DNA Ladder Research Articles

Biomaterial-Interrelated Bacterial Sweeper: Simplified Self-Assembled Octapeptides with Double-Layered Trp Zipper Induces Membrane Destabilization and Bacterial Apoptosis-Like Death.

Treatment of microbial-associated infections continues to be hampered by impaired antibacterial efficiency and the variability in nanomedicines. Herein, an octapeptide library with a double-layered zipper, constructed via a systematic arrangement, simplifying the sequence and optimizing the structure (diverse motifs including surfactant-like, central-bola, and end-bola), is assessed in terms of biological efficiency and self-assembly properties. The results indicate that peptides with double-layered Trp zipper exhibit significant antimicrobial activity. Extracellularly, affinity interactions between micelles and bacteria induce the lateral flow of the membrane and electric potential perturbation. Intracellularly, lead molecules cause apoptosis-like death, as indicated by excessive accumulation of reactive oxygen species, generation of a DNA ladder, and upregulation of mazEF expression. Among them, RW-1 performs the best in vivo and in vitro. The intersecting combination of Trp zipper and surfactants possesses overwhelming superiority with respect to bacterial sweepers (therapeutic index [TI] = 52.89), nanostructures (micelles), and bacterial damage compared to RW-2 (central-bola) and RW-3 (end-bola). These findings confirm that the combination of double-layered Trp zipper and surfactants has potential for application as a combined motif for combating microbial infection and connects the vast gap between antimicrobial peptides and self-assembly, such as Jacob's ladder.

Thioredoxin-1 mediates neuroprotection of Schisanhenol against MPP+-induced apoptosis via suppression of ASK1-P38-NF-\u03baB pathway in SH-SY5Y cells

Oxidative stress-induced dopaminergic neuronal loss and apoptosis play a crucial role in the pathogenesis of Parkinson’s disease (PD), and as a vital antioxidant protein, thioredoxin (Trx) exerts neuroprotection against PD. In this study, we investigated the effect of Schisanhenol (Sal), an active component from a traditional Chinese herb Schisandra rubriflora (Franch.), on MPP+-induced apoptosis and its association with thioredoxin-1 (Trx1) in SH-SY5Y cells. The protein levels of Trx1 and apoptosis-related proteins were detected by Western blot, the expression of Trx1 mRNA by real time qPCR, and apoptosis was detected by fluorescence microscopy and flow cytometry. Pretreatment with Sal (1 µM, 10 µM, and 50 µM) dose-dependently ameliorated MPP+-induced neuronal injury, confirmed by the improvement of the viability and morphological changes. Sal decreased the apoptosis rate of cells, suppressed the production of DNA ladder and sub-G1 peak, inhibited the Caspase-3 activity and the expression of apoptosis-related proteins. Sal enhanced the expression of Trx1 both in the protein and mRNA levels. However, the Trx1 inhibitor PX-12 suppressed the protective effects of Sal. In addition, Sal inhibited NF-κB translocation and activation. These results suggest that Sal has a protective effect against MPP+-induced apoptosis in SH-SY5Y cells via up-regulation of Trx1 expression and suppression of ASK1-P38-NF-κB pathway.

Qing Yan Li Ge Tang, a Chinese Herbal Formula, Induces Autophagic Cell Death through the PI3K/Akt/mTOR Pathway in Nasopharyngeal Carcinoma Cells In Vitro.

Since a portion of patients with nasopharyngeal carcinoma (NPC) do not benefit much from current standard treatments, it is still needed to discover new therapeutic drugs to improve the prognosis of the patients. Considering that Chinese traditional medicine plays a role in inhibiting tumor progression, in this study, we aimed to investigate whether a Chinese herbal formula, Qing Yan Li Ge Tang (QYLGT), has the anticancer activity in NPC cells and explore the underlying mechanism as well. MTT assay, colony formation assay, immunoblotting assay, and DNA laddering assay were performed to assess cell viability, cell colony formation, protein expression, and DNA fragmentation, respectively. Results show that QYLGT was able to inhibit the cell viability and decrease colony formation ability in NPC cells. QYLGT could also increase the formation of intracellular vacuoles and induce the autophagy-related protein expressions, including Atg3, Atg6, and Atg12-Atg5 conjugate in NPC cells. Treatment with an autophagy inhibitor, 3-methyladenine, could significantly recover QYLGT-inhibited cell viability of NPC cells. In addition, QYLGT did not significantly induce apoptosis in NPC cells. We also found that QYLGT had the ability to activate phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway. Treatment with PI3K inhibitors, LY294002 and wortmannin, or mTOR inhibitors, rapamycin and Torin 1, could not only recover QYLGT-inhibited cell viability of NPC cells but also inhibit Atg3 expression. Taken together, our results demonstrated that QYLGT could induce autophagic cell death in NPC cells through the PI3K/Akt/mTOR pathway.

Sesamol Induces Apoptosis-Like Cell Death in Leishmania donovani.

BackgroundVisceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani (L. donovani), is the most severe form of leishmaniasis. It is largely responsible for significant morbidity and mortality in tropical and subtropical countries. Currently, available therapeutics have lots of limitations including high-cost, adverse side-effects, painful route of administration, less efficacy, and resistance. Therefore, it is time to search for cheap and effective antileishmanial agents. In the present work, we evaluated the antileishmanial potential of sesamol against promastigotes as well as intracellular amastigotes. Further, we tried to work out its mechanism of antileishmanial action on parasites through different assays.Methodology In vitro and ex vivo antileishmanial assays were performed to evaluate the antileishmanial potential of sesamol on L. donovani. Cytotoxicity was determined by MTT assay on human THP-1-derived macrophages. Sesamol-induced morphological and ultrastructural changes were determined by electron microscopy. H2DCFDA staining, JC-1dye staining, and MitoSOX red staining were performed for reactive oxygen assay (ROS), mitochondrial membrane potential, and mitochondrial superoxide, respectively. Annexin V/PI staining for apoptosis, TUNEL assay, and DNA laddering for studying sesamol-induced DNA fragmentation were performed.ConclusionsSesamol inhibited the growth and proliferation of L. donovani promastigotes in a dose-dependent manner. It also reduced the intracellular parasite load without causing significant toxicity on host-macrophages. Overall, it showed antileishmanial effects through induction of ROS, mitochondrial dysfunction, DNA fragmentation, cell cycle arrest, and apoptosis-like cell death to parasites. Our results suggested the possible use of sesamol for the treatment of leishmaniasis after further in vivo validations.

Novel β‑hydroxy ketones: Synthesis, spectroscopic characterization, molecular docking, and anticancer activity studies

In this study, a series of novel β‑hydroxy ketone derivatives 3a-o were designed, synthesized, and evaluated for their anticancer activity. The structure of these compounds were characterized by IR, 1H and 13C NMR, mass spectrometry and elemental analysis methods. All the synthesized compounds were screened for anticancer activity against MCF-7 and U87 cells. Among them, compound 3l was appeared to be the most potent compound on both cancer cells; and IC50 dose was determined as 145 µM for MCF-7 cells and 6,6 µM for U87 cells. DNA ladder and Annexin V apoptotic marker tests were used and as a result, 3l caused the initiation of apoptosis on U87 cells. VDAC protein expression increased dramatically after U87 glioblastoma brain cancer cells were treated with compound 3l Additionally, the molecular modeling of these compounds was studied in FLT3 receptor, Estrogen receptor, and PARP2 receptor for the treatment of Acute Myeloid Leukemia (AML), breast cancer, and Glioblastoma (GBM) respectively. Their binding motifs and drug-like properties were investigated, and the results are highlighted in the discussion. Based on the results, compound 3l may have a potential drug candidate profile that can reverse the drug resistance profile.

Multiplasmid DNA in agarose gel electrophoresis: Suitable dye and temperature is essential for prominent profiles

Nucleic acids dye Goldview is widely used in agarose gel electrophoresis (AGE). However, in this study, a sample of multiplasmid DNA (multi-pDNA) stained with Goldview analyzed by AGE showed its instability at low temperature. Three types of DNA samples were analyzed, including linear DNA (ladder), single-plasmid DNA (single-pDNA), and multi-pDNA, electrophoretic conditions were optimized by adjusting the dye, the buffer, and the temperature (1-50°C). The results showed that the light intensity of Gelred is 2.2-times higher than that of Goldview in staining multi-pDNA. Compared with the single-pDNA and the linear DNA, the multi-pDNA stained with Goldview was greatly affected by temperature. This short communication indicated that Gelred is a highly applicable dye for analyzing multiplasmid samples. The degree and the way of binding of Goldview to multi-pDNA are greatly affected by temperature.

Construction of DNA ladder for determination of small size DNA fragments

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.

Embilica officinalis L. inhibits the growth and proliferation of Leishmania donovani through the induction of ultrastructural changes, mitochondrial dysfunction, oxidative stress and apoptosis-like cell death

Visceral leishmaniasis (VL) is caused by a protozoan parasite, Leishmania donovani (L. donovani). It affects around 1–2 million people around the world annually. There is an urgent need to investigate new medicament of it due to difficult method of drug administration, long period of treatment, high cost of the drug, adverse side-effects, low efficacy and development of parasite resistance to the available drugs. Medicinal plants have also been used for the treatment of different diseases in traditional system of medicines due to their holistic effects. The Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland has already started the program for identification of potential medicinal plant and plant products having antileishmanial potential. Keeping all these in consideration, we planned to study the antileishmanial activity of one of the medicinal plant, Embilica officinalis L. (EO) fruit extract. EO fruit extract inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes in dose-dependent manner. EO fruit extract induced morphological and ultrastructural changes in parasites as observed under Electron Microscope. It also induced the oxidative stress, mitochondrial dysfunction, DNA laddering and apotosis-like cell death in parasites. Here, we for the first time reported such a detailed mechanism of action of antileishmanial activity of EO fruit extract. Our results suggested that EO fruit extract could be used for the development of new phytomedicine against leishmaniasis.

Analysis of cf-mtDNA and cf-nDNA fragment size distribution using different isolation methods in BV-2 cell supernatant of starvation-induced autophagy

Fragment size distribution, the important biological properties of cell-free DNA (cfDNA), provides useful information required for diagnostic assay development. However, besides methodological discrepancies, it varies due to the complicated origins and occurrences of in vivo cfDNA. In addition, limited data are available concerning the cfDNA associated with autophagy and distributional difference between cf-mitochondrial DNA (cf-mtDNA) and cf-nuclear DNA (cf-nDNA) fragments. Here we developed an in vitro model of mouse microglial cell (BV-2) with starvation-induced autophagy, in which cfDNA was isolated from the cell supernatant by ultrafiltration (UF) and column-based commercial kit (CC), respectively. Using Agilent 2100 Bioanalyzer, a DNA ladder pattern as the presence of peaks corresponding to mono-, di- and tri-nucleosomes was clearly visualized both in isolation products of UF and CC. However, we also detected shorter fragments than mono-nucleosome by UF. In comparing the UF and CC, we found that the former produced the higher recovery efficiency for spiked-in DNA of shorter fragments than mono-nucleosome in both water and medium, but the latter was superior for spiked-in DNA fragments which were longer than or equal to mono-nucleosome in medium. Combined with these two isolation methods, we have observed that autophagy-associated cf-mtDNA and cf-nDNA were both highly enriched in <mono-nucleosomes fragments more than 71%, and showed no significant differences in the relative percentages for these four fragment sizes. These results have improved our understanding of the fragment size distribution of autophagy-derived cf-mtDNA and cf-nDNA in vitro, and might further develop application of cfDNA as a diagnostic tool.

DNA Adduct Detection after Post-Labeling Technique with PCR Amplification (DNA-ADAPT-qPCR) Identifies the Pre-ribosomal RNA Gene as a Direct Target of Platinum-Acridine Anticancer Agents.

To study the DNA damage caused by a potent platinum-acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click chemistry-enabled, azide-modified derivative (APA) was developed. The method involves biotinylation, affinity capture, and bead-based enrichment of APA-modified genomic DNA. The key steps of the assay were validated and optimized in model duplexes, including full-length plasmids, restriction fragments, and a DNA ladder. Native DNA treated with APA and subsequently subjected to post-labeling with a biotin affinity tag was enzymatically digested and fragments were analyzed by in-line LC-MS and MS/MS. The monofunctional-intercalative adducts formed by APA in 5'-pyrimidine/guanine sequences in double-stranded DNA were quantitatively biotinylated by strain-promoted 1,3-dipolar cycloaddition chemistry. When applied to DNA extracted from A549 lung cancer cells, the assay in combination with qPCR amplification demonstrates that platinum-acridines form adducts in the gene sequences encoding pre-ribosomal RNA, a potential pharmacological target of these agents.

Assessment of antimitotic and programmed cell death stimulation potentials of Galium sinaicum (Delile ex Decne) Boiss. at cellular level

Galium sinaicum is a wild medicinal plant in saint Catherine, Egypt. To distinguish apoptotic effect of G. sinaicum ethanol extract (GsEE), we examined the role of GsEE in inducing programmed cell death (PCD) of Allium cepa root meristematic cell (AcR). Cells was subjected to GsEE in definite concentrations (0.1,0.3, 0.5%) and duration (6, 12h), then PCD induction was assessed. Application of GsEE arrested the mitotic division of AcR with metaphase accumulation. Electron microscopy analysis demonstrated ultrastructural alterations of organelles verifying PCD hallmarks. Protein electrophoresis analysis of AcR revealed a change in protein profile of Allium cepa root, also quantitative analysis showed significant increase in nuclease activity enzymes that stimulated DNA laddering fragmentation. Additionally, cell proliferation of MCF-7 and BHK21 was arrested by GsEE. Apoptotic effect of G. sinaicum may be attributed to the presence of potent phenolic compounds such as querectin and rutin as established by HPLC phenolic fingerprint analysis.

The protective role of resveratrol against sulfoxaflor-induced toxicity in testis of adult male rats.

This work was designed to explore the protective role of resveratrol (RES) against sulfoxaflor (Sulfx)-induced reproductive toxicity in adult male rats. The animals were divided into six groups: Control group, Sulfx treated groups (79.5 and 205 mg/kg/day), RES treated group (20 mg/kg/day), RES + Sulfx treated groups (20 mg/kg Res + 79.5 or 205 mg/kg Sulfx) orally for 28 consecutive days. Testicular samples were collected from all groups at the end of the treatment period. Tissue supernatants were isolated for oxidative stress and cellular energy parameters; tissue samples were prepared for histopathological examination. In addition, caspase-3 activity was calculated to assess spermatogenesis. Finally, DNA laddering assay was performed to detect DNA fragmentation as a hallmark of apoptosis. Our results showed that Sulfx treatment induced a significant increase in testicular levels of MDA, NOx, GSSG and reduced GSH level and cellular energy parameters in a dose-dependent manner compared to the control group. The results were confirmed by histopathological study which showed pathological changes in Sulfx treated groups. A significant increase in caspase 3 and DNA fragmentation was also observed. However, concomitant administration of RES to Sulfx -treated rats showed significant modulation against Sulfx-induced reproductive toxicity and attenuated the biochemical, apoptotic and histopathological changes. In conclusion, our results suggest that exposure to Sulfx at the two selected doses induces testicular toxicity and these effects can be ameliorated by supplementation of RES.

Sclareol Inhibits Hypoxia-Inducible Factor-1α Accumulation and Induces Apoptosis in Hypoxic Cancer Cells.

Purpose: The hypoxia in solid tumors is associated with the resistance to chemo/radiotherapy. Hypoxia-inducible factor-1 (HIF-1) plays a key role in cell remodeling to hypoxia. Therefore, the inhibition of HIF-1 accumulation is considered a hopeful strategy for the treatment of cancer. Here, we aimed to evaluate the geno- and cytotoxicity properties of sclareol, a natural bicyclic diterpene alcohol, on A549 cells in CoCl2-induced hypoxia. Methods: The cytotoxicity and apoptosis-inducing properties of sclareol on the A549 cell were evaluated using MTT assay and Annexin V/PI staining, respectively in hypoxia. DAPI staining, DNA ladder, and comet assay were used to evaluate the genotoxicity. Further, the qPCR technique was employed to assess the expression of HIF-1α, HIF-1β, and downstream target genes (GluT1, and Eno1). Finally, the level of HIF-1α protein was evaluated through Western blotting in sclareol-treated cells in hypoxia. Results: The inhibitory concentration (IC50) of sclareol against A549 cells was 8 μg/mL at 48 hours in hypoxia. The genotoxicity of sclareol was confirmed in the cells treated with sclareol in hypoxia. Sclareol induced ~46% apoptosis and also necrosis in the hypoxic condition. The qPCR analyses showed an enhanced suppression of HIF-1α, HIF-1β, GluT1, and Eno1 due to the sclareol treatment in the hypoxia. Moreover, protein quantification analysis showed dose-dependently degradation of HIF-1α in hypoxia upon treatment with sclareol. Conclusion: The results obtained here indicate that sclareol possesses dose-dependent cytotoxicity effects against A549 cells in hypoxia through inhibition of HIF-1α protein accumulation, increasing cell sensitivity to intracellular oxygen levels, and disruption of cell adaptation to hypoxia.

Chemical Composition, α-Glucosidase Inhibitory and Anticancer Activity of Essential Oil of Thymus vulgaris Leaves

Dietary herbs are used for imparting flavour, aroma, and piquancy to foods. Besides their role in improving food quality, spices also have health benefits, such as anticancer, antidiabetic, antimicrobial, antioxidant, hypolipidemic, analgesic, immunostimulant, etc. Thymus vulgaris (family: Lamiaceae) is a valuable medicinal herb known for its health benefits. The essential oil from T. vulgaris was isolated by the hydrodistillation method, and chemical constituents in it were determined by GC-MS. Anticancer activity was determined using a DNA ladder assay, and antidiabetic activity was accessed by α-glucosidase inhibition. The GC-MS analysis showed the presence of 16 chemical constituents, and thymol (55.88 %), linalool (13.71 %), carvacrol (8.36 %), and p-cymene (6.00 %) were found as major components. The antidiabetic activity of T. vulgaris essential oil showed potent inhibition of α-glucosidase enzyme with an IC50 value 125.1±4.25 μg/mL. The anticancer activity results revealed that treating MCF-7 cells with 100 μg/mL essential oil of thyme produces DNA fragmentation and promotes apoptosis. The current study provides a scientific basis for its possible use in controlling diabetes and breast cancer. The study further expands the knowledge of T. vulgaris chemical composition and provides evidence of ethnomedicinal use of the plant by exploring antidiabetic and anticancer activities.

The Protective Role of Wheat Germ Oil Against Adverse Effect of Deltamethrin on Reproductive Aspects of Male Albino Rats

Deltamethrin (DLM) is a synthetic pyrethroid type-II known to cause many adverse effects in experimental animals such as endocrine disruption, reproductive toxicity, and negative impact on gametogenesis. The present study was conducted to evaluate the protective role ofwheat germ oil against reproductive toxicity induced by Deltamethrin (DLM) in male albino rats through evaluation of semen picture, measuring the testosterone levels,DNA fragmentation percentage and pathologic changes of the testes. Thirty male rats were divided into three groups (10 each); the first group (C) was administered with normal saline, the second group was given (DLM) at a dose of (6 mg/kg. B.W. of rats) andthe third group (DLM+WGO) was treated with wheat germ oil (1.5 ml/Kg. B.W. of rats),and after one h DLM (6 mg/kg. B.W. of rats) was administered. All treatments were orally given using stomach gavage for 30 days. The rats were left for 65 days of treatment to complete time of one spermatogenesis and maturation of sperms in epididymis then sacrificed and blood samples were collected, testes and epididymis were removed, weighted, and examined histopathologically. DLM administration significantly decreased the weight of testes and adversely affected the semen quality. Testosterone levels were significantly reduced. There is a significant elevation in the DNA laddering percentage in the DLM group compared to the C group. Histopathological examination of the testes revealed that DLM induced changes in the form of distorted seminiferous tubules with intraluminal sloughing degenerated spermatogenic cells. Also there is marked interstitial edema and congestion. The co-administration of DLM with wheat germ showed improvement in weight of testes, semen pictures, testosterone level and a significant reduction in the DNA laddering percentage compared to DLM group. It can be concluded that, the wheat germ oil can mitigate the adverse effects of DLM on male fertility, and leads to improvement of male reproductive performance.

Isolation and molecular detection of avian mycoplasmosis in selected areas of Mymensingh district in Bangladesh

Avian mycoplasmosis caused by several species of Mycoplasma including Mycoplasma gallisepticum, M synoviae, M. meleagridis and M. iowae. Among these Mycoplasma gallisepticum is the most important poultry pathogen in Bangladesh. For effective control of Mycoplasmosis, proper early diagnosis is the corner stone. The research work was designed, a total of 20 samples, lung exudates, swabs from trachea and air sacs were collected from dead birds of different poultry farms in Mymensingh district during October-December, 2007. Samples were collected in 10% buffered formalin for histopathological study. Swabs were collected in mycoplasma broth supplemented with supplement-G. Additionally Kanamycin solution was added to prevent the growth of gram–Ve bacteria and then the organisms were transferred into mycoplasma agar for isolation. Histopathological studies were conducted using routine procedure in Hematoxylin and Eosin stain. Isolated Mycoplasma were subjected to DNA extraction, Nested PCR was done using a commercial PCR kit. The histopathological study revealed the presence of mycoplasmal related tissue changes, such as severe congestion and infiltration of mononuclear cells in different organs. The extracted DNA accumulated at the upper position of DNA ladder as band without any smear formation. The DNA from avian mycoplasmas was amplified and gave amplified product of 975 bp by outer primer and 395 bp by inner primer which was much smaller than the expected size. In this study, preliminary results from field samples suggest that culture using mycoplasma agar and broth supplement with Supplement-G and Kanamycin solution could be useful for the isolation of pathogenic avian mycoplasmas. Asian J. Med. Biol. Res. 2021, 7 (2), 182-190

Biological activity and molecular docking studies of some new quinolines as potent anticancer agents.

The objective of this study is to investigate the antiproliferative and cytotoxic properties and theaction mechanism of substituted quinoline and tetrahydroquinolines 3, 4, 5, 7, and 8 against rat glioblastoma (C6), human cervical cancer (HeLa), human adenocarcinoma (HT29) cancer cell lines by BrdU Cell Proliferation ELISA, Lactate Dehydrogenase, DNA laddering and Topoisomerase I assays. The results of the study showed that 6,8-dibromotetrahydroquinoline 3 possess in vitro antiproliferative activity against C6, HeLa, and HT29 cell lines while morpholine/piperazine substituted quinoline 7 and 8 showed selective antiproliferative activity on C6 cell line with IC50 values 47.5 and 46.3µg/mL, respectively. Moreover, 6,8-dibromoTHQ 3 caused DNA fragmentation while it did not inhibit the Topoisomerase I (Topo I) enzyme. On the other hand, compound8 did not cause DNA laddering while 8 inhibited the Topo I enzyme. According to these results, 6,8-dibromoTHQ 3 stimulates apoptosis on the C6 cell line while 6,8-dibromo-3-morhonilylquinoline (8) inhibits the Topo I enzyme to cause antiproliferative activity.

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6–48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

Overexpressed exocyst complex component 3-like 1 spontaneously induces apoptosis.

Exocyst complex component 3-like 1 (EXOC3L1), which regulates insulin secretion, is ubiquitously present in heart, lung, liver, spleen, kidney, muscle, cerebellum, pituitary, adrenal grand, and pancreatic islets. Its deduced amino acid sequence has 31% identity and 53% similarity with Sec6, so they are considered isoforms. Since Sec6 suppresses apoptosis via HSP27, we investigated the involvement of EXOC3L1 expression in apoptosis. We found that overexpressed EXOC3L1 in Chinese hamster ovary cells significantly reduced cultured cell numbers. It also significantly increased apoptotic DNA ladder, caspase 3 activity, and cleavage of caspase 3 compared with the control. Thus, although Sec6 reduces apoptosis by increasing HSP27 phosphorylation, overexpressed EXOC3L1 alone can spontaneously induce apoptosis without apoptotic stimulators or inducers.

Inhibition of MMP2-PEX by a novel ester of dihydroxy cinnamic and linoleic acid from the seagrass Cymodocea serrulata

Matrix metalloproteinases (MMPs) are pivotal for cancer cell migration and metastasis which are generally over-expressed in such cell types. Many drugs targeting MMPs do so by binding to the conserved catalytic domains and thus exhibit poor selectivity due to domain-similarities with other proteases. We report herein the binding of a novel compound [3-(E-3,4-dihydroxycinnamaoyloxyl)-2-hydroxypropyl 9Z, 12Z-octadeca-9, 12-dienoate; Mol. wt: 516.67 Da], (C1), isolated from a seagrass, Cymodocea serrulata to the unconserved hemopexin-like (PEX) domain of MMP2 (− 9.258 kcal/mol). MD simulations for 25 ns, suggest stable ligand-target binding. In addition, C1 killed an ovarian cancer cell line, PA1 at IC50: 5.8 μM (lesser than Doxorubicin: 8.6 µM) and formed micronuclei, apoptotic bodies and nucleoplasmic bridges whilst causing DNA laddering, S and G2/M phase dual arrests and MMP disturbance, suggesting intrinsic apoptosis. The molecule increased mRNA transcripts of BAX and BAD and down-regulated cell survival genes, Bcl-xL, Bcl-2, MMP2 and MMP9. The chemical and structural details of C1 were deduced through FT-IR, GC–MS, ESI–MS, 1H and 13C NMR [both 1D and 2D] spectra.