DNA Ladder Research Articles

Caspase-Independent Apoptosis Induced by Reperfusion Following Ischemia without Bile Duct Occlusion in Rat Liver.

The contribution of caspases to hepatic ischemia/reperfusion (I/R)-induced apoptosis has not been completely understood yet. Several studies have demonstrated increased caspase activity during I/R and the protective effect of caspase inhibitors against I/R injuries. However, reports with opposing results also exist. Herein, we examined the contribution of caspases to the I/R-induced hepatic apoptosis in rats using caspase inhibitors and specific substrates of caspases. Hepatic I/R was induced via a 2-h occlusion of the portal vein and the hepatic artery, without conducting bile duct occlusion. DNA laddering and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were increased at 3 h after reperfusion. Pretreatment with caspase inhibitors (Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-cmk) 2 or 10 mg/kg intravenously (i.v.), 20 mg/kg intraperitoneally (i.p.), Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) 3 mg/kg i.v.) failed to reduce apoptosis induced by I/R. Interestingly, apoptosis induced by the portal triad (hepatic artery, portal vein, and bile duct) occlusion/reperfusion could be marginally attenuated using Z-Asp-cmk (2 mg/kg i.v.). The cleavage activity for Ac-DEVD-α-(4-methylcoumaryl-7-amide) (MCA), a caspase-3/7/8/9 substrate, was significantly increased by I/R. Conversely, the cleavage activities for Ac-DNLD-MCA and MCA-VDQVDGW[K-DNP]-NH2, specific substrates for caspase-3 and -7 respectively, were decreased by I/R. Protein expression of the cellular inhibitor of apoptosis protein 2 (c-IAP2), an endogenous caspase inhibitor, was increased by ischemia. Nuclear translocation of the apoptosis-inducing factor (AIF), an initiator protein of caspase-independent apoptosis, was also increased during I/R. These results suggest that caspases are inhibited by c-IAP2 induced during ischemia and that AIF may be involved in initiation of apoptosis induced by hepatic I/R without bile duct occlusion.

Inhibition of Candida species via Proteosome Inhibitor MG-262 (ZL3B)

Backround: Candida species take the fourth place among the microorganisms which cause hospital infections. New therapeutic agents are needed because of rapidly increasing resistance rates. Boric acid preparations are preferred in local treatment especially in resistant cases while azole formulations are the first choice in oral treatment. The only boric acid analog that was approved by FDA (Food and Drug Administration) for systemic use is Bortezomib (Velcade). Bortezomib is a proteasome inhibitor and proteasome inhibitors cause apoptosis of eukaryotic cells. Although the yeast also has a well preserved 20S proteasome region, the studies on the effects of proteasome inhibitors on the yeast are limited. In this study, the effects of the one of the most potent proteasome inhibitor, MG-262, on Candida spp. were investigated. Methods: C. albicans ATCC 90028, C. krusei ATCC 6258 and C. glabrata (clinical strain) strains were used in our study. Antifungal susceptibilities of the strains against MG-262 and effects of MG-262 on oxidative metabolism, proteasome activity and apoptosis as well as virulence factors such as hyphal transformation, pseudohypha, germ tube and biofilm formation were investigated. CLSI (Clinical and Laboratory Standards Institute) guidelines were used for antifungal susceptibility tests. The effect on hypha and pseudohypha formation and germ tube were determined with conventional tests. Biofilm formation was detected according to the microplate method. Oxidative metabolism experiments, proteosome activity and apoptosis tests were performed with Alamarblue dye, fluorogenic peptides and DNA ladder assays, respectively. Findings: MG-262 exhibited minimum inhibitory activity at 25 μg/ml concentration for C. albicans ATCC 90028 and at 50 μg/ml concentration for C. krusei ATCC 6258 and C. glabrata. Minimum fungicidal concentrations for all species were 50 μg/ml. Hypha, pseudohypha, germ tube and biofilm formation, proteosome activity and oxidative metabolism were all inhibited at 50 μg/ml and higher concentrations. The apoptosis was detected at 200 μg/ml. Conclusion: MG-262 caused apoptosis by inhibiting oxidative metabolism and showed fungicidal activity. It also inhibited the virulence factors such as hypha, pseudohypha germ tube and biofilm formation. Inhibition of proteasome is promising in terms of discovery of a new generation antifungal agent.

Hispolon Suppresses LPS- or LTA-Induced iNOS/NO Production and Apoptosis in BV-2 Microglial Cells.

Hispolon (HIS) is an active polyphenol compound derived from Phellinus linteus (Berkeley & Curtis), and our previous study showed that HIS effectively inhibited inflammatory responses in macrophages [Yang, L.Y., S.C. Shen, K.T. Cheng, G.V. Subbaraju, C.C. Chien and Y.C. Chen. Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages. J. Ethnopharmacol. 156: 61-72, 2014]; however, its effect on neuronal inflammation is still undefined. In this study, HIS concentration- and time-dependently inhibited lipopolysaccharide (LPS)- and lipoteichoic acid (LTA)-induced inducible nitric oxide (NO) synthase (iNOS)/NO production with increased heme oxygenase (HO)-1 proteins in BV-2 microglial cells. Accordingly, HIS protected BV-2 cells from LPS- or LTA-induced apoptosis, characterized by decreased DNA ladder formation, and caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage in BV-2 cells. Similarly, the NOS inhibitor, N-nitro-L-arginine methyl ester (NAME), inhibited LPS- or LTA-induced apoptosis of BV-2 cells, but neither NAME nor HIS showed any inhibition of NO production or cell death induced by the NO donor, sodium nitroprusside (SNP), indicating the involvement of NO in the inflammatory apoptosis of microglial cells. Activation of c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-[Formula: see text]B contributed to LPS- or LTA-induced iNOS/NO production and apoptosis of BV-2 cells, and that was suppressed by HIS. Additionally, HIS possesses activity to induce HO-1 protein expression via activation of extracellular signal-regulated kinase (ERK) in BV-2 cells, and application of the HO inhibitor, tin protoporphyrin (SnPP), or knockdown of HO-1 protein by HO-1 small interfering (si)RNA significantly reversed HIS inhibition of NO production and cell death in BV-2 cells stimulated by LPS. Results of an analysis of the effects of HIS and two structurally related chemicals, i.e. dehydroxy-HIS (D-HIS) and HIS-methyl ester (HIS-ME), showed that HIS expressed the most potent inhibitory effects on iNOS/NO production, JNK activation, and apoptosis in BV-2 microglial cells activated by LPS with increased HO-1 protein expression. Overall these results suggested that HIS possesses inhibitory activity against LPS- or LTA-induced inflammatory responses including iNOS/NO production and apoptosis in BV-2 microglial cells and that the mechanisms involve upregulation of the HO-1 protein and downregulation of JNK/NF-[Formula: see text]B activation. A critical role of hydroxyl at position C3 in the anti-inflammatory actions of HIS against activated BV-2 microglial cells was suggested.

Proteoglycans Isolated from Bramble Shark Cartilage (Echinorhinus brucus) Inhibits Proliferation of MCF-7 Human Breast Cancer Cells by Inducing Apoptosis

Proteoglycans (PGs) were isolated from deep sea shark Echinorhinus brucus (bramble shark) cartilage and their anti-proliferative activity against MCF-7 cell lines was evaluated. To establish apoptosis involvement for cytotoxicity, the following assays were performed i.e., caspases 3 and 9 assay, double fluorescent staining and DNA laddering assay. PGs were isolated by DEAE-sephacel ion exchange chromatography and identification and purity confirmed by chemical composition such as total carbohydrate, total protein, hexosamine, uronic aicd, sulfated GAG, wavelength scan and modified AGE. To confirm apoptosis pathway expression of genes BAX, Bcl-2 and p53 were carried out. PGs (100 μg/mL) induced a significant rise in activity of caspase 3 and 9, major apoptotic markers. A significant rise in the expression was also observed for Bcl-2 and p53. Whereas, low BAX expression was observed for PGs (100 μg/mL) treated sample. Significant anti-proliferative effect was observed and the mechanism behind cell death was concluded to be apoptosis.

Beta radiation induces apoptosis in human histiocytic lymphoma cells

Objective: Lu-177 has a great potential for use as theranostics radiopharmaceuticals for management of cancer, evidenced by its increasing use over the past decade in nuclear medicine. The detail mechanisms of cell toxicity induced by the beta-radiation emitted from Lu-177 are not well understood. Hence, to explore the lurking mechanism of cell death, different parameters were assessed after treatment of human histiocytic lymphoma cells (U937) with Lu-177. Materials and Methods: U937 cells (1 × 106) were exposed to 3.7 and 37 MBq of Lu-177 and incubated for 24 and 48 h in humidified CO2incubator. The cell viability and apoptosis were estimated in treated and control cells by trypan blue dye exclusion and electrophoretic DNA ladder assay, respectively. Reverse transcriptase polymerase chain reaction was carried out to study the expression of anti-apoptotic genes. Results: It was found that the cell death and apoptosis were high in the cells which were exposed for a longer duration of time with 37MBq of Lu-177. These results were further confirmed by observation of downregulation of anti-apoptotic genes such as BCL-2 and BCLXL. Conclusion: It is concluded from the study that the Lu-177 induced apoptotic cell death in human histiocytic lymphoma cells by downregulation of anti-apoptotic genes.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells.

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

Evodiamine Prevents Glioma Growth, Induces Glioblastoma Cell Apoptosis and Cell Cycle Arrest through JNK Activation.

Evodiamine (EVO) is an active medicinal compound derived from the traditional herbal medicine Evodia rutaecarpa. It has been reported that evodiamine has several beneficial biological properties, including anticancer and anti-inflammatory activities. However, the in vitro and in vivo anticancer activities of EVO against the growth of glioblastoma cells remain undefined. EVO induced significant decreases in the viability of U87 and C6 glioma cells, but not of primary astrocytes, according with the occurrence of apoptotic characteristics including DNA ladders, caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage, and hypodiploid cells. The disruption of the mitochondrial membrane potential (MMP) was detected, and it was found that the peptidyl caspase-9 inhibitor, Z-LEHD-FMK, significantly prevented glioma cells from EVO-induced apoptosis. Increased c-Jun N-terminal kinase (JNK) protein phosphorylation by EVO was observed, and the addition of JNK inhibitors, SP600125 and JNKI inhibited the EVO-induced apoptosis was inhibited. Additionally, EVO treatment induced G2/M arrest with increased polymerized tubulin protein expression in U87 and C6 cells. Elevated expressions of the cyclin B1, p53, and phosphorylated (p)-p53 proteins were detected in EVO-treated glioma cells, and these were inhibited by JNK inhibitors. An in vivo study showed that EVO significantly reduced the growth of gliomas elicited by the subcutaneous injection of U87 cells with increases in cyclin B1, p53, and p-p53 protein expressions in tumors. An analysis of eight EVO-related chemicals showed that alkyl groups at position 14 in EVO are important for its anti-glioma effects which involve both apoptosis and G2/M arrest. Evidence is provided that supports EVO induction of apoptosis and G2/M arrest via the activation of JNK-mediated gene expression and disruption of MMP in glioblastoma cells. EVO was shown to penetrate the blood-brain barrier; EVO is therefore predicted to be a promising compound for the chemotherapy of glioblastomas and deserves further investigations.

Antioxidant therapy impresses in oxidative stress-induced kidney cells

Renal cell cancer (RCC) is not a single entity, but consists of different types of tumors derived from various parts of the nephron (epithelium or renal tubules). It is known that cancer initiation and progression is related to the balance between oxidants and antioxidants directly. Interestingly, advanced stages of cancer such as metastasis, angiogenesis are associated with cell oxidative capacity. Familiar antioxidative substances such as carotenes and vitamin C inhibit oxidation of other molecules during carcinogenesis. They can define the distinction between cancer and normal cells, destroying cancer cells while stabilizing healthy cells. apoptotic activities of kidney cells were measured with caspase Elisa kits. DNA laddering test was used to show DNA damage in H2O2 condition. For tumor mechanism, they act as pro-oxidants, producing hydrogen peroxide that attacks the cancer, whereas, in normal conditions they act as protective antioxidants. The unlike reaction of specific antioxidants should be known at different cell stages. The aim of this study was to assess the antioxidative roles of alpha lipoic acid on kidney cancers during oxidative stress induction (Tab. 1, Fig. 7, Ref. 27).

Biologically Synthesized Gold Nanoparticles using Ocimum sanctum (Tulsi Leaf Extract) Induced Anti-Tumor Response in a T Cell Daltons Lymphoma

The characterizations of green synthesized gold nanoparticles (AuNPs) prepared using Ocimum sanctum leaf extract were done by UV-Spectrophotometer at 500-540 nm. The XRD data obtained were found similar to gold JCPDS File No- 04-0784. SEM and TEM analysis of AuNPs revealed spherical shape and size of 200 nm. Further FT-IR data indicated the various biomolecules present in Ocimum sanctum leaf extract provides stability to gold nanoparticles synthesis. The AuNPs were studied for their anti-cancer activity on Dalton’s lymphoma (DL) cells and the results obtained with IC50 value of < 50 ng/ml performed by MTT assay. Further, to confirm anti-tumor potential and the mode of action of the synthesized AuNPs, cell viability assay, nuclear morphology, DNA fragmentation assay, mitochondrial membrane potential (ΔΨm) analysis, and cell cycle analysis were done using DL cells. DL cells treated with the AuNPs showed reduced cell viability, altered nuclear morphology, typical apoptotic DNA ladder formation and apoptosis. From the above finding it can be concluded that the AuNPs have potential to decrease the proliferation of tumor cells and enhanced the production of ROS. Gold nanoparticles used in cancer detection and diagnosis/treatment are mainly in preclinical stages of cancer development.

Genotyping Of ABO gene In Iraqi Population Sample By Using PCR-RFLP Technique

The aim of this study was the molecular detection of ABO gene and identify the patterns of alleles in Iraqi population sample. The study consisted of 100 unrelated individuals who appear to be healthy. The blood samples were used for detecting of blood groups and genotyping of ABO gene allele by using PCR-RFLP technique. The DNA was extracted by using extraction kit which gave relatively high yield concentration (40- 110 ng/µl), And purity within the optimal range. DNA concentration was adjusted to the proper concentration of PCR reaction. ABO gene was amplified by using two sets of specific primers for ABO locus with required alleles. Two separate segments of the glycosyltransferase gene containing nucleotide 261 in exon 6 and nucleotide 703 in exon 7 of ABO gene were amplified. The first set was used for the amplification of 468pb DNA fragment, Which contain the nucleotide 261. The second set was used to amplify 298bp DNA fragment, Which contain the nucleotide 703. Digestion products were run on 2% agarose gel, All fragments were compared with DNA ladder and there sizes were determined. The electrophoresis patterns of 100 samples showed that ABO genotypes were 5(4.0218%) AA, 23(23.9782%) AO, 2(3.3191%) BB, 24(22.6809%) BO, 8(8%) AB and 38(38%) OO. The O2 allele was encountered in this study. The study also showed that there is no difference when comparing the Phenotype with the Genotype.

Major triterpenoids in Chinese hawthorn “Crataegus pinnatifida” and their effects on cell proliferation and apoptosis induction in MDA-MB-231 cancer cells

The cytotoxicity and antiproliferative effect of phytochemicals presenting in the fruits of Chinese hawthorn (Crataegus pinnatifida) were evaluated. Shanlihong (Crataegus pinnatifida Bge. var. major N.E.Br.) variety possessed significant levels of flavonoids and triterpenoids, and showed potent antiproliferative effect against HepG2, MCF-7 and MDA-MB- 231 human cancer cells lines. Triterpenoids-enriched fraction (S9) prepared by Semi-preparative HPLC, and its predominant ingredient ursolic acid (UA) demonstrated remarkably antiproliferative activities for all the tested cancer cell lines. DNA flow cytometric analysis showed that S9 fraction and UA significantly induced G1 arrest in MDA-MB-231 cells in a dose-dependent manner. Western blotting analysis revealed that S9 fraction and UA significantly induced PCNA, CDK4, and Cyclin D1 downregulation in MDA-MB-231 cells, followed by p21Waf1/Cip1 up-regulation. Additionally, flow cytometer and DNA ladder assays indicated that S9 fraction and UA significantly induced MDA-MB-231 cells apoptosis. Mitochondrial death pathway was involved in this apoptosis as significantly induced caspase-9 and caspase-3 activation. These results suggested that triterpenoids-enriched fraction and UA exhibited antiproliferative activity through the cell cycle arrest and apoptosis induction, and was majorly responsible for the potent anticancer activity of Chinese hawthorn.

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection

Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary – hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments.

Active extracellular substances of Bacillus thuringiensis ITRI-G1 induce microalgae self-disruption for microalgal biofuel.

Microalgal cultures are a clean and sustainable means to use solar energy for CO2 fixation and fuel production. Microalgae grow efficiently and are rich in oil, but recovering that oil is typically expensive and consumes much energy. Therefore, effective and low-cost techniques for microalgal disruption and oil or lipid extraction are required by the algal biofuel industry. This study introduces a novel technique that uses active extracellular substances to induce microalgal cell disruption. A bacterium indigenous to Taiwan, Bacillus thuringiensis, was used to produce the active extracellular substances, which were volatile compounds with high thermal stability. Approximately 74% of fresh microalgal cells were disrupted after a 12-h treatment with the active extracellular substances. Algal lipid extraction efficiency was improved and the oil extraction time was decreased by approximately 37.5% compared with the control treatment. The substances effectively disrupted fresh microalgal cells but not dehydrated microalgal cells. An analysis of microalgal DNA from fresh cells after disruption treatment demonstrated typical DNA laddering, indicating that disruption may have resulted from programmed cell death. This study revealed that biological treatments are environmentally friendly methods for increasing microalgal lipid extraction efficiency, and introduced a microalgal cell self-disruption mechanism.

Chloral Hydrate Treatment Induced Apoptosis of Macrophages via Fas Signaling Pathway.

BackgroundThere are recent reports on several anesthetics that have anti-inflammatory and anti-infective effects apart from their uses for pain relief and muscle relaxation. Chloral hydrate is a clinical anesthetic drug and sedative that has also been reported to attenuate inflammatory response, but the mechanisms are not clearly understood.Material/MethodsThis study investigated the effect of chloral hydrate treatment on the apoptosis of macrophages and explored the underlying mechanisms. RAW264.7 macrophages were treated with various concentrations of chloral hydrate for various lengths of time. Morphological changes were observed under a light microscope and apoptosis was detected with annexin-V-FITC/PI double-staining assay, Hochest 33258 and DNA ladder assay, the expression of Fas/FasL was detected with a flow cytometer, and the Fas signaling pathway was assessed by Western blotting.ResultsThe results showed that chloral hydrate treatment induced the morphology of RAW264.7 macrophages to change shape from typical fusiform to round in a concentration- and time-dependent manner, and was finally suspended in the supernatant. For the induction of apoptosis, chloral hydrate treatment induced the apoptosis of RAW264.7 macrophages from early-to-late stage apoptosis in a concentration- and time-dependent manner. For the mechanism, chloral hydrate treatment induced higher expression of Fas on RAW264.7 macrophages, and was also associated with changes in the expression of proteins involved in Fas signaling pathways.ConclusionsChloral hydrate treatment can induce the apoptosis of RAW264.7 macrophages through the Fas signaling pathway, which may provide new options for adjunctive treatment of acute inflammation.

Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing.

The implementation of liquid biopsy for biomarker testing and response to treatment monitoring in cancer patients would presumable increase laboratory throughput, requiring the development of automated methods for circulating free DNA (cfDNA) isolation. The present study compares the MagNA Pure Compact (MPC) Nucleic Acid Isolation Kit I and Maxwell® RSC (MR) ccfDNA Plasma Kit and the later with QIAamp Circulating Nucleid Acid (QCNA) Kit using 57 plasma samples from cancer patients. cfDNA concentration was measured using the Qubit fluorometer. DNA fragments lengt were assessed using the Agilent 2100 Bioanalyzer. Circulating tumor DNA (ctDNA) was quantified by digital PCR (dPCR). Firstly, we observed that MPC method significantly extracted less cfDNA than MR (P<0.0001). However, there were no significant differences in extraction yields of QCNA and MR kits. cfDNA isolation yield was also associated with tumor stage but not with tumor location. Secondly, an oligonucleosomal DNA ladder pattern was observed in 88% of the samples and significant differences in the recovery of mono-, di- and tri-nucleosomes DNA fragments were observed between MPC and MR methodologies. Finally, tumor mutation quantification on cfDNA was performed on 38 paired samples using digital PCR. Mutant allele fractions (MAFs) between paired samples were not significantly different. Methods for isolation of cfDNA can affect DNA yield and molecular weight fractions recovery. These observations should be taken into account for cfDNA analysis in routine clinical practice.

A novel L-fucose-binding lectin from Fenneropenaeus indicus induced cytotoxicity in breast cancer cells.

Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.

Resveratrol analogue (E)-8-acetoxy-2-[2-(3,4-diacetoxyphenyl)ethenyl]-quinazoline induces apoptosis via Fas-mediated pathway in HL-60 human leukemia cells.

Previously, we reported that (E)-8-acetoxy-2-[2-(3,4-diacetoxyphenyl)ethenyl]-quinazoline (8-ADEQ), a synthetic analogue of resveratrol had anti-inflammatory and G2/M cell cycle arrest activities, but the underlying molecular mechanism of cytotoxic effects of this compound was not determined. In this study, 8-ADEQ displayed potent cytotoxicity and triggered apoptosis in HL-60 cells as evidenced by DNA fragmentation, DNA ladder formation, and the externalization of AnnexinV-targeted phosphatidylserine residues in HL-60 cells. In addition, 8-ADEQ triggered activation of caspases-8, -9, -6 and -3 and cleavage of their substrates such as poly(ADP-ribose) polymerase (PARP). Moreover, 8-ADEQ induced loss of mitochondrial membrane potential (MMP) and release of cytochromec to the cytosol. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD‑fmk) significantly suppressed the 8-ADEQ-induced DNA fragmentation. Interestingly, pretreatment with z-IETD-fmk, a caspase-8 inhibitor, completely abolished 8-ADEQ-induced caspase-3 and -9 activation, and subsequent DNA fragmentation. 8-ADEQ also increased the expression of Fas, Fas-associated death domain (FADD) and FasL, and formation of death-inducing signaling complex (DISC). Further analysis revealed that 8-ADEQ-induced apoptosis was mediated by upregulation of reactive oxidative species (ROS) generation. Taken together, our data indicated that 8-ADEQ-stimulated apoptosis in HL-60 leukemia cells is due to a Fas-mediated caspase-8-dependent pathway via ROS generation, but also, to a lesser extent cytochromec release and caspase-9 activation.

Greater than X kb: a quantitative assessment of preservation conditions on genomic DNA quality, and a proposed standard for genome-quality DNA.

Advances in biodiversity genomic sequencing will increasingly depend on the availability of DNA samples—and their quantifiable metadata—preserved in large institutional biorepositories that are discoverable to the scientific community. Improvements in sequencing technology constantly provide longer reads, such that longer fragment length, higher molecular weight, and overall “genome-quality” DNA (gDNA) will be desirable. Ideally, biorepositories should publish numerical scale measurements of DNA quality useful to the user community. However, the most widely used technique to evaluate DNA quality, the classic agarose gel, has yet to be quantified. Here we propose a simple and economical method using open source image analysis software to make gDNA gel images quantifiable, and propose percentage of gDNA “greater than X kb” as a standard of comparison, where X is a band from any widely used DNA ladder with desirably large band sizes. We employ two metadata standards (“DNA Threshold” and “Percent above Threshold”) introduced as part of the Global Genome Biodiversity Network (GGBN) Darwin Core extension. We illustrate the method using the traditionally used HindIII ladder and the 9,416 base-pair (bp) band as a standard. We also present data, for two taxa, a vertebrate (fish) and an invertebrate (crab), on how gDNA quality varies with seven tissue preservation methods, time since death, preservation method (i.e. buffers vs. cold temperatures), and storage temperature of various buffers over time. Our results suggest that putting tissue into a buffer prior to freezing may be better than directly into ultra-cold conditions.

Interferon α sensitizes human osteosarcoma cells to doxorubicin-induced apoptosis through p53-dependent pathway

Objective: To investigate the effect of IFNα on doxorubicin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for improving the treatment of osteosarcoma. Methods: Osteosacoma U2OS and MG63 cells were treated with IFNα and Doxorubicin, alone or in combination, for 72 h . Cytotoxicity was determined with MTT. Apoptosis was evaluated through fluorescence-activated cell sorting, Hoechst33258 staining and DNA ladder assay. The expression of p53, Bax, Bcl-2, Mdm2, p21, caspase-3 and PARP was determined with Western blot. siRNA interference was used to silence p53. Results: IFNα treatment for 72 h did not induce cytotoxicity but greatly enhanced doxorubicin-induced cytotoxicity and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells. Compared with other groups, the combination of IFNα and doxorubicin induced more obvious apoptotic morphological changes and DNA ladder. IFNα did not alter the expression of the indicate genes. The expression of p53, Bax, Mdm2 and p21 was up-regulated by doxorubicin and further increased in response to combination. The expression of Bcl-2 was down-regulated by doxorubicin and further decreased in response to combination.There were no differences among groups in MG63 cells. The expression of p53 was effectively blocked by p53-siRNA in U2OS cells. The p53 silencing greatly reduced the cytotoxicity mediated by combination for 72 h, compared with non- and control-siRNA groups. The activation of caspase-3 and PARP mediated by combination was largely suppressed by p53 silence. Conclusion: IFNα sensitizes human osteosarcoma cells to doxorubicin-induced apoptosis through p53-dependent pathway. The combination of IFNα and traditional chemotherapy can be used in osteosarcoma treatment.

Evaluation of the cytotoxic, apoptosis inducing activity and molecular docking of spiroquinazolinone benzamide derivatives in MCF-7 breast cancer cells

Previous studies have suggested that quinazolinone derivatives are potent apoptosis-inducing agents in various cancer cell lines. In the present study, we have investigated cytotoxic, apoptosis induction, and molecular docking activities of the spiroquinazolinone benzamide derivatives family on MCF-7 human breast cancer cells. The MTT cytotoxicity assays and docking studies showed that 4t-CHQB was the most active compound among the prepared spiroquinazolinone benzamide compounds with IC50 of 50 ± 1.2 μM and was selected for further assessments. Apoptosis, as the mechanism of cell death, was assessed morphologically by acridine orange/ethidium bromide (AO/EtBr) double staining, evaluation of the cell surface phosphatidylserine (PS) expression through annexin V/PI technique and, the formation of DNA ladder. Down regulation of survivin was evaluated in protein level after cell treatment with 4t-CHQB using western blotting method. Molecular modeling experiments involving 4t-CHQB binding site of survivin showed several strong hydrogen bonds and hydrophobic interactions between many important amino acid residues. Overall, the obtained data suggest that the assessed spiroquinazolinone benzamide compounds may provide a novel therapeutic approach for further evaluation, as an effective chemotherapeutic family acting through down regulation of survivin and apoptosis induction in breast cancer.