Methyl Thiazol Tetrazolium Assay Research Articles

Telocinobufagin Has Antitumor Effects in Non-Small-Cell Lung Cancer by Inhibiting STAT3 Signaling.

Non-small-cell lung carcer (NSCLC), the main histological subtype of lung cancer, is responsible for significant morbidity and mortality worldwide. Telocinobufagin, an active compound of the Chinese traditional medicine ChanSu, has antitumor effects, but its mechanism of action remains unknown. Therefore, we investigated the effect of telocinobufagin on NSCLC growth and metastasis and its possible mechanism of action, in vitro and in vivo. Cell proliferation, migration, and apoptosis were measured by methyl thiazol tetrazolium assay, colony formation, 5-ethynyl-2'-deoxyuridine incorporation, Transwell migration, wound healing, and flow cytometry analysis. A mouse xenograft model was used to evaluate tumor formation in vivo. Telocinobufagin was found to suppress proliferation and metastasis and induce apoptosis in human NSCLC cells. Moreover, telocinobufagin was able to significantly inhibit STAT3 phosphorylation at tyrosine 705 (Y705) and its downstream targets. Additionally, telocinobufagin also impaired the IL-6-induced nuclear translocation of STAT3. Consistent with the in vitro experiments, telocinobufagin reduced the A549 xenograft tumor burden and the levels of P-STAT3Y705, MCL1, BCL2, and cleaved PARP1 in vivo. These results support telocinobufagin as a promising STAT3 signaling inhibitor candidate for the treatment of NSCLC patients.

Leukotriene B4 loaded in microspheres regulate the expression of genes related to odontoblastic differentiation and biomineralization by dental pulp stem cells

BackgroundLeukotriene B4 (LTB4) is a potent lipid mediator that stimulate the immune response. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB4 in inducing differentiation of dental pulp stem cells.MethodsMicrospheres (MS) loaded with LTB4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB4 encapsulation and in vitro LTB4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB4 or MS loaded with LTB4 (0.01 and 0.1 μM). Cytotoxicity and cell viability was determined by lactate dehydrogenase and methylthiazol tetrazolium assays. Gene expression were measured by quantitative reverse transcription polymerase chain reaction after 3, 6, 24, 48 and 72 h. Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB4 in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett’s post-test (α = 0.05).ResultsTreatment with LTB4 or MS loaded with LTB4 (0.01 and 0.1 µm-μM) were not cytotoxic to OD-21 cells. Treatment with LTB4 modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB4 loaded in microspheres (0.1 μM) allowed long term dental pulp cell differentiation and biomineralization.ConclusionLTB4, soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB4 was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.

Oral administration of pH-responsive polyamine modified cyclodextrin nanoparticles for controlled release of anti-tumor drugs

pH-responsive nanoparticles based on polyamine modified β-cyclodextrin (PEHA-β-CD) and sodium dodecylbenzene sulfonate (SDBS) were fabricated through electrostatic interaction and employed to load doxorubicin (DOX) and celastrol (CSL) in the form of DOX(CSL)-loaded PEHA-β-CD/SDBS. The obtained nanoparticles were characterized using Tyndall effect, UV–vis, XRD, 1H NMR, DLS, TEM and zeta potential. The DOX(CSL)-loaded PEHA-β-CD/SDBS with minimal amount released in the acidic pH environment (pH = 1.2) like that of the stomach, while effective release in the basic pH environment (pH = 8.5) like that of the intestine. The release kinetics were investigated using different models to explain drug release mechanism. The pH-controlled release nanoparticles system provides an opportunity to the oral route of drug administration. The Methyl Thiazol Tetrazolium (MTT) assay indicated that DOX(CSL)-loaded PEHA-β-CD/SDBS showed high toxicity to five tumor cells. What's more, it exhibited low cytotoxicity to normal BEAS-2B epithelial cells. The flow cytometry analysis showed that DOX(CSL)-loaded PEHA-β-CD/SDBS could inhibit tumor cells growth through inducing cells apoptosis. These results indicated that the pH-responsive nanoparticles held a great promise to be developed as an oral administration drugs delivery system with sustained release and target specificity.

Effect of nano-selenium loaded with lycium barbarum polysaccharide on the proliferation of lens epithelial cells after UVB damage in vitro.

To investigate the effect of nano-selenium loaded with different concentrations of lycium barbarum polysaccharide (LBP-SeNPs) on the proliferation of human lens epithelial cells (HLECs) from UV irradiation. LBP-SeNPs were prepared and their particle size was detected. HLECs (SRA01/04) were irradiated with UVB for different time (0, 10, 20, 30, 40, 50, 60min) to construct a damaged model, the survival rate of cells was determined by methylthiazol tetrazolium (MTT) assay. The 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) staining was used to observe the status of cell nucleus and drug entering cytoplasm through cell membrane in SRA01/04 cells after adding LBP-SENPS loaded with coumarin fluorescence agent 24h under fluorescence microscope. SRA01/04 normal and UVB-damaged cells were treated with different amounts of LBP-SeNPs at different concentrations, cells proliferation were observed. The particle size of LBP-SeNPs was stable in the range of 150-200 nm. The survival rate changes with time after UVB irradiation were statistically significant. The 10min of UVB exposure as the time was chosen to construct the cell damage model. With DAPI staining, LBP-SeNPs were observed to enter the cytoplasm through the cell membrane under fluorescence inverted microscope. Cytotoxicity of SRA01/04 at different concentrations of LBP-SeNPs were measured. Cell survival rate was statistically different compared with the control group. The higher the loading concentration of LBP in nano-Se drugs was, the higher the cell proliferation rate was (P<0.05). The lower the concentration of LBP-SeNPs, the higher the cell proliferation rate, showing a negative growth trend (P<0.05). The group with the highest average cell proliferation rate was 0.5 µmol/L 2.0 mg/mL LBP-SeNPs (128.80%). When the 2.0 mg/mL LBP-SeNPs group was selected for cell photography, the cell density was higher at 0.5 µmol/L. With the increase of concentration, SRA01/04 cells appeared more cytoplasm dehydration, cell shrinkage and apoptotic bodies, and cell density decreased. LBP-SeNPs has moderate particle size and good stability. LBP-SeNPs can protect HLECs (SRA01/04) from UVB-induced damage, and the cell proliferation rate is further increased with increasing the amount of loaded LBP and decreasing nano-selenium concentration.

Induction of apoptosis by Ruta chalepensis L. essential oil in human breast cancer cells (MCF-7)

Context: Recent scientific studies have reported that essential oils induce apoptosis in various cancer cell types by interfering with intracellular signaling pathways. Aims: To evaluate the cytotoxicity, the apoptotic activity of essential oil (EO) of Ruta chalepensis L. against MCF-7 cell line. Methods: Cytotoxicity was determined using methyl thiazol tetrazolium assay. The apoptotic activity of EO was analyzed using annexin V-fluorescein isothiocyanate/propidium iodide binding flow cytometry. The cell morphology was inspected under an inverted microscope. DAPI staining assay was used for the morphological observation. Activation of caspases-3/7, -8, and-9 was assessed using a caspase assay kit. Results: Ruta chalepensis essential oil significantly inhibited the proliferation of MCF-7 cells at 72 h. Moreover, the results showed that cell death is associated with the apoptotic process, and the number of apoptotic cells was significantly increased in the groups treated with EO than in control cells. The main morphological hallmarks of apoptosis in the nucleus were membrane blebbing, chromatin condensation, and nuclear fragmentation. Also, R. chalepensis EO-induced apoptosis in the MCF-7 cell line was via the extrinsic caspase-8 dependent pathway in a dose and time-dependent manner. Conclusions: Ruta chalepensis essential oil demonstrated significant apoptotic activity against experimental breast carcinoma. Therefore, it could be introduced as a suitable candidate for breast cancer therapy after further investigation.

Development of Chitosan‐Assisted Fe3O4@SiO2 Magnetic Nanostructures Functionalized with Nisin as a Topical Combating System against Vancomycin‐Intermediate Staphylococcus aureus (VISA) Skin Wound Infection in Mice

The clinical significance of vancomycin‐intermediate S. aureus (VISA) infections is intensified by its tendency to develop resistance to antimicrobials and persistent infections. The decreasing effectiveness of the antimicrobials available is now seriously compromised; thus, there is an emergent need to invent new classes of antimicrobial agents that can rapidly and efficiently eradicate infections. Fe3O4@SiO2@chitosan (CS) nanocomposites were successfully synthesized and then decorated with nisin to gain Fe3O4/SiO2/CS/nisin‐based magnetic nanostructures (Fe3O4@SiO2@CS‐NISIN). The nanomaterials were characterized comparatively via FTIR (Fourier transform infrared spectroscopy), XRD (X‐ray diffraction), FE‐SEM (field emission scanning electron microscopes), DLS (dynamic light scattering), and VSM (vibrating sample magnetometer) methods. The methyl thiazol tetrazolium (MTT) assay was performed to determine the inhibitory effects of antibacterial agents on the cell viability. The in vitro bactericidal effect of all compounds was characterized using the microdilution assay. Finally, the topical antibacterial efficacy of free nisin, Fe3O4@SiO2@CS, and Fe3O4@SiO2@CS‐NISIN nanocomposites against murine superficial wound infection models was determined. The functionalized nanocomposites were more efficient in suppressing bacterial growth in vitro and in vivo compared to the same quantities of untargeted nanocomposites. MTT results showed acceptable biocompatibility of all nanoformulations, and no substantial difference in the cell viability was recorded between treated cells and untreated control. These results suggest that Fe3O4@SiO2@CS‐NISIN nanocomposites can be served as an alternative antimicrobial agent in innovative and emerging technologies to treat a variety of staphylococcal infections.

Synergistic Cytotoxic Effect of Honey Bee Venom and Cisplatin on Tongue Squamous Cell Carcinoma Cell Line

BACKGROUND: Tongue cancer is one of the most common head and neck cancers in the world. Nowadays, natural compounds are important resources of many anti-cancer drugs. Venom from honey bees possesses potent anti-cancer activities. Cisplatin is a chemotherapeutic drug that has been used for decades to treat cancer cells. Recently, combination therapy has been a popular treatment choice for cancer patients. AIM: This study was conducted to evaluate the synergistic cytotoxic effect of honey bee venom (BV) and cisplatin on tongue squamous cell carcinoma 25 (SCC-25) cell lines. METHODS: The cytotoxic effect was determined using methyl thiazol tetrazolium assay, microscopic examination, real-time polymerase chain reaction (RT-PCR), and statistical analysis. RESULTS: The findings revealed that the cytotoxic potential of the tested drugs on SCC-25 cells was dose-dependent. Microscopic examination showed that BV and cisplatin alone and in combination mainly produced apoptotic cell death. Regarding RT-PCR results, P53 and caspase-3 expression levels were significantly increased in SCC-25-treated cells (p = 0.0001). CONCLUSION: The combined use of BV and cisplatin induced a marked synergistic cytotoxic effect on SCC-25 cell line.

Thermosensitive hydrogel for in situ-controlled methotrexate delivery

Abstract Methotrexate (MTX) is widely used for the treatment of various types of cancer; however, it has drawbacks such as low solubility, lack of selectivity, premature degradation, and side effects. To solve these weaknesses, a hydrogel with the ability to contain and release MTX under physiological conditions without burst release was synthesized. The hydrogel was fabricated with a poly(ɛ-caprolactone)-b-poly(ethylene glycol)-b-poly(ɛ-caprolactone) (PCL–PEG–PCL) triblock copolymer, synthesized by ring-opening polymerization. The characterizations by proton nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectrometry confirmed the copolymer assembly, whereas the molecular weight analysis validated the PCL2000–PEG1000–PCL2000 structure. The copolymer aqueous solution exhibited sol–gel phase transition at 37°C and injection capacity. The hydrogel supported a load of 1,000 μg MTX·mL−1, showing a gradual and sustained release profile of the drug for 14 days, with a delivery up to 92% at pH 6.7. The cytotoxicity of the MTX-loaded hydrogel was performed by the methyl thiazole tetrazolium assay, showing a mean inhibitory concentration of 50% of MCF-7 cells (IC50) at 43 µg MTX·mL−1.

In Vitro Anti-Biofilm Activity of Bacteriophage K (ATCC 19685-B1) and Daptomycin against Staphylococci.

The purpose of the present study was to investigate anti-staphylococcal activity of daptomycin and bacteriophage K, alone or in combination, against biofilm-producers and non-producers S. aureus and S. epidermidis strains, under biofilm forming and cells’ proliferation conditions. Daptomycin and bacteriophage K (ATCC 19685B1), in different concentrations, were tested against 10 Staphylococcus aureus and 10 S. epidermidis, characterized by phenotypes and genotypes. The quantitative microtiter plate (crystal violet, CV), methylthiazoltetrazolium (MTT), and growth curve (GC) assays were performed. No statistically significant difference was found between species, whereas daptomycin alone performed better using medium and high concentrations of the drug and bacteriophage K was more active against strains with higher susceptibility, by CV and MTT assays. Best results were achieved using both agents combined in high concentrations. Bacteriophage K was effective within 3.8 and 2.4 h, depending on the concentration used, by the GC assay. Combination of daptomycin with bacteriophage K was more effective against staphylococci, depending on the concentrations used and strains’ susceptibility. Further studies are needed to evaluate if this approach might be a choice for prevention or therapy of biofilm-associated infections.

Synergistic Antibacterial and Antibiofilm Activity of the MreB Inhibitor A22 Hydrochloride in Combination with Conventional Antibiotics against Pseudomonas aeruginosa and Escherichia coli Clinical Isolates.

In the era of antibiotic resistance, the bacterial cytoskeletal protein MreB is presented as a potential target for the development of novel antimicrobials. Combined treatments of clinical antibiotics with anti-MreB compounds may be promising candidates in combating the resistance crisis, but also in preserving the potency of many conventional drugs. This study aimed to evaluate the synergistic antibacterial and antibiofilm activities of the MreB inhibitor A22 hydrochloride in combination with various antibiotics. The minimum inhibitory concentration (MIC) values of the individual compounds were determined by the broth microdilution method against 66 clinical isolates of Gram-negative bacteria. Synergy was assessed by the checkerboard assay. The fractional inhibitory concentration index was calculated for each of the A22-antibiotic combination. Bactericidal activity of the combinations was evaluated by time-kill curve assays. The antibiofilm activity of the most synergistic combinations was determined by crystal violet stain, methyl thiazol tetrazolium assay, and confocal laser scanning microscopy analysis. The combined cytotoxic and hemolytic activity was also evaluated toward human cells. According to our results, Pseudomonas aeruginosa and Escherichia coli isolates were resistant to conventional antibiotics to varying degrees. A22 inhibited the bacterial growth in a dose-dependent manner with MIC values ranging between 2 and 64 μg/mL. In combination studies, synergism occurred most frequently with A22-ceftazidime and A22-meropemen against Pseudomonas aeruginosa and A22-cefoxitin and A22-azithromycin against Escherichia coli. No antagonism was observed. In time-kill studies, synergism was observed with all expected combinations. Synergistic combinations even at the lowest tested concentrations were able to inhibit biofilm formation and eradicate mature biofilms in both strains. Cytotoxic and hemolytic effects of the same combinations toward human cells were not observed. The findings of the present study support previous research regarding the use of MreB as a novel antibiotic target. The obtained data expand the existing knowledge about the antimicrobial and antibiofilm activity of the A22 inhibitor, and they indicate that A22 can serve as a leading compound for studying potential synergism between MreB inhibitors and antibiotics in the future.

Antileukemic Activity and Molecular Docking Study of a Polyphenolic Extract from Coriander Seeds.

Leukemia is a group of hematological neoplastic disorders linked to high mortality rates worldwide, but increasing resistance has led to the therapeutic failure of conventional chemotherapy. This study aimed to evaluate in vitro the antileukemic activity and potential mechanism of action of a polyphenolic extract obtained from the seeds of Coriandrum sativum L. (CSP). A methylthiazoletetrazolium assay was performed to assess the CSP cytotoxicity on chronic (K562) and acute (HL60) myeloid leukemia cell lines and on normal Vero cell line. CSP toxicity was also evaluated in vivo using the OECD 423 acute toxicity model on Swiss albino mice. The results demonstrated a remarkable antitumoral activity against K562 and HL60 cell lines (IC50 = 16.86 µM and 11.75 µM, respectively) although no cytotoxicity was observed for the Vero cells or mice. A silico study was performed on the following receptors that are highly implicated in the development of leukemia: ABL kinase, ABL1, BCL2, and FLT3. The molecular docking demonstrated a high affinity interaction between the principal CSP components and the receptors. Our findings demonstrated that CSP extract has remarkable antileukemic activity, which is mainly mediated by the flavonoids, catechins, and rutin, all of which showed the highest binding affinity for the targeted receptors. This study revealed a promising active compound alternative research-oriented biopharmacists to explore.

Investigation the Effect of Encapsulated Bromelain Enzyme in Magnetic Carbon Nanotubes on Colorectal Cancer Cells

Background: Bromelain (BL) is an enzyme extracted from Ananas comosus, which has been known for its therapeutic properties. Objectives: The anticarcinogenic activity of BL was examined with and without the presence of magnetic carbon nanotubes (MCNTs) against HT-29 colorectal cancer cells. Methods: The operational factors affecting BL adsorption, such as contact time (30, 60, 90, 120, and 180 min), adsorbent dosage (1 g/L and 5 g/L), initial bromelain concentration (50, 150, and 300 mg/L), and temperature (35 and 50°C) were studied in details. Then, cancer cells were exposed to various BL concentrations (0.1, 1, 10, and 100 μg/mL), and the cell viability was determined by methylthiazol tetrazolium (MTT) assay after 24, 48, and 72 h. Results: The highest adsorption of BL on nanotubes was at 41.62 mg/L and achieved at 35°C and 90 min at the initial concentration of 50 mg/L and 1 g/L of MCNTs. The adsorption followed the Freundlich model and second-order kinetics. The results indicated that MCNTs could be a potential effective adsorbent for the removal of BL. Conclusions: MTT assay indicated that BL at a concentration of 100 μg/mL alone and in combination with MCNTs efficiently inhibited the HT-29 cancerous cells. However, encapsulated BL had a considerable advantage of slow delivery, which is favorable for cancer treatment.

Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury.

The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B1 (AFB1 ) on human renal cells. Hek293 cells were exposed to AFB1 (0-100 μM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50 ) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50 ) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1 -treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated renal cells. The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.

Ginsenoside Rg3 suppresses ovarian cancer cell proliferation and invasion by inhibiting the expression of lncRNA H19.

Ovarian cancer (OC) is the most malignant disease of the female reproductive system and accounts for a large proportion of gynecological cancer-related deaths. Emerging evidence has indicated that ginsenoside Rg3, one of the tetracyclic triterpenoid saponins in ginseng, plays crucial roles in regulating cancer progression, yet its role and mechanisms in regulating the proliferation and invasion of OC are still elusive. In this study, the cell viability, proliferation, migration and invasion of OC were assessed by using methyl thiazol tetrazolium (MTT), colony formation, wound healing and Transwell assays, respectively. The protein levels of E-cadherin and N-cadherin were analyzed by Western blot assay. The expression of long noncoding RNA (lncRNA) H19 was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR). The results revealed that ginsenoside Rg3 significantly inhibited the viability of OC cells (SKOV3 and A2780) in a concentration‑dependent manner. Ginsenoside Rg3 (50 μg/ml) had almost no significant effect on the activity of human ovarian epithelial cells (HOSEpiCs). Thus, this dose was selected for the subsequent experiments. Furthermore, Rg3 markedly decreased the colony formation, migration and invasion of OC cells. In addition, the expression of N-cadherin was downregulated, and the expression of E-cadherin was upregulated with Rg3 treatment. Moreover, lncRNA H19 was upregulated in OC cells, and Rg3 negatively regulated H19 expression in a concentration-dependent manner. In terms of the mechanism, knockdown of H19 inhibited cell proliferation, migration and invasion, while overexpression of H19 reversed the inhibitory effect of Rg3 on the OC cells. In conclusion, ginsenoside Rg3 suppresses the proliferation, migration and invasion of OC cells by partially inhibiting the expression of lncRNA H19.

Bioactivity, nutritional property, and rapid chemical characterization of aqueous extract of Annona muricata leaf from Mexico

Purpose: To investigate the bioactive and nutritional properties, as well as rapid chemical characterization of aqueous extract of Annona muricata leaf from Mexico&#x0D; Methods: The crude aqueous extract of A. muricata leaf was obtained by decoction. Cytotoxicity was tested against cervicouterine cancer cells (HeLa) using methyl thiazol tetrazolium (MTT) assay. Antioxidant activity was evaluated using 2, 2-diphenylpicrylhydrazyl (DPPH) assay. Nutritional evaluation was carried out according to Association of Official Analytical Chemists (AOAC) procedures. Rapid qualitative chemical characterization of the extract was carried out by direct analysis in real-time mass spectrometry (DART-MS) method.&#x0D; Results: The aqueous extract of A. muricata leaf showed cytotoxicity against HeLa cells and also antioxidant activity in a concentration-dependent manner. Nutritional analysis revealed the presence of carbohydrates, vitamin C, Na, and Fe in the aqueous extract. DART-MS spectra showed the presence of alkaloids and phenols as the major components.&#x0D; Conclusion: The cytotoxic and antioxidant activities of the aqueous extract of A. muricata leaf lend some support for its traditional uses as anti-cancer remedy. These activities are probably due to its active secondary metabolites. Thus, the aqueous extract is a source of healthy nutritional components as well as a potential anti-cancer agent for humans.

Is Enoxaparin Sodium Exactly Safe For Subcutaneous Fibroblast?: A Cell Culture Study

Aim: Despite relatively low amount in the subcutaneous tissue, fibroblasts play a critical role in the continuity of intercellular connections, maintenance of tissue integrity, and forming a balanced fascial network. Enoxaparin sodium is widely used in the prophylaxis and treatment of deep vein thrombosis. In the present study, we aimed to examine the cytotoxic and apoptotic effects of enoxaparin sodium on fibroblast cells in an in vitro model. Material and Methods: In a mouse model, L929 fibroblast cells were treated with enoxaparin sodium 4000 IU, 2000 IU, 1000 IU, 500 IU, and 250 IU. At 48 hours, cell morphology was evaluated; cell viability was analyzed through methylthiazole tetrazolium assay and apoptosis was assessed by propidium iodide/ acridine orange staining. Results: The test results showed that high doses (4000 IU, 2000 IU) exerted cytotoxic effects and induced apoptotic morphology. Compared to the control group, there was no significant difference in the cell viability in Dilutions III, IV, and V. Conclusion: Based on our results, despite prophylactic dose in the in vitro setting, high-dose enoxaparin showed cytotoxic and apoptotic effects. Long-term high-dose enoxaparin sodium may affect the number of subcutaneous fibroblasts, impairing the skin integrity and subcutaneous tissue healing

Fabrication, characterization, and in vitro bioactivity evaluation of freeze-cast highly porous hardystonite ceramic reinforced by graphene oxide as a novel bone scaffold

One of the major challenges in bone tissue engineering is the preparation of highly interconnected porous scaffolds with suitable mechanical properties. Synthetic scaffolds used in medicine are usually made of single-phase of ceramic or polymer. However, the combination of these materials with graphene-based nanofillers can produce scaffolds with improved mechanical and biological properties. In this research, we synthesize highly porous (up to 85%) and lamellar hardystonite-graphene oxide (0–1.5 wt% GO) composite scaffolds through the freeze-casting technique and then sintering it for 5 h at 1150 °C. The results of microstructural observations showed using higher amounts of GO leads to an increase in the porosity and a decrease in the shrinkage level. The optimum mechanical properties among the studied samples are related to HT-1 wt% GO (E = 71.77 ± 2.40 MPa, σ = 1.8 ± 016 MPa, and K = 47.87 MJ/m3). Therefore, biological tests were performed on the HT-1 wt% GO scaffold and HT scaffold as the optimal and control samples, respectively. In vitro bioactivity experiments confirm the formation of apatite on surfaces of HT and HT-1 wt% GO specimens after soaking them in SBF for 14 days in static circumstances. Based on the cell studies, the HT-1 wt% GO scaffold sample showed the best attachment and proliferation of osteoblastic cells. The methyl thiazole tetrazolium (MTT) assays were used to characterize the biocompatibility of the HT-1 wt% GO composites in vitro. Also, the alkaline phosphatase (ALP) activity and proliferation rate of cells on the HT-1 wt% GO composite was higher compared with the pure HT ceramics. Overall, it is concluded that the HT-1 wt% GO scaffold with enhanced biological and mechanical features is suitable for use as a novel bone scaffold.

Anti-proliferative and apoptotic effects of hull-less pumpkin extract on human papillary thyroid carcinoma cell line.

Papillary thyroid carcinoma (PTC) is one of the most common cancers of the endocrine system. Previous studies have shown that the extract of hull-less pumpkin seed (HLPS) has a significant anti-cancer effect. The aim of this study was to evaluate the effect of this plant extract on the proliferation of PTC cells. In this study, an extract of this plant was prepared by soxhlet extraction method and analyzed by Gas Chromatography-Mass Spectrometry. The cytotoxicity of PTX and plant extract was investigated using the methylthiazol tetrazolium (MTT) method. For careful investigation of morphological alteration, we used hematoxylin and eosin and Giemsa stinging. Based on MTT assay test, the IC50 value of paclitaxel (PTX) was significantly less than the hydro-alcoholic extract of HLPS at all of the incubation time. Our results of histological staining showed that HLPS and PTX induced significant morphological alteration in the PTC cultured cell that consistent with cell death. Comparing the groups treated by PTX or HLPS with control group showed significant differences. It seems that HLPS extract has an apparent effect on treatment of PTC, at least in laboratory condition, albeit for realistic decision about the effect of HLPS on PTC, more molecular investigations are necessary.

Apoptosis-inducing effects of Terminalia phanerophlebia leaf extracts on human renal cells

The study investigated the toxicogenic effects and molecular mechanisms of Terminalia phanerophlebia on human renal cells. The methylthiazol tetrazolium (MTT) assay was used to assess the cytotoxicity of a crude leaf extract of Terminalia phanerophlebia in Hek293 cells. Luminometry was used to determine caspase activity, phosphatidylserine externalization and necrosis. The comet assay evaluated DNA fragmentation and the lactate dehydrogenase (LDH) assay was used to assess membrane integrity. Western blotting measured the expression of bcl-2-like protein 4 (Bax), inhibitors of apoptosis proteins (IAP), cleaved poly (ADP-ribose) polymerase (cPARP), p53 and nuclear factor kappa B (NF-κB) in treated Hek293 cells. A reduction in cell viability was observed with a half maximal inhibitory concentration (IC50) of 1.36 mg/ml. At this concentration, Terminalia phanerophlebia decreased initiator and executioner caspase activity. Phosphatidylserine validated apoptosis in IC25-treated cells, while necrosis was observed in all treated cells. Comet formation indicated that there was DNA fragmentation. High levels of p53 resulted in upregulation of Bax in IC25-treated cells, while the opposite was noted in IC50-treated cells. Upregulation of NF-κB is associated with decreased caspase-3 and cPARP and further supported by an increase in IAP indicating that Hek293 cell death was mainly through apoptosis and necrosis. The findings suggest that while Terminalia phanerophlebia is a good extract for tuberculosis therapeutic intervention, it may exert toxic effects on kidney cells via the apoptotic pathway.

Deoxynivalenol downregulates NRF2-induced cytoprotective response in human hepatocellular carcinoma (HepG2) cells

Deoxynivalenol (DON) commonly infects agricultural foods; it exhibits toxicity by inducing oxidative stress and inhibiting protein synthesis. Nuclear factor erythroid 2-related factor 2 (NRF2) regulates the cellular antioxidant response. We investigated the cytotoxicity of DON and its effect on the NRF2 antioxidant response in HepG2 cells. The Methyl Thiazol Tetrazolium (MTT), glutathione (GSH) and ATP assays evaluated toxicity, whilst lipid peroxidation and membrane damage were assessed using the Thiobarbituric acid reactive substance (TBARS) and lactate dehydrogenase (LDH) assays. Protein expression of NRF2, phosphorylated (p-ser40) NRF2, catalase (CAT), superoxide dismutase 2 (SOD2), and Sirtuin 3 (Sirt3) were quantified by Western Blotting. Gene expression of glutathione peroxidase (GPx), CAT and SOD2 was determined using qPCR. DON decreased cell viability, GSH concentrations and ATP levels and increased lipid peroxidation and membrane damage. DON significantly decreased total NRF2 and increased p-NRF2 and downregulated the transcription and translation of NRF2 target antioxidant enzymes. Further, expression of the mitochondrial stress response protein, Sirt3 was significantly decreased. In conclusion, DON induced oxidative stress and downregulated NRF2-induced cytoprotection by suppressing the antioxidant signalling mechanism in HepG2 cells.