Late Apoptotic Rate Research Articles

Cytomodulatory characteristics of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) against cypermethrin on skin fibroblast cells (HFF-1)

The hematopoietic factor granulocyte macrophage-colony stimulating factor (GM-CSF) has been identified via its capacity to promote bone marrow progenitors' development and differentiation into granulocytes and macrophages. Extensive pre-clinical research has established its promise as a critical therapeutic target in an assortment of inflammatory and autoimmune disorders. Despite the broad literature on GM-CSF as hematopoietic of stem cells, the cyto/geno protective aspects remain unknown. This study aimed to assess the cyto/geno protective possessions of GM-CSF on cypermethrin-induced cellular toxicity on HFF-1 cells as an in vitro model. In pre-treatment culture, cells were exposed to various GM-CSF concentrations (5, 10, 20, and 40 ng/mL) with cypermethrin at IC50 (5.13 ng/mL). Cytotoxicity, apoptotic rates, and genotoxicity were measured using the MTT, Annexin V-FITC/PI staining via flow-cytometry, and the comet assay. Cypermethrin at 5.13 ng/mL revealed cytotoxicity, apoptosis, oxidative stress, and genotoxicity while highlighting GM-CSF's protective properties on HFF-1. GM-CSF markedly attenuated cypermethrin-induced apoptotic cell death (early and late apoptotic rates). GM-CSF considerably regulated oxidative stress and genotoxicity by reducing the ROS and LPO levels, maintaining the status of GSH and activity of SOD, and suppressing genotoxicity in the comet assay parameters. Therefore, GM-CSF could be promising as an antioxidant, anti-apoptotic, genoprotective and cytomodulating agent.

Modulatory effect of amifostine (WR-1065) against genotoxicity and oxidative stress induced by methotrexate in human umbilical vein endothelial cells (HUVECs)

Amifostine is used in chemotherapy and radiotherapy as a cytoprotective adjuvant alongside DNA-binding chemotherapeutic agents. It functions by reducing free radicals and detoxifying harmful metabolites. Methotrexate, as an antimetabolite drug has been considered for treating various cancers and autoimmune diseases. However, the cytotoxic effects of methotrexate extend beyond tumor cells to crucial organs, including the heart. This study applied the HUVEC cell line as a reference in vitro model for researching the characteristics of vascular endothelium and cardiotoxicity. The current study aimed to assess amifostine’s potential cytoprotective properties against methotrexate-induced cellular damage. Cytotoxicity was measured using the MTT assay. Apoptotic rates were evaluated by Annexin V-FITC/PI staining via flow cytometry. The genoprotective effect of amifostine was determined using the comet assay. Cells were exposed to various amifostine doses (10–200 μg/mL) and methotrexate (2.5 μM) in pretreatment culture condition. Methotrexate at 2.5 μM revealed cytotoxicity, apoptosis, oxidative stress and genotoxicity while highlighting amifostine’s cyto/geno protective properties on HUVECs. Amifostine significantly decreased the levels of ROS and LPO while preserving the status of GSH and SOD activity. Furthermore, it inhibited genotoxicity (tail length, %DNA in tail, and tail moment) in the comet assay. Amifostine markedly attenuated methotrexate-induced apoptotic cell death (early and late apoptotic rates). These findings convey that amifostine can operate as a cytoprotectant agent.

Genome characterization of Hirame novirhabdovirus (HIRRV) isolate CNPo2015 and transcriptome analysis of Hirame natural embryo (HINAE) cells infected with CNPo2015

Hirame novirhabdovirus (HIRRV) is a fish rhabdovirus belonging to family Rhabdoviridae, genus Novirhabdovirus, which is highly contagious and virulent, and causes hemorrhagic disease in many fish species. In the present work, the whole genome sequence of HIRRV strain CNPo2015 that previously isolated from cultured flounders was obtained using high-throughput sequencing. It consists of 10,998 nucleotides and encodes six viral proteins arranged in order of 3′-N-P-M-G-NV-L-5′. Among Novirhabdovirus, L protein of CNPo2015 possessed the lowest amino acid sequence divergence with HIRRV isolate CA 9703 and HIRRV 080113, and the highest with Snakehead rhabdovirus. Furthermore, the immune response of Hirame natural embryo (HINAE) cell line to HIRRV infection was characterized by RNA-seq, and the results showed that 1976 differentially expressed genes (DEGs) including 1219 up-regulated and 727 down-regulated genes were identified in the HINAE cells infected with HIRRV at 48 h post infection (hpi). Several KEGG pathways were significantly enriched in the viral infected cells, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, cell cycle, apoptosis, RIG-I-like receptors signaling pathway and P13K-AKT signaling pathway. Post viral infection, the flow cytometric Annexin V/PI assay found that apoptotic rate of HINAE cells showed a slight increase within 3 days and then the early and late apoptotic rate were significantly increased to 41 ± 2.65% and 12.37 ± 2.61% at day 4, respectively. Meanwhile, qRT-PCR results also showed that six apoptosis-related genes (BCL2L1, CASPASE 3, CASPASE 10, FAS, AKT and CDK1) were significantly upregulated. This investigation has not only enriched our knowledge of sequence difference characteristics between CNPo2015 and other Novirhabdoviruses, but also provided a data basis for deeper understanding of immune responses in flounder cells post viral infection.

New Pyrimidine-5-Carbonitriles as COX-2 Inhibitors: Design, Synthesis, Anticancer Screening, Molecular Docking, and In Silico ADME Profile Studies.

Two series of cyanopyrimidine hybrids were synthesized bearing either benzo[d]imidazole, benzo[d]oxazole, benzo[d]thiazole, and benzo[b]thiophene derivatives via methylene amino linker 3a-3d (Formula A) or various sulphonamide phenyl moieties 5a-5d (Formula B) at the C-2 position. All compounds' cyclooxygenase COX-2 inhibitory activities were evaluated, and all synthesized compounds demonstrated potent activity at minimal concentrations, with IC50 values in the submicromolar range. Compounds 3b, 5b, and 5d were discovered to be the most active pyrimidine derivatives, with the highest COX-2 percent inhibition and IC50 values being nearly equal to Celecoxib and approximately 4.7-, 9.3-, and 10.5-fold higher than Nimesulide. Furthermore, the pyrimidine derivatives 3b, 5b, and 5d demonstrated anticancer activity comparable to or better than doxorubicin against four cell lines, i.e., MCF-7, A549, A498, and HepG2, with IC50 values in nanomolar in addition to low cytotoxicity on the normal W38-I cell line. The effect of compound 5d on cell cycle progression and apoptosis induction was investigated, and it was found that compound 5d could seize cell growth at the sub-G1 and G2/M phases, as well as increase the proportion of early and late apoptotic rates in MCF-7 cells by nearly 13- and 60-fold, respectively. Moreover, in silico studies for compounds 3b, 5b, and 5d revealed promising findings, such as strong GIT absorption, absence of BBB permeability, nil-to-low drug-drug interactions, good oral bioavailability, and optimal physicochemical properties, indicating their potential as promising therapeutic candidates.

Roles of calpain in the apoptosis of Eimeria tenella host cells at the middle and late developmental stages.

This study investigated the role of calpain in Eimeria tenella-induced host cell apoptosis. Chick embryo cecal epithelial cell culture technology, flow cytometry, enzyme-linked immunosorbent assays, and fluorescence quantitative PCR were used to detect the E. tenella host cell apoptotic rate, Bax and Bid expression levels, and calpain activity. The results demonstrated that Bax, Bid, and calpain levels were upregulated and apoptosis was increased following E. tenella infection at 24-120h. Calpain levels were reduced by pharmacological inhibition of calpain using SJA6017 or by blocking Ca2+ entry into the cell using BAPTA/AM at 24-120h. The mRNA and protein levels of Bax and Bid, the E. tenella infection rate, and the early apoptotic and late apoptotic (necrosis) rates were decreased by using SJA6017 at 24-120h. These results indicated that E. tenella-promoted host cell apoptosis is regulated by calpain via Bid and Bax at 24-120h. Thus, manipulation of calpain levels could be used to manage E. tenella infection in chickens in the middle and late developmental stages.

Cytotoxic effects of different detergent containing children's toothpastes on human gingival epithelial cells

BackgroundThis study aimed to evaluate possible cytotoxic effects to gingival epithelial cells exposed to children toothpastes containing different detergent.MethodsTissues required for the isolation of human gingival epithelial cells were obtained by biopsy during the extraction of the impacted third molar tooth. Toothpaste solutions of different concentrations were prepared from five different children’s toothpastes with different detergent contents. Isolated gingival epithelial cells were stimulated with experimental groups consisting of toothpaste solutions (Colgate, Sensodyne, Splat, Nenedent, Perlodent) at different concentrations and a control group consisting of complete Dulbecco’s modified eagle medium. After the experiments, cell viability was evaluated using flow cytometry. 2 Way ANOVA was used to see the interaction effect of the main effects of toothpaste solution and concentration factors. Pairwise comparisons were made by Tukey post hoc tests. In the study, the significance level was taken as 0.05.ResultsAs a result of the analysis, it was seen that the toothpaste solution and concentration factors and the interactions of these 2 factors were effective on the viable, early apoptotic, late apoptotic and necrotic cell rates. The statistically highest live cell ratios were detected in Splat’s toothpaste solutions (90.14% at 0.4% concentration) after the control group (90.82%) and the group with the lowest viability values was determined in Colgate group (75.74% at 0.4% concentration) (p < 0.05).ConclusionsAccording to the results of the study, it was observed that toothpastes containing SLS affected the viability of cells more negatively than toothpastes with other detergent contents.

Tanshinone Iia Inhibits Megakaryopoiesis in Immune Vasculitis

Introduction Tanshinone IIA, an active component of Danshen (Salvia miltiorrhiza), has been used for centuries to treat hypercoagulation-related diseases, which attributed to its anti-platelet and anti-inflammatory effects. However, the role of Tanshinone IIA in megakaryocytes, the precursor of platelet within the bone marrow, remains unclear. Therefore, the present study established a rabbit model with immune vasculitis to examine the effect of Tanshinone IIA on megakaryopoiesis and to identify the underlying mechanism(s).Methods Immune vasculitis was established in rabbits (3-4 weeks old) by two intravenous injection of 10% bovine serum albumin (2.5 ml/kg) at two-week interval. Those rabbits were randomly treated with Tanshinone IIA (5 mg/kg/d, 7 d, iv) or aspirin (100 mg/kg/d, 7 d, ig). Megakaryocyte count and CFU-MK formation were measured by Wright's and AChE staining, respectively. Human megakaryotic cell lines Meg-01 and CHRF-288-11 were used to examine the effect of Tanshinone IIA on apoptosis by Annexin V-FITC/PI, mitochondrial membrane potential/JC-1 and Caspase-3 activity assays using flow cytometry.ResultsIn rabbits with immune vasculitis, the platelet count, platelet aggregation and the serum levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly increased when compared to their healthy controls. After 7 days of Tanshinone IIA treatment, all these parameters were significantly reduced, with the inhibitions comparable to those caused by aspirin. In addition, the number of megakaryocytes and the formation of CFU-MK were also statistically increased in rabbits with immune vasculitis, which could be significantly reduced by Tanshinone IIA. In vitro, Tanshinone IIA (1, 3, 10 and 30 μg/ml) also significantly inhibited the formation of CFU-MK of bone marrow cells of BALB/c mice (6-10 weeks) in a dose-dependent manner. In human megakaryocytic cell line Meg-01, Tanshinone ⅡA (10 μg/ml, 72 h) induced apoptosis; both early and late apoptotic rates were significantly increased. In another human megakaryocytic cell line CHRF-288-11, Tanshinone ⅡA (10 μg/ml, 72 h) statistically increased the proportion of depolarized cells, from 9.70% to 14.13%, according to mitochondrial membrane potential using JC-1 assay. The expression of active Caspase-3 in CHRF-288-11 was also significantly increased by Tanshinone ⅡA (10 μg/ml, 72 h) from 5.25% to 15.86%.Conclusion The present study shows that Tanshinone IIA ameliorates immune vasculitis by inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes, which might explain the anti-platelet and anti-inflammatory effects of Tanshinone IIA. DisclosuresNo relevant conflicts of interest to declare.

Effects of hnRNP E1 on expression of early genes E2, E6 of HPV16 and biological function in cervical cancer cells

Objective: To explore the effects of hnRNP E1 on the expression of early genes E2, E6 of HPV16 and the biological function in cervical cancer SiHa cell lines. Methods: The cell experiments in vitro were carried out in cervical cancer cell lines SiHa. The expression levels of E2, E6 mRNA and protein of HPV16 were detected by Real-time PCR and Western blot, respectively, before and after up-regulating hnRNP E1. Meanwhile, the cell proliferation, cycle and apoptosis were evaluated by CCK-8 and flow cytometry. Data analyses were performed using SPSS 22.0 and Graphpad Prism 7.0 software. Results: Compared with the blank and the blank plasmid group, the cells activity and proliferation decreased at 24, 48 and 72 h after up-regulating hnRNP E1 (P<0.05), while the percentage of cells in G0/G1 phase increased and the percentage in S and G2/M phase and proliferation index decreased (P<0.05). Moreover, the late apoptotic rate and the total apoptotic rate increased (P<0.05). The expression levels of E6 mRNA and protein of HPV16 in hnRNP E1 up-regulated group were significantly lower than that in both blank group and blank plasmid group, the differences were significant (P<0.05), showing the tendency of cells proliferation index decrease and total apoptotic rate increase with decreased HPV16 E6 expression. There were no significant differences in the expression of E2 mRNA of HPV16 among the three groups (P=0.427), and no E2 protein of HPV16 was detected. Conclusions: hnRNP E1 could inhibit the transcription and translation of E6 oncogene of HPV16 and further inhibit the proliferation and promote apoptosis of cervical cancer cells, suggesting that hnRNP E1 might be a potential target marker to prevent cervical lesions. But no association between hnRNP E1 and HPV16 E2 was found in SiHa cells.

Potential antiviral and anticancer effect of imidazoles and bridgehead imidazoles generated by HPV-Induced cervical carcinomas via reactivating the P53/ pRb pathway and inhibition of CA IX

Human papillomaviruses E6 and E7 oncoproteins are crucial to viral-induced cervical cancers and targeting E6 / E7 leads to safer and better treatment for cervical cancer. Hence, a simple and green solvent-free protocol was applied for the synthesis of novel imidazoles and bridgehead imidazoles (purine analogs) via versatile straightforward synthetic routes. All the synthesized compounds have been characterized by IR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analyses and then have been estimated for their in vitro antiviral activity against HPV, genotype 18. Two compounds 4 and 12 were the most promising HR- HPV inhibitors with a percentage of 95.00 and 96.84 %, respectively. Both compounds demonstrated a substantial down-regulation of HPV oncoproteins E6 and E7 with up-regulation of tumor suppressor proteins, p53, and pRb, respectively using western blot technique. Furthermore, the cytotoxicity of compounds 4 and 12 against the cervical cancer Hela cell line was further examined. Compound 4 exhibited strong anticancer activity with IC50 0.08 μM, which is equivalent to 5-FU (IC50 0.09 μM). The cell cycle analysis was performed for investigating the potential mechanism of compound 4, resulting in a significant accumulation of the cell population in both pre-G1 and G0-G1 phases and arrest the cell cycle at G1 phase. Besides, compound 4 induced apoptosis, triggering cell death via increasing the early and late apoptotic rates by approximately 16 and 188 folds compared with the control. The most cytotoxic agent 4 revealed a remarkable inhibition of the targeted carbonic anhydrase IX enzyme with an IC50 value of 0.12 μM comparable to the standard drug. In addition, the ADME profiles of the most highly successful derivatives have been studied in order to determine their ability to be produced as good drug candidates.

Effect of cell cycle synchronization on cadmium-induced apoptosis and necrosis in NRK-52E cells

ABSTRACT Heavy metal pollution is a problem that cannot be ignored. Due to the prevalence of cadmium in the environment and its harmful effects on humans, cadmium pollution has become a research hotspot recently. The mechanism of cadmium-induced toxicity has also drawn much attention and most studies have been conducted using whole cells, but the toxicological mechanism of cadmium remains unclear. In this study, we aimed to obtain NRK-52E cells at different growth stages by various methods and analyze the differences in cadmium toxicity. The results show that the cadmium sensitivity of cells in each phase was different and the late apoptotic rate was increased significantly after 5 µM Cd treatment. In addition, cadmium easily induces apoptosis of G0- and S-phase cells, as well as necrosis of S- and M-phase cells, but has no significant effect on G1-phase cells. Overall, we first explored the differences in the effects of cadmium on NRK-52E cells at various growth phases. Besides, the findings of this study might provide a theoretical basis for further exploration of the toxicological mechanism of cadmium.Abbreviations Cd: cadmium; CDK: cyclin-dependent kinases; DAPI 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; TBST: Tris-buffered saline with Tween-20; PI: propidium iodide; DMEM: Dulbecco’s Modified Eagle Medium; BCA: bicinchoninic acid

Effective inhibition of cancer cells by recombinant adenovirus expressing EGFR-targeting artificial microRNA and reversed-caspase-3.

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 μg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 μg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.

Antiproliferative and caspase-mediated apoptosis inducing effects of Murraya koenigii seeds against cancer cells

Murraya koenigii (Rutaceae), a tropical to the sub-tropical tree is well disseminated in the eastern region of India and known for its curative potential against ample diseases and disorders. In this study, the methanolic extract of Murraya koenigii seeds (MEMS) was screened for the first time to determine the in vitro cytotoxicity and caspase-mediated apoptosis-inducing effects against breast and colon cancer cells. Both in vitro assay and in silico molecular docking study were performed to screen the anti-proliferative potential of MEMS. Cicer arietinum seeds germination and Allium cepa root tip inhibition assay were used to study the anti-mitotic effect of the MEMS. Angiogenesis inhibition and cytotoxicity of MEMS were determined using chicken egg chorioallantoic membrane (CAM) assay and brine shrimp lethality bioassay respectively. MTT assay was used to exhibit the cell viability while apoptotic cells were determined using annexin V-fluorescein isothiocyanate assay. The activity of caspase-3 and caspase-7 was performed using the luminometric assay. SeDeM diagram and Heckel plot were generated for MEMS to predict their compression suitability. The MEMS significantly reduced the growth of Cicer arietinum seeds sprout (P<0.05) compared to control and on a par with methotrexate treated groups. Cell division in Allium cepa roots was diminished by 7.73 fold compared to control at the dose of 1000 µg/mL. MEMS showed moderate anti-angiogenic activity (14.81%) on CAM and considerable cytotoxicity (LD50= 40.860 μg/mL) to brine shrimps. MEMS elicited the prominent cytotoxic effect against DLD-1 colorectal cancer cells compared to MCF-7 breast cancer cells. It showed caspase-mediated apoptosis with reduced mitochondrial membrane potential. Early and late apoptotic rates were observed to be 7.13 and 2.57 fold higher respectively, than control. In silico docking analysis corroborated that neplanocin A and girinimbine interacted with proteolytic enzymes (caspase-3 and caspase-7) mainly by hydrogen bonding, aromatic, hydrophobic, and Van der Waals interactions. SeDeM diagram and Heckel plot revealed the poor compression behavior of MEMS. However, MEMS is having enormous potential and can be processed for further in vivo experiments.

Relationship between autophagy, apoptosis and endoplasmic reticulum stress induced by melatonin in osteoblasts by septin7 expression.

Melatonin secreted by the pineal body is associated with the occurrence and development of idiopathic scoliosis. Melatonin has a concentration-dependent dual effect on osteoblast proliferation, in which higher concentrations can inhibit osteoblast proliferation and induce apoptosis; however, the underlying mechanism remains unclear. In the present study, flow cytometry was used to demonstrate that osteoblast cells treated with melatonin exhibited significantly increased early and late stage apoptotic rates as the concentration increased. Chromatin condensation in the nucleus and apoptotic body formation could be observed using fluorescent microscopy in osteoblast cells treated with 2 mM melatonin. Western blotting results showed that there was an upregulation in the expression of apoptosis marker proteins [poly (ADP-ribose) polymerase 1 (PARP-1)], endoplasmic reticulum stress [ERS; C/EBP homologous protein (CHOP) and glucose-regulated protein, 78 kDa (GRP78)] and autophagy [microtubule-associated protein 1 light chain 3β (LC3)-I/LC3II]. PARP-1 expression was not altered when treated with ERS inhibitor 4PBA and autophagy inhibitor 3MA, whereas 4PBA or 3MA in combination with 2 mM melatonin (or the three together) significantly increased PARP-1 expression. Furthermore, the use of septin7 small interfering RNA confirmed that increased expression of GRP78 and CHOP was related to septin7, and melatonin- mediated ERS was necessary for septin7 activation. These findings suggest that ERS and autophagy might occur in the early stage of treatment with a high concentration of melatonin, and each might play a protective role in promoting survival; in a later stage, ERS and autophagy might interact and contribute to the induction of apoptosis. Overall, the results indicated that septin7 may be a target protein of melatonin-induced ERS.

Ethanol extraction technology of Epimedii Folium and protective effect of ethanol extract on chondrocyte

Uniform design-comprehensive scoring method was used to investigate the effects of ethanol dosage, ethanol concentration and extraction time, based on the evaluation index from transfer rates of epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ, which are the main active components in Epimedii Folium. Furthermore, the optimum conditions for the ethanol extraction process were determined by multiple linear stepwise regression and empirical test. Then, the ethanol extract of Epimedii Folium prepared according to the optimized technological conditions was used to intervene the injury model of chondrocyte induced by interleukin-1 beta(IL-1β). Annexin V-FITC/PI staining flow cytometry was used to detect the apoptotic rate of chondrocyte and analyze the effect of ethanol extract of Epimedii Folium on chondrocyte injury model. The optimum conditions of ethanol extraction were as follows. Crude powder of Epimedii Folium was added with 18 times of 70% ethanol solution, and extracted for twice in the refluxing process, for 60 minutes each time. Under the conditions, the extraction rates of the above five active components were 94.21%, 94.76%, 93.85%, 96.17% and 96.85%, respectively. The optimized ethanol extraction process of Epimedii Folium was reasonable, feasible and reproducible. This ethanol extract could significantly reduce the early apoptotic rate, late apoptotic and necrotic rate, total apoptotic rate(P&lt;0.05 or P&lt;0.01) of chondrocyte injury model induced by IL-1β, suggesting that the ethanol extract of Epimedii Folium can inhibit the apoptosis of chondrocytes induced by IL-1β to a certain extent, which lays a foundation for further study on its prevention and treatment of osteoarthritis.

Preparation and antitumor activity of sulfated exopolysaccharide from Rhizopus nigricans

To explore the effect of the composition ratio on substitution of sulfate group in sulfated exopolysaccharide (EPS) from Rhizopus nigricans and how sulfate modification affects the anti-tumor activity of EPS. We used a chlorosulfonic acid-pyridine method to modify EPS and analyzed the effect of esterification ratio on the degree of sulfate substitution using barium chloride turbidimetry. The sulfate groups binding with EPS were analyzed with infrared spectrum analysis. CCK-8 assay was used to evaluate the inhibitory effect of EPS sulfate (SEPS) on the proliferation of human colon cancer HCT 116 cells, and annexin V-FITC/PI double staining was used to assess the pro-apoptotic effect of SEPS in the cells. The esterifying agent and EPS at the composition ratios of 1:1 and 2:1 resulted in sulfate substitution of 0.98% (SEPS-1) and 1.18% (SEPS-2), respectively, and the substitution was improved by increasing the ratio of the esterifying agent (P < 0.05). Infrared spectrum analysis showed that the S=O stretching vibration absorption peak of -OSO3- appeared near 1249 cm-1, indicating that the sulfate group combined with EPS to form sulfate. CCK-8 assay showed that SEPS-1 produced stronger inhibitory effects on the proliferation of HCT 116 cells than EPS within the concentration range of 0.02-0.10 mg/L (P < 0.05). At the concentrations of 0.04-0.08 mg/L, SEPS-2 showed a lower anti-tumor activity than SEPS-1 (P < 0.05). SEPS-1 also showed stronger pro-apoptotic effect than EPS, and as its concentration increased, SEPS-1 dose-dependently increased the ratio of early apoptotic cells and necrotic cells; the cells treated with 0.06, 0.08 and 0.10 mg/mL SEPS-1 showed early apoptotic rates of 6.38%, 11.8% and 12.5%, and late apoptotic and necrotic rates of 5.26%, 8.04% and 6.80%, respectively. The composition ratio of the esterifying agent has a direct impact on the degree of substitution of EPS, which can be improved by increasing the ratio of the esterifying agent. Sulfate modification of EPS can enhance its antitumor activity, which, however, is not directly related with the degree of substitution.

Highly biocompatible graphene quantum dots: green synthesis, toxicity comparison and fluorescence imaging

Graphene quantum dots (GQDs) have tremendous potential in biological imaging due to their bright visible photoluminescence mission. However, the tedious preparation procedures and potential toxicity of GQDs greatly limit their application in biological field. Here, highly biocompatible GQDs (HGQDs) have been successfully prepared only by glucose in aqueous solution. Compared with GQDs prepared from conventional methods (CGQDs), the cytotoxicity of HGQDs reduced by more than 60%, and the flow cytometric analysis of the normal cells treated with HGQDs showed that the early and late apoptotic rate reduced by more than 72% and 40%, respectively. In vitro fluorescence imaging showed that both cells and bacteria could be imaged by HGQDs, and the morphology of cells and bacteria could be kept to a maximum extent. A long-term in vivo study revealed that no obvious organ (heart, liver, spleen, lung and kidney) damage or lesions were observed, and the blood–brain barrier (BBB) could be overcome, which provides the possibility for treatment and diagnosis of brain-related diseases. With adequate studies of biocompatibility, both in vitro and in vivo, HGQDs may be considered for further biological application.

Effects of Saccharomyces cerevisiae on alleviating cytotoxicity of porcine jejunal epithelia cells induced by deoxynivalenol

Deoxynivalenol (DON) is one of the mycotoxins most frequently encountering in cereal-based foods throughout the world. Saccharomyces cerevisiae was used to alleviate porcine jejunal epithelia cell (IPEC-J2) injury induced by DON in this study. The results indicated that cell viability and proliferation rates were significantly decreased when DON concentrations were increased from 0 to 64 µM after 24 h incubation (p < 0.05). The longer incubation time and higher DON concentrations would cause more serious effects on cell viability. S. cerevisiae could significantly degrade DON and decrease lactic dehydrogenase (LDH) release in the cells induced by DON (p < 0.05). DON (4 µM) could increase necrotic and apoptotic cell rates as well as decrease viable cell rates, compared with the control group (p < 0.05). However, S. cerevisiae addition in the DON group could decrease necrotic, late apoptotic and early apoptotic cell rates by 38.05%, 46.37% and 44.78% respectively, increase viable cell rates by 2.35%, compared with the single DON group (p < 0.05). In addition, S. cerevisiae addition could up-regulate mRNA abundances of IL-6, IL-8 and IL-10 in IPEC-J2 cells (p < 0.05), but down-regulate mRNA abundances of tight junction proteins (TJP-1) and occludin by 36.13% and 50.18% at 1 µM of DON (p < 0.05). It could be concluded that S. cerevisiae was able to alleviate IPEC-J2 cell damage exposed to DON.

Effect of microRNA-181 on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10

Objective To explore effect of microRNA (miRNA, miR)-181b on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10 (PTEN). Methods miR-181b inhibitor and miR-181b NC were transfected into HepG2 cell by liposome Lipofectamine™3000. The expression of miR-181b was detected by real-time quantitative polymerase chain reaction (Real-time PCR). Cell viability was measured by methyl thiazol tetrazolium (MTT) assay. Cell clone ability was detected by cell clone formation test. Cell apoptosis and cell cycle was detected by flow cytometry. The expression of PTEN protein and mRNA was measured by Western blotting and Real-time PCR. Luciferase reporter analysis was performed. Results The expression of miR-181b in miR-181b inhibitor group (0.23±0.02) was lower than that in miR-181b NC group (1.00±0.01, P<0.05). Cell viability in miR-181b inhibitor group (0.37±0.03) was lower than that in miR-181b NC group (0.59±0.06, P<0.05). Clone formation test showed cell clone number in miR-181b inhibitor group (37.64±3.70) was lower than that in miR-181b NC group (132.56±12.37, P<0.05). Cell early apoptotic rate and late apoptotic rate in miR-181b inhibitor group was higher than that in miR-181b NC group (P<0.05). At the same time, compared with miR-181b NC group, cell cycle was made arrested at G1 phase (P<0.05), the expression of PTEN protein and mRNA was up-regulated in miR-181b inhibitor group (P<0.05), miR-181b inhibitor targeted regulation the expression of PTEN. Conclusion miR-181b inhibitor could inhibit HepG2 cell proliferation and induce cell apoptosis by targeting up-regulation expression of PTEN. Key words: Hepatocellular carcinoma; MicroRNA-181b; Phosphatase and tensin ho-molog deleted from chromosome 10; Proliferation; Apoptosis

Effect of microRNA-26a on proliferation and migration of breast cancer cell via targeting regulation expressions of E2F7

Objective To explore effect of microRNA (miRNA, miR)-26a on proliferation and migration of breast cancer cell via targeting regulation expressions of nuclear transcription factor (E2F7). Methods The expression of miR-26a and E2F7 in BT549, MDA-MB-231, MDA-MB-435, MCF-7 breast cancer cell and MCF-10A normal mammary epithelial cell was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting, respectively. miR-26a mimics and miR-26a NC were transfected into MDA-MB-231 cell by liposome Lipofectamine™3000. The expression of miR-26a, cell viability, cell apoptotic rate, cell migration ability, the expression of E2F7 protein and mRNA was detected. Luciferase reporter analysis was performed. Results The expression of miR-26a in BT549 (0.40±0.04), MDA-MB-231 (0.35±0.02), MDA-MB-435 (0.42±0.03), MCF-7 (0.55±0.05) breast cancer cell was lower than that in MCF-10A (1.46±0.14) normal mammary epithelial cell (P<0.01). The expression of E2F7 in BT549 (1.25±0.12), MDA-MB-231 (3.10±0.31), MDA-MB-435 (2.18±0.21), MCF-7 (0.36±0.02) breast cancer cell was higher than that in MCF-10A (0.15±0.01) normal mammary epithelial cell (P<0.01). The expression of miR-26a in miR-26a mimics group (1.84±0.18) was higher than that in miR-26a NC group (0.36±0.03) (P<0.01). Cells’ viability in miR-26a mimics group (0.383±0.036) was lower than that in miR-26a NC group (0.589±0.060) (P<0.01). Cells’ early apoptotic rate [(16.51±1.65)%] and late apoptotic rate [(23.47±2.35)%] in miR-26a mimics group was higher than that in miR-26a NC group [ (2.58±0.26)%, (3.20±0.32)%] (P<0.01). Cells’ migration ability in miR-26a mimics group (42.36±4.23) was lower than that in miR-26a NC group (189.53±15.64) (P<0.01). The expression of E2F7 protein and mRNA in miR-26a mimics group was lower than that in miR-26a NC group (P<0.01), miR-26a mimics targeted regulation the expression of E2F7. Conclusion Over-expression of miR-26a could inhibit MDA-MB-231 cell proliferation and migration by targeting down-regulation expression of E2F7. Key words: Breast cancer; MicroRNA-26a; Nuclear transcription factor; Proliferation; Migration

The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas

The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as CgSmac) was identified from oyster Crassostrea gigas. The open reading frame of CgSmac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. CgSmac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that CgSmac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSmac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of CgSmac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of CgSmac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of CgSmac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in dsCgSmac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of CgSmac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in dsCgSmac + LPS group than those of dsEGFP + LPS group. Collectively, CgSmac could activate mitochondrial apoptosis pathway by promoting caspase-3 activity in oyster hemocytes against exogenous LPS invasion. These results provided new insights on oyster apoptosis and the immune defense mechanisms in invertebrates.