells (K562/ADR). The cytotoxicity of PGG was evaluated using a CCK-8 assay, and cell apoptosis was assessed using flow cytometry. Western blotting was used to analyze protein expression levels. P-glycoprotein (P-gp) activity was evaluated by monitoring the kinetics of P-gp-mediated efflux of pirarubicin (THP). Finally, molecular docking, molecular dynamics simulation, and molecular mechanics with generalized Born and surface area solvation (MM-GBSA) calculation were conducted to investigate drug-protein interactions. We found that PGG selectively induced cytotoxicity in K562/ADR cells and enhanced sensitivity to doxorubicin (DOX), indicating its potential as a reversal agent. PGG reduced the expression of P-gp and its gene transcript levels. Additionally, PGG inhibited P-gp-mediated efflux and increased intracellular drug accumulation in drug-resistant cells. Molecular dynamics simulations and MM-GBSA calculation provided insights into the binding affinity of PGG to P-gp, suggesting that PGG binds tightly to both the substrate and the ATP binding sites of P-gp. These findings support the potential of PGG to target P-gp, reverse drug resistance, and enhance the efficacy of anticancer therapies.

Bioinformatic analysis of highly consumed phytochemicals as P-gp binders to overcome drug-resistance.

P-glycoprotein (P-gp) is an adenosine triphosphate (ATP)-dependent membrane efflux pump for protecting cells against xenobiotic compounds. Unfortunately, overexpressed P-gp in neoplastic cells prevents cell entry of numerous chemotherapeutic agents leading to multidrug resistance (MDR). MDR cells may be re-sensitized to chemotherapeutic drugs via P-gp inhibition/modulation. Side effects of synthetic P-gp inhibitors encouraged the development of natural products. Molecular docking and density functional theory (DFT) calculations were used as fast and accurate computational methods to explore a structure binding relationship of some dietary phytochemicals inside distinctive P-gp binding sites (modulatory/inhibitory). For this purpose, top-scored docked conformations were subjected to per-residue energy decomposition analysis in the B3LYP level of theory with a 6-31g (d, p) basis set by Gaussian98 package. Consecutive application of computational techniques revealed binding modes/affinities of nutritive phytochemicals within dominant binding sites of P-gp. Blind docking scores for best-ranked compounds were superior to verapamil and rhodamine-123. Pairwise amino acid decomposition of superior docked conformations revealed Tyr303 as an important P-gp binding residue. DFT-based induced polarization analysis revealed major electrostatic fluctuations at the atomistic level and confirmed larger effects for amino acids with energy-favored binding interactions. Conformational analysis exhibited that auraptene and 7,4',7'',4'''-tetra-O-methylamentoflavone might not necessarily interact to P-gp binding sites through minimum energy conformations. Although there are still many hurdles to overcome, obtained results may propose a few nutritive phytochemicals as potential P-gp binding agents. Moreover; top-scored derivatives may have the chance to exhibit tumor chemo-sensitizing effects.

Ganoderma lucidum extract reverses hepatocellular carcinoma multidrug resistance via inhibiting the function of P-glycoprotein in vitro and in vivo

Cancer is a leading cause of death globally. Chemotherapy still plays an indispensable role in the clinical treatment of cancer. However, the emergence of multidrug resistance (MDR) has greatly obstructed the further application of chemotherapy agents. Ganoderma lucidum (G. lucidum) is a traditional Chinese medicine as well as an edible mushroom. In this study, we first explored the effect of six extract samples derived from G. lucidum on the cell viability of adriamycin-resistant human hepatocellular carcinoma multidrug-resistant cell subline (HepG-2/ADM). All these samples showed no obvious toxicity to cells; however, only G. lucidum ethanol extract could reverse resistance to doxorubicin and paclitaxel. Then the P-glycoprotein (P-gp) function in hepatocellular carcinoma G2 (HepG-2) and HepG-2/ADM cells was determined after incubated with these samples, and we found that G. lucidum ethanol extract could inhibit P-gp function in vitro. Furthermore, G. lucidum ethanol extract could reverse resistance to paclitaxel in HepG-2/ADM tumor-bearing mice in vivo, while the protein expression level of P-gp was unchanged. Taken together, these results indicated the potential role of G. lucidum ethanol extract in reversing MDR in the clinical treatment of hepatocellular carcinoma.

Transferrin-Decorated PLGA Nanoparticles Loaded with an Organoselenium Compound as an Innovative Approach to Sensitize MDR Tumor Cells: An In Vitro Study Using 2D and 3D Cell Models.

Multidrug resistance (MDR) is the main challenge in cancer treatment. In this sense, we designed transferrin (Tf)-conjugated PLGA nanoparticles (NPs) containing an organoselenium compound as an alternative to enhance the efficacy of cancer therapy and sensitize MDR tumor cells. Cytotoxicity studies were performed on different sensitive tumor cell lines and on an MDR tumor cell line, and the Tf-conjugated NPs presented significantly higher antiproliferative activity than the nontargeted counterparts in all tested cell lines. Due to the promising antitumor activity of the Tf-decorated NPs, further studies were performed using the MDR cells (NCI/ADR-RES cell line) comparatively to one sensitive cell line (HeLa). The cytotoxicity of NPs was evaluated in 3D tumor spheroids and, similarly to the results achieved in the 2D assays, the Tf-conjugated NPs were more effective at reducing the spheroid's growth. The targeted Tf-NPs were also able to inhibit tumor cell migration, presented a higher cell internalization and induced a greater number of apoptotic events in both cell lines. Therefore, these findings evidenced the advantages of Tf-decorated NPs over the nontargeted counterparts, with the Tf-conjugated NPs containing an organoselenium compound representing a promising drug delivery system to overcome MDR and enhance the efficacy of cancer therapy.

Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells

The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investigation. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immunofluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents.

Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Preparation of liposomes co-encapsulating doxorubicin and mifepristone for treating multidrug-resistant cancer

The emergence of multidrug resistance (MDR) is well-known to decrease the therapeutic effects of anticancer drugs. To increase anticancer drug efficacy and decrease adverse effects, we prepared liposomes co-encapsulating doxorubicin (DXR) and mifepristone (MIF) (DXR/MIF-LPs) for treating MDR cancer and evaluated the MDR reversal effect in DXR-resistant Colon-26 cancer cells (C26/DXR). Liposomal encapsulation was performed using remote loading methods: amphipathic DXR was encapsulated using the ammonium sulfate gradient method, whereas hydrophobic MIF was encapsulated in preformed liposomes by incubating with water-miscible dimethyl sulfoxide. Both drugs were successfully encapsulated in liposomes by simultaneously incubating with blank liposomes without remarkably altering the particle size. The release of each drug from DXR/MIF-LPs corresponded to that from liposomes encapsulating a single drug, indicating that drug encapsulation did not influence drug release. Treatment with MIF did not impact the overexpression of P-glycoprotein (P-gp) in C26/DXR cells. However, DXR/MIF-LPs increased the cellular association of DXR and enhanced cytotoxicity compared with liposomes encapsulating DXR against C26/DXR cells, indicating that MIF inhibits P-gp function and increases the cytotoxicity of DXR in MDR cells. Collectively, our findings suggest that the co-delivery of DXR and MIF using a liposomal formulation is a promising approach for reversing MDR.

Hypophyllanthin and Phyllanthin from Phyllanthus niruri Synergize Doxorubicin Anticancer Properties against Resistant Breast Cancer Cells.

Doxorubicin (DOX) is a cornerstone chemotherapeutic agent for the treatment of several malignancies such as breast cancer; however, its activity is ameliorated by the development of a resistant phenotype. Phyllanthus species have been studied previously for their potential anticancer properties. The current work is aimed to study the potential cytotoxicity and chemomodulatory effects of hypophyllanthin (PN4) and phyllanthin (PN5) isolated from Phyllanthus niruri to DOX against the adriamycin multidrug-resistant breast cancer cells (MCF-7ADR) and elucidate their mechanism of action. The major compounds of the active methylene chloride fraction were isolated and assessed for their potential cytotoxicity and chemomodulatory effects on DOX against naïve (MCF-7) and resistant breast (MCF-7ADR) cancer cells. The mechanism of action of both compounds in terms of their impacts on programmed/non-programmed cell death (apoptosis and autophagy/necrosis), cell cycle progression/arrest, and tumor cell migration/invasion was investigated. Both compounds PN4 and PN5 showed a moderate but similar potency against MCF-7 as well as MCF-7ADR and significantly synergized DOX-induced anticancer properties against MCF-7ADR. The chemomodulatory effect of both compounds to DOX was found to be via potentiating DOX-induced cell cycle interference and apoptosis induction. It was found that PN4 and PN5 blocked the apoptosis-escape autophagy pathway in MCF-7ADR. On the molecular level, both compounds interfered with SIRT1 expression and consequently suppressed Akt phosphorylation, and PN5 blocked apoptosis escape. Furthermore, PN4 and PN5 showed promising antimigratory and anti-invasive effects against MCF-7ADR, as confirmed by suppression of N-cadherin/β-catenin expression. In conclusion, for the first time, hypophyllanthin and phyllanthin isolated from P. niruri showed promising chemomodulatory effects to the DOX-induced chemotherapeutic activity against MCF-7ADR. Both compounds significantly synergized DOX-induced anticancer properties against MCF-7ADR. This enhanced activity was explained by further promoting DOX-induced apoptosis and suppressing the apoptosis-escape autophagy feature of the resistant breast cancer cells. Both compounds (hypophyllanthin and phyllanthin) interfered with the SIRT1/Akt pathway and suppressed the N-cadherin/β-catenin axis, confirming the observed antiproliferative, cytotoxic, and anti-invasive effects of hypophyllanthin and phyllanthin.

Optimization of a Luteolin-Loaded TPGS/Poloxamer 407 Nanomicelle: The Effects of Copolymers, Hydration Temperature and Duration, and Freezing Temperature on Encapsulation Efficiency, Particle Size, and Solubility.

Luteolin is a flavonoid compound that has been widely studied for its various anti-cancer properties and sensitization to multidrug-resistant cells. However, the limited solubility and bioavailability of Lut hindered its potential clinical use. Theoretically, the combination of this compound with vitamin E TPGS and poloxamer 407 can produce a synergistic effect to enhance tumor apoptosis and P-glycoprotein inhibition. This study aimed to develop and optimize vitamin E TPGS/Poloxamer 407 micelles loaded with luteolin through investigating certain factors that can affect the encapsulation efficiency and particle size of the micelle. A micelle was prepared using the film hydration method, and the micellar solution was lyophilized. The cake formed was analyzed. The factors investigated include the concentrations of the surfactants, ratio of vitamin E TPGS/Poloxamer 407, temperature of the hydrating solution, duration of hydration, and freezing temperature before lyophilization. The effects of these factors on the encapsulation efficiency and particle size of the micelle were also studied. The encapsulation efficiency was measured using a UV-Vis spectrophotometer, while particle size was measured using dynamic light scattering. The optimized micelle was found to have 90% encapsulation efficiency with a particle size of less than 40 nm, which was achieved using a 10% concentration of surfactants at a vitamin E TPGS/Poloxamer 407 ratio of 3:1. The optimized temperature for hydrating the micellar film was 40 °C, the optimized mixing time was 1 h, and the optimized freezing temperature was -80 °C. The solubility of the luteolin-loaded micelles increased 459-fold compared to pure Lut in water. The critical micelle concentration of the vitamin E TPGS/Poloxamer 407 micelle was 0.001 mg/mL, and the release study showed that luteolin-loaded micelles exhibited sustained release behavior. The release of luteolin from a micelle was found to be higher in pH 6.8 compared to pH 7.4, which signified that luteolin could be accumulated more in a tumor microenvironment compared to blood. This study demonstrated that several factors need to be considered when developing such nanoparticles in order to obtain a well-optimized micelle.

Functional Nucleic Acids-Engineered Bio-Barcode Nanoplatforms for Targeted Synergistic Therapy of Multidrug-Resistant Cancer.

Rational design of multifunctional nanomedicines has revolutionized the therapeutic efficacy of cancers. Herein, we have constructed the functional nucleic acids (FNAs)-engineered nanoplatforms based on the concept of a bio-barcode (BBC) for synergistic targeted therapy of multidrug-resistant (MDR) cancer. In this study, the platinum(IV) prodrug is synthesized to covalently link two kinds of FNAs at a rational ratio to fabricate three-dimensional BBC-like DNA nanoscaffolds, accompanied by the one-pot encapsulation of ZnO nanoparticles (NPs) through electrostatic interaction. The multivalent AS1411 aptamers equipped in ZnO@BBCs facilitate specific and efficient endocytosis into MDR human lung adenocarcinoma cells (A549/DDP). In response to the intracellular environment of A549/DDP cells, such as the lysosome-acidic pH and overexpressed GSH, the ZnO NPs are degraded into Zn2+ ions for generating reactive oxygen species (ROS), while the Pt(IV) prodrugs are reduced into Pt(II) active species by glutathione (GSH), followed by the release of therapeutic DNAzymes for chemotherapy and gene therapy. In particular, the designed system plays an important role in remodeling the intracellular environment to reverse cancer MDR. On the one hand, the depletion of GSH promotes the downregulation of glutathione peroxidase 4 (GPX4) for amplifying oxidative stress and increasing lipid peroxidation (LPO), resulting in the activation of ferroptosis. On the other hand, the silence of early growth response protein 1 (Egr-1) mRNA by Zn2+-dependent DNAzymes directly inhibits the proliferation and migration of MDR cells, which further suppresses the P-glycoprotein (P-gp)-mediated drug efflux. Thus, the proposed nanoplatforms show great promise for the development of versatile therapeutic tools and personalized nanomedicines for MDR cancers.

Isoliensinine augments the therapeutic potential of paclitaxel in multidrug-resistant colon cancer stem cells and induced mitochondria-mediated cell death.

Previously we have reported the isoliensinine (ISO) potentates the therapeutic potential of cisplatin in cisplatin resistant colorectal cancer stem cells. The present study evaluates the chemo-sensitizing potential of the combinatorial regimen of ISO and Paclitaxcel (PTX) on multidrug-resistant (MDR)-HCT-15 cells to reduce the dose requirement of both ISO and PTX. The results of the present study suggest that treatment with the combinatorial regimen of ISO and PTX enhanced the cytotoxic effect with resultant increase in apoptosis in MDR-HCT-15 cells as evident from the altered cellular morphology, G2/M cell cycle arrest, propidium iodideuptake, Annexin V, increased intracellular Ca2+ accumulation, decreased mitochondrial membrane potential, diminished ATP production, PARP-1 cleavage, altered expression of ERK1/2, and apoptotic proteins. Treatment with combinatorial regimen of ISO and PTX also modulated the expression of the transcription factors SOX2, OCT4 which determine the stemness of cancer cells. Thus, results of the present study suggest that ISO and PTX combination regimen induces apoptosis in MDR-HCT-15 in a synergistic manner.

Decursinol Angelate Inhibits Glutamate Dehydrogenase 1 Activity and Induces Intrinsic Apoptosis in MDR-CRC Cells.

Colorectal cancer (CRC) was the second most commonly diagnosed cancer worldwide and the second most common cause of cancer-related deaths in Europe in 2020. After CRC patients' recovery, in many cases a patient's tumor returns and develops chemoresistance, which has remained a major challenge worldwide. We previously published our novel findings on the role of DA in inhibiting the activity of GDH1 using in silico and enzymatic assays. No studies have been conducted so far to explain the inhibitory role of DA against glutamate dehydrogenase in MDR-CRC cells. We developed a multidrug-resistant colorectal cancer cell line, HCT-116MDR, after treatment with cisplatin and 5-fluorouracil. We confirmed the MDR phenotype by evaluating the expression of MDR1, ABCB5, extracellular vesicles, polyploidy, DNA damage response markers and GDH1 in comparison with parental HCT-116WT (HCT-116 wild type). Following confirmation, we determined the IC50 and performed clonogenic assay for the efficacy of decursinol angelate (DA) against HCT-116MDR (HCT-116 multidrug resistant). Subsequently, we evaluated the novel interactions of DA with GDH1 and the expression of important markers regulating redox homeostasis and cell death. DA treatment markedly downregulated the expression of GDH1 at 50 and 75 μM after 36 h, which directly correlated with reduced expression of the Krebs cycle metabolites α-ketoglutarate and fumarate. We also observed a systematic dose-dependent downregulation of MDR1, ABCB5, TERT, ERCC1 and γH2AX. Similarly, the expression of important antioxidant markers was also downregulated. The markers for intrinsic apoptosis were notably upregulated in a dose-dependent manner. The results were further validated by flow cytometry and TUNEL assay. Additionally, GDH1 knockdown on both HCT-116WT and HCT-116MDR corresponded to a decreased expression of γH2AX, catalase, SOD1 and Gpx-1, and an eventual increase in apoptosis markers. In conclusion, inhibition of GDH1 increased ROS production, decreased cell proliferation and increased cell death.

PP2A modulation overcomes multidrug resistance in chronic lymphocytic leukemia via mPTP-dependent apoptosis.

Targeted therapies such as venetoclax (VEN) (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple antiapoptotic proteins and display resistance to proapoptotic agents. Here, we demonstrated that multidrug-resistant CLL cells in vivo exhibited apoptosis restriction at a pre-mitochondrial level due to insufficient activation of the Bax and Bak (Bax/Bak) proteins. Co-immunoprecipitation analyses with selective BH domain antagonists revealed that the pleiotropic proapoptotic protein (Bim) was prevented from activating Bax/Bak by "switching" interactions to other upregulated antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that small-molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug-resistant leukemia and lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug-resistant CLL cells through induction of mitochondrial permeability transition pores, independent of Bax/Bak. DT-061 inhibited the growth of wild-type and Bax/Bak double-knockout, multidrug-resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug-resistant CLL cells in patients and validated a pharmacologically tractable pathway to deplete this reservoir.

The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells

Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and recently emerged in a new role in the treatment of cancer. ZINC253504760, a synthetic cardenolide that is structurally similar to well-known GCs, digitoxin and digoxin, has not been investigated yet. This study aims to investigate the cytotoxicity of ZINC253504760 on MDR cell lines and its molecular mode of action for cancer treatment. Four drug-resistant cell lines (P-glycoprotein-, ABCB5-, and EGFR-overexpressing cells, and TP53-knockout cells) did not show cross-resistance to ZINC253504760 except BCRP-overexpressing cells. Transcriptomic profiling indicated that cell death and survival as well as cell cycle (G2/M damage) were the top cellular functions affected by ZINC253504760 in CCRF-CEM cells, while CDK1 was linked with the downregulation of MEK and ERK. With flow cytometry, ZINC253504760 induced G2/M phase arrest. Interestingly, ZINC253504760 induced a novel state-of-the-art mode of cell death (parthanatos) through PARP and PAR overexpression as shown by western blotting, apoptosis-inducing factor (AIF) translocation by immunofluorescence, DNA damage by comet assay, and mitochondrial membrane potential collapse by flow cytometry. These results were ROS-independent. Furthermore, ZINC253504760 is an ATP-competitive MEK inhibitor evidenced by its interaction with the MEK phosphorylation site as shown by molecular docking in silico and binding to recombinant MEK by microscale thermophoresis in vitro. To the best of our knowledge, this is the first time to describe a cardenolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug resistance in cancer.Graphical A cardiac glycoside compound ZINC253504760 displayed cytotoxicity against different multidrug-resistant cell lines. ZINC253504760 exhibited cytotoxicity in CCRF-CEM leukemia cells by predominantly inducing a new mode of cell death (parthanatos). ZINC253504760 downregulated MEK1/2 phosphorylation and further affected ERK activation, which induced G2/M phase arrest.

Early in vitro evidence indicates that deacetylated sialic acids modulate multi-drug resistance in colon and lung cancers via breast cancer resistance protein.

Cancers utilize sugar residues to engage in multidrug resistance. The underlying mechanism of action involving glycans, specifically the glycan sialic acid (Sia) and its various functional group alterations, has not been explored. ATP-binding cassette (ABC) transporter proteins, key proteins utilized by cancers to engage in multidrug resistant (MDR) pathways, contain Sias in their extracellular domains. The core structure of Sia can contain a variety of functional groups, including O-acetylation on the C6 tail. Modulating the expression of acetylated-Sias on Breast Cancer Resistance Protein (BCRP), a significant ABC transporter implicated in MDR, in lung and colon cancer cells directly impacted the ability of cancer cells to either retain or efflux chemotherapeutics. Via CRISPR-Cas-9 gene editing, acetylation was modulated by the removal of CAS1 Domain-containing protein (CASD1) and Sialate O-Acetyl esterase (SIAE) genes. Using western blot, immunofluorescence, gene expression, and drug sensitivity analysis, we confirmed that deacetylated Sias regulated a MDR pathway in colon and lung cancer in early in vitro models. When deacetylated Sias were expressed on BCRP, colon and lung cancer cells were able to export high levels of BCRP to the cell's surface, resulting in an increased BCRP efflux activity, reduced sensitivity to the anticancer drug Mitoxantrone, and high proliferation relative to control cells. These observations correlated with increased levels of cell survival proteins, BcL-2 and PARP1. Further studies also implicated the lysosomal pathway for the observed variation in BCRP levels among the cell variants. RNASeq data analysis of clinical samples revealed higher CASD1 expression as a favorable marker of survival in lung adenocarcinoma. Collectively, our findings indicate that deacetylated Sia is utilized by colon and lung cancers to engage in MDR via overexpression and efflux action of BCRP.

In Vivo Imaging Guided GMBP1 Modified Nanoplatform for Real Time Monitoring and Photothermal Therapy of Multidrug Resistance in Gastric Cancer

Chemotherapy for gastric cancer (GC) is easy to cause multidrug resistance. Accordingly, it is of great significance to accurately monitor multidrug resistance for individualized treatment in GC. Molecular imaging technology provides a powerful means for the detection of multidrug resistance in GC. Research suggests that GMBP1 can specifically combined with overexpressed GRP78 receptor on GC multidrug resistance cells. Therefore, we constructed a dual-mode imaging nanocomplex based on GMBP1 (Au@bMSN-Cy7.5-GMBP1) that enables real-time multidrug resistance monitoring In Vivo through fluorescence imaging and photoacoustic imaging. In addition, Au@bMSN-Cy7.5-GMBP1 has a good photothermal effect so that it can be used for In Vivo photothermal therapy of multidrug resistance of GC. Au@bMSN-Cy7.5-GMBP1 has good stability and photothermal effect and its particle size is about 100 nm. Au@bMSN-Cy7.5-GMBP1 has low cytotoxicity and can effectively inhibit tumor cell growth under laser irradiation. Au@bMSN-Cy7.5-GMBP1 has low hematotoxicity and good biosafety. Au@bMSN-Cy7.5-GMBP1 can be observed to accumulate gradually in the tumor site through fluorescence imaging and photoacoustic imaging, so as to realize real-time monitoring of GC multidrug resistance. In Vivo photothermal therapy results show that Au@bMSN-Cy7.5-GMBP1 can effectively inhibit tumor growth under laser irradiation, and realize photothermal treatment of multidrug resistance in GC. Au@bMSN-Cy7.5-GMBP1 provides an excellent nano-platform for individualized treatment and multidrug resistance monitoring of GC.

Medicago Sativa Defensin1 as a tumor sensitizer for improving chemotherapy: translation from anti-fungal agent to a potential anti-cancer agent.

Plant defensins including Medicago Sativa defensin 1 (MsDef1) are cysteine-rich antifungal peptides which are known for potent broad-spectrum antifungal activity against bacterial or fungal pathogens of plants. The antimicrobial activities of these cationic defensins are attributed to their capacity to bind to cell membranes to create potentially structural defects tin the cell membranes to interact with intracellular target (s) and mediates cytotoxic effects. Our earlier work identified Glucosylceramide (GlcCer) of fungus F. graminearum as a potential target for biological activity. Multi-drug resistant (MDR) cancer cells overexpress GlcCer on the surface of plasma membrane. Hence, MsDef1 may have a potential to bind to GlcCer of MDR cancer cells to induce cell death. We have characterized the three-dimensional structure of MsDef1 and the solution dynamics using of 15N-labeled MsDef1 nuclear magnetic resonance (NMR) spectroscopy which showed that GlcCer binds MsDef1 at two specific sites on the peptide molecule. The ability of MsDef1 to permeate MDR cancer cells was demonstrated by measuring the release of apoptotic ceramide in drug resistant MCF-7R cells. It was also shown that MsDef1 activated dual cell death pathways ceramide and Apoptosis Stimulating Kinase ASK1 by disintegrating GlcCer and oxidizing tumor specific biomarker thioredoxin (Trx) respectively. As a result, MsDef1 sensitizes MDR cancer cells to evoke a better response from Doxorubicin, a front-line chemotherapy for triple negative breast cancer (TNBC) treatment. The combination of MsDef1 and Doxorubicin induced 5 to10-fold greater apoptosis in vitro MDR cells MDA-MB-231R compared to either MsDef1 or Doxorubicin alone. Confocal microscopy revealed that MsDef1 facilitates a) influx of Doxorubicin in MDR cancer cells, b) preferential uptake by MDR cells but not by normal fibroblasts and breast epithelial cells (MCF-10A). These results suggest that MsDef1 targets MDR cancer cells and may find utility as a neoadjuvant chemotherapy. Hence, the extension of antifungal properties of MsDef1 to cancer my result in addressing the MDR problems in cancer.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

Design, Synthesis, and Antiproliferative Activity of Benzopyran-4-One-Isoxazole Hybrid Compounds.

The biological significance of benzopyran-4-ones as cytotoxic agents against multi-drug resistant cancer cell lines and isoxazoles as anti-inflammatory agents in cellular assays prompted us to design and synthesize their hybrid compounds and explore their antiproliferative activity against a panel of six cancer cell lines and two normal cell lines. Compounds 5a-d displayed significant antiproliferative activities against all the cancer cell lines tested, and IC50 values were in the range of 5.2-22.2 μM against MDA-MB-231 cancer cells, while they were minimally cytotoxic to the HEK-293 and LLC-PK1 normal cell lines. The IC50 values of 5a-d against normal HEK-293 cells were in the range of 102.4-293.2 μM. Compound 5a was screened for kinase inhibitory activity, proteolytic human serum stability, and apoptotic activity. The compound was found inactive towards different kinases, while it completely degraded after 2 h of incubation with human serum. At 5 μM concentration, it induced apoptosis in MDA-MB-231 by 50.8%. Overall, these findings suggest that new benzopyran-4-one-isoxazole hybrid compounds, particularly 5a-d, are selective anticancer agents, potentially safe for human cells, and could be synthesized at low cost. Additionally, Compound 5a exhibits potential anticancer activity mediated via inhibition of cancer cell proliferation and induction of apoptosis.