Discovery of a Flavonoid FM04 as a Potent Inhibitor to Reverse P-Glycoprotein-Mediated Drug Resistance in Xenografts and Improve Oral Bioavailability of Paclitaxel

Biotransformation of flavonoid dimer FD18 resulted in an active metabolite FM04. It was more druggable because of its improved physicochemical properties. FM04 (EC₅₀ = 83 nM) was 1.8-fold more potent than FD18 in reversing P-glycoprotein (P-gp)-mediated paclitaxel (PTX) resistance in vitro. Similar to FD18, FM04 chemosensitized LCC6MDR cells towards multiple anticancer drugs by inhibiting the transport activity of P-gp and restoring intracellular drug levels. It stimulated the P-gp ATPase by 3.3-fold at 100 μM. Different from FD18, FM04 itself was not a transport substrate of P-gp and presumably, it cannot work as a competitive inhibitor. In the human melanoma MDA435/LCC6MDR xenograft, the co-administration of FM04 (28 mg/kg, I.P.) with PTX (12 mg/kg, I.V.) directly modulated P-gp-mediated PTX resistance and caused a 56% (*, p < 0.05) reduction in tumor volume without toxicity or animal death. When FM04 was administered orally at 45 mg/kg as a dual inhibitor of P-gp/CYP2C8 or 3A4 enzymes in the intestine, it increased the intestinal absorption of PTX from 0.2% to 14% in mice and caused about 57- to 66-fold improvement of AUC as compared to a single oral dose of PTX. Oral co-administration of FM04 (45 mg/kg) with PTX (40, 60 or 70 mg/kg) suppressed the human melanoma MDA435/LCC6 tumor growth with at least a 73% (***, p < 0.001) reduction in tumor volume without serious toxicity. Therefore, FM04 can be developed into a novel combination chemotherapy to treat cancer by directly targeting the P-gp overexpressed tumors or potentiating the oral bioavailability of P-gp substrate drugs.     

Stereoselective Modulation of P‐Glycoprotein by Chiral Small Molecules

Inhibition of drug efflux pumps such as P‐glycoprotein (P‐gp) is an approach toward combating multidrug resistance, which is a significant hurdle in current cancer treatments. To address this, N‐substituted aryloxymethyl pyrrolidines were designed and synthesized in their homochiral forms in order to investigate the stereochemical requirements for the binding site of P‐gp. Our study provides evidence that the chiral property of molecules could be a strategy for improving the capacity for interacting with P‐gp, as the most active compounds of the series stereoselectively modulated this efflux pump. The naphthalene‐1‐yl analogue (R)‐2‐[(2,3‐dichlorophenoxy)methyl]‐1‐(naphthalen‐1‐ylmethyl)pyrrolidine) [(R)‐ 7 a ] emerged foremost for its potency and stereoselectivity toward P‐gp, with the S enantiomer being nearly inactive. The modulation of P‐gp by (R)‐ 7 a involved consumption of ATP, thus demonstrating that the compound behaves as a P‐gp substrate.     

The Role of Metalloproteinases and Their Tissue Inhibitors on Ocular Diseases: Focusing on Potential Mechanisms

Eye diseases are associated with visual impairment, reduced quality of life, and may even lead to vision loss. The efficacy of available treatment of eye diseases is not satisfactory. The unique environment of the eye related to anatomical and physiological barriers and constraints limits the bioavailability of existing agents. In turn, complex ethiopathogenesis of ocular disorders that used drugs generally are non-disease specific and do not act causally. Therefore, there is a need for the development of a new therapeutic and preventive approach. It seems that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have a significant role in the development and progression of eye diseases and could be used in the therapy of these disorders as pharmacological targets. MMPs and TIMPs play an important role in the angiogenesis, epithelial-mesenchymal transition, cell invasion, and migration, which occur in ocular diseases. In this review, we aim to describe the participation of MMPs and TIMPs in the eye diseases, such as age-related macular degeneration, cataract, diabetic retinopathy, dry eye syndrome, glaucoma, and ocular cancers, posterior capsule opacification focusing on potential mechanisms.     

S-Adenosylmethionine Increases the Sensitivity of Human Colorectal Cancer Cells to 5-Fluorouracil by Inhibiting P-Glycoprotein Expression and NF-κB Activation

Colorectal cancer (CRC) is the second deadliest cancer worldwide despite significant advances in both diagnosis and therapy. The high incidence of CRC and its poor prognosis, partially attributed to multi-drug resistance and antiapoptotic activity of cancer cells, arouse strong interest in the identification and development of new treatments. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and proapoptotic effects as well as for its potential in overcoming drug resistance in many kinds of human tumors. Here, we report that AdoMet enhanced the antitumor activity of 5-Fluorouracil (5-FU) in HCT 116^p53+/+ and in LoVo CRC cells through the inhibition of autophagy, induced by 5-FU as a cell defense mechanism to escape the drug cytotoxicity. Multiple drug resistance is mainly due to the overexpression of drug efflux pumps, such as P-glycoprotein (P-gp). We demonstrate here that AdoMet was able to revert the 5-FU-induced upregulation of P-gp expression and to decrease levels of acetylated NF-κB, the activated form of NF-κB, the major antiapoptotic factor involved in P-gp-related chemoresistance. Overall, our data show that AdoMet, was able to overcome 5-FU chemoresistance in CRC cells by targeting multiple pathways such as autophagy, P-gp expression, and NF-κB signaling activation and provided important implications for the development of new adjuvant therapies to improve CRC treatment and patient outcomes.     

5-Arylidenerhodanines as P-gp Modulators: An Interesting Effect of the Carboxyl Group on ABCB1 Function in Multidrug-Resistant Cancer Cells

Multidrug resistance (MDR) is considered one of the major mechanisms responsible for the failure of numerous anticancer and antiviral chemotherapies. Various strategies to overcome the MDR phenomenon have been developed, and one of the most attractive research directions is focused on the inhibition of MDR transporters, membrane proteins that extrude cytotoxic drugs from living cells. Here, we report the results of our studies on a series newly synthesized of 5-arylidenerhodanines and their ability to inhibit the ABCB1 efflux pump in mouse T-lymphoma cancer cells. In the series, compounds possessing a triphenylamine moiety and the carboxyl group in their structure were of particular interest. These amphiphilic compounds showed over 17-fold stronger efflux pump inhibitory effects than verapamil. The cytotoxic and antiproliferative effects of target rhodanines on T-lymphoma cells were also investigated. A putative binding mode for 11, one of the most potent P-gp inhibitors tested here, was predicted by molecular docking studies and discussed with regard to the binding mode of verapamil.     

A Potential Daidzein Derivative Enhances Cytotoxicity of Epirubicin on Human Colon Adenocarcinoma Caco-2 Cells

In this study, we evaluated the effects of 8-hydroxydaidzein (8HD), an isoflavone isolated from fermented soy germ koji, and epirubicin (Epi), an antineoplastic agent, on the production of reactive oxygen species (ROS). We subsequently correlated the ROS levels to the anticancer mechanisms of Epi and 8HD in human colon adenocarcinoma Caco-2 cells. 8HD enhanced cytotoxicity of Epi and generated a synergistic effect. Epi and/or 8HD treatments increased the hydrogen peroxide and superoxide levels. Combined treatment markedly decreased mRNA expression levels of multidrug resistance protein 1 (MDR1), MDR-associated protein (MRP) 1, and MRP2. 8HD significantly intensified Epi intracellular accumulation in Caco-2 cells. 8HD and/or Epi-induced apoptosis, as indicated by the reduced mitochondrial membrane potential and increased sub-G1 phase in cell cycle. Moreover, 8HD and Epi significantly enhanced the mRNA expressions of Bax, p53, caspases-3, -8, and -9. To our best knowledge, this study verifies for the first time that 8HD effectively circumvents MDR in Caco-2 cells through the ROS-dependent inhibition of efflux transporters and p53-mediated activation of both death receptor and mitochondrial pathways of apoptosis. Our findings of 8HD shed light on the future search for potential biotransformed isoflavones to intensify the cytotoxicity of anticancer drugs through simultaneous reversal of pump and nonpump resistance.     

MMPs and TIMPs Expression Levels in the Periodontal Ligament during Orthodontic Tooth Movement: A Systematic Review of In Vitro and In Vivo Studies

Background: During orthodontic tooth movement (OTM), applied orthodontic forces cause an extensive remodeling of the extracellular matrix (ECM) in the periodontal ligament (PDL). This is mainly orchestrated by different types of matrix metalloproteinases (MMPs) and their tissue inhibitors of matrix metalloproteinases (TIMPs), which are both secreted by periodontal ligament (PDL) fibroblasts. Multiple in vitro and in vivo studies already investigated the influence of applied orthodontic forces on the expression of MMPs and TIMPs. The aim of this systematic review was to explore the expression levels of MMPs and TIMPs during OTM and the influence of specific orthodontic force-related parameters. Methods: Electronic article search was performed on PubMed and Web of Science until 31 January 2021. Screenings of titles, abstracts and full texts were performed according to PRISMA, whereas eligibility criteria were defined for in vitro and in vivo studies, respectively, according to the PICO schema. Risk of bias assessment for in vitro studies was verified by specific methodological and reporting criteria. For in vivo studies, risk of bias assessment was adapted from the Joanna Briggs Institute Critical Appraisal Checklist for analytical cross-sectional study. Results: Electronic article search identified 3266 records, from which 28 in vitro and 12 in vivo studies were included. The studies showed that orthodontic forces mainly caused increased MMPs and TIMPs expression levels, whereas the exact effect may depend on various intervention and sample parameters and subject characteristics. Conclusion: This systematic review revealed that orthodontic forces induce a significant effect on MMPs and TIMPs in the PDL. This connection may contribute to the controlled depletion and formation of the PDLs’ ECM at the compression and tension site, respectively, and finally to the highly regulated OTM.     

Semisynthesis and screening of a small library of pro-apoptotic squamocin analogues: selection and study of a benzoquinone hybrid with an improved biological profile

Acetogenins of Annonaceae, including squamocin (1), exert spectacular cytotoxicity and the most potent inhibition of NADH:ubiquinone oxidoreductase known so far. Cell death induced by these natural products was identified as apoptosis and was thought to be linked to alterations in mitochondrial function. Quinone-squamocin hybrid compounds were semisynthesized and evaluated for their pro-apoptotic properties with a screening method based on dissipation of the mitochondrial transmembrane potential (DeltaPsim). Herein, we report a short one-step synthesis of a squamocin carboxylic acid analogue. For the first time on a natural product, the radical decarboxylation and quinone addition reaction has enabled preparation of a library of squamocin-quinone hybrids and four other analogues. Squamoquinone, tenfold more potent than squamocin as an inducer of apoptosis, emerged as a promising compound, as it induces apoptosis through a mitochondrial caspase-dependent pathway.     

beta'-Hydroxy-alpha,beta-unsaturated ketones: A new pharmacophore for the design of anticancer drugs. Part 2

Novel antiproliferative beta'-acyloxy-alpha,beta-unsaturated ketones were obtained by means of an iron(III)-catalyzed multicomponent domino process (ABB' 3CR). The most active derivatives displayed GI(50) values in the range of 0.5-3.9 muM against a panel of representative human solid tumor cell lines: A2780, SW1573, HBL-100, T-47D and WiDr. Analysis of cells following 24 h exposure to these drugs showed cell cycle arrest in the S and G(2)/M phase, in a dose-dependent manner. Our data indicate that the beta'-acyloxy-alpha,beta-unsaturated ketones cause permanent damage to the cells and induce apoptosis.     

Study of the anticancer properties of tin(IV) carboxylate complexes on a panel of human tumor cell lines

A group of organotin(IV) complexes were prepared: [SnCy₃(DMNI)] ( 1 ), [SnCy₃(BZDO)] ( 2 ), [SnCy₃(DMFU)] ( 3 ), and [SnPh₂(BZDO)₂] ( 4 ), for which DMNIH=2,6‐dimethoxynicotinic acid, BZDOH=1,4‐benzodioxane‐6‐carboxylic acid, and DMFUH=2,5‐dimethyl‐3‐furoic acid. The cytotoxic activities of compounds 1 – 4 were tested against pancreatic carcinoma (PANC‐1), erythroleukemia (K562), and two glioblastoma multiform (U87 and LN‐229) human cell lines; they show very high antiproliferative activity, with IC₅₀ values in the 150–700 nM range after incubation for 72 h. Distribution of cellular DNA upon treatment with 1 – 4 revealed that whereas compounds 1 – 3 induce apoptosis in most of the cell lines, compound 4 does not affect cell viability in any cell line tested, indicating a possible difference in cytotoxic mechanism. Studies with the daunomycin‐resistant K562/R cell line expressing P‐glycoprotein (Pgp) showed that compounds 1 – 4 are not substrates of this protein efflux pump, indicating that these compounds do not induce acquisition of multidrug resistance, which is associated with the overexpression of Pgp.     

3-substituted 2-phenylimidazo[2,1-b]benzothiazoles: synthesis, anticancer activity, and inhibition of tubulin polymerization

A new series of 3‐substituted 2‐phenylimidazo[2,1‐b]benzothiazoles ( 3 a – h ) were synthesized by C‐arylation of 2‐arylimidazo[2,1‐b]benzothiazoles using palladium acetate as catalyst, and the resulting compounds were evaluated for their anticancer activity. Compounds 3 a , 3 e , and 3 h exhibited good antiproliferative activity, with GI₅₀ values in the range of 0.19–83.1 μM . Compound 3 h showed potent anticancer efficacy against 60 human cancer cell lines, with a mean GI₅₀ value of 0.88 μM . This compound also induced cell‐cycle arrest in the G₂/M phase and inhibited tubulin polymerization followed by activation of caspase‐3 and apoptosis. A high‐throughput tubulin polymerization assay showed that the level of inhibition for compound 3 h is similar to that of combretastatin A‐4. Molecular modeling studies provided a molecular basis for the favorable binding of compounds 3 a , 3 e , and 3 h to the colchicine binding pocket of tubulin.     

Synthesis and biological activities of a 3'-azido analogue of Doxorubicin against drug-resistant cancer cells

Doxorubicin (DOX), an anthracycline antibiotic, is one of the most active anticancer chemotherapeutic agents. The clinical use of DOX, however, is limited by the dose-dependant P-glycoprotein (P-gp)-mediated resistance. Herein, a 3'-azido analogue of DOX (ADOX) was prepared from daunorubicin (DNR). ADOX exhibited potent antitumor activities in drug-sensitive (MCF-7 and K562) and drug-resistant cell lines (MCF-7/DNR, K562/DOX), respectively. The drug resistance index (DRI) values of ADOX were much lower than that of DOX. The cytotoxicity experiments of ADOX or DOX against K562/DOX, with or without P-gp inhibitor, indicated that ADOX circumvents resistance by abolishing the P-gp recognition. This conclusion was further supported by drug influx/efflux flow cytometry experiments, as well as by molecular docking of ADOX to P-gp. In vivo animal tests, ADOX exhibited higher activity and less toxicity than DOX. The current data warranted ADOX for additional pre-clinical evaluations for new drug development.     

The Use of a Liposomal Formulation Incorporating an Antimicrobial Peptide from Tilapia as a New Adjuvant to Epirubicin in Human Squamous Cell Carcinoma and Pluripotent Testicular Embryonic Carcinoma Cells

This study aims to explore the effects and mechanisms of hepcidin, a potential antimicrobial peptide from Tilapia, and epirubicin (Epi), an antineoplastic agent, on the generation of reactive oxygen species (ROS) and link the ROS levels to the reversal mechanisms of multidrug resistance (MDR) by epirubicin and hepcidin in human squamous cell carcinoma SCC15 and human embryonal carcinoma NT2D1 cells. The cells, pretreated with hepcidin, epirubicin, or a combination of these compounds in PEGylated liposomes, were used to validate the molecular mechanisms involved in inhibiting efflux transporters and inducing apoptosis as evaluated by cytotoxicity, intracellular accumulation, mRNA levels, cell cycle distribution, and caspase activity of this combination. We found that hepcidin significantly enhanced the cytotoxicity of epirubicin in liposomes. The co-incubation of epirubicin with hepcidin in liposomes intensified the ROS production, including hydrogen peroxide and superoxide free radicals. Hepcidin significantly increased epirubicin intracellular uptake into NT2D1 and SCC15 cells, as supported by the diminished mRNA expressions of MDR1, MDR-associated protein (MRP) 1, and MRP2. Hepcidin and/or epirubicin in liposomes triggered apoptosis, as verified by the reduced mitochondrial membrane potential, increased sub-G1 phase of cell cycle, incremental populations of apoptosis using annexin V/PI assay, and chromatin condensation. As far as we know, this is the first example showing that PEGylated liposomal TH1-5 and epirubicin gives rise to cell death in human squamous carcinoma and testicular embryonic carcinoma cells through the reduced epirubicin efflux via ROS-mediated suppression of P-gp and MRPs and concomitant initiation of mitochondrial apoptosis pathway. Hence, hepcidin in PEGylated liposomes may function as an adjuvant to anticancer drugs, thus demonstrating a novel strategy for reversing MDR.     

Theories and Molecular Basis of Vascular Aging: A Review of the Literature from VascAgeNet Group on Pathophysiological Mechanisms of Vascular Aging

Vascular aging, characterized by structural and functional alterations of the vascular wall, is a hallmark of aging and is tightly related to the development of cardiovascular mortality and age-associated vascular pathologies. Over the last years, extensive and ongoing research has highlighted several sophisticated molecular mechanisms that are involved in the pathophysiology of vascular aging. A more thorough understanding of these mechanisms could help to provide a new insight into the complex biology of this non-reversible vascular process and direct future interventions to improve longevity. In this review, we discuss the role of the most important molecular pathways involved in vascular ageing including oxidative stress, vascular inflammation, extracellular matrix metalloproteinases activity, epigenetic regulation, telomere shortening, senescence and autophagy.     

Incorporation of EGCG into an etch-and-rinse adhesive system: mechanical properties and bond strength to caries affected dentin

This study evaluated how a matrix metalloproteinase (MMP) inhibitor (EGCG) incorporated into an etch-and-rinse adhesive system (AS) affects the long-term microtensile bond strength (MTBS) to caries affected dentin (CAD) and flexural strength (FS) of AS. Forty CAD surfaces received acid conditioning and were randomly divided into four groups (n?=?10): EGCG-PRE – dentin treatment with 200?μg/mL EGCG solution; EGCG-INC – 200?μg/mL EGCG solution incorporated into SA; CHX-PRE – dentin treatment with 2% digluconate solution; NT – no treatment. The AS (Adper Single Bond 2, 3M ESPE) was applied and resin composite blocks were built on the dentin. Beam-shaped specimens (0.8?mm²) were obtained submitted to MTBS testing following 24?h, 6 months or 1 year of water storage (WS). Adhesive interface was analyzed by SEM. FS testing was performed by preparing specimens with the adhesive system, with or without the EGCG solution (200?μg/mL) (n?=?10). Tests were carried out in a universal machine (0.5?mm/min). MTBS data were submitted to two-way ANOVA and Tukey’s test, whereas FS data, to the Student’s t-test (α?=?0.05). MTBS values were not affected by EGCG application, either incorporated or used as a dentin treatment agent, regardless of the WS period; however, CHX negatively affected MTBS to CAD (p?<?0.001). WS significantly reduced MTBS values (p?<?0.001) from the 6-months time point. FS was not affected by EGCG incorporation into the SA (p?=?0.2527). EGCG incorporation into AS was unable to maintain bond stability to CAD over time, whereas it did not compromise adhesive’s property.     

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.     

1,4-Naphthoquinone (CNN1) Induces Apoptosis through DNA Damage and Promotes Upregulation of H2AFX in Leukemia Multidrug Resistant Cell Line

The multidrug resistance (MDR) phenotype is one of the major obstacles in the treatment of chronic myeloid leukemia (CML) in advantage stages such as blast crisis. In this scenario, more patients develop resistance mechanisms during the course of the disease, making tyrosine kinase inhibitors (TKIs) target therapies ineffective. Therefore, the aim of the study was to examine the pharmacological role of CNN1, a para-naphthoquinone, in a leukemia multidrug resistant cell line. First, the in vitro cytotoxic activity of Imatinib Mesylate (IM) in K-562 and FEPS cell lines was evaluated. Subsequently, membrane integrity and mitochondrial membrane potential assays were performed to assess the cytotoxic effects of CNN1 in K-562 and FEPS cell lines, followed by cell cycle, alkaline comet assay and annexin V-Alexa Fluor^® 488/propidium iodide assays (Annexin/PI) using flow cytometry. RT-qPCR was used to evaluate the H2AFX gene expression. The results demonstrate that CNN1 was able to induce apoptosis, cell membrane rupture and mitochondrial membrane depolarization in leukemia cell lines. In addition, CNN1 also induced genotoxic effects and caused DNA fragmentation, cell cycle arrest at the G2/M phase in leukemia cells. No genotoxicity was observed on peripheral blood mononuclear cells (PBMC). Additionally, CNN1 increased mRNA levels of H2AFX. Therefore, CNN1 presented anticancer properties against leukemia multidrug resistant cell line being a potential anticancer agent for the treatment of resistant CML.     

Acryloylphenylcarboxamides: A New Class of Breast Cancer Resistance Protein (ABCG2) Modulators

Chalcones are easily synthesized natural precursors of secondary plant metabolites, and their derivatives show various biological activities including inhibition of ABC transporters. Especially, their role as inhibitors of ABCG2, the most recently discovered ABC transporter involved in multidrug resistance, inspired the synthesis of new structurally diverse derivatives. Therefore, we combined the typical chalcone moiety with several acid chlorides by using an amide linker at position 2′, 3′, or 4′ on ring A of the chalcone. The resulting 35 compounds covered a wide spectrum of substitution patterns, which allowed development of structure–activity relationships and to find the optimal structural features for further investigations. Synthesized acryloylphenylcarboxamides were investigated for their inhibitory activity against ABCG2 and their behavior toward ABCB1 and ABCC1. Furthermore, for the most promising compounds, their intrinsic cytotoxicity and their ability to reverse ABCG2‐mediated multidrug resistance were determined.     

The Potential of Antibiotics and Nanomaterial Combinations as Therapeutic Strategies in the Management of Multidrug-Resistant Infections: A Review

Antibiotic resistance has become a major public health concern around the world. This is exacerbated by the non-discovery of novel drugs, the development of resistance mechanisms in most of the clinical isolates of bacteria, as well as recurring infections, hindering disease treatment efficacy. In vitro data has shown that antibiotic combinations can be effective when microorganisms are resistant to individual drugs. Recently, advances in the direction of combination therapy for the treatment of multidrug-resistant (MDR) bacterial infections have embraced antibiotic combinations and the use of nanoparticles conjugated with antibiotics. Nanoparticles (NPs) can penetrate the cellular membrane of disease-causing organisms and obstruct essential molecular pathways, showing unique antibacterial mechanisms. Combined with the optimal drugs, NPs have established synergy and may assist in regulating the general threat of emergent bacterial resistance. This review comprises a general overview of antibiotic combinations strategies for the treatment of microbial infections. The potential of antibiotic combinations with NPs as new entrants in the antimicrobial therapy domain is discussed.