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Effect of apigenin dimer 9d on pentamidine accumulation in Leishmania parasites. (a) Determination of pentamidine accumulation. One millilitre of 4-day-old promastigotes (Le wild-type and LePentR50) at a cell density of 2Â10 7 cells/mL was used for the pentamidine accumulation assay. The cells were washed with PBS and then incubated at 278C for 3 h with increasing concentrations of 9d (0, 15, 30 and 60 mM). Each concentration of 9d was tested in triplicate and repeated twice in separate experiments. The amount of pentamidine accumulated was determined by HPLC and is presented as mM pentamidine per mg of protein (mean+SEM, n¼2). #Dose-dependent pentamidine accumulation in LePentR50 has been reported previously 34 and is included in this figure for comparison. (b) Cytotoxic effect of 9d on Leishmania parasites. Promastigotes were treated as in (a). Each concentration of 9d was tested in triplicate and repeated twice in separate experiments. The percentage of survivors was determined by MTS proliferation assay (mean+SEM, n ¼2).
Source publication
The aim of this study was to investigate the synergistic effect of quinacrine and a novel apigenin dimer (compound 9d) on reversing pentamidine resistance of Leishmania parasites.
Pentamidine-resistant cell lines, LePentR50 and LdAG83PentR50, were generated by gradually increasing pentamidine pressure on wild-type promastigotes. We tested the effec...
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... higher concentration was used because a shorter incu- bation time of 3 h was used in the accumulation assay. We found that 9d can increase pentamidine accumulation in both Le wild-type and LePentR50 in a dose-dependent manner (Figure 4a). Treatment of Le wild-type with 60 mM 9d can increase pentamidine accumulation from 2.5 to 27 mM/mg of protein (11-fold increase). ...
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... of Le wild-type with 60 mM 9d can increase pentamidine accumulation from 2.5 to 27 mM/mg of protein (11-fold increase). We have reported similar results for LePentR50 where 60 mM 9d can increase pentamidine accumu- lation of LePentR50 cells from 2.0 to 27 mM/mg of protein (14-fold increase) (Figure 4a). 34 This result suggests that 9d may be modulating a pentamidine efflux transporter in both Le and LePentR50. ...
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... This result suggests that 9d may be modulating a pentamidine efflux transporter in both Le and LePentR50. Incubation of Le wild-type and LePentR50 with the same concentrations of 9d for the same period of time did not result in any significant cytotoxicity (Figure 4b). The dose- dependent increase in pentamidine accumulation was, therefore, due to the modulatory effect of 9d, and not due to a cytotoxic effect on Le wild-type or LePentR50. ...
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... found that quinacrine did not increase pentamidine accumulation (Figure 3a). Interestingly, apigenin dimer 9d can sensitize both Le wild-type and LePentR50 by increasing the intracellular accumulation of pentamidine in a dose-dependent manner (Figure 4a) Figure 5. Cytotoxicity of quinacrine and apigenin dimer 9d combination towards LePentR50. Effects of combinations of quinacrine and 9d on the cytotoxicity towards LePentR50 were tested by MTS proliferation assay. ...
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... 9d can sensitize both Le and LePentR50 to pentamidine to a similar extent (3.8-and 3.4-fold, respectively, for Le wild-type and LePentR50; Table 1). This same level of sensitization was reflected in the pentamidine accumulation (Figure 4a). These results suggest that the target of 9d, presumably an ABC trans- porter, is present at the same level in both Le and LePentR50. ...
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Citations
... TR degrades trypanothione, which is employed for neutralizing ROS generated by macrophages during infection. 102 In contrast to glutathione (GSH), which is the principal redox defense molecule in mammals, trypanothione is the primary detoxifying pathway for oxidative damage in Trypanosomatid parasites. Pentavalent antimonials are considered prodrugs because their in vivo transformation results in the generation of active as well as toxic trivalent antimonials Sb(III), which causes Leishmania apoptotic death. ...
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... Furthermore in glycosylation, in several plants, API forms dimer molecules to form bioflavonoids and other diverse structural groups. The greatest studied API dimer of pharmacological importance is amentoflavone (3, which is established in recognized medicinal herbs such as St John's wort [61], ginko (Ginkgo biloba L.) [62], and spike mosses [63]. AP is synthesized in the cytoplasmic surface of the endoplasmic reticulum and the reaction is catalyzed through a class of enzymes [64]. ...
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... This first isolated strain of the parasite was distributed to laboratories worldwide and today sold by ATCC ® Number: 50120 ™ . Leishmania enriettii has been used as a model organism to several studies such as experimental infections, cell mediated immune studies, intracellular Leishmania survival studies, as a model to study the dissemination of cutaneous leishmaniosis, disease treatment and more recently to genome of the parasite ( [3][4][5][6][7][8][9][10][11][12][13][14] to cite a few). ...
Abstract Leishmania enriettii has only been found in Curitiba metropolitan region, southern Brazil were it was first observed in Cavia porcellus from the vivarium of Instituto de Biologia e Pesquisas Tecnológicas (IBPT - today named TECPAR) by Medina, 1944. Despite more than a half century from its discovery and several research articles on this species, the natural clinical signs in guinea pigs and the parasite genetic variability is still unclear. The aims of this study were to describe the clinical features, investigate the potential wild reservoirs and, in addition, we intended to understand the polymorphism trait of the species. We analyzed 26 naturally infected guinea pigs from eight Paraná state cities. All animals showed lesions compatible with leishmaniosis, such as skin nodules or ulcers on body extremities. Direct examination of the lesion samples obtained by fine-needle aspiration or punch biopsy was conducted followed by isolation and identification of parasite DNA by random amplification of polymorphic DNA (RAPD)-PCR. Through the direct exam, a large number of intracellular amastigote forms were observed in the lesions. Different strains of the parasite, isolated from the 26 animals, were grouped in 5 clusters of approximately 65% similarity. We looked for L. enriettii in other potential reservoir hosts but the parasite was not observed. These results confirm that distinct strains of L. enriettii circulate in guinea pigs from Paraná state, more specifically in the Atlantic forest region, where we believe it serves as the center for dispersion of the species.
... To overcome the resistance, some flavonoid dimers were synthesized which remarkably reversed the resistance process through increased accumulation of drugs in mitochondria of L. enriettii. The flavonoid dimers, along with quinacrine, showed a synergistic effect on reversing the resistance of PMD in Leishmania (Wong et al., 2009). ...
Parasitic infections have enormous health, social, and economic influence worldwide, predominantly in tropical and subtropical areas. Diseases caused by protozoan parasites, like malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, giardiasis, etc. have major contributions in the global death rate due to infections. The burden of these parasitic protozoans has increased around the globe due to the lack of effective, commercially-availablevaccines, and the production of less efficacious drugs with poor pharmacokinetics. Unfortunately, in areas with endemic protozoan infections, antiparasitic drugs use and efficacy have been compromised by drug resistance. The development of resistance has been found to be associated with decreased drug uptake, the export of drugs from parasites, genetic modifications, loss of drug activity, and alteration of the drug target. Recently, the isolation and characterization of resistance-related genes and proteins have increased the knowledge about drug resistance greatly and provided a way forward for the identification of new drug candidates. In this chapter, our focus will be on the mode of action and mechanism of resistance development of those drugs used to treat protozoal infections in human including malaria, toxoplasmosis, trypanosomiasis, giardiasis, cystoisosporiasis, babesiosis, and leishmaniasis.
... Chemical synthesis. Compounds 5,6,9,10,11,12,14 to 22,25 to 34, and 49-hydroxyflavone were prepared according to reported procedure (16,17,(34)(35)(36)(37). Nuclear magnetic resonance (NMR) spectra were recorded by Bruker MHz DPX400 spectrometer at 400.13 MHz for 1 H and 100.62 MHz for 13 C. ...
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FM09h
, was highly active with IC50 of 0.3 μM against L. amazonensis, L. tropica and L. braziliensis amastigotes. It was metabolically stable (39% and 66% of
FM09h
remaining after 30-minute incubation with human and rat liver microsomes respectively). In L. amazonensis LV78 cutaneous leishmaniasis mouse model, intralesional injection of
FM09h
(10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).
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Microbes broadly constitute several organisms like viruses, protozoa, bacteria, and fungi present in our biosphere. Fast-paced environmental changes have influenced contact of human populations with newly identified microbes resulting in diseases that can spread quickly. These microbes can cause infections like HIV, SARS-CoV2, malaria, nosocomial Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), or Candida infection for which there are no available vaccines/drugs or are less efficient to prevent or treat these infections. In the pursuit to find potential safe agents for therapy of microbial infections, natural biflavonoids like amentoflavone, tetrahydroamentoflavone, ginkgetin, bilobetin, morelloflavone, agathisflavone, hinokiflavone, Garcinia biflavones 1 (GB1), Garcinia biflavones 2 (GB2), robustaflavone, strychnobiflavone, ochnaflavone, dulcisbiflavonoid C, tetramethoxy-6,6′′-bigenkwanin and other derivatives isolated from several species of plants can provide effective starting points and become a source of future drugs. These biflavonoids show activity against influenza, severe acute respiratory syndrome (SARS), dengue, HIV-AIDS, coxsackieviral, hepatitis, HSV, Epstein-Barr virus (EBV), protozoal (Leishmaniasis, Malaria) infections, bacterial and fungal infections. Some of the biflavonoids can provide antiviral and protozoal activity by inhibition of neuraminidase, chymotrypsin-like protease, DV‑NS5 RNA dependant RNA polymerase, reverse transcriptase (RT), fatty acid synthase, DNA polymerase, UL54 gene expression, Epstein−Barr virus early antigen activation, recombinant cysteine protease type 2.8 (r-CPB2.8), Plasmodium falciparum enoyl-acyl carrier protein (ACP) reductase or cause depolarization of parasitic mitochondrial membranes. They may also provide anti-inflammatory therapeutic activity against the infection-induced cytokine storm. Considering the varied bioactivity of these biflavonoids against these organisms, their structure-activity relationships are derived and wherever possible compared with monoflavones. Overall, this review aims to highlight these natural biflavonoids and briefly discuss their sources, reported mechanism of action, pharmacological uses, and comment on resistance mechanism, flavopiridol repurposing and the bioavailability aspects to provide a starting point for anti-microbial research in this area.
... These synthetic flavonoids are reversal agents for overcoming PMD resistance in parasite Leishmania. The same dimers showed synergistic effect with quinacrine on reversing PMD resistance of Leishmania parasites [92,93]. ...
Protozoan diseases continue to be a worldwide social and economic health problem. Increased drug resistance, emerging cross resistance, and lack of new drugs with novel mechanisms of action significantly reduce the effectiveness of current antiprotozoal therapies. While drug resistance associated to anti-infective agents is a reality, society seems to remain unaware of its proportions and consequences. Parasites usually develops ingenious and innovative mechanisms to achieve drug resistance, which requires more research and investment to fight it. In this review, drug resistance developed by protozoan parasites Plasmodium, Leishmania, and Trypanosoma will be discussed.
... Mouse peritoneal elicited macrophages (PEMs) were obtained as 314 previously described [24]. PEM cells were seeded into a 96-well flat bottom 315 microtiter plate at 2 × 10 4 cells/well in 100 μL DMEM supplemented with 10% 316 Cry3Aa-DS1 on LV78 and HU3 amastigotes were calculated using Prism 333 GraphPad 6.0 software. ...
Antimicrobial peptides (AMPs) have recently attracted great attention due to their rapid action, broad spectrum
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intracellular infections, however, remains a challenge because of their low penetration efciency into the pathogen's intracellular niche. Herein, we report that sub-micrometer-sized crystals of the protein Cry3Aa formed
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... A rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes was also observed upon treatment of these parasites with the drug [111]. Pentamidine may also disrupt mitochondrial activity by interfering with mitochondrial DNA topoisomerase II, much as antimonials do to nuclear DNA [112]. ...
Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. Pentavalent antimonials are currently the first line of defense for the treatment of both visceral (VL) and cutaneous leishmaniasis (CL). However, clinical resistance to this class of drug is a major impediment to treatment. Amphotericin B, pentamidine, and miltefosine offer significant promise in the treatment of VL and CL, including antimony-resistant cases. Several other drugs, for example, allopurinol, atovaquone, fluconazole, paromomycin, and sitamaquine, are in various stages of clinical trials. Application of many of these drugs has resulted in the development of either clinical- or laboratory-induced drug resistance. Understanding the mechanisms of resistance is important for the development of newer generation of drugs to provide better treatment of the disease.
... The mitochondrial apoptosis pathway is considered to be the major target by which anti-cancer drugs induce apoptosis in many cancer cells, causing DNA damage, influencing the proteolysis or dephosphorylation of apoptosis-promoting proteins, activating caspases, and leading to the characteristic changes of apoptosis. 28 In this study, Hoechst 33258 staining revealed the characteristic changes of apoptosis in the nucleus of GSCs after miR-423-5p inhibitor + apigenin treatment. The anti-apoptotic protein of Bcl-2 was detected and found to be decreased, whereas the pro-apoptotic protein Bax was increased with the combined treatment. ...
This study aimed to investigate the effect of miR-423-5p on the sensitivity of glioma stem cells to apigenin and to explore the potential mechanism. Previous research indicated that apigenin can effectively inhibit the proliferation of many cancer cells, including glioma cells, though our data unexpectedly showed that apigenin had no effect on glioma stem cell apoptosis. As many studies have reported that malignant transformation and progression of glioma are due to glioma stem cells, an anti-glioma stem cell approach has become an important direction for glioma treatment. In this study, we found miR-423-5p to be overexpressed in glioma tissues and corresponding glioma stem cells. Downregulation of miR-423-5p repressed glioma stem cell growth but did not cause apoptosis. Based on the concept of “Pharmaco-miR,” this study further demonstrated that the combination of miR-423-5p knockdown and apigenin had a notable additive effect on inhibiting proliferation and promoting apoptosis in glioma stem cells. Hoechst staining showed higher apoptosis rates and typical apoptotic morphological changes of the cell nucleus, and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocya-nine iodide) staining revealed reduced mitochondrial membrane potential. Further research demonstrated that the mechanism is associated with a shift in the Bax/Bcl-2 ratio, an increased cytochrome c level, Apaf-1 induction, and caspase-3 activation. In conclusion, this study indicates that downregulation of miR-423-5p enhances the sensitivity of glioma stem cells to apigenin through the mitochondrial pathway.
