Cellular Viability Test Research Articles

Development and Characterization of Quercetin-Loaded Polymeric Liposomes with Gelatin-Poly(ethylene glycol)-Folic Acid Coating to Increase Their Long-Circulating and Anticancer Activity.

Liposomes as drug-delivery systems have been researched and applied in multiple scientific reports and introduced as patented products with interesting therapeutic properties. Despite various advantages, this drug carrier faces major difficulties in its innate stability, cancer cell specificity, and control over the release of hydrophobic drugs, particularly quercetin, a naturally derived drug that carries many desirable characteristics for anticancer treatment. To improve the effectiveness of liposomes to deliver quercetin by tackling and mitigating the mentioned hurdles, we developed a strategy to establish the ability to passively target cancerous cells, as well as to increase the bioavailability of loaded drugs by incorporating poly(ethylene glycol), gelatin, and folic acid moieties to modify the liposomal system's surface. This research developed a chemically synthesized gelatin, poly(ethylene glycol), and folic acid as a single polymer to coat drug-loaded liposome systems. Liposomes were coated with gelatin-poly(ethylene glycol)-folic acid by electrostatic interaction, characterized by their size, morphology, ζ potential, drug loading efficiency, infrared structures, differential scanning calorimetry spectra, and drug-releasing profiles, and then evaluated for their cytotoxicity to MCF-7 breast cancer cells, as well as cellular uptake, analyzed by confocal imaging to further elaborate on the in vitro behavior of the coated liposome. The results indicated an unusual change in size with increased coating materials, followed by increased colloidal stability, ζ potential, and improved cytotoxicity to cancer cells, as shown by the cellular viability test with MCF-7. Cellular uptake also confirmed these results, providing data for the effects of biopolymer coating, while confirming that folic acid can increase the uptake of liposome by cancer cells. In consideration of such results, the modified gelatin-poly(ethylene glycol)-folic acid-coated liposome can be a potential system in delivering the assigned anticancer compound. This modified biopolymer showed excellent properties as a coating material and should be considered for further practical applications in the future.

Morphological, Thermal, and Cytotoxicity Features Assessment of Starch–Myristic Acid Complex‐Derived Nanostructured Materials

AbstractThe present study investigates the morphological and cytotoxic properties of starch–myristic acid complex (SMC) derivatives. The study attempts to prepare a SMC by frying. By liquid–liquid extraction, starch–myristic acid complex nanostructured materials are isolated. X‐ray diffractometry and transmission electron microscopy are used to determine the morphology and crystallization of the extract. The study performs various assays to assess the toxicological profile of starch–myristic acid complex‐derived nanostructured materials (SMC‐NMs), including cellular viability tests, cellular and nuclear morphological examinations, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) measurements. Transmission electron microscopy (TEM) images reveal nanoscale particles between 30 and 90 nm in water fraction‐based SMC‐NMs (WSMC‐NMs) and 160 and 250 nm in methanol fraction‐based SMC‐NMs (MSMC‐NMs). The WSMC‐NMs and MSMC‐NMs decrease the cell viability by 77% and 91% at 250 µg mL−1, respectively. The SMC‐NMs enhance necrotic cell death by increasing the level of ROS in human mesenchymal stem cells (hMSCs). The SMC‐NMs alter gene expression within 24 h. Gene expression of GSTM3 and GSR has explicitly upregulated in a dose‐dependent manner in WSMC‐NMs and MSMC‐NMs exposed cells. The study finds that nanoscale particles may develop during cooking, increasing the risk of diabetes, cardiovascular diseases, and obesity.

Influence of ZnO Nanoparticles on the Properties of Ibuprofen-Loaded Alginate-Based Biocomposite Hydrogels with Potential Antimicrobial and Anti-Inflammatory Effects.

Hydrogels are a favorable alternative to accelerate the burn wound healing process and skin regeneration owing to their capability of absorbing contaminated exudates. The bacterial infections that occur in burn wounds might be treated using different topically applied materials, but bacterial resistance to antibiotics has become a major problem worldwide. Therefore, the use of non-antibiotic treatments represents a major interest in current research. In this study, new antibiocomposite hydrogels with anti-inflammatory and antimicrobial properties based on hyaluronic acid (HA) and sodium alginate (AG) were obtained using 4-(4,6-dimethoxy-1,3,5-triazinyl-2)-4-methylmorpholinium chloride as an activator. The combination of Ibuprofen, a non-steroidal anti-inflammatory drug commonly used to reduce inflammation, fever and pain in the body, with zinc oxide nanoparticles (ZnO NPs) was used in this study aimed at creating a complex hydrogel with anti-inflammatory and antimicrobial action and capable of improving the healing process of wounds caused by burns. FTIR spectra confirmed the cross-linking of AG with HA as well as the successful incorporation of ZnO NPs. Using electronic microscopy, it was noticed that the morphology of hydrogels is influenced by the incorporation of ZnO nanoparticles. Moreover, the incorporation of ZnO nanoparticles into hydrogels also has an influence on the swelling behavior at both pH 7.4 and 5.4. In fact, the swelling rate is lower when the amounts of the activator, HA and ZnO NPs are high. A drug release rate of almost 100% was observed for hydrogels without ZnO NPs, whereas the addition of nanoparticles to hydrogels led to a decrease in the release rate to 68% during 24 h. Cellular viability tests demonstrated the non-cytotoxic behavior of the hydrogels without the ZnO NPs, whereas a weak to moderate cytotoxic effect was noticed for hydrogels with ZnO NPs. The hydrogels containing 4% and 5% ZnO NPs, respectively, showed good antimicrobial activity against the S. aureus strain. These preliminary data prove that these types of hydrogels can be of interest as biomaterials for the treatment of burn wounds.

Photoprotective effect of 18β-glycyrrhetinic acid derivatives against ultra violet (UV)-B-Induced skin aging

Excessive exposure to sun can harm the skin, causing sunburn, photo-aging, and even skin cancer. Different benzylidene derivatives (A02-A18 and A19-A34) of 18β-Glycyrrhetinic acid (A01) were designed and synthesized in an effort to discover photo-protective compounds against UV-B -induced skin aging. The synthesized derivatives were subjected to cellular viability test using MTT assay in primary Human Dermal Fibroblasts (HDFs). The results indicate A01, A05, A15, A22, A23, A25, A26, A28, A29, A32, A33, and A34 significantly enhanced cell viability of HDFs. Compound A33 at 10 and 25 μM showed a significant photo-protective effect against UV-B (10 mJ/cm2) –induced damage in HDFs. A33 at 25 μM significantly restored the UV-B -induced damage via its potent anti-oxidant, anti-apoptotic effects and ability to prevent collagen degradation. These findings pave the way for further development of A33 as a photo-protective skin agent.

Effect on viability and cellular proliferation of rhBMP-2 immobilized on TEMPO modified cellulose hydrogel

BMP´s are signaling proteins that belong to the Transforming Growth Factor-β (TGF-β) superfamily. These proteins promote the recruitment and differentiation of mesenchymal progenitor cells into bone forming cells, the osteoblasts and increase the rate of bone formation. The carrier systems to release rhBMP-2 to the action site are based on the use of free and soluble BMP incorporated into biopolymers such as collagen, gelatin, chitosan, hyaluronic acid and silk. The fused rhBMP-2-thioredoxin could be an interesting approach for new advances in the field of carrying systems of these growth factors. The fused protein thioredoxin can be useful as a coupling agent of BMP-2 to the carrier system, binding it to the surface of the matrix and it is one of the main aims of this work. The recombinant protein rhBMP-2 was produced by IPTG induction obtaining a soluble protein without the need for refolding process. The immobilization of rhBMP-2 at the surface of the TEMPO modified cellulose nanofibrils was indicated by FTIR spectroscopy. The cellular viability tests indicated increased proliferative behavior of both, C2C12 and stem cells from rats, when seeded in presence of rhBMP2 when compared to the free rhBMP2 substrate. The calcified extracellular matrix confirmed the increased activity of the rhBMP2-cellulose substrate, indicating the success of the proposed method. The cell proliferation assays indicated the method used to immobilize rhBMP2 onto the surface of the TEMPO modified cellulose was successful. The cells growth increased when compared to the reference sample free of rhBMP2.

CPEB4-Promoted Paclitaxel Resistance in Ovarian Cancer In Vitro Relies on Translational Regulation of CSAG2.

Background: Drug resistance is a major obstacle in chemotherapy for ovarian cancer, wherein the up regulation of drug-resistant genes plays an important role. The cytoplasmic polyadenylation element binding protein 4 (CPEB4) is an RNA binding protein that controls mRNA cytoplasmic polyadenylation and translation. Methods: The expression of CPEB4 in paclitaxel-resistant ovarian cancer cell lines and recurrent ovarian tumors relative to counterparts was determined by qRT-PCR, Western blotting and immunohistochemistry. The response to paclitaxel treatment was evaluated by cellular viability test and colony formation assay. RNA immunoprecipitation and poly(A) tail test were applied to examine the levels of RNA binding and cytoplasmic polyadenylation. Results: CPEB4 is elevated in paclitaxel-resistant ovarian cancer cells and recurrent ovarian tumors treated with paclitaxel-based chemotherapy. In addition, CPEB4 overexpression promotes paclitaxel resistance in ovarian cancer cells in vitro, and vice versa, CPEB4 knockdown restores paclitaxel sensitivity, indicating that CPEB4 confers paclitaxel resistance in ovarian cancer cells. Mechanistically, CPEB4 binds with the taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) mRNAs and induces its expression at a translational level. Moreover, CSAG2 expression is upregulated in paclitaxel-resistant ovarian carcinoma and cancer cell lines, and more importantly, siRNA-mediated CSAG2 knockdown overtly attenuates CPEB4-mediated paclitaxel resistance. Conclusion: This study suggests that the drug-resistant protein CSAG2 is translationally induced by CPEB4, which underlies CPEB4-promoted paclitaxel resistance in ovarian cancer in vitro. Thus, interfering CPEB4/CSAG2 axis might be of benefit to overcome paclitaxel-resistant ovarian cancer.

Biological Evaluation of Black Chokeberry Extract Free and Embedded in Two Mesoporous Silica-Type Matrices.

Black chokeberry fruits possess a wide range of biological activities, among which the most important that are frequently mentioned in the literature are their antioxidant, anti-inflammatory, anti-proliferative, and antimicrobial properties. The present paper reports, for the first time, the encapsulation of the ethanolic extract of Aronia melanocarpa L. fruits into two mesoporous silica-type matrices (i.e., pristine MCM-41 and MCM-41 silica decorated with zinc oxide nanoparticles). The aim of this work was to evaluate the antiradicalic capacity, the antimicrobial potential, and the effects on the cell viability on a cancer cell line (i.e., A375 human melanoma cell line) versus normal cells (i.e., HaCaT human keratinocytes) of black chokeberry extract loaded on silica-type matrices in comparison to that of the extract alone. The ethanolic polyphenolic extract obtained by conventional extraction was characterized by high-performance liquid chromatography with a photodiode array detector (HPLC–PDA) and spectrophotometric methods. The extract was found to contain high amounts of polyphenols and flavonoids, as well as good radical scavenging activity. The extract-loaded materials were investigated by Fourier transform infrared spectroscopy, N2 adsorption–desorption isotherms, thermal analysis, and radical scavenger activity on solid samples. The black chokeberry extract, both free and loaded onto mesoporous silica-type matrices, exhibited a significant antioxidant capacity. Antibacterial activity was recorded only for Gram-positive bacteria, with a more potent antibacterial effect being observed for the extract loaded onto the ZnO-modified MCM-41 silica-type support than for the free extract, probably due to the synergistic effect of the ZnO nanoparticles that decorate the pore walls of silica. The cellular viability test (i.e., MTT assay) showed dose- and time-dependent activity regarding the melanoma cell line. The healthy cells were less affected than the cancer cells, with all tested samples showing good cytocompatibility at doses of up to 100 µg/mL. Improved in vitro antiproliferative and antimigratory (i.e., scratch assay) potential was demonstrated through the loading of black chokeberry extract into mesoporous silica-type matrices, and the screened samples exhibited low selectivity against the tested non-tumor cell line. Based on presented results, one can conclude that mesoporous silica-type matrices are good hosts for black chokeberry extract, increasing its antioxidant, antibacterial (on the screened strains), and in vitro antitumor (on the screened cell line) properties.

Biogenic silver nanoparticles reduce adherence, infection, and proliferation of toxoplasma gondii RH strain in HeLa cells without inflammatory mediators induction

The highlights of biogenic silver nanoparticles (AgNp-Bio) include low toxicity – depending on size and concentration – and efficient antiparasitic activity. Therefore, the objective of this study was to assess the action of the AgNp-Bio on HeLa cells in an infection with strain of RH Toxoplasma gondii. Firstly, we performed a cellular viability test and characterized the AgNp-Bio to proceed with the infection of HeLa cells with T. gondii to be treated using AgNp-Bio or conventional drugs. Subsequently, we determined the level of standard cytokines Th1/Th2 as well as the content of nitric oxide (NO) and reactive oxygen species (ROS). Results indicated a mean size of 69 nm in diameter for the AgNp-Bio and obtained a dose-dependent toxicity. In addition, the concentrations of 3 and 6 μM promoted a significant decrease in adherence, infection, and intracellular proliferation. We also found lower IL-8 and production of inflammatory mediators. Thus, the nanoparticles reduced the adherence, infection, and proliferation of ROS and NO, in addition to immunomodulating the IL-8. Therefore, our data proved relevant to introduce a promising therapeutic alternative to toxoplasmosis.

PTP1B phosphatase as a novel target of oleuropein activity in MCF-7 breast cancer model

Phosphatase PTP1B has become a therapeutic target for the treatment of type 2-diabetes, whereas recent studies have revealed that PTP1B plays a pivotal role in pathophysiology and development of breast cancer. Oleuropein is a natural, phenolic compound with anticancer activity. The aim of this study was to address the question whether PTP1B constitutes a target for oleuropein in breast cancer MCF-7 cells.The cellular MCF-7 breast cancer model was used in the study. The experiments were performed using cellular viability tests, Elisa assays, immunoprecipitation, flow cytometry analyses and computer modelling.Herein, we evidenced that the reduced activity of phosphatase PTP1B after treatment with oleuropein is strictly correlated with decreased MCF-7 cellular viability and cell cycle arrest. These results provide new insight into further research on oleuropein and possible role of the compound in adjuvant treatment of breast cancer.

Co-delivery of Dual Toll-Like Receptor Agonists and Antigen in Poly(Lactic-Co-Glycolic) Acid/Polyethylenimine Cationic Hybrid Nanoparticles Promote Efficient In Vivo Immune Responses.

Strategies to design delivery vehicles are critical in modern vaccine-adjuvant development. Nanoparticles (NPs) encapsulating antigen(s) and adjuvant(s) are promising vehicles to deliver antigen(s) and adjuvant(s) to antigen-presenting cells (APCs), allowing optimal immune responses against a specific pathogen. In this study, we developed a novel adjuvant delivery approach for induction of efficient in vivo immune responses. Polyethylenimine (PEI) was physically conjugated to poly(lactic-co-glycolic) acid (PLGA) to form PLGA/PEI NPs. This complex was encapsulated with resiquimod (R848) as toll-like receptor (TLR) 7/8 agonist, or monophosphoryl lipid A (MPLA) as TLR4 agonist and co-assembled with cytosine–phosphorothioate–guanine oligodeoxynucleotide (CpG ODN) as TLR9 agonist to form a tripartite formulation [two TLR agonists (inside and outside NPs) and PLGA/PEI NPs as delivery system]. The physicochemical characteristics, cytotoxicity and cellular uptake of these synthesized delivery vehicles were investigated. Cellular viability test revealed no pronounced cytotoxicity as well as increased cellular uptake compared to control groups in murine macrophage cells (J774 cell line). In the next step, PLGA (MPLA or R848)/PEI (CpG ODN) were co-delivered with ovalbumin (OVA) encapsulated into PLGA NPs to enhance the induction of immune responses. The immunogenicity properties of these co-delivery formulations were examined in vivo by evaluating the cytokine (IFN-γ, IL-4, and IL-1β) secretion and antibody (IgG1, IgG2a) production. Robust and efficient immune responses were achieved after in vivo administration of PLGA (MPLA or R848)/PEI (CpG ODN) co-delivered with OVA encapsulated in PLGA NPs in BALB/c mice. Our results demonstrate a rational design of using dual TLR agonists in a context-dependent manner for efficient nanoparticulate adjuvant-vaccine development.

Abstract 2058: Co-treatment of NVP-BSK805 highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition

Abstract The high expression of P-glycoprotein (P-gp) is consistently observed in multidrug resistance (MDR) cancer cells. The purpose of our study is to identify conditions that could increase the sensitivity of P-gp-overexpressing the drug-resistant KBV20C cancer cell line. We have used the commercially-available epigenetic library, which includes 128 compounds. Using the cellular viability test, we also identified that NVP-BSK805 is highly sensitized to KBV20C-resistant cells and vincristine treatment. However, NVP-BSK805-induced sensitization was not observed in vincristine-treated sensitive KB parent cells, suggesting that the effects are specific to resistant cancer cells. Using FACS analysis, western blot, and Annexin V staining, we identified that NVP-BSK805 sensitized vincristine-treated KBV20C cells arrest via apoptosis and G2. Furthermore, sensitization potency of NVP-BSK805, in combination with vincristine, was measured in KBV20C cells when comparing them with P-gp inhibitor verapamil or another Jak2 inhibitor CEP-33779. We further investigated the mechanisms of NVP-BSK805 against sensitization of vincristine-treated KBV20C cells. The sensitization mechanism of NVP-BSK805 was mainly dependent on the inhibition of P-gp. In addition, ATPase activity was conducted to identify its potential role in NVP-BSK805 for P-gp inhibition. Both NVP-BSK805 and CEP-33779 showed high binding affinity docking scores -9.1 and -7.4, respectively, against the ABCB1 receptor protein. Collectively, inhibition of P-gp by NVP-BSK805 can increase vincristine-sensitivity in MDR cancer cells through increased apoptosis and G2 arrest. Our findings indicate that the Jak2 inhibitor may be a promising target in the treatment of patients resistant to antimitotic drugs. Citation Format: Yu Jin Park, Ji Hyun Cheon, Ji Yeon Son, Sung Pil Yoon, Hyung Sik Kim. Co-treatment of NVP-BSK805 highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2017-2058

Abstract 2059: Co-treatment of XL019, a selective Jak2 inhibitor, highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition

Abstract 1 Co-treatment of XL019, a selective Jak2 inhibitor, highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition Ji Hyun Cheon1, Yu Jin Park, Ji Yeon Son, Sung Pil Yoon, and Hyung Sik Kim Lab of molecular of toxicology, School of Pharmacy, Sungkyunkwan University, Suwon, South Korea A high expression of P-glycoprotein (P-gp) is consistently observed in multidrug resistance (MDR) cancer cells. The purpose of our study was to identify conditions that would increase the sensitivity of P-gp-overexpressing drug-resistant KBV20C cancer cell line. We used commercially available epigenetic library, which includes 128 compounds. Using cellular viability test, we identified that XL019 highly sensitized KBV20C-resistant cells to vincristine treatment. However, XL019-induced sensitization was not observed in vincristine-treated sensitive KB parent cells, suggesting that these effects are specific to resistant cancer cells. Using FACS analysis, western-blot, and annexin V staining, we identified that XL109 sensitized vincristine-treated KBV20C cells via apoptosis and G2 arrest. Furthermore, sensitization potency of XL109 in combination with vincristine was measured in KBV20C cells when comparing with P-gp inhibitor verapamil or another Jak2 inhibitor CEP-33779. We further investigated the mechanisms of XL109 against sensitization of vincristine-treated KBV20C cells. The sensitization mechanism of XL109 was mainly depends on the inhibition of P-gp. In addition, ATPase activity was also conducted to identify as potential role of XL019 in P-gp inhibition. In the docking modeling, XL019 and CEP-33779 showed high binding affinity docking score against ABCB1 receptor protein by -10.590 and -9.104, respectively. Collectively, inhibition of P-gp by XL109 can increase the vincristine-sensitivity in MDR cancer cells through increased apoptosis and G2 arrest. Our findings indicate that Jak2 inhibitor may be a promising target in the treatment of patients resistant to anti-mitotic drug. Note: This abstract was not presented at the meeting. Citation Format: Ji Hyun Cheon, Yu Jin Park, Ji Yeon Son, Sung Pil Yoon, Hyung Sik Kim. Co-treatment of XL019, a selective Jak2 inhibitor, highly sensitizes drug-resistant cancer cells by increasing P-gp inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2017-2059

Effect of Ultraviolet Irradiation on the Osseointegration of a Titanium Alloy with Bone.

Introduction:Attempt has been made to analyze the potential of titanium (Ti) alloy for osteointegration by the effect of surface photo functionalization in different aspects as follows: in Ringer's solution, in vitro cell growth, and in vivo study on rabbit. The present study was aimed to investigate the influence of ultraviolet (UV) light on surface topography, corrosion behavior, and bioactivity of indigenously manufactured samples of Ti alloy mini-implant.Materials and Methods:The study includes surface modification of Ti samples by UV treatment, corrosion testing of the specimens using Potentiostat (GAMRY System), qualitative examination of modified surface topography using scanning electron microscope, and cellular viability test on Ti alloy surface (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ASSAY). To find the effect of UV light on implant bone integration, biochemical test was performed on the femur of rabbits.Results and Discussion:Corrosion resistance of untreated Ti alloy in Ringer's solution was found to be less, whereas corrosion rate was more. Corrosion resistance of UV-treated samples was found to increase significantly, thereby lowering the corrosion rate. Cell growth in UV-treated specimen was observed to be higher than that in untreated samples. It is important to mention that cell growth was significantly enhanced on samples which were UV treated for longer duration of time.Conclusions:There was a marked improvement in cell growth on UV-treated Ti alloy samples. Hence, it is expected that it would enhance the process of osseointegration of Ti with bone. Another important finding obtained was that the removal torque values of UV-treated implants were higher than that of untreated implants. The overall result reveals that UV treatment of implants does help us in speeding up the osseointegration process.

Viability of Human Gingival Fibroblast (FGH) Treated with Ethanolic "Aroeira" Extract (Myracrodruon urundeuva Allemão)

The aim of this study was to evaluate the effect of ethanolic aroeira (Myracrodruon urundeuva) extract on the viability of human gingival fibroblast. For this, fibroblasts (2x103 cells/well) were plated in a 96-well plate and incubated for 24 h; the medium (Eagle's medium modified by Dulbecco - DMEM) supplemented with 10% fetal bovine serum was replaced by DMEM with different ethanolic extract concentration (0, 0.1, 1, 10, 100, and 1000μg / mL). The fibroblast viability was analyzed after 48,72, and 96 h by the neutral red capture test and violet crystal. The aroeira extract, at high concentrations (100 and 1000 µg/mL) caused decrease in both cellular viability tests (p<0.05). However, dilutions between 0.1 and 10 µg/mL did not affect the viability of the cells. It was concluded that aroeira extract was able to change the gingival fibroblast viability, and this effect was concentration dependent.

Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation

A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.

Dichloroacetate attenuates hypoxia-induced resistance to 5-fluorouracil in gastric cancer through the regulation of glucose metabolism

In this study, we investigated whether gastric cancer with hypoxia-induced resistance to 5-fluorouracil (5-FU) could be re-sensitized following treatment with low-dose dichloroacetate (DCA), an inhibitor of the glycolytic pathway. The expression profiles of hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase-1 (PDK-1) were analyzed in tissues from 10 patients with gastric cancer who had different responses to adjuvant 5-FU treatment. For the in vitro assays, cell viability and apoptosis were evaluated with and without treatment with 20mM DCA in the AGS and MKN45 cell lines, as well as in PDK1 knockdown cell lines. The expression levels of HIF-1α and PDK-1 were both elevated in the tumor tissues relative to the normal gastric tissues of most patients who showed recurrence after adjuvant 5-FU treatment. Cellular viability tests showed that these cell lines had a lower sensitivity to 5-FU under hypoxic conditions compared to normoxic conditions. Moreover, the addition of 20mM DCA only increased the sensitivity of these cells to 5-FU under hypoxic conditions, and the resistance to 5-FU under hypoxia was also attenuated in PDK1 knockdown cell lines. In conclusion, DCA treatment was able to re-sensitize gastric cancer cells with hypoxia-induced resistance to 5-FU through the alteration of glucose metabolism.

연근(蓮根)의 신경 보호 효과 및 기전연구

Objectives : There is a possibility LRE as remedy in Alzheimer disease (AD), but it's nerve protection effect and mechanism have to be elucidate. In this research, we applied LRE on <TEX>$A{\beta}_{25-35}$</TEX> pre-treated SH-SY5Y cells, to find out the nerve protection effect and mechanism in AD cell model. Methods : We tried to confirm that effect by experimenting with 20, 50, and <TEX>$100{\mu}g/ml$</TEX> concentration of LRE as a medicine. Next experiment, we assessed damage effect which induced <TEX>$A{\beta}_{25-35}$</TEX>, known to cause AD, on SH-SY5Y cell. In addition, cellular viability test is executed under <TEX>$H_2O_2$</TEX> treatment condition in a SH-SY5Y cell. Results : 1. In <TEX>$A{\beta}_{25-35}$</TEX> treated SH-SY5Y cell, LRE exhibited an anti-phosphorylation effect about tau protein, JNK, and IKB. 2. LRE prevent nerve cell apoptosis, which indued <TEX>$A{\beta}_{25-35}$</TEX> and oxidative stress, modify JNK engaged synaptic structure and <TEX>$NF{\kappa}B$</TEX> induced p75-neurotrophin receptor polymorphism. Conclusions : We found that LRE prevented oxidative stress-induced cellular destruction, for example, increased SOD activity of <TEX>$A{\beta}_{25-35}$</TEX> treated SH-SY5Y cell and reduced toxicity of oxygen free radical. Consequently, the ingredients of LRE have a role as a catalyzer for <TEX>$A{\beta}_{25-35}$</TEX> clearance and as scavenger for active oxygen free radical.

Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives

Quinoxaline is a chemical compound that presents a structure that is similar to quinolone antibiotics. The present work reports the study of the antimicrobial activity of quinoxaline N,N-dioxide and some derivatives against bacterial and yeast strains. The compounds studied were quinoxaline-1,4-dioxide (QNX), 2-methylquinoxaline-1,4-dioxide (2MQNX), 2-methyl-3-benzoylquinoxaline-1,4-dioxide (2M3BenzoylQNX), 2-methyl-3-benzylquinoxaline-1,4-dioxide (2M3BQNX), 2-amino-3-cyanoquinoxaline-1,4-dioxide (2A3CQNX), 3-methyl-2-quinoxalinecarboxamide-1,4-dioxide (3M2QNXC), 2-hydroxyphenazine-N,N-dioxide (2HF) and 3-methyl-N-(2-methylphenyl)quinoxalinecarboxamide-1,4-dioxide (3MN(2MF)QNXC). The prokaryotic strains used were Staphylococcus aureus ATCC 6538, S. aureus ATCC 6538P, S. aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli S3R9, E. coli S3R22, E. coli TEM-1 CTX-M9, E. coli TEM-1, E. coli AmpC Mox-2, E. coli CTX-M2 e E. coli CTX-M9. The Candida albicans ATCC 10231 and Saccharomyces cerevisiae PYCC 4072 were used as eukaryotic strains. For the compounds that presented activity using the disk diffusion method, the minimum inhibitory concentration (MIC) was determined. The alterations of cellular viability were evaluated in a time-course assay. Death curves for bacteria and growth curves for S. cerevisiae PYCC 4072 were also accessed. The results obtained suggest potential new drugs for antimicrobial activity chemotherapy since the MIC's determined present low values and cellular viability tests show the complete elimination of the bacterial strain. Also, the cellular viability tests for the eukaryotic model, S. cerevisiae, indicate low toxicity for the compounds tested.

Evaluation of naproxen and cromolyn activities against cancer cells viability, proliferation, apoptosis, p53 and gene expression of survivin and caspase-3

We previously reported the inhibitory profiles of naproxen and cromolyn against glycogen synthase kinase-3β, which partly explain the molecular mechanisms of their anti-cancer properties. In this study, we performed a detailed biochemical evaluation of the two drugs against colorectal adenocarcinoma (Caco2), hepatocellular carcinoma (HepG2), mammary gland carcinoma (MCF7), epitheloid cervix carcinoma (Hela), lung carcinoma (A5W9) and epidermoid larynx carcinoma (Hep2) cell lines. Additionally, we performed cellular viability tests using trypan blue, proliferation MTT assay, apoptosis, p53 and real-time polymerase chain reaction for gene expression of survivin and caspase-3. Not only the two drugs were found to significantly reduce the viability of different cell lines, but they also were shown to have potent dose-dependent reduction of cellular proliferation. They exhibited cytotoxicity IC50 values of 3.69 and 4.16 μM for naproxen and cromolyn, respectively. Viability and proliferation results clearly correlated with apoptosis and p53 experiments in showing that both drugs significantly raised apoptotic percentages. Furthermore, we observed a significant reduction in survivin and elevation of caspase-3 gene expression upon exposure to the two drugs. It can be concluded that both naproxen and cromolyn have significant anti-cancer properties.

Effects of Fetal Bovine Serum deprivation in cell cultures on the production of Anticarsia gemmatalis Multinucleopolyhedrovirus

BackgroundAnticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum.ResultsCulture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells.ConclusionsUFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery.