Sulforhodamine B Assay Research Articles

Major Sesquiterpenoids in Psychotria laui Leaf Essential oil Exhibit Anti-Inflammatory and Cytotoxic Properties

Objective: This study aims to investigate the chemical constituents and biological properties of the essential oil derived from Psychotria laui, a plant recognized for its medicinal applications, particularly in traditional Vietnamese medicine, where it is utilized for the treatment of digestive and inflammatory disorders. Methods: Fresh leaves of Psychotria laui were gathered in Quangtri, Vietnam, and subjected to hydro-distillation using a Clevenger-type apparatus to obtain the essential oil. The chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS). The antioxidant potential was measured using the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, while the inhibition of nitric oxide production was tested in lipopolysaccharide-stimulated RAW264.7 cells. Cytotoxicity against the SK-LU-1 cell line (human lung adenocarcinoma) was evaluated using the sulforhodamine B (SRB) assay. Results: The essential oil from Psychotria laui contained 78 distinct constituents. The major compounds identified were spathulenol (10.4%), thujopsan-2- α-ol (9.5%), 7- epi- α-eudesmol (6.5%), 14-hydroxy-9- epi-( E)-caryophyllene (4.4%), epi- α-cadinol (3.9%), α-muurolol (3.8%), 1- epi-cubenol (3.5%), and δ-cadinene (3.2%). The essential oil showed no antioxidant activity, with an IC50 (half-maximal inhibitory concentration) value greater than 500 µg/mL, in contrast to the positive control, L-ascorbic acid, which had an IC50 of 7.78 ± 0.39 µg/mL in the DPPH assay. However, its anti-inflammatory activity was evident by its ability to inhibit nitric oxide production, with an IC50 of 19.48 ± 1.53 µg/mL in lipopolysaccharide-stimulated RAW264.7 macrophages. Moreover, the essential oil exhibited potent cytotoxicity against the SK-LU-1 cancer cell line, with an IC50 of 8.49 ± 0.73 µg/mL in the SRB assay. Conclusion: The essential oil from Psychotria laui leaves contains numerous bioactive compounds and exhibits significant anti-inflammatory and cytotoxic properties, although it lacks antioxidant activity.

Design, Synthesis, Characterization, Molecular Docking Studies, In vitro and In vivo Cervical Cancer Activity of Novel N-Substituted Pyrazole Derivatives

The aim of this study is to design, synthesize, characterize few novel N-substituted pyrazole derivatives and evaluated for their anticancer capabilities; these compounds were selected based on their ability to inhibit the HPV E6 protein. The IR, 1H & 13C NMR, mass spectral and elemental analysis were used to determine the structural details of the newly synthesized substances. The molecular docking was performed using Auto Dock Vina, and the protein-ligand interaction was analyzed using Discovery Studio. The sulforhodamine B (SRB) assay was used to determine the in vitro anticancer activity against the HeLa cell line. Body weight analysis, mean survival time and % increase in life span approaches were used to assess in vivo anticancer effectiveness in Swiss albino mice bearing Dalton’s Lymphoma Ascites (DAL) tumor model. Synthesized compounds have excellent docking energies, according to docking experiments. Compounds I, II and III were selected for anticancer activity in vivo DAL-bearing mice based on the findings of their in vitro and SRB assays exhibiting the antiproliferative activity. Pyrazole derivatives, compounds I and III, demonstrated significant potential as anticancer agents.

Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

Evaluation of volatile components from the tuber, fibrous roots, bud, stem and leaf tissues of Bletilla striata for its anti-colon cancer activity.

Bletilla striata (Thunb.) Rchb.f., a medicinal plant in the Orchidaceae family, is mainly found in East Asia and has extensive pharmacological activities. Plant's volatile components are important active ingredients with a wide range of physiological activities, and B. striata has a special odor and unique volatile components. Yet it has received little attention, hindering a full understanding of its phytochemical components. Employing the ultrasonic-assisted extraction method, the volatile components of B. striata's fibrous root, bud, aerial part and tuber were extracted, resulting in yields of 0.06%, 0.64%, 3.38% and 4.47%, respectively. A total of 78 compounds were identified from their chemical profiles using gas chromatography-mass spectrometry (GC-MS), including 45 components with the main compounds of linoleic acid (content accounting for 31.23%), n-hexadecanoic acid (13.53%), and octadecanoic acid (9.5%) from the tuber, 34 components with the main compounds of eicosane, 2-methyl- (28.42%), linoelaidic acid (10.43%), linoleic acid (4.53%), and n-hexadecanoic acid (6.91%) from the fibrous root, 38 components with the main compounds of pentadeca-6,9-dien-1-ol (9.29%), n-hexadecanoic acid (11%), eicosane,2-methyl- (23.43%), and linoleic acid (23.53%) from the bud, and 27 components with the main compounds of linoelaidic acid (5.97%), n-hexadecanoic acid (15.99%), and linolenic acid ethyl ester (18.9%) from the aerial part. Additionally, the growth inhibition activity against colon cancer HCT116 cells was evaluated using sulforhodamine B (SRB) assay and the thiazolyl blue tetrazolium bromide (MTT) assay, and the accumulation of reactive oxygen species (ROS) was determined using dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining and fluorescence intensity analysis. The volatile extracts exhibited significant growth inhibitory efficacy against HCT116 cells, with half-maximal inhibitory concentration (IC50) values of 3.65, 2.32, 2.42 and 3.89mg/mL in the SRB assay, and 3.55, 2.58, 3.12 and 4.80mg/mL in the MTT assay for the root, bud, aerial part, and tuber, respectively. Notably, treatment with the aerial part extract caused morphological changes in the cells and significantly raised the intracellular ROS level. In summary, the chemical profiles of the volatile components of B. striata were revealed for the first time, demonstrating a certain tissue specificity. Additionally, it demonstrated for the first time that these volatile extracts possess potent anti-colon cancer activity, highlighting the importance of these volatile components in B. striata's medicinal properties.

Design, Synthesis and Anticancer Activity of Substituted 1, 3-Thiazolidin-4-One Derivatives

Background: Cancer is the global cause of death worldwide. Anticancer drug development is the need in today’s scenario. Thiazolidine is the nucleus that shows several pharmacological activities like anticancer, anti-inflammatory, antioxidant, antibacterial, antifungal, antidiabetic, antihyperlipidemic, and antiarthritic activity. In the present work molecular docking Glide module (Schrodinger Inc., USA) has been used for ligand docking against the Polo-like kinase-1. The series of substituted 3-Benzothiazol-2-yl-2-phenyl)-thiazolidin-4-ones were synthesized by the microwave-assisted synthesis system (CEM, USA) and characterized by melting point, FT-IR, 1H NMR, 13C NMR, and HR-MS analysis. Results: Molecular docking studies shows good docking score as well as interactions. Among the synthesized compounds, BG2 had the highest docking score of -8.381, followed by BG8 (-8.19) and BG1 (-8.156). All the newly synthesized compounds were examined for their in vitro anticancer activity against breast cancer cell line MCF-7 by Sulforhodamine B (SRB) assay. Conclusion: BG1, BG2, BG3, BG4, BG5, BG8, BG9, BG10, BG11, BG12, and BG13 (GI50: <80 µg/ml) exhibited significant cell growth inhibitory activity. These results indicate that compounds showing in-vitro activity by molecular docking studies and SRB assay could be lead compounds for further development of anticancer agents and suitable candidates for in-vivo anticancer activity.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5957.

Abstract 3090: Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community

Abstract The NCI60 screen was established in the 1980s and introduced in the 1990s as a screening service for the worldwide cancer research community. The screen features 60 human tumor cell lines that represent 9 cancer types including non-small cell lung, colon, central nervous system, ovarian, renal, prostate and breast, as well as leukemia and melanoma. On a weekly basis, synthetic molecules, natural products, and biologics are screened against the 60-cell line panel to evaluate their anticancer potential. On average, more than 13,000 compounds are screened annually for investigators throughout the world. Cell growth and cytotoxicity were assessed by the sulforhodamine B (SRB) assay, performed in a 96-well format, that measures cellular protein content with an absorbance readout and enabled unprecedented throughput in the 1980s. Although the SRB assay supported a robust screening platform for three decades, its sensitivity and throughput are limited. Recently the NCI’s DCTD introduced the HTS384 NCI60 as a modernized screening platform. While the same 60 cell lines are evaluated, the HTS384 NCI60 is fully automated and performed in a 384-well format to enable a higher throughput. Additionally, the 48-h test agent exposure period of the SRB assay was increased to 72 h, which may result in lower 50% growth inhibition values (GI50) for some agents. A highly sensitive CellTiter-Glo luminescence readout for cell viability, based on cellular ATP content, was selected to enable comparisons of the NCI60 monolayer data to 3D cell culture models where the same assay chemistry can be applied. The HTS384 NCI60 was evaluated by screening a library of 1,003 U.S. Food and Drug Administration-approved and investigational oncology agents at five concentrations. Assay performance of the 60 cell lines was assessed from the controls of 30 - 60 microplates per cell line. The average doubling time of the 60 cell lines during the 72-h exposure period ranged between 19 h for NCI-H460 (17 - 23 h, n = 57) to 117 h for MDA-MB-468 (43 - 272 h, n = 60). The average signal-to-background ratio of all 60 cell lines was 89 (3,413 microplates) and ranged from 6 for OVCAR-5 (3 - 13, n = 54) to 229 for SF-539 (141 - 566, n = 60). The average coefficient of variation for microplate vehicle controls (n = 14) of all 60 cell lines (n = 3,413) was 5.2% and ranged from 3.2% for SK-MEL-28 (1.4 - 7.7%, n = 60) to 11.1% for HL-60(TB) (5.2 - 20.2%, n = 59). The average Z’-factor value of all 60 cell lines (n = 3,413) was 0.82 and ranged between 0.66 for HL-60(TB) (0.39 - 0.84, n = 59) to 0.89 for MALME-3M (0.79 - 0.95, n = 59), SF-295 (0.49 - 0.97, n = 59), SK-MEL-28 (0.74 - 0.95, n = 60), and UO-31 (0.8 - 0.94, n = 54). The development and performance of the HTS384 NCI60 screen will be described. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Eric M. Jones, Thomas Silvers, John R. Britt, Ronald C. Taylor, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3090.

Determination of the optimal pH for doxorubicin encapsulation in polymeric micelles

HypothesisThe anticancer drug doxorubicin hydrochloride (DX) shows a high solubility in aqueous media thanks to the positive charge in the ammonium group. This feature, however, affects the drug encapsulation in the hydrophobic domains of polymeric micelles (PMs) used for the targeted delivery of the drug. At basic pH, DX deprotonates but also acquires a negative charge in the phenolic groups of the anthracycline structure. Both the efficiency and the rate of encapsulation will be increased by choosing an appropriate pH such that the drug molecule is in neutral form. ExperimentsAn optimal pH for the encapsulation of the DX in PMs based on commercial poloxamers and on the diblock copolymer methoxy-poly(ethylene glycol)17-b-poly(ε-caprolactone)9 was determined by fluorescence spectroscopy, following the time evolution of both the intensity ratio of the first and the second emission bands of DX and its fluorescence lifetime, both sensitive to the environment polarity. Intracellular delivery of PMs encapsulated drug was followed by Confocal Scanning Laser Microscopy (CSLM). Cell viability was assessed with the sulforhodamine B (SRB) assay. FindingsBy adjusting pH to 8.1 a high yield of incorporation of DX in the PMs was achieved coupled to an appreciable increase (one order of magnitude) in the drug encapsulation rate. In-vitro tests in selected cancer cell lines showed the slow release of the drug and a delay in the cytotoxic response in comparison to free DX as detected by CSLM and SRB assay. The proposed methodology paves the way for a greener, faster and more efficient encapsulation of DX in PMs.

The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time

The present study validates the potential of the in vitro H9c2(2-1) cell-based sulforhodamine B (SRB) assay to evaluate the temporal variability of wastewater quality. The impact of effluent disposal on water quality and the efficiency of the wastewater treatment process were also assessed. To correlate standard analytical method results with in vitro results, a total of 16 physicochemical parameters, such as nutrients, pH, chemical oxygen demand, total suspended solids and metals, were determined in both raw and treated wastewater samples. Results revealed that the H9c2(2-1) cell-based SRB assay has an enormous potential to evaluate municipal wastewater quality over time and to discriminate influent and effluent toxic characteristics, as well as for water quality monitoring and surveillance of the efficacy of treatment processes. Finally, the gathered results alerted to the impact of phosphates in a biological system, leading us to recommend the selection of this parameter as a potential environmental health indicator.

Human Gingival Fibroblasts Response to Different Endodontic Sealers: An In Vitro Study

Endodontic treatment aims to eliminate infection of the root canals and fill the dental pulp space. The biocompatibility studies of the sealers used in root canals obturation are crucial since they are applied in direct contact with periradicular tissues. Objective: The aim of this study was to evaluate the cytotoxicity of three root canal sealers—AH Plus, Bio MTA+, and Bio C sealer—on immortalized human gingival fibroblasts. Methods: AH Plus, Bio MTA+, and Bio C sealers were evaluated through incubation in real-time and material-conditioned media. Cells were incubated for 24 h and 72 h, at three different concentrations (1, 10, and 100 mg/mL) of each sealer. The cytotoxic activity of the sealers was assessed by Methyl tetrazolium (MTT) and Sulforhodamine B (SRB) assays. Cell morphology and cytogenetic alterations were studied microscopically. Results: MTT and SRB assays revealed similar results within both approaches. Cell culture exposed to sealers through incubation in real-time revealed a cytotoxic effect of AH Plus at 100 mg/mL. Material-conditioned media study revealed a cytotoxic effect of Bio MTA+ and Bio C, increasing with higher compound concentration and reaching 50% with 100 mg/mL. Regarding the cell’s morphology, Bio C sealer revealed a decrease in cell confluence and several morphological changes. AH Plus and Bio MTA+ did not seem to affect the cell confluence however morphology alterations were observed. In the cytogenetic study, a severe decrease of the mitotic index and a large number of chromosomal aberrations were observed. The present study represents an advance in the understanding of the biocompatibility of AH Plus, Bio MTA+, and Bio C sealers. These sealers demonstrated some cytotoxicity, depending on the concentration used. Although more validation studies are still needed, this study brings very relevant results in terms of cytotoxicity, cell morphology, and cytogenetic alterations. Conclusions: These results could help in the selection of the most appropriate compounds to be used in clinical practice as well as to determine the maximum recommended amounts of each sealer. Clinical Relevance: This study highlights the potential cytotoxic effects of three commonly used root canal sealers on human gingival fibroblasts, with varying degrees of impact depending on the concentration used. The results emphasize the importance of careful consideration when selecting and applying these materials in clinical practice.

Green Synthesis of Silver and Titanium Oxide Nanoparticles Using Tea and Eggshell Wastes, Their Characterization, and Biocompatibility Evaluation

Using biodegradable wastes represents a viable alternative to creating a sustainable economy that benefits all humans. The present study aimed to use daily used waste products, tea (TE) and eggshell (ES) wastes, to synthesize silver (AgNPs) and titanium oxide (TiO2NPs) nanoparticles, respectively. Firstly, the green-synthesized nanoparticles were characterized using an ultraviolet-visible spectrophotometer (UV-VIS), Scanning (SEM), transmission electron microscope (TEM), Dynamic light scattering (DLS), zeta potential analysis, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Then, followed by their cytotoxic assessment against normal human skin fibroblast (HSF) cells using sulforhodamine B (SRB) assay, AgNPs_TE (300 and 470 nm) and TiO2NPs_ESE (320 nm) formation was confirmed using UV-vis spectra. SEM and XRD showed their crystalline shape. TEM images determined the nano-size of AgNPs_TE (25 nm) and TiO2NPs_ESE (120 nm), which appeared smaller in comparison with DLS analysis (299.8 and 742.9 nm), with zeta potentials of −20.5 mV and −12.6 mV, respectively. There was a great difference in both NPs’ sizes using TEM and DLS measurements because DLS is known to be more sensitive to larger particles due to their light scattering. FTIR detected the functional groups found in TE and ESE that were responsible for the synthesis, capping, and stabilization of the synthesized AgNPs and TiO2NPs. The SRB assay reveals the safety of TiO2NPs on normal HSF cells with an IC50 > 100, while AgNPs have a high cytotoxic effect with an IC50 = 54.99 μg/mL.

Green approach for the synthesis of ZnO nanoparticles using Cymbopogon citratus aqueous leaf extract: characterization and evaluation of their biological activities

BackgroundThe green synthesis of metal and metal oxide nanoparticles (NPs), notably from plants, has attracted increasing attention in recent years. Although the increased popularity use of Cymbopogon citratus as a therapeutic substance, to date, there has not been any research on the chemistry of C. citratus aqueous leaf extract (ALE) or synthesis of ZnO NPs utilizing an extract from it. The ecologically safe ALE of C. citratus was employed in this study as a bio-reducing and capping agent to synthesize ZnO NPs.ResultsThe novelty of the current study is the investigation of the antioxidant, anti-inflammatory, anti-microbial, and cytotoxic potencies of biosynthesized ZnO NPs utilizing C. citratus ALE. Zinc acetate dihydrate was used as the precursor and the leaf extract serves as the reducing agent. ZnO NPs from ALE of C. citratus were characterized by the spherical in form by using high-resolution transmission electron microscopy (HR-TEM) and the Scherrer formula was used to calculate the size of the crystalline structure. The presence of numerous functional groups in both the ALE and the NPs is confirmed by FTIR analysis. The highest absorption peak is observed at 370 nm. The stability and particle size of the biosynthesized ZnO NPs are demonstrated by dynamic light scattering (DLS) analysis. The biosynthesized ZnO NPs exhibited excellent antioxidant activity with an IC50 value of 45.67 ± 0.1 μg/mL and exerted interesting anti-inflammatory activity (98.1% ± 0.04) when compared to the standard indomethacin (92.1% ± 0.07) at 1 mg/mL. They also showed anti-microbial activity for both bacterial and fungal which growth rates for both significantly decreased with the increase in ZnO NPs concentration compared to the control. The anticancer activity of biosynthesized ZnO NPs and C. citratus ALE was in vitro tested against seven human cancer cell lines (HCCL) (i.e. H1299, MDA-MB-468, HNO97, HEK, HCT116, HuH7, and HEPG2) compared to normal cells (HSF) using the sulforhodamine-B (SRB) assay. More interestingly, the biosynthesized ZnO NPs displayed remarkable selective cytotoxicity against all tested cancer cell lines without any effect on normal cells. In contrast, the cancer cell lines were not affected by the ALE of C. citratus at any concentrations tested.ConclusionsAll the findings confirm that the ZnO NPs biosynthesized in the current work are promising candidates for a variety of biological activities, and as a result, they can be helpful to the medical sector, environmental and agricultural applications.Graphical

Cytotoxicity Effects of Zanthoxylum simulans Hance. Fruit Bark Extract

Zanthoxylum simulans Hance. is known in folklore as a spicy herb, commonly used to treat cold-induced diseases in the body. In this study, we evaluated the cytotoxic effects of Zanthoxylum simulans fruit bark extract. Samples of Zanthoxylum simulans were extracted with 70% ethanol and subsequently fractionated with n-Hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH) solvents. To evaluate the cytotoxic effect, we performed SRB (Sulforhodamine B) assay on the three cell lines human liver HepG2, lung SK-LU-1, and cervical carcinoma Hela. The results showed that the EtOH total extract had the strongest cytotoxicity effects on liver cancer cells with an IC50 of 63.68±3.97 mg/mL, but showed no toxicity to SK-LU-1 lung cancer or Hela cervical cancer cells. The results of cytotoxic effects on HepG2 liver cancer cells of these fractions showed that the n-Hexane and EtOAc fractions exhibited activity with IC50 values of 17.12±0.94 and 28.45±2.85 µg/mL, respectively, while the n-BuOH and H2O fractions showed no activity. Our results showed that Zanthoxylum simulans fruit bark extract has a strong cytotoxicity effect on liver cancer cells.

Metabolic Effects of New Glucose Transporter (GLUT-1) and Lactate Dehydrogenase-A (LDH-A) Inhibitors against Chemoresistant Malignant Mesothelioma.

Malignant mesothelioma (MM) is a highly aggressive and resistant tumor. The prognostic role of key effectors of glycolytic metabolism in MM prompted our studies on the cytotoxicity of new inhibitors of glucose transporter type 1 (GLUT-1) and lactate dehydrogenase-A (LDH-A) in relation to ATP/NAD+ metabolism, glycolysis and mitochondrial respiration. The antiproliferative activity of GLUT-1 (PGL13, PGL14) and LDH-A (NHI-1, NHI-2) inhibitors, alone and in combination, were tested with the sulforhodamine-B assay in peritoneal (MESO-II, STO) and pleural (NCI-H2052 and NCI-H28) MM and non-cancerous (HMEC-1) cells. Effects on energy metabolism were measured by both analysis of nucleotides using RP-HPLC and evaluation of glycolysis and respiration parameters using a Seahorse Analyzer system. All compounds reduced the growth of MM cells in the µmolar range. Interestingly, in H2052 cells, PGL14 decreased ATP concentration from 37 to 23 and NAD+ from 6.5 to 2.3 nmol/mg protein. NHI-2 reduced the ATP/ADP ratio by 76%. The metabolic effects of the inhibitors were stronger in pleural MM and in combination, while in HMEC-1 ATP reduction was 10% lower compared to that of the H2052 cells, and we observed a minor influence on mitochondrial respiration. To conclude, both inhibitors showed cytotoxicity in MM cells, associated with a decrease in ATP and NAD+, and were synergistic in the cells with the highest metabolic modulation. This underlines cellular energy metabolism as a potential target for combined treatments in selected cases of MM.

Characterization, antibacterial and anticancer study of silk fibroin hydrogel

Purpose: Protein-based hydrogels such as silk fibroin hydrogel, are used in tissue engineering and regenerative medicine applications as they showed striking characteristics like biocompatibility and offered various benefits as biomaterials. The current study sought to prepare silk fibroin hydrogel and characterise it in order to assess its antibacterial and anticancer activity.
 Methodology: Silk fibroin hydrogel was prepared and characterized by using different microscopy methods, namely Scanning Electron Microscopy (SEM), Phase Contrast Electron (PCM) microscopy, and foldscope analysis. Further, it was characterized through 1H-NMR analysis, Fourier Transform Infrared spectroscopy (FTIR) analysis, and swelling properties. A Current study also covers an antimicrobial and anticancer analysis of silk fibroin hydrogel by disk diffusion method and SRB (Sulforhodamine B) assay respectively.
 Results: The antibacterial study confirmed that SF hydrogel has a moderate antibacterial activity for Streptococcus mutans, and Salmonella typhi. Additionally, the SRB assay test showed that silk fibroin hydrogels had moderate anticancer activity against the human lung cancer cell line (A549).
 Conclusion: The current study unequivocally demonstrates that silk fibroin hydrogel has antibacterial and anti-cancerous properties, making it a suitable scaffold for future studies that seek to target a specific drug delivery site.
 Keywords: Silk fibroin; Swelling behaviour; Anticancer; Drug carrier; Wound healing

Chemical Profiling, Antiproliferative and Antimigratory Capacity of Haberlea rhodopensis Extracts in an In Vitro Platform of Various Human Cancer Cell Lines.

Haberlea rhodopensis is a Balkan endemic plant that belongs to the Gesneriaceae family, and is believed to have medicinal use and health-promoting properties. This study aimed to (i) prepare aqueous (HAE) and ethanolic (HEE) extracts from the leaves of H. rhodopensis from in vitro propagated plants, (ii) screen for their potential antiproliferative and antimigratory activities, and (iii) chemically characterize both HAE and HEE by identifying compounds which may contribute to their observed bioactivity thereby further supporting their potential use in biomedical applications. The antiproliferative activity of both extracts was assessed against six human cancer cell lines by employing the sulforhodamine-B (SRB) assay. HEE was found to be more potent in inhibiting cancer cell growth as compared to HAE. Therefore, HEE's antimigratory effects were further studied in hepatocellular carcinoma (HepG2) and non-small cell lung adenocarcinoma (A459) cell lines as they were among the most sensitive ones to its antiproliferative activity. HEE was found to exert significant antimigratory concentration-dependent effects in both cell lines assessed with the wound healing assay. Chemical characterization by UPLC-MS/MS analysis identified that HEE contains higher levels of flavonoids, phenolic compounds, pigments (chlorophyll-/-b, lycopene, and β-carotene), monoterpenoids, and condensed tannins compared to HAE, while HAE, contains higher levels of soluble protein and sugars. Furthermore, HEE demonstrated remarkable antioxidant activity evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH●), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS●+) and ferric reducing/antioxidant power (FRAP) assays. We have obtained comprehensive results highlighting the potential of HEE as a source of bioactive compounds with anticancer properties. Future studies should aim at identifying the chemical constituents responsible for the bioactivities observed, and focus on investigating HEE's effects, in in vivo preclinical cancer models.

Discovery of anticancer agents with c-Met inhibitory potential by virtual and experimental screening of a chemical library

c-Met receptor tyrosine kinase has recently emerged as an important target with therapeutic implications in pancreatic cancer. In this study, we carried out a docking virtual screening on an in-house library of 441 synthesized compounds and selected the compounds with the best interactions with the c-Met protein to be subjected to experimental tests. Ten compounds belonging to 3 different classes of chemical structures were selected for this purpose and their antiproliferative effects were studied against 4 pancreatic ductal adenocarcinoma (PDAC) cell lines including AsPC-1, Suit-2, Panc-1 and Mia-Paca-2 cells, primary PDAC cells and also c-Met amplified EBC-1 cell line by sulforhodamine-B assay. Apoptosis induction was examined by Hoechst 33258 staining and annexin V-FITC/propidium iodide flow cytometric assay. The best compound was also assayed in three-dimensional cultures of AsPC-1 cells and its c-Met inhibitory potential was studied by immunoblotting and a homogenous time resolved fluorescence (HTRF) assay. The compound with a phenanthrotriazine hydrazinyl scaffold bearing nitrophenyl pendant (PhTH) was the most active derivative, with IC50 values in the range of 5–8 μM. This compound exerted antiproliferative effect against AsPC-1 cells also in the presence of hepatocyte growth factor (HGF). PhTH induced apoptosis, dose-dependently inhibited spheroid growth, inhibited c-Met activity in cell-free HTRF assay and also inhibited the phosphorylation of c-Met and its downstream effector ERK1/2 in AsPC-1 cells. Molecular docking and dynamics simulation and MM-PBSA analysis confirmed close interactions of PhTH with c-Met kinase domain. Some of the tested compounds in this study seem to be potential c-Met inhibitors with promising activities against PDAC cells.

Composite Nanoarchitectonics of Magnetic Silicon Dioxide-Modified Chitosan for Doxorubicin Delivery and In Vitro Cytotoxicity Assay

Developing drug delivery carriers for highly selective, controlled, and sustained release of the anti-cancer drugs is one of the crucial issues in the cancer strive. We herein report the synthesis of Fe3O4 (M) and SiO2 (S) nanoparticles and their nanocomposites with chitosan (CS) for high loading efficiency and subsequent release potentiality of Doxorubicin (DOX) anticancer drug. The as-synthesized nanostructures were characterized using Fourier transform infrared (FT-IR), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM), and thermal analysis techniques. The average crystallite sizes of the as-prepared M, S, CS/M, CS/S, and CS/M/S nanostructures were found to be 5, 15, 70, 22, and 29 nm, respectively. The loading and cumulative release of Doxorubicin for the produced nanostructures were examined, and the results exhibited loading efficacy of 71%, 95%, 96%, 79%, 17%, and 42% for M, S, CS, CS/M, CS/S, and CS/M/S nanostructures, respectively. The Doxorubicin releasing results revealed a promising cumulative release percentages at pH 4.2 and pH 5 compared with those at pH 7.4. At pH 4.2, the cumulative release percentages for DOX-M, DOX-S, DOX-CS, DOX/M, and DOX/CS/M/S were 94%, 96%, 92%, 95%, and 98%, respectively. While the corresponding percentages at pH 5 were 97%, 90%, 46%, 43%, and 70%. The percentage for DOX-CS/S was 60% at pH 5, though. The in-vitro cytotoxicity of M-DOX, CS-DOX, and M/CS-DOX was explored against two human cancer cell lines (MCF-7 and Hep-G2) using SRB (Sulforhodamine B) assay. The DOX-loaded M/CS exhibited the highest cytotoxicity and its IC50 values were 2.65 and 2.25 μg/mL against Hep-G2 and MCF-7 cell lines, respectively, compared to the corresponding values of 5.1 and 4.5 μg/mL for free DOX. The results indicated that M/CS nanocomposite is a good candidate as drug delivery nano-carrier for the Doxorubicin anti-cancer drug.

Phytochemical-rich Fractions from Foxtail Millet (Setaria italica (L.) P. Beauv) Seeds Exhibited Antioxidant Activity and Reduced the Viability of Breast Cancer Cells In Vitro by Inducing DNA Fragmentation and Promoting Cell Cycle Arrest.

The demand for millet-based diets has increased significantly in recent years due to their beneficial effects on human health. Foxtail Millet (Setaria italica (L.) P. Beauv, previously known as Panicum italicum L., referred as FTM in this manuscript) seeds have been not only used as astringent and diuretic agents, but they are also used to treat dyspepsia and rheumatism. Recent studies have shown that solvent extracts from FTM seeds exhibited antioxidant and antiinflammatory activities. However, the nature and antiproliferative potential of phytochemical constituents of solvent extracts are not much explored. Major objectives of this study are to generate and characterize the phytochemical-rich fractions from Foxtail millet seeds, test the antioxidant activity, and antiproliferative potential against cell lines representing carcinomas of the breast, and determine the mechanisms of cell growth inhibition. Phytochemical-rich fractions were generated by extracting the seeds using 70% ethanol (FTM-FP) and 10% alkali (FTM-BP). Antioxidant potential was determined by ferric reducing antioxidant power (FRAP) assay and DPPH radical scavenging activity assays. The antiproliferative potential was determined using sulforhodamine-B assay. The impact on cell cycle and DNA fragmentation was analyzed by staining the cells with DAPI followed by analyzing the stained cells using NC-3000. Analysis of the results showed the presence of phenolics and flavonoids in the FTM-FP and FTM-BP fractions. Both fractions exhibited antiproliferative potential against breast cancer cell lines. Mechanistically, both fractions induced G2/M cell cycle arrest and increased the fragmentation of DNA, which lead to the accumulation of cells in the Sub-G1 phase. In summary, results of this study demonstrated the potential of foxtail millet phytochemical fractions for retarding the proliferative potential of breast cancer cells.

Evaluation of Antiproliferative Activity of Lac Dye Fractions against MDA-MB-231 and SiHa Cell Lines.

Laccifer lacca (Kerr) produces a mixture of polyhydroxy anthraquinones (laccaic acid) known as lac dye. Literature suggests that these laccaic acids have a structural resemblance with the anticancer drug Adriamycin (ADR). Hence, they may possess potential anticancer activity. This study was designed to explore the in vitro anticancer activity of the three fractions of lac dye, i.e., chloroform (C), methanol (M), and water (W) fractions, and isolation of constituents from bioactive fraction. SRB (Sulforhodamine B) assay method was employed to evaluate the inhibitory action of all three fractions. However, only methanolic showed promising inhibitory action with GI50 <10; this runs in parallel with Adriamycin inhibition (GI50 <10). Two active constituents of the methanolic extract were isolated using column chromatography and were characterized using UV (UV visible spectrophotometer), IR (Infrared spectroscopy), NMR (nuclear magnetic resonance), and mass spectrometry methods. The final structure of the isolated constituents (laccaic acid D and laccaic acid B) was confirmed by 13C and 2D NMR data. Conclusively, only the methanol fraction (M) showed promising anticancer activity against in vitro MDAMB- 231 and SiHa cell lines compared to the standard adriamycin.