Non-specific SIRT inhibition as a mechanism for the cytotoxicity of ginkgolic acids and urushiols

Ginkgolic acids and urushiols are natural alkylphenols known for their mutagenic, carcinogenic and genotoxic potential. However, the mechanism of toxicity of these compounds has not been thoroughly elucidated so far. Considering that the SIRT inhibitory potential of anacardic acids has been hypothesized by in silico techniques, we herein demonstrated through both in vitro and computational methods that structurally related compounds such as ginkgolic acids and urushiols are able to modulate SIRT activity. Moreover, their SIRT inhibitory profile and cytotoxicity were comparable to sirtinol, a non-specific SIRT inhibitor (SIRT1 and SIRT2), and different from EX-527, a SIRT1 specific inhibitor. This is the first report on the SIRT inhibition of ginkgolic acids and urushiols. The results reported here are in line with previously observed effects on the induction of apoptosis by this class of compounds, and the non-specific SIRT inhibition is suggested as a new mechanism for their in vitro cytotoxicity.     

Numerical simulations of in vitro nanoparticle toxicity – The case of poly(amido amine) dendrimers

A phenomenological rate equation model is constructed to numerically simulate nanoparticle uptake and subsequent cellular response. Polyamidoamine dendrimers (generations 4–6) are modelled and the temporal evolution of the intracellular cascade of; increased levels of reactive oxygen species, intracellular antioxidant species, caspase activation, mitochondrial membrane potential decay, tumour necrosis factor and interleukin generation is simulated, based on experimental observations.The dose and generation dependence of several of these response factors are seen to well represent experimental observations at a range of time points. The model indicates that variations between responses of different cell-lines, including murine macrophages, human keratinocytes and colon cells, can be simulated and understood in terms of different intracellular antioxidant levels, and, within a given cell-line, varying responses of different cytotoxicity assays can be understood in terms of their sensitivities to different intracellular cascade events.The model serves as a tool to interpolate and visualise the range of dose and temporal dependences and elucidate the mechanisms underlying the in vitro cytotoxic response to nanoparticle exposure and describes the interaction in terms of independent nanoparticle properties and cellular parameters, based on reaction rates. Such an approach could be a valid alternative to that of effective concentrations for classification of nanotoxicity and may lay the foundation for future quantitative structure activity relationships and predictive nanotoxicity models.     

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NFκB

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor κB (NFκB).METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNFα and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NFκB.RESULTS: E3330 decreased NFκB levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNFα, Fas ligand, and E3330increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability.CONCLUSION: Inhibition of NFκB sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.     

Extracellular directed ag NPs formation and investigation of their antimicrobial and cytotoxic properties

The use of microbial cell culture a valuable tool for the biosynthesis of nanoparticles is considered a green technology as it is eco-friendly, inexpensive and simple. Here, the synthesis of nanosilver particle (AgNP) from the yeast, Saccharomyces cerevisiae, gram (+), Bacillus subtilis and gram (?), Escherichia coli was shown. In this field we are the first to study their the antimicrobial effects of the microorganisms mentioned above against pathogens and anticancer activity on MCF-7 cell line. Silver nanoparticles in the size range of 126–323?nm were synthesized extracellularly by the microorganisms, which have different cell structures. Optical absorption, scanning electron microscopy, and zetasizer analysis confirmed the silver nanoparticles formation. Antimicrobial activity of AgNPs was evaluated the minimum inhibition concentration and disc diffusion methods. AgNPs inhibited nearly 90% the growth of Gram-positive Listeria monocytogenes, Streptococcus pneumoniae and Gram-negative Haemophilus influenzae, Klebsiella pneumoniae, Neisseria meningitidis bacterial pathogens. Anticancer potentials of AgNPs were investigated by MTT method. The synthesized AgNPs exhibited excellent high toxicity on MCF-7 cells and had a dose-dependent effect on cell viability. Especially AgNP 2 eliminated 67% of the MCF-7 cells at the concentration of 3.125?μg/mL. We found that extracellular synthesis of nanoparticles from microbial culture may be ‘green’ alternative to physical and chemical methods from the point of view of synthesis in large amounts and easy process.     

Clinical features of aflatoxin B1-exposed patients with liver cancer and the molecular mechanism of aflatoxin B1 on liver cancer cells

Aflatoxin B1 (AFB1) induces hepatocellular carcinoma (HCC) through consumption of contaminated food in Southern China. Aldo-keto reductase-7A (AKR7A) functionally plays a potent role in the biodetoxification in the liver. In addition, hepatocellular lipid disorder has found to be closely linked to the development of HCC. This study was, therefore, designed to investigate the potent bioeffect of AKR7A on the lipid metabolism in AFB1-exposed hepatocellular carcinoma cells through assaying human cancerous samples and cell culture. In the baseline data, the HCC patients showed increased contents of AFB1 in sera and cancerous samples. In the clinical parameters, the HCC patients demonstrated changed lipid settings in sera. As revealed by immunostaining and immunoblotting, AFB1-elevated HCC sections showed marked down-regulation of AKR7A expression, accompanied with reduced ApoB expression and increased CD36, S6K1 expressions in the HCC. Studies in the human hepatocarcinoma line HepG2 also showed AFB1-exposure to increase ApoA1, LDL, TC, and TG contents; induce cell proliferation; and reduce hepatocellular AKR7A expression. Furthermore, AKR7A bioactivity was inactivated after treatment with perfluorooctane sulfonate (PFOS), an ApoB activator, in AFB1-dosed HepG2 cells. Collectively, our current findings suggest that hepatocellular AKR7A has a protective role against AFB1-induced cytotoxicity through the regulation of CD36, S6K1 and ApoB expression through the reduction of lipid utilization in malignant liver cells.     

Investigation of the differential transport mechanism of cinnabar and mercury containing compounds

BackgroundCinnabar has a long history of uses in Chinese traditional medicines as an ingredient in various remedies. However, the detailed mechanism of cinnabar in medication remains unclear, and the toxicity of cinnabar has been a debate due to its containing mercury sulfide. This study was designed to investigate the differential transport mechanism of cinnabar and other Hg-containing compounds HgCl₂, MeHg and HgS, and to determine if organic anion transporters OAT1 and OAT3 were involved in the differential transport mechanism.Materials and methodsThe 293T cells were employed to investigate and compare the differential transport mechanism of cinnabar and HgCl₂, MeHg and HgS. Cells were incubated with a low dose (5 μM HgCl₂ and MeHg, 200 μM HgS and cinnabar), medium dose (10 μM HgCl₂ and MeHg, 400 μM HgS and cinnabar), and high dose (20 μM HgCl₂ and MeHg, 800 μM HgS and cinnabar) of HgCl₂, MeHg, HgS and cinnabar for 24 h. Following treatment, the cells were collected and the cell viability was determined by MTT assay. The intracellular mercury content was measured at 1, 4, and 24 h after treatment with 10 μM of the tested agents by an atomic fluorescence spectrophotometer. The effect of these tested agents on mitochondrial respiration was determined in a high-resolution oxygraphyat 24 h following treatment. Furthermore, the effect of modulation of expression of transporters OAT1 and OAT3 on the transport and cytotoxicity of the tested agents was evaluated. The up and down regulation of OAT1 and OAT3 were achieved by overexpression and siRNA transfection, respectively.ResultsCompared with HgCl₂ and MeHg, the cytotoxicity of cinnabar and HgS was lower, with cell viability at the high dose cinnabar and HgS being about 65%, while MeHg and HgCl₂ were 40% and 20%, respectively. The intracellular mercury accumulation was time-dependent. At 24 h the intracellular concentrations of HgCl₂ and MeHg were about 7 and 5 times higher, respectively, than that of cinnabar. No significant difference was found in the intracellular mercury content in cells treated with cinnabar compared to HgS. The knockdown and overexpression of the transporter OAT1 resulted in significant reduction and increase, respectively, in mercury accumulation in HgCl₂ -treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar, MeHg, and HgS. In addition, the knockdown and overexpression of the transporter OAT3 caused significant reduction and increase, respectively, in mercury accumulation in both HgCl₂ and MeHg-treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar and HgS. Furthermore, it was found that cells transfected with siOAT1 caused significant resistance to the cytotoxicity induced by HgCl₂, while no noticeable changes in cell viability were observed in cells treated with other tested agents. Additionally, cells transfected with OAT3 did not change cell sensitivity to cytotoxicity induced by all of the four tested agents.ConclusionThis study demonstrates that differential transport and accumulation of mercury in 293T cells exists among cinnabar and the three mercury-containing compounds HgCl₂, MeHg and HgS, leading to distinct sensitivity to mercury induced cytotoxicity. The kidney organic anion transporters OAT1 and OAT3 are partially involved in the regulation of the transport of HgCl₂ and MeHg, but not in the regulation of the transport of cinnabar.     

抗艰难梭菌rakicidin B1新衍生物的设计、合成及生物学活性评价

目的 以rakicidin B1为起始原料,经2步反应合成得到4个全新结构的rakicidin B1衍生物,并对其进行生物学活性研究,以期获得低细胞毒、高效抗艰难梭菌活性的化合物。方法 课题组前期首次发现rakicidin B1具有较强的抗艰难梭菌活性,在其结构基础上进行修饰和优化,通过氨基甲酸酯连接基团引入含氮杂环,设计合成得到4个全新目标化合物。所有化合物结构经高分辩质谱和核磁确证,并经抗艰难梭菌活性测试和细胞毒性活性测试。结果 在合成的4个化合物中,有3个化合物具有与先导化合物更强或相当的抗艰难梭菌活性;同时,MTT测试结果表明,3个化合物的细胞毒性降低,以3b的细胞毒性降低最多。结论 通过对rakicidin B1母核结构进行修饰和优化,获得全新结构的rakicidin B1衍生物并对其进行抗菌和细胞毒活性筛选。其中,化合物3b保留潜在抗艰难梭菌活性且细胞毒性降低最多,作为全新结构类型的抗艰难梭菌活性化合物,有潜在的开发价值。     

抗菌肽Maximin衍生物的体外杀精效果研究

目的比较抗菌肽Maximin衍生物(M1~M19)的体外杀精效果和细胞毒性作用,筛选有进一步研究价值的候选体外杀精剂。方法利用Sander-Cramer方法评价抗菌肽的体外杀精效果;CCK-8试剂盒测定抗菌肽对Hela-229的细胞毒性,筛选出具有较强杀精作用和低细胞毒性的抗菌肽。结果 19种抗菌肽衍生物中,M1、M7、M11、M15和M17五种抗菌肽在2 000mg/L时均能完全制动精子,其中100%制动精子的最低浓度EC100分别是:M7=M11=2 000mg/L,M1=M15=M17=1 500mg/L;利用Hela-229细胞分析细胞毒性时,M11的细胞毒性明显低于其他4种抗菌肽,表现出较高的安全性。结论对19种抗菌肽衍生物的体外研究表明,抗菌肽M11具有较强的杀精作用,且细胞毒性低,有望成为体外杀精剂的候选药物。     

Apoptotic mechanisms of the biotechnologically produced arylnaphtalene lignan justicidin B in the acute myeloid leukemia-derived cell line HL-60

BackgroundThe present study aimed at optimization of the biotechnological production of the lignan justicidin B by genetically transformed cultures of Linum leonii and the pharmacological evaluation of the pro-apoptotic effects of the compound in HL-60 cells.MethodsA rapidly growing selected root line of L. leonii was grown in 2-L bioreactor for period of 40 days and the protocols for obtaining of the compound have been optimized. The pharmacological study included evaluation of the cytotoxicity of the compound in HL-60 cells (MTT-assay), its apoptogenic effects and its effects on caspase 3,8 and 9 activation.ResultsAfter 40 days of sterile run scale up of hairy root culture in bioreactor, 27.2 g/L dry weight of root biomass was harvested from the bioreactor culture vessel, recording about nine times increase over initial inoculum (3.0 g), with 1.55% ± 0.07 Justicidin B, greater than yields from 300 ml flasks. Our findings are the first work toward the scale up of L. leonii hairy roots-based biotechnological production of Justicidin B, employing bioreactors for high biomass production to meet the industrial requirement. The results from the pharmacological evaluation have shown that the tested arylnaphtalene lignan is a potent cytotoxic and proapoptotic agent against HL-60. The induction of apoptosis proceeds via activation of the intrinsic mitochondrial cell-death signaling pathways.ConclusionThe potent activity at low micromolar concentration and the feasibility of biotechnological production of justicidin B implies that there is enormous scope in its further evaluation as possible antineoplastic drug candidate.     

Trigger-responsive,fast-degradable poly(β-amino ester)s for enhanced DNA unpackaging and reduced toxicity

Poly(β-amino ester)s (PBAEs) represent an important class of cationic gene delivery materials which, however, suffer from uncontrolled DNA release due in part to the slow degradation of their polyester backbone. Additionally, PBAEs with high molecular weight (MW) also show considerable toxicities. In this study, we designed and developed PBAEs with trigger-responsive domains built-in polymer backbones that can be rapidly cleaved upon external UV light triggering to promote intracellular DNA release as well as reduce material toxicity. Photo-responsive PBAEs were prepared via polyaddition of (2-nitro-1,3-phenylene)bis(methylene) diacrylate and a bifunctional amine. The nitrobenzene moiety was placed in each repeating unit of the PBAE to allow fast response to external UV irradiation, and thus the ester linkers were cleaved and the polymers were degraded within several minutes upon UV irradiation. Cationic PBAEs with high MWs were able to mediate effective intracellular gene delivery, while upon UV irradiation post-transfection, enhanced DNA unpackaging and reduced material toxicity were observed, which collectively contributed to greatly improved transfection efficiencies in various mammalian cell types tested. This strategy allows precise manipulation of material toxicity and gene release profiles of PBAEs, and thus provides an effective design approach to address critical issues in non-viral gene delivery.