Trypan Blue Dye Exclusion Assay Research Articles

KEFIRAN in vitro biological activity on enterocytes and mesodermal origin cell lines: Focus on adenocarcinoma cells HT29

Kefiran, the exopolysaccharide found in the fermented milk named “kefir” has been receiving attention over the last years due to its interesting biological and functional properties. It is a non-digestible branched glucogalactan with a molecular weight higher than 1 106 Da. Kefiran was isolated from kefir grains and its biological activity was assessed on different cell lines. Proliferation (trypan blue exclusion assay), mitochondrial activity (MTT assay), viability (Alive-Vulnerable-Dead model, AVD) and cell death were studied. It was found that proliferation was not inhibited in mesodermal cell line MC3 (osteoblast cell line) or in not-invasive colonocytes (Caco-2/TC7 cell line); on the contrary, the high invasive HT29 cell line and the UMR106 osteosarcoma cell line were sensitive to kefiran 0.5 mg/mL, and a significant diminishment on cell proliferation was found by trypan blue dye exclusion assay. Kefiran maintained mitochondrial activity while increased cell death processes in a dose-dependent way in HT29 cell line. When viability was assessed by AVD model, it was found that kefiran increased vulnerability status of neoplasic cell line HT29. In summary, in the present work, we shown that kefiran has a different effect on tumor and non-tumor cell lines. Our results contribute to the understanding of kefiran effect on the empirical antitumoral effect attributed to kefir consumption.

Unravelling the tissue regenerative nature of marine polysaccharide chitosan embedded halloysite reinforced poly (vinyl alcohol) nanocomposite films

The study focuses on developing the poly (vinyl alcohol) (PVA) nanocomposite films reinforced with Halloysite Nano-Tubes (HNT) which were surface functionalised with chitosan forming a Schiff base structure. The work was aimed to traverse its way in tissue engineering. Modification of halloysite with sea polysaccharide chitosan enhanced its ability to bind to the PVA matrix. The modification was assisted by FTIR, XRD, FESEM, TEM, AFM and DSC-TGA techniques. The films could withstand a higher temperature and exhibited high ranges of tensile strength and Young’s modulus. In addition, biocompatible studies such as in-vitro swelling, enzymatic degradation, water contact angle and hemolysis presented extremely well compatibilities proving it to be viable in physiological pH (in phosphate buffered saline). The cell adhesion and proliferation studies conducted on NIH3T3 mouse fibroblasts revealed the cell proliferation and tissue regeneration properties of the films. Both the tests performed for cell growth- Trypan blue dye exclusion and Acridine Orange Ethidium bromide assays showed a doubled rate of cell growth on the films which proves its biomedical nature.Graphical

Downregulation of Rad51 Expression and Activity Potentiates the Cytotoxic Effect of Osimertinib in Human Non-Small Cell Lung Cancer Cells.

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has shown significant clinical benefits in patients with EGFR-sensitizing mutations or the EGFR T790M mutation. The homologous recombination (HR) pathway is crucial for repairing DNA double-strand breaks (DSBs). Rad51 plays a central role in HR, facilitating the search for homology and promoting DNA strand exchange between homologous DNA molecules. Rad51 is overexpressed in numerous types of cancer cells. B02, a specific small molecule inhibitor of Rad51, inhibits the DNA strand exchange activity of Rad51. Previous studies have indicated that B02 disrupted Rad51 foci formation in response to DNA damage and inhibited DSBs repair in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. However, the potential therapeutic effects of combining osimertinib with a Rad51 inhibitor are not well understood. The aim of this study was to elucidate whether the downregulation of Rad51 expression and activity can enhance the osimertinib-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells. We used the MTS, trypan blue dye exclusion and colony-formation ability assay to determine whether osimertinib alone or in combination with B02 had cytotoxic effects on NSCLC cell lines. Real-time polymerase chain reaction was conducted to measure the amounts of Rad51 mRNA. The protein levels of phosphorylated AKT and Rad51 were determined by Western blot analysis. We found that osimertinib reduced Rad51 expression by inactivating AKT activity. Rad51 knockdown using small interfering RNA or AKT inactivation through the phosphatidylinositol 3-kinase inhibitor LY294002 or si-AKT RNA transfection enhanced the cytotoxic and growth inhibitory effects of osimertinib. In contrast, AKT-CA (a constitutively active form of AKT) vector-enforced expression could mitigate the cytotoxic and cell growth inhibitory effects of osimertinib. Furthermore, B02 significantly enhanced the cytotoxic and cell growth inhibitory effects of osimertinib in NSCLC cells. Compared to parental cells, the activation of AKT and Rad51 expression in osimertinib-resistant cells could not be significantly inhibited by osimertinib treatment. Moreover, the increased expression of Rad51 is associated with the resistance mechanism in osimertinib-resistant H1975 and A549 cells. Collectively, the downregulation of Rad51 expression and activity enhances the cytotoxic effect of osimertinib in human NSCLC cells.

PEMF Potentiates Doxorubicin-induced Late G2 Arrest in MDA-MB-231 Breast Cancer Cells.

Pulsed electromagnetic field (PEMF) stimulation enhances the efficacy of several anticancer drugs. Doxorubicin is an anticancer drug used to treat various types of cancer, including breast cancer. However, the effect of PEMF stimulation on the efficacy of doxorubicin and the underlying mechanisms remain unclear. Thus, this study aimed to investigate the effect of PEMF stimulation on the anticancer activity of doxorubicin in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were seeded and allowed to incubate for 48 h. The cells were treated with doxorubicin, cisplatin, 5-fluorouracil, or paclitaxel for 48 h. Subsequently, the cells were stimulated with a 60-min PEMF session thrice a day (with an interval of 4 h between each session) for 24 or 48 h. Cell viability was assessed by trypan blue dye exclusion assay and cell-cycle analysis was analyzed by flow cytometry. Molecular mechanisms involved in late G2 arrest were confirmed by a western blot assay and confocal microscopy. MDA-MB-231 cells treated with a combination of doxorubicin and PEMF had remarkably lower viability than those treated with doxorubicin alone. PEMF stimulation increased doxorubicin-induced cell-cycle arrest in the late G2 phase by suppressing cyclin-dependent kinase 1 (CDK1) activity through the enhancement of myelin transcription factor 1 (MYT1) expression, cell division cycle 25C (CDC25C) phosphorylation, and stratifin (14-3-3σ) expression. PEMF also increased doxorubicin-induced DNA damage by inhibiting DNA topoisomerase II alpha (TOP2A). These findings support the use of PEMF stimulation as an adjuvant to strengthen the antiproliferative effect of doxorubicin on breast cancer cells.

The Role of Insulin-like Growth Factor Binding Protein (IGFBP)-2 in DNA Repair and Chemoresistance in Breast Cancer Cells.

The role if insulin-like growth factor binding protein-2 (IGFBP-2) in mediating chemoresistance in breast cancer cells has been demonstrated, but the mechanism of action is unclear. This study aimed to further investigate the role of IGFBP-2 in the DNA damage response induced by etoposide in MCF-7, T47D (ER+ve), and MDA-MB-231 (ER-ve) breast cancer cell lines. In the presence or absence of etoposide, IGFBP-2 was silenced using siRNA in the ER-positive cell lines, or exogenous IGFBP-2 was added to the ER-negative MDA-MB-231 cells. Cell number and death were assessed using trypan blue dye exclusion assay, changes in abundance of proteins were monitored using Western blotting of whole cell lysates, and localization and abundance were determined using immunofluorescence and cell fractionation. Results from ER-positive cell lines demonstrated that upon exposure to etoposide, loss of IGFBP-2 enhanced cell death, and this was associated with a reduction in P-DNA-PKcs and an increase in γH2AX. Conversely, with ER-negative cells, the addition of IGFBP-2 in the presence of etoposide resulted in cell survival, an increase in P-DNA-PKcs, and a reduction in γH2AX. In summary, IGFBP-2 is a survival factor for breast cancer cells that is associated with enhancement of the DNA repair mechanism.

Pulsed Electromagnetic Field Enhances Caffeic Acid Phenethyl Ester-induced Death of MCF-7 Breast Cancer Cells.

Caffeic acid phenethyl ester (CAPE) exerts anticancer effects against several cancer types, including breast cancer. Pulsed electromagnetic field (PEMF) improves the efficiency of some chemotherapeutic drugs. In this study, we examined the effects of PEMF stimulation on the anticancer activity of CAPE in MCF-7 breast cancer cells and the underlying signal transduction pathways. MCF-7 cells were seeded and incubated for 24 h. Each of the drugs (5-fluorouracil, paclitaxel, gefitinib, or CAPE) was added to the cells on day 0. Then, cells were immediately stimulated with a 60-min PEMF session thrice a day (with 4-h interval between sessions) for 1-3 days. Cell death and viability were assessed by flow cytometry and trypan blue dye exclusion assay. Molecular mechanisms involved in cell death were confirmed by western blot assay. Compared with treatment with CAPE alone, co-treatment with CAPE and PEMF more strongly reduced the viability of MCF-7 cells, further increased the percentage of the sub-G1 population, poly (ADP-ribose) polymerase (PARP) cleavage, activation of apoptotic caspases, up-regulation of pro-apoptotic proteins, such as Fas cell surface death receptor (FAS) and BCL2 associated X, apoptosis regulator (BAX), and reduced the expression of anti-apoptotic proteins, such as BCL-2 apoptosis regulator (BCL-2), MCL-1 apoptosis regulator, BCL-2 family member (MCL-1), and survivin. PEMF stimulation also increased CAPE-induced phosphorylation of p53, and inhibition of p53 partially restored the PEMF-reduced viability of CAPE-treated MCF-7 cells. PEMF stimulation enhanced CAPE-induced cell death by activating p53, which regulates the expression of apoptosis-related molecules, subsequently activating the caspase-dependent apoptotic pathway in MCF-7 cells, suggesting that PEMF can be utilized as an adjuvant to enhance the effect of CAPE on breast cancer cells.

Effect of dose-rate on nitric oxide-induced apoptosis in A375 human melanoma cells

Purpose: To investigate the effect of dose-rate on nitric oxide (NO)-induced apoptosis in A375 human melanoma cells. Methods: The A375 cells were exposed to NO at a steady-state concentration of 7 μM, similar to the level estimated to occur in vivo in inflamed tissues, and delivered by diffusion through silastic tubing. Trypan blue dye exclusion assay was used to assess cell proliferation and viability. Cell cycle was studied by propidium iodide (PI) staining while Annexin V-FITC and PI assays were used to evaluate apoptosis. Western blotting assay was carried out to determine protein levels of p53, Bax, DR4, DR5, Fas (CD95), Procaspase 8, Procaspase 9 and Procaspase 3. Results: Viability of A375 cells was reduced by 22 % after 24 h leading to extensive apoptosis and cell cycle arrest following exposure to a cumulative dose of 3360 μM/min NO. Treatment with NO stimulated p53 and triggered mitochondrial apoptotic events by inducing conformational changes in Bax. Also, activation of caspase 9 and 3, DR4, DR5, Fas (CD95) and caspase 8 appeared to be mediated concurrently by death receptor processing and downstream caspases. Conclusion: Nitric oxide (NO) induces programmed cell death, thus indicating that it could serve as an effective inhibitor to halt the progression of melanoma or as an enhancer to improve therapeutic strategies for treatment.

Agar-tragacanth/silk fibroin hydrogel containing Zn-based MOF as a novel nanobiocomposite with biological activity

In this study, a novel nanobiocomposite consisting of agar (Ag), tragacanth gum (TG), silk fibroin (SF), and MOF-5 was synthesized and extensively investigated by various analytical techniques and basic biological assays for potential biomedical applications. The performed Trypan blue dye exclusion assay indicated that the proliferation percentage of HEK293T cells was 71.19%, while the proliferation of cancer cells (K-562 and MCF-7) was significantly lower, at 10.74% and 3.33%. Furthermore, the Ag-TG hydrogel/SF/MOF-5 nanobiocomposite exhibited significant antimicrobial activity against both E. coli and S. aureus strains, with growth inhibition rates of 76.08% and 69.19% respectively. Additionally, the hemolytic index of fabricated nanobiocomposite was found approximately 19%. These findings suggest that the nanobiocomposite exhibits significant potential for application in cancer therapy and wound healing.

Parkin Enhances Gefitinib-induced Anoikis in HeLa Cervical Cancer Cells.

Gefitinib exhibits anticancer activity against cervical cancer cells via anoikis, a type of apoptosis induced by cell detachment from the extracellular matrix. Previous studies have reported that Parkin expression affects the efficacy of anticancer drugs. However, the impact of Parkin expression on the therapeutic effects of gefitinib in human cervical cancer remains unclear. Thus, this study aimed to evaluate whether Parkin over-expression improves the therapeutic effects of gefitinib against HeLa cervical cancer cells. Cell viability and apoptotic death of HeLa cells were measured by trypan blue dye exclusion assay and flow cytometry. Cell detachment, adhesion, spreading, and cell-cell interaction were observed by inverted microscopy. Alteration of adhesion-related molecules was evaluated by confocal microscopy and western blot assay. Parkin expression potentiated gefitinib-induced cell detachment by affecting the organization of the actin cytoskeleton. In addition, Parkin expression induced a further reduction in the reattachment of and interaction between detached cells. The therapeutic efficacy of low-dose gefitinib combined with Parkin expression was equivalent to that of high-dose gefitinib alone. Parkin expression promotes gefitinib-induced anoikis, consequently increasing the efficacy of gefitinib against HeLa human cervical cancer cells. Based on our results, we propose that Parkin can be used to increase the anti-cancer effect of gefitinib on cervical cancer cells.

Anticancer Activity and GC-MS Profiling of Bioactive Constituents in the Methanolic Extracts of Spatoglossum variabile and Gracilaria corticata

Background: Marine algae stand out as repositories of bioactive metabolites, widely harnessed in treating diverse diseases. Given the often-challenging side effects associated with conventional cancer treatments, researchers are increasingly turning their attention to marine algae as a promising source for discovering novel and potent anticancer compounds. In this study, we meticulously explored the potential anticancer activity of Spatoglossum variabile and Gracilaria corticata sourced from the Indian Ocean. background: Marine algae are rich in bioactive metabolites that are extensively used to treat various diseases. Researchers have considered marine algae for novel and promising anticancer compounds due to the adverse effects of standard cancer treatments Materials and Methods: The secondary metabolites were extracted using methanol solvent according to the standard protocol. The extract was subjected to GC-MS analysis and evaluated for its short- and long-term cytotoxicity and anti-proliferative ability in the HCT116 cell line using MTT, clonogenic, and trypan blue dye exclusion assays, respectively. Results and Discussion: The methanolic extract of Spatoglossum variabile contained 21 unique anticancer compounds, except for the other bioactive compounds. The results indicate that increasing the concentration of algal extracts decreased the cell viability significantly, with an IC50 of 51.49 μg/mL and 37.50 μg /mL in the HCT116 cell line. The results showed a significant reduction in colorectal cancer colony formation and a gradual reduction in cell proliferation. These results underscore the potential for targeted, low-toxicity biomedicines due to their selective cytotoxicity against cancer cells. method: The extract was subjected to GC-MS analysis and evaluated for its short- and long-term cytotoxicity and anti-proliferative ability in HCT116 cell line using MTT, clonogenic, and trypan blue dye exclusion assays, respectively. Conclusion: Our findings show that Spatoglosuum variabile & Gracilaria corticata extracts contain various bioactive compounds, display long-term and short-term cytotoxicity and antiproliferative effects, which indicates that their constituent compounds might be further refined into effective anti-colorectal cancer medications. result: The methanolic extract of Spatoglossum variabile contained 21 unique anti-cancer compounds, in addition to other bioactive compounds. The results indicate that increasing the concentration of algal extracts decreased the cell viability significantly, with an IC50 of 51.49 µg/mL and 37.50 µg /ml in the HCT116 cell line. The results showed a significant reduction in colony formation and a gradual reduction in cell proliferation

Improving the power of drug toxicity measurements by quantitative nuclei imaging

Imaging-based anticancer drug screens are becoming more prevalent due to development of automated fluorescent microscopes and imaging stations, as well as rapid advancements in image processing software. Automated cell imaging provides many benefits such as their ability to provide high-content data, modularity, dynamics recording and the fact that imaging is the most direct way to access cell viability and cell proliferation. However, currently most publicly available large-scale anticancer drugs screens, such as GDSC, CTRP and NCI-60, provide cell viability data measured by assays based on colorimetric or luminometric measurements of NADH or ATP levels. Although such datasets provide valuable data, it is unclear how well drug toxicity measurements can be integrated with imaging data. Here we explored the relations between drug toxicity data obtained by XTT assay, two quantitative nuclei imaging methods and trypan blue dye exclusion assay using a set of four cancer cell lines with different morphologies and 30 drugs with different mechanisms of action. We show that imaging-based approaches provide high accuracy and the differences between results obtained by different methods highly depend on drug mechanism of action. Selecting AUC metrics over IC50 or comparing data where significantly drugs reduced cell numbers noticeably improves consistency between methods. Using automated cell segmentation protocols we analyzed mitochondria activity in more than 11 thousand drug-treated cells and showed that XTT assay produces unreliable data for CDK4/6, Aurora A, VEGFR and PARP inhibitors due induced cell size growth and increase in individual mitochondria activity. We also explored several benefits of image-based analysis such as ability to monitor cell number dynamics, dissect changes in total and individual mitochondria activity from cell proliferation, and ability to identify chromatin remodeling drugs. Finally, we provide a web tool that allows comparing results obtained by different methods.

Comparative Efficacy of the Dimethyl Sulfoxide, Glycerol and Methanol on the Post-Thaw Cell Viability of HeLa Cells

Cryoprotectants are used to protect cells during freezing. The concentration, type, and freeze-thaw conditions of these substances vary depending on the type of cell to be used. It is very important to determine the appropriate cryopreservation method for the particular cell. This study aims to provide insights into the optimal cryopreservation method for HeLa cells by comparing the performance of different cryoprotectants and evaluating their effectiveness under various freezing and storage conditions. Cell suspensions were frozen with a freezing media composed of cryoprotectant + fetal bovine serum + medium at a ratio of 5:10:85 (v:v:v) and stored under the following conditions: 3 months (-20 °C), 1 month (-80 °C), and 6 months (-80 °C). Cell viability and recovery rates were analyzed immediately post-thaw and after 48 h using the trypan blue dye exclusion assay. In 3 months (-20 °C), viability and recovery rates were higher in the methanol group. Glycerol showed better performance in 1 month (-80 °C). DMSO was the most efficient in 6 months (-80 °C). Methanol failed at -80 °C storage temperature. This study demonstrates the effect of these cryoprotectants in HeLa cells on cell viability and cell recovery rates immediately after thawing and after 48 hours of cultivation.

Characterization of a hemolytic and antibiotic-resistant Pseudomonas aeruginosa strain S3 pathogenic to fish isolated from Mahananda River in India.

Virulent strain Pseudomonas aeruginosa isolated from Mahananda River exhibited the highest hemolytic activity and virulence factors and was pathogenic to fish as clinical signs of hemorrhagic spots, loss of scales, and fin erosions were found. S3 was cytotoxic to the human liver cell line (WRL-68) in the trypan blue dye exclusion assay. Genotype characterization using whole genome analysis showed that S3 was similar to P. aeruginosa PAO1. The draft genome sequence had an estimated length of 62,69,783 bp, a GC content of 66.3%, and contained 5916 coding sequences. Eight genes across the genome were predicted to be related to hemolysin action. Antibiotic resistance genes such as class C and class D beta-lactamases, fosA, APH, and catB were detected, along with the strong presence of multiple efflux system genes. This study shows that river water is contaminated by pathogenic P. aeruginosa harboring an array of virulence and antibiotic resistance genes which warrants periodic monitoring to prevent disease outbreaks.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell–cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Effects of Imatinib and Artemisia vulgaris extracts in combination on leukemia cell proliferation

Resistance to imatinib develops gradually and presents major complications in patients with chronic myeloid leukemia (CML) disease, necessitating additional study for therapy improvement. Artemisia vulgaris is a member of the genus Artemisia which is traditionally used in herbal medicine. Historically, this plant was used to cure a variety of many human diseases including cancer. The goal of this study was to investigate the combined effect of imatinib and A. vulgaris extract on CML cell lines. For this objective, TCCY cells were treated for 72 hours with Imatinib, A. vulgaris extract, and the combinations at various concentrations. The proliferation of cells was determined by using the Trypan Blue dye exclusion assay. The combined efficacy of extract and Imatinib was determined using a nonlinear isobologram model at IC80 levels. To find discrepancies between observed and predicted data, Friedman tests and Nemenyi post-hoc analysis were used. To confirm the combined impact, the Wilcoxon signed-ranks test was utilized. The results suggested that the effect of Imatinib and A. vulgaris in combination was additive to synergistic. As a result, it was found that taking A. vulgaris extract can make Imatinib more effective against the CML cell line TCCY.

Synthesis and Biological Evaluation of Novel Benzylidene Thiazolo Pyrimidin-3(5H)-One Derivatives

Starting compound 1 was synthesized according to reference. 1 Benzylidene thiazole pyrimidin-3(5H)-ones were synthesized reactions of 1 with bromoacetic acid and various aryl-aldehydes in the same vessel via one-step, unlike studies in the literature. Quantum chemical parameters and full geometry optimizations for all compounds were computed using DFT based on B3LYP. Cytotoxic action potential of synthesized compounds was evaluated using trypan blue dye exclusion and MTT assays in different cell lines including adenocarcinoma alveolar basal epithelial-like adherent A549 cells, the colon adenocarcinoma HT-29 cells, prostate adenocarcinoma DU-145 cells, and diploid ARPE-19 retinal pigment epithelial cells. Embryotoxicity and genotoxicity assessments were performed on pluripotent human embryonal carcinoma NT2 and human lymphocyte cells, respectively. Compound A1 exhibited good anticancer activity on A549 and DU-145 cell lines, and the compounds including A3, 4, 6, and 9 induced cytotoxicity on A549 cells. The compounds A1-10 also showed a good biosafety profile at relatively lower concentrations.

Bioassay Techniques in Entomological Research

BIOASSAY (Bio - life, Assay - determination) is the measurement of the potency of any stimulus (physical, chemical, biological, and psychological) and the reactions that it produces in living matter. In Entomology, to know about the potency, Specificity of any compounds, standardization of doses, and identify modes of action bioassay is needed. In addition to the basic bioassay methods such as poison food, dry film techniques, etc., depending upon the researcher’s necessity, they are modified or newly developed. Diet covering and diet incorporation for lepidopterans and homopterans have been developed as advancements over the poison food assay method, by using insecticide-incorporated artificial diet. WHO cone, Wire ball test, Susceptibility test, CDC bottle assay and MCD assay developed for mosquito resistance monitoring and Larval Packet Technique (LPT) for cattle ticks, Sandwich method, TIBS, and Adult vial test are advancements over dry film technique. In Thrips Insecticide Bioassay System (TIBS), treated vials can be stored for up to 3 weeks at 4˚C for future efficacy assays at the field level. Adult Vial Tests have been developed for pentatomid and mirid adult bug bioassays. Larval Immersion Technique (LIT), Adult Immersion Technique (AIT) for cattle ticks, and MEBS have been developed as advancements over topical bioassay. In Mite Egg Bioassay System (MEBS), entomopathogenic fungi have been tested against Tetranychus cinnabarinus. MTT colorimetric assay and Trypan Blue Dye Exclusion assay helps to identify cell viability in insect cell line cultures. Spodoptera frugiperda cell line, Sf – 21 was tested against a scorpion, Leiurus quinquestriatus toxin β– BUTX – LQQ1a and the toxicity was determined through the viability of cell lines. Various basic and advanced bioassay techniques are discussed elaborately in this review.

The cytotoxicity effect of 7-hydroxy-3,4-dihydrocadalene from Heterotheca inuloides and semisynthetic cadalenes derivates towards breast cancer cells: involvement of oxidative stress-mediated apoptosis.

Heterotheca inuloides, traditionally employed in Mexico, has demonstrated anticancer activities. Although it has been proven that the cytotoxic effect is attributed to cadinane-type sesquiterpenes such as 7-hydroxy-3,4-dihydrocadalene, the mechanism of action by which these agents act in tumor lines and their regulation remain unknown. This study was undertaken to investigate for first time the cytotoxic activity and mechanism of action of 7-hydroxy-3,4-dihydrocadalene and two semi-synthetic cadinanes derivatives towards breast cancer cells. Cell viability and proliferation were assayed by thiazolyl blue tetrazolium bromide (MTT) assay and Trypan blue dye exclusion assay. Cell migration measure was tested by wound-healing assay. Moreover, the reactive oxygen species (ROS) and lipid peroxidation generation were measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay and thiobarbituric acid reactive substance (TBARS) assay, respectively. Furthermore, expression of caspase-3, Bcl-2 and GAPDH were analyzed by western blot. The results showed that 7-hydroxy-3,4-dihydrocadalene inhibited MCF7 cell viability in a concentration and time dependent manner. The cytotoxic potency of semisynthetic derivatives 7-(phenylcarbamate)-3,4-dihydrocadalene and 7-(phenylcarbamate)-cadalene was remarkably lower. Moreover, in silico studies showed that 7-hydroxy-3,4-dihydrocadalene, and not so the semi-synthetic derivatives, has optimal physical-chemical properties to lead a promising cytotoxic agent. Further examination on the action mechanism of 7-hydroxy-3,4-dihydrocadalene suggested that this natural product exerted cytotoxicity via oxidative stress as evidenced in a significantly increase of intracellular ROS levels and in an induction of lipid peroxidation. Furthermore, the compound increased caspase-3 and caspase-9 activities and slightly inhibited Bcl-2 levels. Interestingly, it also reduced mitochondrial ATP synthesis and induced mitochondrial uncoupling. Taken together, 7-hydroxy-3,4-dihydrocadalene is a promising cytotoxic compound against breast cancer via oxidative stress-induction.

Lactoferrin functionalized concave cube Au nanoparticles as biocompatible antibacterial agent

Gold nanoparticles (AuNPs) are one of the most extensively studied nanomaterials and their distinct physicochemical properties make them suitable for versatile applications. Herein, we synthesized concave cube-shaped gold nanoparticles (CCAuNPs) and functionalized them with lactoferrin (Lf), a natural antimicrobial protein, through electrostatic interaction as well as weak covalent formation. The functionalization of CCAuNPs was confirmed through UV–Visible (i.e., bathochromic shift of the surface plasmon resonance peak by 7 nm), Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectroscopy, and their surface zeta potential. The concave cusp of CCAuNPs was confirmed through atomic force microscopy (AFM). The Lf-functionalized CCAuNPs (Lf-CCAuNPs) exhibited superior antibacterial propensity against a series of bacteria when compared to that of CCAuNPs. However, they didn't demonstrate any antibacterial activity against Lactobacillus plantarum, one of the key beneficial gut bacteria. The lipid peroxidation (LPO) assay confirmed the oxidation of fatty acids in the bacterial membrane upon interaction with AuNPs, which made the bacterial membrane porous. The resultant membrane-impaired dead bacteria were visualized through CellTox™ Green assay as well as the Trypan Blue dye exclusion assay. Both the nanoparticles demonstrated excellent hemocompatibility as well as biocompatibility (both in vitro and in vivo) as confirmed by MTT assay and the levels of important functional biomarkers of liver (e.g., ALT, AST, and ALP) and kidney (e.g., creatinine, uric acid, and BUN) in the serum. Overall, Lf-CCAuNPs with excellent hemocompatibility, and biocompatibility can be deployed as potential antibacterial agents to tackle the menace of pathogenic bacteria.

Cytotoxic activity of unique synthesized five-membered heterocyclic compounds coordinated with tiopronin monovalent.

We recently synthesized a compound in which 5-mercapto-1-methyltetrazole (MM4) was coordinated to tiopronin monovalent (TPN-Au(Ⅰ)) and reported its cytotoxic activity against human leukemia cells in vitro. Objective: We further synthesized other heterocyclic compounds coordinated with TPN-Au(Ⅰ) and assessed their cytotoxic activity against hepatocellular carcinoma HepG2 and lung cancer cell line H1299 in vitro. Seven kinds of compounds were synthesized by introducing a five-membered heterocyclic compound into TPN-Au(Ⅰ). The number of viable cells was counted by a trypan blue dye exclusion assay. Fluorescence conjugated-Annexin V and propidium iodide were used for the apoptosis analysis. Seven compounds were successfully synthesized. Among these compounds, TPN-Au(Ⅰ)-MTZ (3-mercapto-1,2,4-triazole), TPN-Au(Ⅰ)-MMT (2-mercapto-5-methyl-1,3,4-thiadiazole), and TPN-Au(Ⅰ)-MMTT (2-mercapto-5-methylthio-1,3,4-thiadiazole) effectively suppressed the proliferation and induced apoptosis in HepG2 cells. In addition, TPN-Au(Ⅰ)-MMTT and TPN-Au(Ⅰ)-MMT also showed effective cytotoxicity against H1299 cells. Conclusion: The present results showed that introduction of some five-membered heterocyclic compounds, especially MMT and MMTT, to TPN-Au(Ⅰ) improved the cytotoxicity against solid cancer cells.