Long-term Cytotoxicity Research Articles

Assessment of Cytotoxicity on Moringa Olifera Against Ehrlich Ascites Carcinoma in Swiss Albino Mice

Objective: The present study determines the potent cytotoxic and antitumor properties of methanolic extract of Moringa oleifera leaves.Material and Method: The leaves were collected from in and around Tiruchengode of Nammakal district of Tamilnadu. The collected leaves were shade dried and powdered and the Methanolic extract was extracted using the soxhlet apparatus. In-vitro cytotoxicity study was done by Tryphan Blue Dye Exclusion method and MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5- diphenyltetrazolium bromide) assay. In short-term cytotoxicity study by Tryphan Blue Dye Exclusion method the IC50 value against EAC cell lines was found to be 163.91 µg/ml. In Long-term cytotoxicity study by MTT assay the IC50 value against Normal Mouse Embryonic Fibroblast (NIH 3T3) was found to be 260.85 µg/ml for Human Cervical Cancer cells (HeLA) was 182.41 µg/ml for Human Laryngeal Epithelial Carcinoma (HeP-2) was 195.93. µg/ml and for Human Liver Cancer cells (HepG2) was 168.61 µg/ml. The MST of the control group was 17.33 ± 1.53 days, whereas it was 43.67 ± 1.15, 23.67 ± 1.53 and 33.33 ± 1.53 days for the groups treated with MMO (200 and 400mg/kg) and 5 FU respectively (0.001 & 0.001). The increase in lifespan of Tumour-bearing mice treated with MMO and 5-FU was found to be 36. 75, 92.78 and 152.84 respectively.Result: Haematological parameters of a tumour bearing mice on the day 14 were showed significant changes when compared to normal mice. The total WBC count, protein and PCV were found to increase with a reduction in the hemoglobin content of RBC. At the same time interval, MMO (200 and 400mg/kg) treatment could change these parameters near normal. Maximum alternation occurred in the MMO treatment at the dose of (400mg/kg). There was the significant reduction in the Tumour volume of mice treated with MMO (200 and 400 mg/kg/p.o.). The Tumour volume of control animals was 2. 92 ± 0.12 ml where it was 2.55 ± 0.11 ml and 1.82 ± 0.04 ml for the groups treated with MMO (200 and 400mg/kg/p.o) respectively (P < 0.01 & 0.005).Coclusion: The present study provides clear evidence, that the extract of Moringa olifera shows effective cytotoxicity against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice. A further study about the active principles and mechanism of action of the Methanolic extract of Moringa oleifera at the molecular level was needed.

Oxidized low-density lipoprotein modulates differentiation of murine memory CD8+ T cell subpopulations

To investigate effect of oxidized low-density lipoprotein (ox-LDL) on memory CD8+ T cell subpopulation differentiation in mice with autoimmune diabetes. Cultured splenic CD8+ T cells from pre-diabetic NOD mice isolated with magnetic beads were treated with 30 µg/mL ox-LDL and 10 U/mL interleukin-2 (IL-2) for 24 h and the control cells were treated with IL-2 only. Flow cytometry was used to determine the percentage of splenic CD8+IFN-γ+ T cells, expressions of CD8, CD44 and CD62L on the T cells, and the activation of T cell factor-1 (TCF-1) and STAT-3. The CD127+ memory T cells were purified and transplanted into the pre-diabetic NOD mice via the tail vein, and the blood glucose was recorded weekly and survival time of the mice was monitored. Treatment with ox-LDL significantly reduced islet β cell-specific cytotoxic CD8+T cells as compared with the control group [(0.7∓0.03)% vs (2.7∓0.14)%, P<0.01]. The percentage of effector memory CD8+T cells (Tem) in the total memory CD8+T cells was reduced [(10.3∓0.71)% vs (30.3∓1.36)%, P<0.01] and that of stem cell-like memory T cells was significantly increased [(72.3∓3.8)% vs (55.1∓2.61)%, P<0.05] following ox-LDL treatment, which also resulted in significantly decreased activation of TCF-1 [(14.5∓0.82)% vs (34.2∓1.23)%, P<0.01] and pSTAT-3 [(3.3∓0.12)% vs (22.1∓1.1)%, P<0.01]. Transplantation of ox-LDL-treated memory T cells in pre-diabetic NOD mice obviously inhibited the increase of blood glucose and prolonged the survival time of the mice (P<0.05). Ox-LDL decreases the activation of transcriptional factors TCF-1 and phosphorylation of STAT-3, inhibits the formation of effector memory CD8+ T cells with long-term cytotoxicity, but promote the generation of stem cell-like memory CD8+ T cells, which result in suppression of islet β cell-specific effector cytotoxic CD8+ T cell differentiation to lessen autoimmune injury to the islet β cells.

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

BackgroundThree-dimensional (3D) spheroids are frequently used in toxicological study because their morphology and function closely resemble those of tissue. As these properties are maintained over a long term, repeated treatment of the spheroids with a test object is possible. Generally, in the repeated treatment test to assess cytotoxicity in the spheroids, ATP assay, colorimetric measurement using pigments or high-content imaging analysis is performed. However, continuous assessment of cytotoxicity in the same spheroids using the above assays or analysis is impossible because the spheroids must be disrupted or killed. To overcome this technical limitation, we constructed a simple monitoring system in which cytotoxicity in the spheroids can be continuously monitored by nondestructive bioluminescence measurement.ResultsMouse primary hepatocytes were isolated from transchromosomic (Tc) mice harboring a mouse artificial chromosome (MAC) vector expressing beetle luciferase Emerald Luc (ELuc) under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter/rabbit β-globin intron II (CAG) promoter, and used in 3D cultures. We confirmed that both luminescence and albumin secretion from the spheroids seeded in the 96-well format Cell-ableTM were maintained for approximately 1 month. Finally, we repetitively treated the luminescent 3D spheroids with representative hepatotoxicants for approximately 1 month, and continuously and nondestructively measured bioluminescence every day. We successfully obtained daily changes of the dose-response bioluminescence curves for the respective toxicants.ConclusionsIn this study, we constructed a monitoring system in which cytotoxicity in the same 3D spheroids was continuously and sensitively monitored over a long term. Because this system can be easily applied to other cells, such as human primary cells or stem cells, it is expected to serve as the preferred platform for simple and cost-effective long-term monitoring of cellular events, including cytotoxicity.

Carbon nanotubes as cancer therapeutic carriers and mediators.

Carbon nanotubes (CNTs) have received increasing attention in biomedical fields because of their unique structures and properties, including high aspect ratios, large surface areas, rich surface chemical functionalities, and size stability on the nanoscale. Particularly, they are attractive as carriers and mediators for cancer therapy. Through appropriate functionalization, CNTs have been used as nanocarriers for anticancer drugs including doxorubicin, camptothecin, carboplatin, cisplatin, paclitaxel, Pt(II), and Pt(IV), and genes including plasmid DNA, small-interfering RNA, oligonucleotides, and RNA/DNA aptamers. CNTs can also deliver proteins and immunotherapy components. Using combinations of light energy, they have also been applied as mediators for photothermal therapy and photodynamic therapy to directly destroy cancer cells without severely damaging normal tissue. If limitations such as a long-term cytotoxicity in the body, lack of size uniformity during the synthetic process, loading deviations for drug–CNT complexes, and release controllability at the target point are overcome, CNTs will become one of the strongest tools that are available for various other biomedical fields as well as for cancer therapy.

Hyaluronic Acid-Coated Silver Nanoparticles As a Nanoplatform for in Vivo Imaging Applications.

An efficient chemical reduction protocol has been developed for the synthesis of hyaluronic acid-coated silver nanoparticles (HA-Ag NPs) that are spherical, ultrasmall and monodisperse. The as-synthesized HA-Ag NPs not only exhibited excellent long-term stability and low cytotoxicity but also could be used as a nanoplatform for X-ray computed tomography (CT) and single-photon emission computed tomography (SPECT) imaging after being radiolabeled with 99mTc.

Long-Term Cytotoxicity, pH and Dissolution Rate of AH Plus and MTA Fillapex

The aim of the present study was to verify the long-term cytotoxic effects of the MTA Fillapex and to compare them with AH Plus. Dissolution rate and pH were also evaluated. Human osteoblast cells were incubated with elutes of fresh specimens from AH Plus and MTA Fillapex, and with elutes of the same specimens for 4 successive weeks. Elute's pH was evaluated at each time point. A multiparametric cell viability assay was performed. For dissolution rate, ISO methodology was used. The results were analyzed by one-way analysis of variance, complemented with the Tukey post-test (p<0.05). No significant difference was found among the materials when fresh mixed (p>0.05). After 1 week, AH Plus became non-cytotoxic on all three evaluated parameters. Conversely, MTA Fillapex remained severely and mildly cytotoxic over the entire experimental period (p<0.05). The dissolution rate of AH Plus was significantly lower than MTA Fillapex at all time points (p>0.05). The pH of AH Plus was significantly lower than MTA Fillapex at the second and third week (p<0.05). In the other tested time points no statistical difference was observed. In conclusion, MTA Fillapex remained cytotoxic after 4 weeks and its cytotoxicity may be related to the high dissolution rate of this material.

Fabrication of silver nanoparticle-doped hydroxyapatite coatings with oriented block arrays for enhancing bactericidal effect and osteoinductivity

Implant-associated infection is a common postoperative complication and remains a serious problem in orthopedic surgery. This work describes the synthesis of silver nanoparticle-doped hydroxyapatite coatings with oriented block arrays (AgNP-BHAC). The resulting nanostructure was investigated using scanning electron microscopy, energy-dispersive spectrometry, transmission electron microscopy, X-ray powder diffraction, and Fourier transform infrared spectroscopy. AgNP-BHAC exhibited excellent antimicrobial activity toward gram-negative Escherichia coli and gram-positive Staphylococcus aureus owing to the antibacterial effects of the silver nanoparticles. Human bone marrow stromal cells (hBMSC) culture revealed that the AgNP-BHAC exhibited better biocompatibility, and permitted improved cell proliferation, attachment, and osteoinductivity than uncoated Ti–6Al–4V titanium alloy, the favored material for biomedical applications. In summary, this study presents a convenient and effective method for the incorporation of silver into HA coatings with block morphology. This method can be utilized to modify a variety of metallic implant surfaces to improve their antimicrobial effects and reduce potential long-term cytotoxicity.

Molecular targeted photoimmunotherapy for HER2-positive human gastric cancer in combination with chemotherapy results in improved treatment outcomes through different cytotoxic mechanisms.

BackgroundPhotoimmunotherapy (PIT) is a novel type of molecular optical imaging-guided cancer phototherapy based on a monoclonal antibody conjugated to a photosensitizer, IR700, in combination with near-infrared (NIR) light. PIT rapidly causes target-specific cell death by inducing cell membrane damages and appears to be highly effective; however, we have previously demonstrated that tumor recurrences were eventually seen in PIT-treated mice, likely owing to inhomogeneous mAb-IR700 conjugate distribution in the tumor, thus limiting the effectiveness of PIT as a monotherapy. Here, we examined the effects of human epidermal growth factor-2 (HER2)-targeted PIT in combination with 5-fluorouracil (5-FU) compared to PIT alone for HER2-expressing human gastric cancer cells.MethodsNCI-N87 cells, HER2-positive human gastric cancer cells, were used for the experiments. Trastuzumab, a monoclonal antibody directed against HER2, was conjugated to IR700. To assess the short-term cytotoxicity and examine the apoptotic effects upon addition of 5-FU in vitro, we performed LIVE/DEAD and caspase-3 activity assays. Additionally, to explore the effects on long-term growth inhibition, trypan blue dye exclusion assay was performed. NCI-N87 tumor xenograft models were prepared for in vivo treatment studies and the tumor-bearing mice were randomized into various treatment groups.ResultsCompared to PIT alone, the combination of HER2-targeted PIT and 5-FU rapidly induced significant cytotoxicity in both the short-term and long-term cytotoxicity assays. While both 5-FU and/or trastuzumab-IR700 conjugate treatment induced an increase in caspase-3 activity, there was no additional increase in caspase-3 activity upon NIR light irradiation after incubation with 5-FU and/or trastuzumab-IR700. The combination of HER2-targeted PIT and 5-FU resulted in greater and longer tumor growth inhibition than PIT monotherapy in vivo. This combined effect of PIT and 5-FU is likely owing to their different mechanisms of inducing tumor cell death, namely necrotic membrane damage by PIT and apoptotic cell death by 5-FU and trastuzumab.ConclusionsPIT in combination with 5-FU resulted in enhanced antitumor effects compared to PIT alone for HER2-expressing human gastric cancer in vitro and in vivo. This combination photoimmunochemotherapy represents a practical method for treating human gastric cancer and should be investigated further in the clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2072-0) contains supplementary material, which is available to authorized users.

In situ deposition of a personalized nanofibrous dressing via a handy electrospinning device for skin wound care.

Current strategies for wound care provide limited relief to millions of patients who suffer from burns, chronic skin ulcers or surgical-related wounds. The goal of this work is to develop an in situ deposition of a personalized nanofibrous dressing via a handy electrospinning (e-spinning) device and evaluate its properties related to skin wound care. MCM-41 type mesoporous silica nanoparticles decorated with silver nanoparticles (Ag-MSNs) were prepared by a facile and environmentally friendly approach, which possessed long-term antibacterial activity and low cytotoxicity. Poly-ε-caprolactone (PCL) incorporated with Ag-MSNs was successfully electrospun (e-spun) into nanofibrous membranes. These in situ e-spun nanofibrous membranes allowed the continuous release of Ag ions and showed broad-spectrum antimicrobial activity against two common types of pathogens, Staphylococcus aureus and Escherichia coli. In addition, the in vivo studies revealed that these antibacterial nanofibrous membranes could reduce the inflammatory response and accelerate wound healing in Wistar rats. The above results strongly demonstrate that such patient-specific dressings could be broadly applied in emergency medical transport, hospitals, clinics and at the patients' home in the near future.

Photo-bactericidal Properties of Hydroxyapatite Implant Surface Coating

Implant-associated infection is a common postoperative complication and remains a serious problem in the surgery. This work describes the development of hydroxyapatite implants with photo-bactericidal properties by the surface coating. The unique coating as crystalline nanoparticles of IR photosensitizers of implant was investigated using infrared spectroscopy. It has been proved that by the interaction of nanoparticles covering implant with the polar solvent, which simulates the interaction of the implant with the biocomponents in vivo (fast proliferating and with immune-competent cells), IR photosensitizers nanoparticles change the spectroscopic properties, becoming fluorescent and phototoxic. Thus, the developed coating based on crystalline IR photosensitizer nanoparticles with studied specific properties should have antibacterial, anti-inflammatory effect by the photodynamic treatment in the near implant area. This research opens the prospect of such technology application in order to provide the local inflammatory and autoimmune reactions prevention in the area of implantation that will improve implant antimicrobial effects and reduce potential long-term cytotoxicity. The results of the study suggest a promising this technology in order to create implants with photo-bactericidal properties.

Lower cytotoxicity, high stability, and long-term antibacterial activity of a poly(methacrylic acid)/isoniazid/rifampin nanogel against multidrug-resistant intestinal Mycobacterium tuberculosis.

To overcome the undesirable side effects and reduce the cytotoxicity of isoniazid (INH) and rifampin (RMP) in the digestive tract, a poly(methacrylic acid) (PMAA) nanogel was developed as a carrier of INH and RMP. This PMAA/INH/RMP nanogel was prepared as a treatment for intestinal tuberculosis caused by multidrug-resistant Mycobacterium tuberculosis (MTB). The morphology, size, and in vitro release properties were evaluated in a simulated gastrointestinal medium, and long-term antibacterial performance, cytotoxicity, stability, and activity of this novel PMAA/INH/RMP nanogel against multidrug-resistant MTB in the intestine were investigated. Our results indicate that the PMAA/INH/RMP nanogel exhibited extended antibacterial activity by virtue of its long-term release of INH and RMP in the simulated gastrointestinal medium. Further, this PMAA/INH/RMP nanogel exhibited lower cytotoxicity than did INH or RMP alone, suggesting that this PMAA/INH/RMP nanogel could be a more useful dosage form than separate doses of INH and RMP for intestinal MTB. The novel aspects of this study include the cytotoxicity study and the three-phase release profile study, which might be useful for other researchers in this field.

Altering the response of intracellular reactive oxygen to magnetic nanoparticles using ultrasound and microbubbles

Engineered iron oxide magnetic nanoparticles (MNPs) are one of the most promising tools in nanomedicine-based diagnostics and therapy. However, increasing evidence suggests that their specific delivery efficiency and potential long-term cytotoxicity remain a great concern. In this study, using 12 nm γ-Fe2O3 MNPs, we investigated three types of uptake pathways for MNPs into HepG2 cells: (1) a conventional incubation endocytic pathway; (2) MNPs co-administrated with microbubbles under ultrasound exposure; and (3) ultrasound delivery of MNPs covalently coated on the surface of microbubbles. The delivery efficiency and intracellular distribution of MNPs were evaluated, and the cytotoxicity induced by reactive oxygen species (ROS) was studied in detail. The results show that MNPs can be delivered into the lysosomes via classical incubation endocytic internalization; however, microbubbles and ultrasound allow the MNPs to pass through the cell membrane and enter the cytosol via a non-internalizing uptake route much more evenly and efficiently. Further, these different delivery routes result in different ROS levels and antioxidant capacities, as well as intracellular glutathione peroxidase activity for HepG2 cells. Our data indicate that the microbubble–ultrasound treatment method can serve as an efficient cytosolic delivery strategy to minimize long-term cytotoxicity of MNPs.

Intracellular Adenosine Triphosphate Deprivation through Lanthanide-Doped Nanoparticles.

Growing interest in lanthanide-doped nanoparticles for biological and medical uses has brought particular attention to their safety concerns. However, the intrinsic toxicity of this new class of optical nanomaterials in biological systems has not been fully evaluated. In this work, we systematically evaluate the long-term cytotoxicity of lanthanide-doped nanoparticles (NaGdF4 and NaYF4) to HeLa cells by monitoring cell viability (mitochondrial activity), adenosine triphosphate (ATP) level, and cell membrane integrity (lactate dehydrogenase release), respectively. Importantly, we find that ligand-free lanthanide-doped nanoparticles induce intracellular ATP deprivation of HeLa cells, resulting in a significant decrease in cell viability after exposure for 7 days. We attribute the particle-induced cell death to two distinct cell death pathways, autophagy and apoptosis, which are primarily mediated via the interaction between the nanoparticle and the phosphate group of cellular ATP. The understanding gained from the investigation of cytotoxicity associated with lanthanide-doped nanoparticles provides keen insights into the safe use of these nanoparticles in biological systems.

Intravenous siRNA Silencing of Survivin Enhances Activity of Mitomycin C in Human Bladder RT4 Xenografts

Survivin inhibits apoptosis and enables tumor cells to escape from therapy induced senescence. High survivin expression is associated with bladder cancer aggressiveness and recurrence. We evaluated whether survivin expression is reduced by siRNA and whether survivin silencing would enhance mitomycin C activity in human RT4 bladder transitional cell tumors in vitro and in vivo. We assessed the effectiveness of siRNA therapy using 2 newly developed pegylated cationic liposome carriers, PCat and PPCat. Each has a fusogenic lipid to destabilize the endosomal membrane. PPCat further contains paclitaxel to enhance in vivo delivery and transfection of survivin siRNA. In vitro antitumor activity was evaluated by short-term MTT and long-term clonogenicity cytotoxicity assays. In vivo intravenous therapy was assessed in mice bearing subcutaneous tumors. Nontarget siRNA showed no antitumor activity in vitro or in vivo. Treatment of cultured cells with mitomycin C at a 50% cytotoxic concentration enhanced survivin mRNA and protein levels. Adding PPCat or PCat containing survivin siRNA reversed survivin induction and enhanced mitomycin C activity (p <0.05). In tumor bearing mice single agent mitomycin C delayed tumor growth and almost tripled the survivin protein level in residual tumors. Adding PPCat-survivin siRNA, which alone resulted in a minor survivin decrease of less than 10%, completely reversed mitomycin C induced survivin and enhanced mitomycin C activity (p <0.05). Results indicate that there is effective in vivo survivin silencing and synergism between mitomycin C and PPCat-survivin siRNA. This combination represents a potentially useful chemo-gene therapy for bladder cancer.

Multichannel lens-free CMOS sensors for real-time monitoring of cell growth.

A low-cost platform is proposed for the growth and real-time monitoring of biological cells. The main components of the platform include a PMMA cell culture microchip and a multichannel lens-free CMOS (complementary metal-oxide-semiconductor) / LED imaging system. The PMMA microchip comprises a three-layer structure and is fabricated using a low-cost CO2 laser ablation technique. The CMOS / LED monitoring system is controlled using a self-written LabVIEW program. The platform has overall dimensions of just 130 × 104 × 115 mm(3) and can therefore be placed within a commercial incubator. The feasibility of the proposed system is demonstrated using HepG2 cancer cell samples with concentrations of 5000, 10 000, 20 000, and 40 000 cells/mL. In addition, cell cytotoxicity tests are performed using 8, 16, and 32 mM cyclophosphamide. For all of the experiments, the cell growth is observed over a period of 48 h. The cell growth rate is found to vary in the range of 44∼52% under normal conditions and from 17.4∼34.5% under cyclophosphamide-treated conditions. In general, the results confirm the long-term cell growth and real-time monitoring ability of the proposed system. Moreover, the magnification provided by the lens-free CMOS / LED observation system is around 40× that provided by a traditional microscope. Consequently, the proposed system has significant potential for long-term cell proliferation and cytotoxicity evaluation investigations.

The use of poly(methacrylic acid) nanogel to control the release of amoxycillin with lower cytotoxicity

In order to control the release of amoxycillin (AM) with lower cytotoxicity and higher activity, ethylene glycol dimethacrylate was used as the cross-linker, and a series of poly(methacrylic acid) (PMAA) nanogels were prepared to load the AM. Then, the morphology, size, in vitro release property, long-term antibacterial performance, cytotoxicity, stability and activity of this novel AM/PMAA nanogel were investigated. The results showed that the AM/PMAA nanogel sustainably released AM with long-term antibacterial activity. Moreover, the AM/PMAA nanogel could improve the stability of AM. More importantly, this AM/PMAA nanogel showed slighter cytotoxicity than AM alone, suggesting that the AM/PMAA nanogel was a more useful dosage form than AM for infectious diseases.

Effect of Physicochemical Properties of Drugs on Morphology and Release of Microspheres

1,3-bis(-2-chloroethyl)-1-nitrosourea (BCNU)-loaded and paclitaxel-loaded poly(D,L-lactide) (PDLLA) microspheres, respectively, were prepared by the spray-drying method. The influence of the physicochemical properties of the two drugs on the morphology, encapsulation efficiency, and in vitro drug release behavior were studied. The results show that the plasticization of BCNU to PDLLA greatly affects the morphology and release behavior of the microspheres. The microparticles prepared with BCNU had a spherical shape when the initial loading amount was less than 10%. With a further increase in the BCNU loading amount, the microspheres began to aggregate and lost their spherical shape. Compared to paclitaxel-loaded microspheres, BCNU-loaded ones exhibited remarkable burst release behavior due to active macromolecule motion. The cytotoxicity experiments show that the long-term cytotoxicity on tumor cells of BCNU-loaded microspheres was similar to that of paclitaxel-loaded microspheres, and that both the drug loaded in microsphere exhibited a superior inhibition effect on tumor cells to their commercial form.

Comparison of the anti-cancer effect of Disulfiram and 5-Aza-CdR on pancreatic cancer cell line PANC-1.

Background:Pancreatic cancer has poor prognosis by surgical and chemotherapy when it is diagnosed, so other anti-cancerous assistant therapeutic drugs are suggested e.g. epigenetic reversal of tumor-suppressor genes on promoter hypermethylation. 5-Aza-CdR is a nucleoside analog of DNMTi but it has long-term cytotoxicity effects. This study compares the anticancer effect of 5-Aza-CdR and Disulfiram potencies on PANC-1 cell line and up-regulation of p21.Materials and Methods:PANC-1 cell line was cultured in DMEM high glucose and treated by 5-Aza-CdR with 5 and 10 μM concentration for four days and 13 μM DSF (Diulfiram) for 24 hours. MS-PCR and RT-PCR were carried out to detect the methylation pattern and estimate the mRNA expression of RASSF1A and p21 in PANC-1.Result:MS-PCR demonstrated partial unmethylation after treatment with 5-Aza-CdR while there was no unmethylated band after DSF treatment. RT-PCR showed significant differences between re-expression of RASSF1A before and after treatment with 10 μM 5-Aza-CdR (P < 0.01) but not after treatment with 13 μM DSF (P > 0.05). The significant correlation was observed between RASSF1A re-expression and p21 up-regulation before and after treatment with 10 μM 5-Aza-CdR (P < 0.01) but not after treatment with 13 μM DSF (P > 0.05), while p21 up-regulation was significantly higher after DSF treatment (P < 0.01).Conclusion:Our findings indicated that 5-Aza-CdR induces the re-expression of RASSF1A and p21 up-regulation in PANC-1. DSF showed no epigenetic reversion while it affected p21 up-regulation.

Two-Photon Ratiometric Fluorescent Sensor Based on Specific Biomolecular Recognition for Selective and Sensitive Detection of Copper Ions in Live Cells

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E2Zn2SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E2Zn2SOD = Cu-free derivative of bovine liver copper-zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu(2+) level with clear red-to-yellow color change based on specific biomolecular recognition of E2Zn2SOD for Cu(2+) ion. The inorganic-organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu(2+) ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu(2+) with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10(-7)-10(-3) M), low detection limit down to ∼10 nM, and excellent selectivity over other metal ions, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu(2+) changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu(2+) in live cells.

(Z)-4-Bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one sensitizes Escherichia coli persister cells to antibiotics

Persisters are a small subpopulation of bacterial cells that are dormant and extremely tolerant to antibiotics. The intrinsic antibiotic tolerance of persisters also facilitates the development of multidrug resistance through acquired mechanisms based on drug resistance genes. In this study, we demonstrate that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can reduce persistence during Escherichia coli growth and revert the antibiotic tolerance of its persister cells. The effects of BF8 were more profound when the pH was increased from 6 to 8.5. Although BF8 is a quorum sensing (QS) inhibitor, similar effects were observed for the wild-type E. coli RP437 and its ΔluxS mutant, suggesting that these effects did not occur solely through inhibition of AI-2-mediated QS. In addition to its effects on planktonic persisters, BF8 was also found to disperse RP437 biofilms and to render associated cells more sensitive to ofloxacin. At the doses that are effective against E. coli persister cells, BF8 appeared to be safe to the tested normal mammalian cells in vitro and exhibited no long-term cytotoxicity to normal mouse tissues in vivo. These findings broadened the activities of brominated furanones and shed new light on persister control.