Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Downregulation of topoisomerase 1 and 2 with acriflavine sensitizes bladder cancer cells to cisplatin-based chemotherapy.

BACKGROUND: Resistance to cisplatin is a major obstacle to effective treatment of bladder cancer (BC). The present study aimed to determine whether a combination of acriflavine (ACF) with cisplatin could potentiate the antitumor property of cisplatin against the BC cells. Furthermore, the molecular mechanism behind the anticancer action of ACF was considered. METHODS AND RESULTS: Two human BC cells (5637 and EJ138) contain mutated form of p53 was culture in standard condition. Cotreatment protocol (simultaneous combination of IC30 value of ACF + various dose of cisplatin for 72 h) and pretreatment protocol (pretreatment with IC15 value of ACF for 24 h + various dose of cisplatin for 48 h) was used to determine the effect of ACF on the cells' sensitivity to main drug cisplatin. To assess the mechanism of action of ACF, real-time PCR was used to evaluate mRNA levels of hypoxia-inducible factor-1α (HIF-1α), Bax, Bcl-2, topoisomerase1 (TOP1) and topoisomerase 2 (TOP2A). Combination of ACF with cisplatin either as cotreatment or opretreatment protocol could significantly reduce the IC50 values of cisplatin as compared to the IC50 of cisplatin when use as a single drug. In addition, ACF could markedly decrease mRNA expression of TOP1 and TOP2A without changing the expression of HIF-1ɑ, Bax and Bcl-2. CONCLUSIONS: Our findings indicate that combination of cisplatin with ACF was able to significantly enhance the sensitivity of BC cells to cisplatin. The antitumor activity of ACF is exerted through the downregulation of TOP1 and TOP2A genes expression. ACF may serve as an adjuvant to boost cisplatin-based chemotherapy.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro.

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. Mpro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of Mpro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against Mpro with IC50 of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 Mpro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of Mpro, and ACF has been previously reported as a PLpro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.

Plasmid stability of potential probiotic Lactobacillus plantarum strains in artificial gastric juice, at elevated temperature, and in the presence of novobiocin and acriflavine.

In this study, the presence of plasmids responsible for carbohydrate fermentation and antibiotic resistance and the stability of these plasmids in artificial gastric juice were investigated in 20 Lactobacillus plantarum strains with probiotic properties. Plasmid curing was performed with novobiocin, acriflavine and elevated incubation temperature to identify plasmids encoded with carbohydrate fermentation and antibiotic resistance genes and to compare them with artificial gastric juice. Plasmid profiling of the strains revealed that 100% of the strains were harbouring plasmids in varying sizes and numbers. The plasmid number of the potential probiotic strains ranged between 1 and 4, and the plasmid size ranged between 5.779 and 16.138 kb. The potential probiotic strains could not survive in the artificial gastric juice at pH 2.0. Although the strains maintained their viability in an artificial gastric juice at pH 2.5 and 3.0, and their derivatives lost their plasmids at a high rate (100%). Similarly, high levels of cured derivatives were obtained with 8 µg/mL novobiocin and 100 µg/mL acriflavine applications, and 24 h incubation at 43 °C. All the experiments were also performed to compare with two L. plantarum-type strains containing plasmids responsible for tetracycline and tetracycline + erythromycin resistances. Artificial gastric juice and other plasmid curing treatments caused a high-frequency loss in the antibiotic resistances of type strains. Determining plasmid stability in artificial gastric juice is a novel approach. Plasmid stability in the gastrointestinal tract is important for maintaining the plasmid-encoded probiotic properties.

Development of Inflammatory Hypoxia and Prevalence of Glycolytic Metabolism in Progressing Herpes Stromal Keratitis Lesions.

Chronic inflammation in tissues often causes the development of hypoxia. Herpes stromal keratitis (HSK) is a corneal chronic inflammatory condition that develops in response to recurrent HSV-1 infection. In this study, we investigated the development of hypoxia, the expression of hypoxia-associated glycolytic genes in HSV-1 infected corneas, and the outcome of blocking hypoxia-inducible factor (HIF) dimerization on the severity of HSK. Our results showed the development of hypoxia, an elevated expression of hypoxia-associated glycolytic genes, and an increased level of lactate in corneas with progressing HSK lesions. The magnitude of hypoxia correlated with the extent of neutrophils infiltrating the infected corneas, and the depletion of neutrophils reduced the development of hypoxia in infected corneas. Additionally, in progressing HSK lesions, nuclear localization of HIF-2α protein was detected in corneal epithelial cells, whereas HIF-1α protein stabilization was observed in infiltrating immune cells. Administration of acriflavine drug to HSV-1-infected mice inhibited nuclear accumulation of HIF-1α and HIF-2α protein in immune cell types and epithelial cells, respectively, in infected corneas. As a result, a decreased influx of CD4 T cells and nongranulocytic myeloid cells, but an increased influx of neutrophils, was noted in developing HSK lesions. Interestingly, acriflavine treatment given during the clinical disease period decreased neovascularization but increased the opacity in HSV-1-infected corneas. Taken together, the results of our study lay the foundation to dissect the role of inflammatory hypoxia and hypoxia-associated genes in the pathogenesis of HSK.

The ATP-dependent chromatin remodelling enzyme Uls1 prevents Topoisomerase II poisoning.

Topoisomerase II (Top2) is an essential enzyme that decatenates DNA via a transient Top2-DNA covalent intermediate. This intermediate can be stabilized by a class of drugs termed Top2 poisons, resulting in massive DNA damage. Thus, Top2 activity is a double-edged sword that needs to be carefully controlled to maintain genome stability. We show that Uls1, an adenosine triphosphate (ATP)-dependent chromatin remodelling (Snf2) enzyme, can alter Top2 chromatin binding and prevent Top2 poisoning in yeast. Deletion mutants of ULS1 are hypersensitive to the Top2 poison acriflavine (ACF), activating the DNA damage checkpoint. We map Uls1's Top2 interaction domain and show that this, together with its ATPase activity, is essential for Uls1 function. By performing ChIP-seq, we show that ACF leads to a general increase in Top2 binding across the genome. We map Uls1 binding sites and identify tRNA genes as key regions where Uls1 associates after ACF treatment. Importantly, the presence of Uls1 at these sites prevents ACF-dependent Top2 accumulation. Our data reveal the effect of Top2 poisons on the global Top2 binding landscape and highlights the role of Uls1 in antagonizing Top2 function. Remodelling Top2 binding is thus an important new means by which Snf2 enzymes promote genome stability.

Acriflavine-loaded solid lipid nanoparticles: preparation, physicochemical characterization, and anti-proliferative properties.

Acriflavine (ACF) is an antiseptic compound with the potential antitumor activity which is used for the fluorescent staining of RNA due to its dominant fluorescent emission at ∼515 nm. Here, solid lipid nanoparticles (SLNs) containing ACF (ACF-SLNs) were prepared and their physicochemical properties, potential geno/cytotoxicity, as well as the fluorescent properties were investigated. FITC-annexin V/PI staining and cell cycle assays were carried out to find the type of cellular death caused. Particle size analysis and SEM images revealed that spherical ACF-SLNs had a homogeneous dispersion with a mean diameter of 106 ± 5.7 nm. Drug loading (DL) of 31.25 ± 4.21 mg/mL and high encapsulation efficiency (EE%) (89.75 ± 5.44) were found. ACF-SLNs physically were relatively stable in terms of dispersion, size, and EE. The uptake study demonstrated the potential use of fluorescent ACF-SLNs in bio-distribution studies. MTT assay showed that plain ACF could induce growth inhibition of A549 cells with IC50 of 8.5, 6, and 4.5 µMol after 24, 48, and 72 hours, respectively, while ACF-SLNs had stable cytotoxic effects after 48 hours. ACF-SLNs induced remarkable apoptosis and even necrosis after 48 h. Conclusively, ACF-SLNs with acceptable physicochemical features showed increased bioimpacts after 48 h compared to plain ACF.

Acriflavine targets oncogenic STAT5 signaling in myeloid leukemia cells.

Acriflavine (ACF) is an antiseptic with anticancer properties, blocking the growth of solid and haematopoietic tumour cells. Moreover, this compound has been also shown to overcome the resistance of cancer cells to chemotherapeutic agents. ACF has been shown to target hypoxia-inducible factors (HIFs) activity, which are key effectors of hypoxia-mediated chemoresistance. In this study, we showed that ACF inhibits the growth and survival of chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cell lines in normoxic conditions. We further demonstrated that ACF down-regulates STAT5 expression in CML and AML cells but activates STAT3 in CML cells in a HIF-independent manner. In addition, we demonstrated that ACF suppresses the resistance of CML cells to tyrosine kinase inhibitors, such as imatinib. Our data suggest that the dual effect of ACF might be exploited to eradicate de novo or acquired resistance of myeloid leukaemia cells to chemotherapy.

Proflavine/acriflavine derivatives with versatile biological activities.

Proflavine derivatives are extremely interesting chemotherapeutic agents, which have shown promising pharmaceutical potential due to their wide range of biological activities. This review summarizes the current state of research into the anticancer, antimicrobial, antimalarial and antileishmanial properties of these attractive compounds. Our attention has focused on new classes of proflavine conjugates, which display significant levels of anticancer activity. Highly promising cytotoxic properties have been identified in proflavine conjugates with imidazolidinones, ureas and thioureas. In particular, proflavine-dialkyldithioureas displayed substantial cytotoxic effect against the human leukemia HL-60 cells with IC50 values from 7.2 to 34.0 µm. As well, palladium complexes with proflavine ligand have important biologic activity. The LC50 values of these complexes were significantly lower than that of cisplatin against the SK-BR-3 cell line.

Ferric Ion Driven Assembly of Catalase-like Supramolecular Photosensitizing Nanozymes for Combating Hypoxic Tumors.

A facile approach to assemble catalase-like photosensitizing nanozymes with a self-oxygen-supplying ability was developed. The process involved Fe3+ -driven self-assembly of fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids. By adding a zinc(II) phthalocyanine-based photosensitizer (ZnPc) and the hypoxia-inducible factor 1 (HIF-1) inhibitor acriflavine (ACF) during the Fe3+ -promoted self-assembly of Fmoc-protected cysteine (Fmoc-Cys), the nanovesicles Fmoc-Cys/Fe@Pc and Fmoc-Cys/Fe@Pc/ACF were prepared, which could be disassembled intracellularly. The released Fe3+ could catalyze the transformation of H2 O2 enriched in cancer cells to oxygen efficiently, thereby ameliorating the hypoxic condition and promoting the photosensitizing activity of the released ZnPc. With an additional therapeutic component, Fmoc-Cys/Fe@Pc/ACF exhibited higher in vitro and in vivo photodynamic activities than Fmoc-Cys/Fe@Pc, demonstrating the synergistic effect of ZnPc and ACF.