Efflux Transporters Research Articles

Enteroids to Study Pediatric Intestinal Drug Transport.

Intestinal maturational changes after birth affect the pharmacokinetics (PK) of drugs, having major implications for drug safety and efficacy. However, little is known about ontogeny-related PK patterns in the intestine. To explore the accuracy of human enteroid monolayers for studying drug transport in the pediatric intestine, we compared the drug transporter functionality and expression in enteroid monolayers and tissue from pediatrics and adults. Enteroid monolayers were cultured of 14 pediatric [median (range) age: 44 weeks (2 days-13 years)] and 5 adult donors, in which bidirectional drug transport experiments were performed. In parallel, we performed similar experiments with tissue explants in Ussing chamber using 11 pediatric [median (range) age: 54 weeks (15 weeks-10 years)] and 6 adult tissues. Enalaprilat, propranolol, talinolol, and rosuvastatin were used to test paracellular, transcellular, and transporter-mediated efflux by P-gp and breast cancer resistance protein (BCRP), respectively. In addition, we compared the expression patterns of ADME-related genes in pediatric and adult enteroid monolayers with tissues using RNA sequencing. Efflux transport by P-gp and BCRP was comparable between the enteroids and tissue. Efflux ratios (ERs) of talinolol and rosuvastatin by P-gp and BCRP, respectively, were higher in enteroid monolayers compared to Ussing chamber, likely caused by experimental differences in model setup and cellular layers present. Explorative statistics on the correlation with age showed trends of increasing ER with age for P-gp in enteroid monolayers; however, it was not significant. In the Ussing chamber setup, lower enalaprilat and propranolol transport was observed with age. Importantly, the RNA sequencing pathway analysis revealed that age-related variation in drug metabolism between neonates and adults was present in both enteroids and intestinal tissue. Age-related differences between 0 and 6 months old and adults were observed in tissue as well as in enteroid monolayers, although to a lesser extent. This study provides the first data for the further development of pediatric enteroids as an in vitro model to study age-related variation in drug transport. Overall, drug transport in enteroids was in line with data obtained from ex vivo tissue (using chamber) experiments. Additionally, pathway analysis showed similar PK-related differences between neonates and adults in both tissue and enteroid monolayers. Given the challenge to elucidate the effect of developmental changes in the pediatric age range in human tissue, intestinal enteroids derived from pediatric patients could provide a versatile experimental platform to study pediatric phenotypes.

Porous Organic Polymer-Based Nanocomposites for Hypoxia Relieving and Enhanced Chemotherapy in Hepatocellular Carcinoma.

Uncontrolled proliferation and altered metabolism of cancer cells result in an imbalance of nutrients and oxygen supply, and persuade hypoxia. Hypoxia, in turn, activates the transcription gene HIF-1α, which eventually upregulates the efflux transporter P-gp and induces multidrug resistance (MDR). Thus, hypoxia leads to the development of resistance to conventional therapies. Therefore, the fabrication of a nanoscale porous system enriched with upconversion nanoparticles to target cancer cells, evade hypoxia, and enhance anticancer therapy is the key goal of this article. Herein, upconversion nanoparticles are embedded in a nanoscale porous organic polymer (POP) and further conjugated with a targeting moiety and a catalase molecule. The nanoscale POP embedded in UCNPs is generated at room temperature. The targeting ligand, lactobionic acid, is attached after polymer coating, which effectively targets liver cancer cells. Then, catalase is grafted effectively to produce oxygen. Endogenously generated oxygen alleviates hypoxia in liver cancer cells. The drug- and catalase-loaded composite exhibit greater cytotoxicity in hypoxic liver cells than in normal cells by overcoming hypoxia and downregulating the hypoxia-inducible factors.

Differential Effect of Simulated Microgravity on the Cellular Uptake of Small Molecules.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

A Revolutionary Approach for Combating Efflux Transporter-mediated Resistant Epilepsy: Advanced Drug Delivery Systems.

Epilepsy is a persistent neurological condition that affects 60 million individuals globally, with recurrent spontaneous seizures affecting 80% of patients. Antiepileptic drugs (AEDs) are the main course of therapy for approximately 65% of epileptic patients, and the remaining 35% develop resistance to medication, which leads to Drug-Resistant Epilepsy (DRE). DRE continues to be an important challenge in clinical epileptology. There are several theories that attempt to explain the neurological causes of pharmacoresistance in epilepsy. The theory that has been studied the most is the transporter hypothesis. Therefore, it is believed that upregulation of multidrug efflux transporters at the blood-brain barrier (BBB), such as P-glycoprotein (P-gp), which extrudes AEDs from their target location, is the major cause, leading to pharmacoresistance in epilepsy. The most effective strategies for managing this DRE are peripheral and central inhibition of P-gp and maintaining an effective concentration of the drug in the brain parenchyma. Presently, no medicinal product that inhibits P-gp is being used in clinical practice. In this review, several innovative and promising treatment methods, including gene therapy, intracranial injections, Pgp inhibitors, nanocarriers, and precision medicine, are discussed. The primary goal of this work is to review the P-gp transporter, its substrates, and the latest novel treatment methods for the management of DRE.

Functional Characterization of Reduced Folate Carrier and Protein-Coupled Folate Transporter for Antifolates Accumulation in Non-Small Cell Lung Cancer Cells.

Antifolates are important for chemotherapy in non-small cell lung cancer (NSCLC). They mainly rely on reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) to enter cells. PCFT is supposed to be the dominant transporter of the two in tumors, as it operates optimally at acidic pH and has limited transport activity at physiological pH, whereas RFC operates optimally at neutral pH. In this study, we found RFC showed a slightly pH-dependent uptake of antifolates, with similar affinity values at pH 7.4 and 6.5. PCFT showed a highly pH-dependent uptake of antifolates, with an optimum pH of 6.0 for pemetrexed and 5.5 for methotrexate. The Michaelis-Menten constant (Km ) value of PCFT for pemetrexed at pH 7.4 was more than 10 times higher than that at pH 6.5. Interestingly, we found that antifolate accumulations mediated by PCFT at acidic pH were significantly affected by the efflux transporter, breast cancer resistance protein (BCRP). The highest pemetrexed concentration was observed at pH 7.0-7.4 after a 60-minute accumulation in PCFT-expressing cells, which was further evidenced by the cytotoxicity of pemetrexed, with the IC50 value of pemetrexed at pH 7.4 being one-third of that at pH 6.5. In addition, the in vivo study indicated that increasing PCFT and RFC expression significantly enhanced the antitumor efficacy of pemetrexed despite the high expression of BCRP. These results suggest that both RFC and PCFT are important for antifolates accumulation in NSCLC, although there is an acidic microenvironment and high BCRP expression in tumors. SIGNIFICANCE STATEMENT: Evaluating the role of reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) on antifolates accumulation in non-small cell lung cancer (NSCLC) is necessary for new drug designs. By using cell models, we found both RFC and PCFT were important for antifolates accumulation in NSCLC. Breast cancer resistance protein (BCRP) significantly affected PCFT-mediated antifolates accumulation at acidic pH but not RFC-mediated pemetrexed accumulation at physiological pH. High expression of PCFT or RFC enhanced the cytotoxicity and antitumor effect of pemetrexed.

Prevalence of qacEΔ1, qacE, oqxA, oqxB, acrA, cepA and zitB genes among multidrug-resistant Klebsiella pneumoniae isolated in a cardiac hospital

Background. Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are the leading cause of mortality worldwide. The widespread use of disinfectants and antiseptics has caused the emergence of K. pneumoniae with reduced sensitivity to them, which, in combination with MDR, can pose a significant epidemiological threat. The aim of the study was to assess the prevalence of efflux pump and transporter genes associated with biocide resistance and their association with antibiotic resistance among K. pneumoniae isolated in a cardiac surgical hospital. Materials and methods. K. pneumoniae isolates (n = 50) from the patients and medical equipment were tested by polymerase chain reaction for the presence of genes of 4 types of efflux pumps (qacEΔ1, qacE, oqxA, oqxB, acrA) and 2 transporters involved in the outflow of cations (cepA) and zinc ions (zitB). Spearman's rank correlation test was used to assess the strength of the association between the efflux pumps, beta-lactamase genes and mobile genetic elements. Results. The occurrence of K. pneumoniae containing qacEΔ1, qacE, oqxA, oqxB, acrA, cepA and zitB was high: 54, 62, 100, 84, 100, 72 и 96% respectively. K. pneumoniae with a combination of all the studied pumps was most often detected (32%), and these isolates were MDR in 100% of cases. The qacE, qacEΔ1 genes were closely associated with resistance to cephalosporins, carbapenems, fluoroquinolones, carbapenemase genes, and integrons. The results of the study showed that the genes of various efflux pumps associated with biocide resistance and their combinations were widely represented among the clinical isolates of MDR K. pneumoniae. Conclusion. The high prevalence of efflux pump genes associated with resistance to quaternary ammonium compounds, chlorhexidine and zinc salts and their significant association with antibiotic resistance in nosocomial K. pneumoniae underlines the importance of further studying the mechanisms of cross-resistance to biocides to improve methods of combating MDR nosocomial pathogens.

Multi-Omics Analyses Uncover the Mechanism Underlying Polyploidization-Enhanced Steviol Glycosides Biosynthesis in Stevia rebaudiana.

Stevia rebaudiana (Bertoni) is a valuable sweetener plant whose sweetness primarily derives from steviol glycosides (SGs), especially rebaudioside A (RA). Polyploidization has the potential to enhance the content of active ingredients in medicinal plants, making this strategy a promising avenue for genetic improvement. However, the underlying regulatory mechanisms that contribute to the fluctuating SGs content between autotetraploid and diploid stevia remain unclear. In this study, we employed metabolic analysis to identify 916 differentially accumulated metabolites (DAMs), with the majority, specifically terpenoids, flavonoids, and lipids, exhibiting upregulation due to polyploidization. Notably, the content of stevia's signature metabolite SGs (including RA, steviolbioside, and rebaudioside C), along with their precursor steviol, increased significantly after polyploidization. Furthermore, a comprehensive analysis of the transcriptome and metabolome revealed that the majority of differentially expressed genes (DEGs) involved in the SG-synthesis pathway (ent-KAH, ent-KS1, UGT73E1, UGT74G1, UGT76G1, UGT85C2, and UGT91D2) were upregulated in autotetraploid stevia, and these DEGs exhibited a positive correlation with the polyploidization-enhanced SGs. Additionally, multi-omics network analysis indicated that several transcription factor families (such as five NACs, four WRKYs, three MYBs, eight bHLHs, and three AP2/ERFs), various transporter genes (four ABC transporters, three triose-phosphate transporters, and two sugar efflux transporters for intercellular exchange), as well as microorganisms (including Ceratobasidium and Flavobacterium) were positively correlated with the accumulation of RA and steviol. Overall, our results indicate the presence of a regulatory circuit orchestrated by polyploidization, which recruits beneficial rhizosphere microbes and modulates the expression of genes associated with SG biosynthesis, ultimately enhancing the SG content in stevia. This finding will provide new insights for promoting the propagation and industrial development of stevia.

Loss of OsMATE6 Function Enhances Drought Resistance Without Yield Penalty by Regulating Stomatal Closure in Rice.

Drought is one of the most severe environmental factors limiting plant growth and crop yield, necessitating the identification of genes that enhance drought resistance for crop improvement. Through screening an ethyl methyl sulfonate-mutagenized rice mutant library, we isolated the PEG tolerance mutant 97-1 (ptm97-1), which displays enhanced resistance to osmotic and drought stress, and increased yield under drought conditions. A point mutation in OsMATE6 was identified as being associated with the drought-resistant phenotype of ptm97-1. The role of OsMATE6 in conferring drought resistance was confirmed by additional OsMATE6 knockout mutants. OsMATE6 is expressed in guard cells, shoots and roots and the OsMATE6-GFP fusion protein predominantly localizes to the plasma membrane. Our ABA efflux assays suggest that OsMATE6 functions as an ABA efflux transporter; mutant protoplasts exhibited a slower ABA release rate compared to the wild type. We hypothesize that OsMATE6 regulates ABA levels in guard cells, influencing stomatal closure and enhancing drought resistance. Notably, OsMATE6 knockout mutants demonstrated greater yields under field drought conditions compared to wild-type plants, highlighting OsMATE6 as a promising candidate for improving crop drought resistance.

Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A.

A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 -3.4 Å resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for "alternate access" to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the "alternate access" mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.

Transcriptomic and functional analyses on a Botrytis cinerea multidrug-resistant (MDR) strain provides new insights into the potential molecular mechanisms of MDR and fitness.

Botrytis cinerea is a notorious pathogen causing pre- and post-harvest spoilage in many economically important crops. Excessive application of site-specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2, encoding an ATP-binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B. cinerea multidrug-resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen.

The role of efflux transporters in cytotoxicity and intracellular concentration of chlorpyrifos and chlorpyrifos oxon in human cell lines

In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK−2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.

Calcium Rescues Streptococcus pneumoniae D39 ΔmntE Manganese-Sensitive Growth Phenotype.

Calcium (Ca2+) functions as a universal signal messenger in eukaryotes but in bacteria, the physiological roles for Ca2+ are limited. Here, we examine the role of Ca2+ in Streptococcus pneumoniae during manganese (Mn2+) intoxication. S. pneumoniae mntE mutants, lacking the Mn2+ efflux transporter, exhibit impaired growth due to accumulation of Mn2+ when exposed to elevated exogenous Mn2+. This Mn2+-sensitive growth defect is restored to wild-type growth level by exogenous Ca2+, in a Ca2+-dependent manner. Despite growth restoration of the mntE mutant to wild-type levels, cellular Mn2+ remains elevated in this strain. Bacterial capsule production is also increased for the mntE mutant, resulting in reduced adherence capacity to surfaces and poor biofilm formation, which is consistent with it experiencing Mn2+ intoxication. Ca2+ presence did not significantly impact bacterial capsule production or biofilm formation. Further analysis of the cell morphology demonstrates that Ca2+ contributes to cell division and reduces cell chain lengths. Together, these data describe the first role of Ca in S. pneumoniae that has potential implications in bacterial virulence since Ca affects cell division and likely Mn2+-associated cellular processes.

Preclinical evaluation of targeted therapies for central nervous system metastases.

The central nervous system (CNS) represents a site of sanctuary for many metastatic tumors when systemic therapies that control the primary tumor cannot effectively penetrate intracranial lesions. Non-small cell lung cancers (NSCLCs) are the most likely of all neoplasms to metastasize to the brain, with up to 60% of patients developing CNS metastases during the disease process. Targeted therapies such as tyrosine kinase inhibitors (TKIs) have helped reduce lung cancer mortality but vary considerably in their capacity to control CNS metastases. The ability of these therapies to effectively target lesions in the CNS depends on several of their pharmacokinetic properties, including blood-brain barrier permeability, affinity for efflux transporters, and binding affinity for both plasma and brain tissue. Despite the existence of numerous preclinical models with which to characterize these properties, many targeted therapies have not been rigorously tested for CNS penetration during the discovery process, whereas some made it through preclinical testing despite poor brain penetration kinetics. Several TKIs have now been engineered with the characteristics of CNS-penetrant drugs, with clinical trials proving these efforts fruitful. This Review outlines the extent and variability of preclinical evidence for the efficacy of NSCLC-targeted therapies, which have been approved by the US Food and Drug Administration (FDA) or are in development, for treating CNS metastases, and how these data correlate with clinical outcomes.

Plant growth: The heavy matter of weight-induced plant stem thickening

As plant stems grow taller and heavier, they adapt by promoting stem thickening. A new study shows that weight-induced radial growth is mediated by the auxin efflux transporter PIN3.

Physiological and Transcriptomic Analyses Demonstrate the Ca2+-Mediated Alleviation of Salt Stress in Magnolia wufengensis.

Magnolia wufengensis, a newly discovered ornamental species in the Magnoliaceae family, is susceptible to salinity. Moreover, Ca2+ is an essential element for plant growth and is receiving increasing attention for its ability to mitigate the negative effects of environmental stress on plants. In the present study, we investigated the effect of Ca2+ on the growth and transcriptome of M. wufengensis under salt stress. The treatments used here were as follows: control, NaCl (150 mmol/L), CaCl2 (5 mmol/L), and NaCl (150 mmol/L) + CaCl2 (5 mmol/L). After a 60-day treatment period, plant growth indices were determined, and leaves were collected for physiological analysis and transcriptome investigation. The combined application of NaCl and CaCl2 alleviated phenotypic damage and restored seedling growth. Moreover, RNA sequencing data revealed that in the Na vs. control group and the NaCa vs. Na group, there were 968 and 2632 differentially expressed genes, respectively, which were both primarily enriched in secondary metabolism, glutathione metabolism, signaling hormone metabolism, glucose metabolism, and amino acid metabolism. These pathways were analyzed to screen key genes: the adenosine triphosphate (ATP)-binding cassette efflux transporter G1 (ABCG1) genes, which are related to transmembrane transport; the calmodulin genes, which are related to signal transmission; and the glutathione S-transferase (GST), glutathione peroxidase (GPX), and peroxidase (POD) genes related to antioxidant enzymes. Lastly, we constructed a hypothesis model of Ca2+-enhanced salt tolerance in M. wufengensis. This study reveals the potential mechanisms by which Ca2+ enhances the salt tolerance of M. wufengensis and provides a theoretical reference for its cultivation in saline areas.

Exploring Kp,uu,BBB values smaller than unity in remoxipride: A physiologically-based CNS model approach highlighting brain metabolism in drugs with passive blood-brain barrier transport

(Aim)Kp,uu,BBB values are crucial indicators of drug distribution into the brain, representing the steady-state relationship between unbound concentrations in plasma and in brain extracellular fluid (brainECF). Kp,uu,BBB values < 1 are often interpreted as indicators of dominant active efflux transport processes at the blood-brain barrier (BBB). However, the potential impact of brain metabolism on this value is typically not addressed. In this study, we investigated the brain distribution of remoxipride, as a paradigm compound for passive BBB transport with yet unexplained brain elimination that was hypothesized to represent brain metabolism. (Methods)The physiologically-based LeiCNS pharmacokinetic predictor (LeiCNS-PK model) was used to compare brain distribution of remoxipride with and without Michaelis-Menten kinetics at the BBB and/or brain cell organelle levels. To that end, multiple in-house (IV 0.7, 3.5, 4, 5.2, 7, 8, 14 and 16 mg kg-1) and external (IV 4 and 8 mg kg-1) rat microdialysis studies plasma and brainECF data were analysed. (Results)The incorporation of active elimination through presumed brain metabolism of remoxipride in the LeiCNS-PK model significantly improved the prediction accuracy of experimentally observed brainECF profiles of this drug. The model integrated with brain metabolism in both barriers and organelles levels is named LeiCNS-PK3.5. (Conclusion)For drugs with Kp,uu,BBB values < 1, not only the current interpretation of dominant BBB efflux transport, but also potential brain metabolism needs to be considered, especially because these may be concentration dependent. This will improve the mechanistic understanding of the processes that determine brain PK profiles.

A novel combinatory treatment against a CDDP-resistant non-small cell lung cancer based on a Ruthenium(II)-cyclopentadienyl compound

The therapeutic approach to many solid tumors, including non-small cell lung cancer (NSCLC), is mainly based on the use of platinum-containing anticancer agents and is often characterized by acquired or intrinsic resistance to the drug. Therefore, the search for safer and more effective drugs is still an open challenge.Two organometallic ruthenium(II)-cyclopentadienyl compounds [Ru(η5-C5H4CHO)(Me2bipy)(PPh3)]+ (RT150) and [Ru(η5-C5H4CH2OH)(Me2bipy)(PPh3)][CF3SO3] (RT151) were tested against a panel of cisplatin-resistant NSCLC cell lines and xenografts. They were more effective than cisplatin in inducing oxidative stress and DNA damage, affecting the cell cycle and causing apoptosis. Importantly, they were found to be inhibitors of drug efflux transporters. Due to this property, the compounds significantly increased the retention and cytotoxicity of cisplatin within NSCLC cells. Notably, they did not display high toxicity in vitro against non-transformed cells (red blood cells, fibroblasts, bronchial epithelial cells, cardiomyocytes, and endothelial cells). Both compounds induced vasorelaxation and reduced endothelial cell migration, suggesting potential anti-angiogenic properties. RT151 confirmed its efficacy against NSCLC xenografts resistant to cisplatin. Either alone or combined with low doses of cisplatin, RT151 showed a good biodistribution profile in the liver, kidney, spleen, lung, and tumor. Hematochemical analysis and post-mortem organ pathology confirmed the safety of the compound in vivo, also when combined with cisplatin.To sum up, we have confirmed the effectiveness of a novel class of drugs against cisplatin-resistant NSCLC. Additionally, the compounds have a good biocompatibility and safety profile.

Human enteroid monolayers as a potential alternative for Ussing chamber and Caco-2 monolayers to study passive permeability and drug efflux.

After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.

Molecular Determinants for Photodynamic Therapy Resistance and Improved Photosensitizer Delivery in Glioma.

Gliomas account for 24% of all the primary brain and Central Nervous System (CNS) tumors. These tumors are diverse in cellular origin, genetic profile, and morphology but collectively have one of the most dismal prognoses of all cancers. Work is constantly underway to discover a new effective form of glioma therapy. Photodynamic therapy (PDT) may be one of them. It involves the local or systemic application of a photosensitive compound-a photosensitizer (PS)-which accumulates in the affected tissues. Photosensitizer molecules absorb light of the appropriate wavelength, initiating the activation processes leading to the formation of reactive oxygen species and the selective destruction of inappropriate cells. Research focusing on the effective use of PDT in glioma therapy is already underway with promising results. In our work, we provide detailed insights into the molecular changes in glioma after photodynamic therapy. We describe a number of molecules that may contribute to the resistance of glioma cells to PDT, such as the adenosine triphosphate (ATP)-binding cassette efflux transporter G2, glutathione, ferrochelatase, heme oxygenase, and hypoxia-inducible factor 1. We identify molecular targets that can be used to improve the photosensitizer delivery to glioma cells, such as the epithelial growth factor receptor, neuropilin-1, low-density lipoprotein receptor, and neuropeptide Y receptors. We note that PDT can increase the expression of some molecules that reduce the effectiveness of therapy, such as Vascular endothelial growth factor (VEGF), glutamate, and nitric oxide. However, the scientific literature lacks clear data on the effects of PDT on many of the molecules described, and the available reports are often contradictory. In our work, we highlight the gaps in this knowledge and point to directions for further research that may enhance the efficacy of PDT in the treatment of glioma.

Cinobufagin treatments suppress tumor growth by enhancing the expression of cuproptosis-related genes in liver cancer.

Cuproptosis is a recently discovered form of regulated cell death triggered by excess copper (Cu) strongly influenced by the import, export, and intracellular utilization of Cu known as Cu homeostasis. Cinobufagin (CB) is a well-known Chinese medicine for its apoptosis-inducing role; however, its function on cuproptosis is poorly understood. To evaluate the effect of CB on inducing cell death through cuproptosis, we used RNA-seq data of HepG2-treated cells with CB to understand Cuproptosis genes. By using CCK-8 assay, Ross assay, GSH assay, and qRT-PCR, we found that CB could enhance cuproptosis in primary liver cancer cell lines, especially by increasing copper transporters CTR1, CTR2, and LIAS and downregulation of copper efflux transporters ATP7A and ATP7B resulted in increased reactive oxygen species (ROS) production, copper ionophores while reduced intracellular copper chelator glutathione (GSH) synthesis. In conclusion, our findings indicated that CB by increasing cuproptosis-related genes can mediate higher cell cytotoxicity against HepG2 and HUH7 and could provide a new insight into mechanisms of CB as an anti-tumor agent for targeting liver cancer.