Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (K_d = 1.59 nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide aptamers.     

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52 ± 2.44E-10 and 5.87 ± 1.3E–9 M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33 ± 1.15E-9 and 4.11 ± 1.09E–9 M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P < 0.05). They also significantly reduced the serum sodium level and increased the urine volume (P < 0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity.     

Novel dammarane saponins from Gynostemma pentaphyllum and their cytotoxic activities against HepG2 cells

Two new dammarane saponins, 2α,3β,12β-trihydroxydammar-20(22),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (1, namely damulin C) and 2α,3β,12β-trihydroxydammar-20(21),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (2, namely damulin D), were isolated from the ethanol extract of Gynostemma pentaphyllum, which had been heat processed by steaming at 125 °C. The NMR spectroscopic data of the novel saponins were completely assigned by using a combination of 2D NMR experiments including ¹H–¹H COSY, HSQC, and HMBC. Their cytotoxic activities of human liver adenocarcinoma HepG2 cells were evaluated in vitro. They showed cytotoxicities against HepG2 cell line with IC₅₀ of 40 ± 0.7 and 38 ± 0.5 μg/ml, respectively.     

A novel cytotoxic aryltetraline lactone from Bupleurum marginatum (Apiaceae)

A detailed phytochemical study of the aerial parts of Bupleurum marginatum Wall. ex DC revealed a novel aryltetraline lactone lignan identified as 9-(benzo[d][1,3]dioxol-5-yl)-6,7,8-trimethoxy-3a,4,9,9a-tetrahydronaphtho[2,3-c]furan-1(3H)-one (marginatoxin) along with nine known compounds and characterization of five other compounds either by GLC/MS or LC/MS techniques. Chemical structures of the isolated compounds were unambiguously elucidated by both 1D, 2D NMR and mass spectrometry techniques.The in vitro cytotoxic activity of both methanol and dichloromethane extracts as well as the isolated compounds was assessed in two human cancer cell lines HepG2 and HeLa using the MTT assay. The new aryltetraline lactone lignan exhibited a potent cytotoxic activity with IC₅₀ values of 12.14 and 16.90 μM after 24 h treatment for HepG2 and HeLa cells, respectively.     

A cell-based single-stranded DNA aptamer specifically targets gastric cancer

Gastric cancer is one of the most prevailing cancers with high morbidity and mortality. Limitations in the current diagnosis and therapy, specially lacking of specific molecular therapeutic targets, ask for the development of new strategies. Aptamer, a newly developed adaptive molecule, could be used in clinical detection and therapy because of its high affinity and specificity. As no aptamer has ever been developed in preventing gastric cancer so far, we were the first who cloned such an aptamer specifically targeting gastric cancer. The aptamer was selected by systematic evolution of ligands by exponential enrichment with gastric cancer cell-line HGC-27 as target cell line and immortalized gastric epithelial cell-line GES-1 as control cell line. The affinity and specificity of candidate aptamers were examined by flow cytometry, confocal imagining and aptamer-based histochemistry staining. After 19 cycles of systematic evolution of ligands by exponential enrichment and subsequent cloning and sequencing, an aptamer with the highest affinity and specificity (nominated as AGC03) among candidates was screened out from a random single-stranded DNA pool. Moreover, AGC03 could not only specifically bind to gastric cancer cells (the equilibrium dissociation constant value was 16.49 ± 0.40 nM) in vitro, but also recognize cancer cells in human cancer tissue. Our most important finding is that AGC03 could even be internalized into cells automatically. In conclusion, we obtained a novel aptamer specifically targeting gastric cancer, which is an effective tool for both gastric cancer diagnosis and drug delivery.     

Human growth hormone-specific aptamer identification using improved oligonucleotide ligand evolution method

LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoiding cross reactions of aptamers with Bacillus subtilis extracellular proteins by negative SELEX, the effects of time and pH on aptamer binding to rhGH were investigated. The highest binding efficiency of aptamers on rhGH-immobilized microparticles was obtained at pH 7.0. The aptamers that interacted with rhGH were eluted by a multi-stage step-up temperature gradient in ΔT = 10 °C increments within the range T = 55–95 °C; and the strongest affinity binding was disrupted at T = 85 °C where C_Apt = 0.16 μM was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K_D = 218 nM rhGH. RhGH was separated from the fermentation broth with 99.8% purity, indicating that the method developed is properly applicable even for an anionic protein.     

A novel disintegrin protein from Naja naja venom induces cytotoxicity and apoptosis in human cancer cell lines in vitro

Animal venoms and toxins are potential bioresources that have been known to mankind as a therapeutic tool for more than a century through folk and traditional medicine. The purified “disintegrin protein” (64 kDa) from the venom of the Indian cobra snake (Naja naja) exhibited cytotoxic effects of various types of human cancer cell lines such as breast cancer (MCF-7), lung cancer (A549) and liver cancer (HepG2). In vitro cytotoxicity, DNA fragmentation, an apoptotic assay and a cell cycle analysis were performed to evaluate the anticancer activity of disintegrin against the above cell lines. The IC₅₀ value of disintegrin was determined to be 2.5 ± 0.5 μg/mL, 3.5 ± 0.5 μg/mL, and 3 ± 0.5 μg/mL for the MCF-7, A549 and HepG2 cell lines respectively. Moreover, the increased distribution of G0/G1 and S phase led to decreased populations of cells in the G2/M phase of MCF-7, HepG2 and A549 cells.     

Genotoxic effect of 6-gingerol on human hepatoma G2 cells

6-Gingerol, a major component of ginger, has antioxidant, anti-apoptotic, and anti-inflammatory activities. However, some dietary phytochemicals possess pro-oxidant effects as well, and the risk of adverse effects is increased by raising the use of doses. The aim of this study was to assess the genotoxic effects of 6-gingerol and to clarify the mechanisms, using human hepatoma G2 (HepG2) cells. Exposure of the cells to 6-gingerol caused significant increase of DNA migration in comet assay, increase of micronuclei frequencies at high concentrations at 20–80 and 20–40 μM, respectively. These results indicate that 6-gingerol caused DNA strand breaks and chromosome damage. To further elucidate the underlying mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH). In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-hydroxydeoxyguanosine (8-OHdG). Results showed that lysosomal membrane stability was reduced after treatment by 6-gingerol (20–80 μM) for 40 min, mitochondrial membrane potential decreased after treatment for 50 min, GSH and ROS levels were significantly increased after treatment for 60 min. These suggest 6-gingerol induces genotoxicity probably by oxidative stress; lysosomal and mitochondrial damage were observed in 6-gingerol-induced toxicity.     

Impact of 1.8-GHz radiofrequency radiation (RFR) on DNA damage and repair induced by doxorubicin in human B-cell lymphoblastoid cells

In the present in vitro study, a comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR, SAR of 2 W/kg) can influence DNA repair in human B-cell lymphoblastoid cells exposed to doxorubicin (DOX) at the doses of 0 μg/ml, 0.05 μg/ml, 0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml. The combinative exposures to RFR with DOX were divided into five categories. DNA damage was detected at 0 h, 6 h, 12 h, 18 h and 24 h after exposure to DOX via the comet assay, and the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage. The results demonstrated that (1) RFR could not directly induce DNA damage of human B-cell lymphoblastoid cells; (2) DOX could significantly induce DNA damage of human B-cell lymphoblastoid cells with the dose–effect relationship, and there were special repair characteristics of DNA damage induced by DOX; (3) E–E–E type (exposure to RFR for 2 h, then simultaneous exposure to RFR and DOX, and exposure to RFR for 6 h, 12 h, 18 h and 24 h after exposure to DOX) combinative exposure could obviously influence DNA repair at 6 h and 12 h after exposure to DOX for four DOX doses (0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml) in human B-cell lymphoblastoid cells.     

Synthesis and synergetic anti-tumor activity evaluation of dihydroartemisinin-organogermanium(IV) compound

Dihydroartemisinin (DHA), a semi-synthetic derivative of the herb artemisinin, has shown commendable bioactivity. In this paper, a novel dihydroartemisinin-organogermanium (DHA-Ge) compound was synthesized, characterized and its potential anti-tumor activity was evaluated by various methods. MTT results demonstrated that DHA-Ge could effectively inhibit the proliferation of HepG2 cells and showed their dose-dependent properties. The IC₅₀ value of inhibition effect on HepG2 cells of DHA-Ge was 10.23 μg/ml which was lower than 39.44 μg/ml of DHA. Flow cytometric results suggested that DHA-Ge could induce apoptosis of HepG2 cells and the apoptosis rate was 20.26% after 24 h treatment with 56.8 μg/ml DHA-Ge concentration. Atomic force microscopy images showed that HepG2 cells were collapsed and the cell nucleus were fragmented after 24 h treatment. All these results together showed that the DHA-Ge possessed desirable synergetic enhanced anti-tumor effects and could be developed as a suitable tumor therapeutic agent.     

Hepatocellular toxicity of clopidogrel: Mechanisms and risk factors

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100 μM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100 μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100 μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100 µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100 µM) in HepG2/CYP3A4 supersome. Co-incubation with 1 μM ketoconazole or 10 mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100 μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100 µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.     

Pencitrin and pencitrinol,two new citrinin derivatives from an endophytic fungus Penicillium citrinum salicorn 46

Two new citrinin derivatives, pencitrin (1) and pencitrinol (2), and a known compound citrinin (3), together with its two known dimers, penicitrinone A (4), penicitrinone E (5), were isolated from an endophytic fungus P. citrinum 46 derived from Salicornia herbacea Torr. by adding CuCl₂ into fermentation medium. The structures of these compounds were elucidated by detailed spectroscopic analysis, and the absolute configurations of 1, 4, and 5 were determined by comparison of quantum chemical time-dependent density functional theory (TDDFT) calculated and electronic circular dichroism (ECD) spectra. Compound 1 exhibited potent cytotoxic activities towards A549 human lung cancer cells and HepG2 human liver cancer cells with IC₅₀ values of 23.73 ± 3.61 and 35.73 ± 2.15 μM, respectively, whereas compound 5 showed moderate cytotoxic activities towards A549 and HepG2 cancer cells with IC₅₀ values of 40.47 ± 4.52 and 53.57 ± 3.24 μM, respectively. The results from the current research highlighted the effectiveness and usefulness of the pipeline to discover novel bioactive fungal secondary metabolites by modification of the culture media.     

Synthesis and in vitro cytotoxic evaluation of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid

A series of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid were designed and synthesized as potent antitumor agents. Structures of the target molecules were characterized using MS, IR, ¹H NMR, ¹³C NMR and elemental analyses. In the in vitro cytotoxic assay, most compounds showed significant cytotoxic activities against two hepatocarcinoma cells (SMMC-7721 and HepG2) and reduced cytotoxicity against noncancerous human hepatocyte (LO2). Among them, compound 7b exhibited the best cytotoxicity against SMMC-7721 cells (IC₅₀: 0.36 ± 0.13 μM), while 7e was most potent to HepG2 cells (IC₅₀: 0.12 ± 0.03 μM). The cell cycle analysis indicated that compound 7b caused cell cycle arrest of SMMC-7721 cells at G2/M phase. Further, compound 7b also induced the apoptosis of SMMC-7721 cells in Annexin V-APC/7-AAD binding assay.     

Tissue factor pathway inhibitor-2 induced hepatocellular carcinoma cell differentiation

To investigate the effect of over-expression of tissue factor pathway inhibitor-2 (TFPI-2) on the differentiation of hepatocellular carcinoma (HCC) cells (Hep3B and HepG2). The TFPI-2 recombinant adenovirus (pAd-TFPI-2) was constructed using the pAdeasy-1 vector system. Transfected by pAd-TFPI-2, the cell proliferation of HCC cells was evaluated by CCK-8 assay, flow cytometry was used to detect cell apoptosis and CD133 expression. Real-time PCR and Western blot were used to detect the expression levels of markers of hepatocellular cancer stem cells (CSC) and hepatocytes. The over-expression of TFPI-2 significantly suppressed cell proliferation, induced apoptosis, and dramatically decreased the percentage of CD133 cells, which was considered as CSC in HCC. Real-time PCR and Western blot showed that the expression of markers of CSC in Hep3B cells and HepG2 cells infected with pAd-TFPI-2 was markedly lower than those of the control group (P < 0.05), while the expression of markers of hepatocytes was significantly increased (P < 0.05). Hence, TFPI-2 could induce the differentiation of hepatocellular carcinoma cells into hepatocytes, and is expected to serve as a novel way for the treatment of HCC.     

Isolation and characterization of enantioselective DNA aptamers for ibuprofen

Single stranded DNA aptamers that can bind to ibuprofen, a widely used anti-inflammation drug, were selected from random DNA library of 10¹⁵ nucleotides by FluMag-SELEX process. Five different sequences were selected and their enantioselectivity and affinity were characterized. Three out of five aptamer candidates did not show any affinity to (S)-ibuprofen, but only to racemic form of ibuprofen, suggesting that they are (R)-ibuprofen specific aptamers. Another two aptamer candidates showed affinity to both racemic form and (S)-ibuprofen, which were considered as (S)-ibuprofen specific aptamers. The affinity of five ssDNA aptamers isolated was in a range of 1.5–5.2 μM. In addition, all of these five aptamers did not show any affinity to analogues of ibuprofen in its profen’s group (fenoprofen, flubiprofen, and naproxen) and the antibiotics of oxytetracycline, another control.     

Design,synthesis and biological activities of tetrandrine and fangchinoline derivatives as antitumer agents

The isolation and modification of natural products is always a very important resources to anti-tumor drugs. Therefore, a novel series of tetrandrine and fangchinoline derivatives were designed and synthesized, and their antiproliferative activities against HepG2, MCF-7 cells were evaluated and described. From the activity result obtained, high to very high activity in vitro has been found, one of the tested compounds (compound 5d) exhibited the most significant cytotoxic effects. Compound 5d increased 29.2, 7.37 times anti-proliferative activity for HepG2 cells and MCF-7 cells compared to sunitinib (IC₅₀ = 16.06 μM and 25.41 μM). Finally flow cytometry determined that compound 5d could indeed inhibit the proliferation of HepG2 cells via inducing apoptosis.     

DNA binding and photocleavage properties and apoptosis-inducing activities of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)2Cl]Cl and its heterometallic derivatives

The interactions of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)₂Cl]Cl (phen = 1,10-phenanthroline, (Py-3′)TPP = 5-(3′-pyridyl-10,15,20-triphenylporphyrin) (1) and its heterometallic derivatives, [Ni(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (2) and [Cu(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (3), with calf thymus DNA have been investigated by spectroscopic and viscosity measurements in this study. The results showed that these synthetic complexes can bind to double strand helix DNA in groove binding mode, and the intrinsic binding constants of complexes 1, 2 and 3, as calculated according to the decay of the Soret absorption, are (1.35 ± 0.5) ×10⁵ M^?1 (s = 4.2), (1.29 ± 0.5) × 10⁵ M^?1 (s = 5.6) and (1.22 ± 0.5) × 10⁵ M^?1 (s = 6.2) (s is the binding-site size), respectively, which are consistent with those obtained from ethidium bromide-quenching experiments. Further investigations on the photocleavage properties of these complexes on plasmid pBR 322 DNA showed that complexes 1, 2 and 3 could cleave single chain DNA and convert DNA molecules from supercoiled form to the nicked form. As determined by MTT assay, the complexes were also identified as potent antiproliferative agents against A375 human melanoma cells, MCF-7 human breast adrenocarcinoma cells, Colo201 human colon adenocarcinoma cells and HepG2 human liver cancer cells. Complex 1 inhibits the growth of A375 cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigation on intracellular mechanisms indicated that Complex 1 induced depletion of mitochondrial membrane potential (ΔΨ_m) in A375 cells through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our results suggest that ruthenium porphyrin complexes could be candidates for further evaluation as chemopreventive and chemotherapeutic agents for human cancers.     

Selection of a DNA aptamer that binds 8-OHdG using GMP-agarose

DNA aptamers, which bind specific molecule, such as 8-OHdG, with high affinity were investigated using an in vitro selection strategy called systematic evolution of ligands by exponential enrichment (SELEX). However, 8-OHdG was difficult to immobilize on a carrier for SELEX. Therefore, a DNA aptamer binding to 8-OHdG was selected using GMP-agarose as an analogue from a library of about 4⁶⁰ random ssDNA sources. As a result, three aptamer candidates were selected. Among the selected DNA aptamers, the No. 22 DNA aptamer exhibited a high affinity for 8-OHdG. The dissociation constant, K_D, of No. 22 DNA aptamer was on the order of 0.1 μmol/L. This result suggests that using an analogue will be a useful new SELEX method for obtaining various aptamers that are difficult to immobilize on a matrix.