Trypan Blue Dye Exclusion Assay Research Articles

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 the 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.

Artesunate, a new antimalarial clinicaldrug, exhibits potent anti-AML activity by targeting the ROS/Bim and TFRC/Fe2+ pathways.

Artesunate, approved by the Food and Drug Administration in 2020 as a new treatment for severe malaria, also shows anti-tumour activity against acute myeloid leukaemia (AML). However, the underlying molecular mechanism(s) of artesunate-induced apoptosis and differentiation of AML is not clearly elucidated. The biological effects of artesunate on AML were explored in vitro, using cells from AML patients and leukaemia cell lines, and in vivo, using female C57BL/6 or nude nu/nu BALB/c mice. Underlying mechanisms in vitro were examined with the Trypan blue dye exclusion assay, western blotting and flow cytometry. Effects of artesunate in C57BL/6 mice intravenously injected with murine AML cells (C1498-GFP) were assessed by numbers of AML cells and by survival. In vitro, artesunate promoted apoptosis and differentiation in both leukaemia cell lines and patient-derived primary leukaemia cells. Mechanistically, artesunate promoted cell apoptosis by triggering reactive oxygen species (ROS) production and increasing expression of the pro-apoptotic protein Bim. Interestingly, transferrin receptor 1 (TFRC)-mediated regulation of intracellular iron homeostasis also played an essential role in AML cell differentiation induced by artesunate. In vivo, artesunate slowed AML progression and prolonged survival in a mouse leukaemia model. Notably, artesunate displayed no apparent toxicity towards healthy haematopoietic stem cells, bone marrow mononuclear cells or experimental animals. Artesunate is a safe agent with significant anti-leukaemia effects in mice and may serve as a promising chemotherapeutic strategy for patients with AML, based on two different mechanisms, targeting the ROS/Bim and the TFRC/Fe2+ pathways.

Novel 1,2,5-Trisubstituted Benzimidazoles Potentiate Apoptosis by Mitochondrial Dysfunction in Panel of Cancer Cells.

Synthetic small molecules have been very effective in decimating cancer cells by targeting various aberrantly overexpressed oncogenic proteins. These small molecules target proteins involved in cell cycle regulation, cell division, migration, invasion, angiogenesis, and other regulatory proteins to induce apoptosis in cancer cells. In this study, we have synthesized a novel 1,2,5-trisubstituted benzimidazole chemical library of small molecules and unveiled their anticancer potential against a panel of cancer cell lines such as Jurkat, K-562, MOLT-4, HeLa, HCT116, and MIA PaCa-2 cancer cells. The MTT assay and Trypan blue dye exclusion assay clearly unveiled the cytotoxic effect of methyl 1-benzyl-2-(4-fluoro-3-nitrophenyl)-1H-benzo[d]imidazole-5-carboxylate (TJ08) and its potential to induce apoptosis with effective IC50 of 1.88 ± 0.51, 1.89 ± 0.55, 2.05 ± 0.72, 2.11 ± 0.62, 3.04 ± 0.8, and 3.82 ± 0.25 μM against Jurkat, K562, MOLT-4, HeLa, HCT116, and MIA PaCa-2 cancer cell lines, respectively. Altered mitochondrial membrane potential was observed in HeLa, HCT116, and Jurkat cells due to TJ08 treatment, which was unveiled by JC10 staining. Induction of early and late apoptosis by TJ08 treatment was also unveiled by apoptotic analysis and immunofluorescence imaging. Cell cycle analysis distribution confirms the accumulation of cells in the S-phase in a dose-dependent manner.

Targeting Cellular Metabolism With CPI-613 Sensitizes Pancreatic Cancer Cells to Radiation Therapy

PurposeLocal tumor progression is a cause of significant morbidity and mortality in patients with pancreatic ductal adenocarcinoma (PDAC) with surgically unresectable disease. Novel and effective approaches to accomplish durable local control are urgently needed. We tested whether CPI-613 (devimistat), a first-in-class investigational small molecule inhibitor of mitochondrial metabolism, was capable of altering cancer cell energy metabolism and sensitizing PDAC cells to radiation therapy (RT). Methods and MaterialsThe effect of a combined treatment of RT with CPI-613 on the viability of, clonogenic potential of, and cell death induction in PDAC cells (MiaPaCa-2 and Panc-1) was determined using a trypan blue dye exclusion assay, a colony formation assay, and a 7–amino-actinomycin D assay, respectively. The synergistic effects of CPI-613-RT and chemotherapeutic agents (gemcitabine or 5-fluorouracil) were measured in MiaPaCa-2 cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and spheroid formation assay. Changes in energy metabolism were determined by profiling metabolites treated with either RT, CPI-613, or both using liquid chromatography-mass spectrometry. ResultsThis study demonstrates that a combination of single-fraction RT (2 and 10 Gy) with CPI-613 significantly inhibits PDAC cell growth compared with RT alone. Molecular analysis revealed inhibition of α-ketoglutarate dehydrogenase at the protein level. In addition, we demonstrate enhanced cell death of PDAC cells when treated with RT-CPI-613 combination. Targeted metabolomic analysis on PDAC cells post–CPI-613-RT treatment revealed alterations in key mitochondrial metabolites, with broader target engagement by the combination treatment, indicating the sensitization of CPI-613–treated PDAC cells to RT. Furthermore, a combination treatment of CPI-613 with either gemcitabine or 5-fluorouracil in the presence of 2 Gy RT synergistically inhibits PDAC cell proliferation. ConclusionsOur results support a novel combination of CPI-613-RT that warrants further preclinical and early-phase clinical investigations. A phase 1 trial designed to identify the maximum tolerated dose of CPI-613 in combination with chemo-RT in patients with PDAC was recently initiated (NCT05325281).

Evaluation of mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity, and acute toxicity of ethanolic extract of Cissus quadrangularis

Recent studies have focused on exploring the efficacy of Cissus quadrangularis extract (EECQ) against various metabolic disorders involving the liver as the prime target organ, suggesting a considerable threat of hepatotoxicity in the person encountering it. Consequently, the current study was aimed to unravel the mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity in HepG2 cells, and acute toxicity of EECQ. MTT, SRB, trypan blue dye exclusion, and lactate dehydrogenase (LDH) assay were performed in HepG2 cell lines to determine the cytotoxicity of the extract. The mutagenic potential was determined by the Ames test using various strains of Salmonella typhimurium. Acute toxicity was done at a dose of 2000 mg/kg in Sprague Dawley rats. MTT and SRB cytotoxicity assays demonstrated dose-dependent cytotoxicity of extract. The three highest noncytotoxic doses from the above assay, investigated by trypan blue dye exclusion and LDH assay, did not reveal cytotoxicity. Besides, mitochondrial dysfunction was determined by measuring cellular and mitochondrial ROS, ATP, NAD, mitochondrial membrane potential, Bax/Bcl2 ratio, mitochondrial and cytoplasmic cytochrome c, and apoptosis-inducing factor, were found to be equivalent in both extract exposed and unexposed cells. Moreover, the apoptotic cell morphology and the expression of pro-apoptotic mRNAs and proteins were equivalent in both the group. In acute toxicity, EECQ in rats did not cause any significant change in body weight, liver index, and liver function test. All-encompassing, the present study unraveled that EECQ is not mutagenic, cytotoxic, nor apoptotic in human hepatic cells, as well as neither acute toxicity.

The effect of natural polyphenols Resveratrol, Gallic acid, and Kuromanin chloride on human telomerase reverse transcriptase (hTERT) expression in HepG2 hepatocellular carcinoma: role of SIRT1/Nrf2 signaling pathway and oxidative stress.

There is evidence that low doses or physiological concentrations of certain natural polyphenols enhance the activity of telomerase. However, the precise mechanism by which natural polyphenols regulate telomerase activity remains unclear. Recent research indicates that NF-E2 related factor 2 (Nrf2) and silent information regulator 1 (SIRT1) are involved in human telomerase reverse transcriptase (hTERT) regulation. Thus, in order to better comprehend the mechanism by which polyphenols regulate hTERT, the present study investigated the effects of the natural polyphenols Resveratrol, Gallic acid, and Kuromanin chloride on hTERT, Nrf2, and SIRT1 expression as well as oxidative stress in HepG2 hepatocellular carcinoma. The trypan blue dye exclusion assay was used to assess cell viability. The level of mRNA for hTERT, Nrf2, and SIRT1 was then determined using real-time PCR. A spectrophotometric analysis was conducted to quantify oxidative stress markers. The results demonstrated that Resveratrol induces the expression of hTERT and the SIRT1/Nrf2 pathway in a dose-dependent manner. Gallic acid at concentrations of 10 and 20μM also increased the expression of the hTERT and SIRT1/Nrf2 pathway. Furthermore, dose-dependent overexpression of hTERT and Nrf2 was induced by Kuromanin chloride at 10 and 20µM. Moreover, we found that Resveratrol and Kuromanin chloride ameliorated oxidative stress, whereas Gallic acid exacerbated it. This study demonstrates that low doses of polyphenols (Resveratrol, Gallic acid, and Kuromanin chloride) upregulate the expression of the hTERT gene in the HepG2 hepatocellular carcinoma cell line, possibly via induction of the SIRT1/Nrf2 signaling pathway. Therefore, by targeting this pathway or hTERT, the anti-cancer effect of polyphenols can be enhanced.

Use of gold nanoparticle-silibinin conjugates: A novel approach against lung cancer cells.

Lung cancer presents one of the most challenging carcinomas with meager 5-year survival rates (less than 20%), high metastasis and high recurrence due to chemo- and radio- resistance. An alternative or complementation to existing prognosis modalities is the use of phytochemicals such as silibinin, which targets essential cytokines, angiogenic factors and transcription factors for a profound anti-tumor effect. However, the problems of low solubility in an aqueous physiological environment, poor penetration, high metabolism and rapid systemic clearance limit the therapeutic use of silibinin. Conjugation of gold nanoparticles (GNPs) with silibinin may overcome the above challenges along with distinct advantages of biocompatibility, optical properties for monitoring and causation of cytotoxicity in cancer cells. The current study thus aims to develop silibinin conjugated gold nanoparticles (Sb-GNPs) with pH responsive release in the cancer microenvironment, optimizing several parameters for its higher activity and further evaluate the nanoplatform for their efficacy in inducing cell death in vitro against A549 lung cancer cells. GNPs was synthesized using trisodium citrate dihydrate as the reducing agent and further used for the conjugation of silibinin. The synthesized GNPs were found to be monodispersed and spherical in shape. The silibinin was successfully conjugated with gold nanoparticles and long-term stability of GNPs and Sb-GNPs nanoconjugates in suspension phase was confirmed by FTIR and DLS. Anticancer properties of Sb-GNPs were confirmed by different assay using MTT, Trypan blue dye exclusion assay and cell cycle analysis assay. After conjugation of silibinin with GNPs, the efficacy of silibinin increased 4–5 times in killing the cancer cells. This is the first report on using silibinin gold nanoconjugate system for lung cancer therapy with promising future applications.