DAPI Nuclear Staining Research Articles

Cytotoxic and genotoxic evaluation of dipyridamole and its alternative therapeutic potential in cancer therapy: an in vitro and in vivo approach.

The molecular mechanism behind the anti-cancer potential of dipyridamole, an antiplatelet drug was investigated by examining the interaction of dipyridamole with DNA as well as the genotoxicity and cytotoxicity associated with dipyridamole . Interaction of dipyridamole with calf thymus DNA (Ct-DNA) was determined in order to ascertain the strength and mode of binding as well as the thermodynamic parameters involved. Dipyridamole interacts with Ct-DNA through a groove binding mechanism. Furthermore, a preliminary assessment of the genotoxic effect of dipyridamole was performed in vitro using the plasmid nicking assay and comet assay. Genotoxicity was also confirmed through DAPI nuclear staining of HeLa and HCT-116 cells. Cytotoxicity was assessed in vitro on HeLa, HCT-116 as well as HEK293 cells using the MTT assay as well as through inverted microscopy. Dipyridamole was selectively more toxic towards cancer cells. Dipyridamole demonstrated induction of micronucleus and chromosomal aberrations only at high concentrations in bone marrow cells of male Wistar rats, suggesting its safe usage at clinically relevant concentrations. These results indicated the existence of an intrinsic genotoxic potential of dipyridamole which may be contributing to its anti-cancer properties.

The PD‐1 Interactome (Adv. Biology 9/2021)

Programmed Death-1 In article 2100758, Boussiotis, Patsoukis, Wang, and Bardhan show that Venus-based biomolecular fluorescence complementation detects Programmed Death-1 (PD-1) dimerization by confocal microscopy. HEK-293 cells are transfected with PD-1-VenusYFP-N-terminus and PD-1-VenusYFP-C-terminus together with SHP-2 and either kinase active (top) or kinase inactive (bottom) Fyn. YFP signal (yellow) from VenusYFP structural complementation, indicating PD-1 dimerization at the plasma membrane, is generated only in the presence of kinase active Fyn (red: Rhodamine Phalloidin; blue: DAPI nuclear staining).

Exploration of Benzo[b]carbazole-6,11-diones as anticancer agents: Synthesis and studies of hTopoIIα inhibition and apoptotic effects

Two series of (hetero)arylamino-naphthoquinones and benzo-fused carbazolequinones were considered for study with the rationale that related structural motifs are present in numerous drugs, clinical trial agents, natural products and hTopoIIα inhibitors. Total 42 compounds were synthesized by reactions including dehydrogenative CN and Pd-catalyzed CC bond forming transformations. These compounds were screened against numerous cancer cells including highly metastatic one (MCF-7, MDA-MB-231, H-357 and HEK293T), and normal cells (MCF 10A). Some of the active compounds were evaluated for clonogenic cell survival and apoptotic effects in cancer cells (DAPI nuclear staining, Comet assay, Annexin-V-FITC/PI dual staining, flow cytometry, and western blot analysis with relevant proteins). All compounds were tested for hTopoIIα inhibitory activity. The investigated series compounds showed important properties like significant apoptotic antiproliferation in cancer cells with cell cycle arrest at S-phase and downregulation of NF- κβ signaling cascade, relatively less cytotoxicity to normal cells, and hTopoIIα inhibition with more efficiency compared to an anticancer drug etoposide.

Cornus Officinalis Induction of Pancreatic β-cell Autophagy as Revealed by Increased LC3 Expression and P62 Phosphorylation

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of pancreatic beta cells, diminishing the body’s ability to produce insulin. Currently, over 1.2 million people are affected by this disease in the United States with the only treatment being exogenous insulin. The ideal preventative treatment of diabetes would be to target the interventional window during the critical pre-diabetic stage to inhibit the complete loss of pancreatic β-cells. Cornus officinalis (CO) is considered a source of novel therapeutics for this purpose due to prior reports and work from our laboratory (Mol. and Cell. Endo.2019:494:110491) demonstrating that CO may protect β-cells from autoimmune mediated cytotoxicity and enhance function. To elucidate the molecular mechanism of how CO may be biologically impacting a human pancreatic β-cell line (1.1B4), we previously performed a global and phosphorylation mass spectrometry analysis revealing an increased phosphorylation of p62, which is an important regulator of autophagy. To validate the mass spectrometry results and determine if CO is truly capable of inducing pancreatic β-cell autophagy, we examined LC3 expression which serves as a typical marker for autophagic membranes. 1.1B4 cells were treated with CO for 6 and 12 hours with at concentrations of 500 and 1000 µg/ml, respectively. Cells were fixed with formaldehyde and permeabilized with Triton X-100 followed by fluorescent detection with an LC3 antibody along with DAPI nuclear staining. The cells and nuclei were then imaged using a florescent microscope and it was discovered that upon treatment of CO, an increase in cytoplasmic LC3 puncta was markedly observed as compared to untreated controls. Western analysis also demonstrated increased expression of intracellular LC3 upon CO induction. We then went on to confirm the autophagic process by immunoblotting of p62, which is a cargo receptor for autophagy. Immunoblotting demonstrated increased expression of phosphorylated p62 in a concentration and time dependent manner after treatment with CO. Altogether, CO appears to increase expression in the critical markers of autophagy and suggests that CO may have potential as a T1D interventional therapy by promoting protective autophagy in the pancreatic β-cells.

Cellular Uptake Heterogeneity of Small Extracellular Vesicles

Exosomes, also known as small extracellular vesicles (sEV), represent a subpopulation of EVs of 30-200nm in diameter that are contained into multivesicular bodies and are released by cells via exocytosis. There has been increased interest to use sEVs for therapeutic purposes due to their immune-tolerance, potential for targeted delivery and increased half-life in circulation, compare to other nanoparticles. However, due to the heterogeneity of sEVs as well as the different phenotypic features of recipient cells, the uptake mechanisms can be highly variable and selective. Studies from our lab and others show that, in vitro, not all the cells in a given cell population take up vesicles with similar kinetics and avidity. Therefore, understanding the uptake of sEVs by cells in different physiologic states (eg, quiescent vs. proliferative) as well as the intracellular fate of sEVs is key for their use as therapeutic tools. As proof of concept approaches, we used the interaction of sEVs from various endothelial cells with PC3-ML prostate cancer cells, a highly metastatic human cancer cell line. Using imaging flow cytometry and super-resolution microscopy, we determined the uptake of sEVs by a heterogeneous population of prostate cancer cells. sEVs were isolated by sequential ultracentrifugation, labeled with lipophilic dyes (DiD or DiO) and following incubation with recipient cells, nuclei were stained using DAPI or Hoechst dyes. Cells incubated with sEVs overnight showed a highly significant negative correlation between nuclear stain fluorescence and the internalization of labeled sEVs. Using two different nuclear dyes, we found concordant data (DAPI r = -.067 p < 0.0001 and Hoechst r = -.119, p < 0.0001).. This result was independently confirmed for DAPI nuclear staining using fluorescence microscopy (r = -.1352 p = 0.0004). We hypothesized that the negative correlation between nuclear staining and internalization may be cell cycle-dependent. Indeed, a binary switch was described from endocytosis being “on” in interphase to “off” in mitosis as cells traverse the G2/M checkpoint. Using clathrin-dependent endocytosis inhibitor dynasore, we found that >80% of the EVs uptake is via endocytosis. Internalization of sEVs was time-dependent and showed the same negative correlation with nuclear staining at each of the experimental time points (0,5, 1, 2, 4 and 24 hours). We also found that following incubation of cells with sEVs for up to 4hrs, co-localization with the cellular lysosomal compartment was <5%, suggesting that the majority of the internalized EVs escape lysosomal degradation following internalization. Further understanding of the uptake mechanisms of sEVs are key to set the premises for their use as therapeutic delivery systems in various diseases.

Antimicrobial activity and cytotoxicity trait of a bioactive peptide purified from Lactococcus garvieae subsp. bovis BSN307T.

A bioactive peptide of 8595Da was purified from the cell free supernatant of Lactococcus garvieaesubsp. bovis BSN307T . MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200µgml-1 inhibited the growth of both Gram-positive and Gram-negative bacteria by 58-89% and a dose of 500µgml-1 scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17µgml-1 of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay.

Norcantharidin combined with 2-deoxy-d-glucose suppresses the hepatocellular carcinoma cells proliferation and migration.

Present study aims to investigate the combined effect of anticancer drug, norcantharidin (NCTD) in combination with glycolytic inhibitor, i.e. 2-deoxy-d-glucose (2-DG) in liver cancer (HepG2 and Hepa 1-6) cells. Cell viability of NCTD and 2-DG exposed cells was determined by MTT assay, whereas, colony-forming efficiency and migration rate was determined by clonogenic assay and wound healing assay, respectively. Nuclear DAPI staining and Annexin V FITC-PI staining were used to study the apoptosis induction in cells. Fluorescence microscopy imaging was performed to detect the intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential by staining with DCFDA and JC-1 dye, respectively. Cell viability assay revealed that NCTD and 2-DG exposure in combination displays more cytotoxic effect than a single drug. Additionally, cells lose their colony formation efficiency, as well as the reduced migration rate ability was also observed upon combined exposure. Increased nuclear condensation and mitochondrial membrane depolarization are considered as key features for apoptosis induction in cancerous cells. Furthermore, oxidative stress produced in cells due to enhanced intracellular ROS generation is also major probability for cellular damage. Thus, from the initial data it can be concluded that further preclinical studies will be needed to prove the efficacy of NCTD and 2-DG inhepatocellular carcinoma therapy.

The VALVE REGEN project. Developing an innovative tissue engineered heart valve, in vitro and in vivo testing – preliminary results

Abstract Background Regenerative Medicine and Tissue Engineering are the grounds on which multidisciplinary teams aspire to obtain the perfect valvular substitute, which overcomes shortcomings of the present prostheses. Purpose Was to obtain a tissue engineered heart valve (TEHV) by repopulating with valvular resident cells – endothelial (EC) and fibroblasts (FB) a decellularized heart valve scaffold. Then their functionality and behavior was assessed in vitro and in vivo. Methods This study is part of a research grant approved by the Ethics Committee of the University. Six ovine pulmonary valves underwent a perfusion based decellularization protocol. Using a sequence of chemical and enzymatic treatment under a pressure gradient, cell removal was achieved and attested by histological investigations (DAPI nuclear staining –4',6-diamidino-2-phenylindol and haematoxylin-eosin) and DNA extraction. Ovine sub-dermal adipose tissue was harvested followed by stem cells isolation and culture. Using Endothelial Cell Growth Supplement and mechanical stimuli EC were differentiated and with Transforming Growth Factor-B1, FB were obtained. FB were internally seeded into cuspis bases using a 22 gauge needle and externally on the adventitia by using a rotator allowing a uniform distribution and seeding of cells. EC were seeded into leaflets pockets and intra-luminal also using the rotator. The repopulated valves were preconditionated in a bioreactor by gradually exposing them to the pulmonary hemodynamic regimen. By using a high speed camera, their behavior was examined when exposed to in vivo conditions. The in vivo testing was performed by surgical implantation in the gold model considered animal – the sheep. By transesophageal ultrasound (TEE US) and epicardic US, their intra-operatory function was evaluated. Post-procedure, evaluation was performed by periodic trans-thoracic (TTE US). Results Six TEHV were obtained. The decellularization histology assessment revealed acellular scaffolds and non-detectable nucleic material at the DNA extraction. Six adipose derived stem cells cultures were obtained and subsequently specialized towards EC and FB lines. The repopulation procedures underwent without incidents. During the bioreactor preconditioning, the TEHV showed complete opening and competent central coaptation. Leaflets presented physiological movement and absence of damage of valvular apparatus. The TEE US evaluation in vivo revealed normal valvular function without signs of stenosis or regurgitation. The periodic TTE US showed preserved valvular function. Conclusions Our preliminary results point out a manufactured TEHV with physiological behavior when tested in vitro and in vivo. Their interaction with a living body will be pointed out only in the explant phase, after histology analysis. The present results appear optimistic but only extended studies and follow-ups will certify their superiority in terms of performances and behavior. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This paper was financed by a grant from the Competitiveness Operational Programme 2014-2020, Tissue engineering technologies for cardiac valve regeneration, valve-regen, id P_37_673, Mysmis code: 103431, contract 50/05.09.2016.

The Antihelminthic Drug, Mebendazole, Induces Apoptosis in Adult T-Cell Leukemia/Lymphoma Cancer Cells: In-Vitro Trial.

Background: Adult T-cell leukemia/lymphoma (ATLL) is a poor prognostic Hematopoietic malignancy with various therapeutic challenges, which had been classified as non-Hodgkin lymphoma. The Drug switching, as a novel, innovative and promising approach, is an opportunity to overcoming on therapeutic challenges of hard-treating disease, e.g. ATLL. Our aim is evaluating the antiproliferative and apoptotic effect of Mebendazole (MBZ) on ATLL cancer cells in in-vitro conditions. Materials and Methods: We used Jurkat cell-line as ATLL cancer cells. After treatment of MBZ in different concentrations on jurkat cells, the cell viabilities were determined by MTT assay. After IC50 value determination, the 24-, 48- and 72-h treatments had been performed in IC50 concentration and control to evaluating the quantitative apoptosis rate by Annexin/PI Flowcytometry and qualitative apoptosis by DAPI Nuclear staining. Also, Glucose spectrophotometry were performed to evaluate the reduced amount of glucose uptake through MBZ treatment. Results: MBZ inhibits proliferation of jurkat cells and IC50 value had been estimated 10 μM (P< 0.01). According to the flowcytometric results, increasing in drug concentration is associated with decrease cell viability and the percentage of full-apoptosis. However, it inversely correlates with percentage of early-apoptosis rate. Also, the microscopic captures of DAPI Nuclear staining confirms the flowcytometry results in qualitative manner. In addition, it was found that inhibition of glucose uptake was inversely correlated with increased MBZ concentration (P< 0.05). Conclusion: MBZ potentially inhibits the proliferation of ATLL cancer cells in in-vitro condition. MBZ inhibits the growth of Jurkat cells by inducing apoptosis. Also, we suggest that indirectly inhibition of Glucose transporting occurs by MBZ, which could induce apoptosis in cancer cells.

Response to growth hormone in patients with RNPC 3 mutations

Corrigendum7 September 2020Open Access Response to growth hormone in patients with RNPC3 mutations Gabriel Á Martos-Moreno Gabriel Á Martos-Moreno Search for more papers by this author Lourdes Travieso-Suárez Lourdes Travieso-Suárez Search for more papers by this author Jesús Pozo-Román Jesús Pozo-Román Search for more papers by this author María T Muñoz-Calvo María T Muñoz-Calvo Search for more papers by this author Julie A Chowen Julie A Chowen Search for more papers by this author Mikko J Frilander Mikko J Frilander Search for more papers by this author Luis A Pérez-Jurado Luis A Pérez-Jurado Search for more papers by this author Federico G Hawkins Federico G Hawkins Search for more papers by this author Jesús Argente Jesús Argente orcid.org/0000-0001-5826-0276 Search for more papers by this author Gabriel Á Martos-Moreno Gabriel Á Martos-Moreno Search for more papers by this author Lourdes Travieso-Suárez Lourdes Travieso-Suárez Search for more papers by this author Jesús Pozo-Román Jesús Pozo-Román Search for more papers by this author María T Muñoz-Calvo María T Muñoz-Calvo Search for more papers by this author Julie A Chowen Julie A Chowen Search for more papers by this author Mikko J Frilander Mikko J Frilander Search for more papers by this author Luis A Pérez-Jurado Luis A Pérez-Jurado Search for more papers by this author Federico G Hawkins Federico G Hawkins Search for more papers by this author Jesús Argente Jesús Argente orcid.org/0000-0001-5826-0276 Search for more papers by this author Author Information Gabriel Á Martos-Moreno, Lourdes Travieso-Suárez, Jesús Pozo-Román, María T Muñoz-Calvo, Julie A Chowen, Mikko J Frilander, Luis A Pérez-Jurado, Federico G Hawkins and Jesús Argente EMBO Mol Med (2020)12:e13132https://doi.org/10.15252/emmm.202013132 This article corrects the following: Response to growth hormone in patients with RNPC3 mutations06 June 2018 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The authors recently noticed that one of the reported mutations, chr1:104093621C>A/p.P474T, was incorrectly listed as c.1320C>A at the cDNA level. The correct mutation is c.1420C>A; the nomenclature at the protein level was correct. This genetic variant is correctly listed in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/587367/), has an allele frequency of 0.00003185 in gnomAD (https://gnomad.broadinstitute.org/variant/1-104093621-C-A?dataset=gnomad_r2_1) and has been reported in a second family with growth hormone deficiency due RNPC3 dysfunction (Verberne et al, 2020). References Verberne EA, Faries S, Mannens MMAM, Postma AV, van Haelst MM (2020) Expanding the phenotype of biallelic RNPC3 variants associated with growth hormone deficiency. Am J Med Genet A https://doi.org/10.1002/ajmg.a.61632Wiley Online LibraryWeb of Science®Google Scholar Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 12,Issue 9,07 September 2020This month's cover highlights the article Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity by Claudia Koch, Andra Kuske, Klaus Pantel and colleagues. New estrogen receptor‐positive breast cancer cell line CTC‐ITB‐01 enables functional studies of circulating tumor cells. Cytoplasmatic keratin (orange) and nuclear estrogen receptor (green) protein expression in CTC‐ITB‐01 cells. DAPI nuclear staining (blue). (Scientific image by Claudia Koch et al., Institute of Tumor Biology, University Medical Center Hamburg, Eppendorf, Hamburg, Germany) Volume 12Issue 97 September 2020In this issue ReferencesRelatedDetailsLoading ...

Abstract 358: Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer

Abstract Tumor necrosis factor alpha (TNF) is a potent cytokine reported to have several functions, including stimulation of inflammation, directing cell death and cell proliferation. The cellular origin of TNF has generally believed to be the inflammatory cells especially because macrophages are prone to induction of TNF. Using in situ hybridization (ISH) based on TNF mRNA probes (RNAscope) we recently reported that TNF mRNA is also seen in cytokeratin positive cancer cells (CC) and is associated with morphologic appearance interpreted as CC branching (Møller et al, IJMS 2019, doi: 10.3390/ijms20081907). To determine the fraction of CC-derived TNF mRNA signal at different areas in colon cancers we developed a cell and color segmentation-based image analysis tool to process fluorescence signals in digital slides. The approach allowed quantification of the TNF mRNA-positive CC and TNF mRNA-positive stromal cells in the digital slides previously reported, that were also stained for cytokeratin, microRNA-21 (miR-21) and DAPI nuclear stain. In the two cases with most abundant TNF mRNA ISH signal, both showing moderate-to-high budding index, the fraction of TNF-mRNA positive CC in the outer periphery (approximately 500 µm) was 8% and 20%, respectively. The frequency of the TNF-positive CC increased towards the invasive front from 7% (33/500 CC per 0.25 mm2) to 35% (48/137 CC), and from 8% (127/1536 CC per 0.50 mm2) to 28% (108/389 CC), respectively. The upregulation is in agreement with initial qualitative evaluation. In the same areas, the fraction of TNF mRNA positive stromal cells among all TNF mRNA-positive cells was only 8% in both cases, indicating that the majority of the TNF mRNA originates from CC. The fraction of the TNF mRNA-positive CC that were also miR-21 positive was 85% and 43%, respectively, which was higher than the prevalence of the overall miR-21 positive CC, 55% and 30%, suggesting frequent co-expression with miR-21. In conclusion, we have developed a method to obtain quantitative estimates from digital multiplex-stained slides with RNAscope and LNA fluorescence ISH and immunofluorescence. More samples will need to be processed to better understand the importance of TNF expression in cancer cell invasion. Citation Format: Boye Schnack Nielsen, Kim Holmstrom. Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 358.

Synthesis of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones as potential cytotoxic agents

The new derivatives based on (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-one scaffold was synthesized and evaluated for their in vitro cytotoxic potential against a panel of cancer cell lines, viz., A549 (human lung cancer), HCT-116 (human colorectal cancer), B16F10 (murine melanoma cancer), BT-474 (human breast cancer), and MDA-MB-231 (human triple-negative breast cancer). Among them, many of the synthesized compounds exhibited promising cytotoxic potential against the panel of tested cancer cell lines with IC50 <30 µM. Based on the preliminary screening results, the structure-activity relationship (SAR) of the compounds was established. Among the synthesized compounds, 15i displayed a potential anti-proliferative activity against HCT-116 cancer cell line with an IC50 value of 1.21 ± 0.14 µM. Flow cytometric analysis revealed that compound 15i arrested the G0/G1 phase of the cell cycle. Moreover, increased reactive oxygen species (ROS) generation, clonogenic assay, acridine orange staining, DAPI nuclear staining, measurement of mitochondrial membrane potential (ΔΨm), and annexin V-FITC assays revealed that compound 15i promoted cell death through apoptosis.

4530 Elucidating the Influence of Chemotherapy (melphalan) and /or C. difficile toxin B Exposure on Beta-catenin Protein Expression in Caco-2 Monolayers

OBJECTIVES/GOALS: We previously reported that genetic polymorphisms in the beta-catenin gene (CTNNB) are associated with the development of Clostridiodes difficile colitis during autologous stem cell transplantation (https://www-ncbi-nlm-nih-gov.proxy.libraries.uc.edu/pubmed/29594489). To biological validate these findings, we sought to evaluate the development of chemotherapy-associated Clostridiodes difficile infections by assessing the effect of C.difficile toxin B (TcdB) and of using melphalan in beta-catenin protein expression in Caco2 cells. METHODS/STUDY POPULATION: To determine the effect of melphalan and/or C.difficile toxin B on expression of Beta-catenin from human gut epithelial cells: Adenocarcinoma cells (Caco-2) cells were seeded and allowed to grow into monolayersMonolayers were treated with PBS, TcdB, melphalan and/or TcdB + melphalan for 24 hours and then washed with PBSImmunofluorescence was measured on the monolayers to visualize three markers -DAPI-Nuclear Stain (blue),Actin-ccytoskeletal stain (red), B-Catenin (green)Analysis of images with ImageJ (NIH). Statistical analysis of the effect of TcdB and/or melphalan on β-catenin protein levels was determined by One-way ANOVA Cells stained with a primary anti-β catenin antibody and an Alexa-488 secondary antibody were evaluated by flow cytometry to quantify the effect of melphalan and/or C. difficile toxin B on Caco2 cells. RESULTS/ANTICIPATED RESULTS: Immunofluorescent intensity was higher in the control (PSS exposed) cells when compared to melphalan, TcdB and mephalan+TcdB exposed cells (p = 0.026, 0.004 and 0.049 respectively) DISCUSSION/SIGNIFICANCE OF IMPACT: A significant difference was seen in β catenin expression in Caco-2 monolayers exposed to TcdB and/or melphalan. These data support the a role of β-catenin in the pathophysiology of CDI during chemotherapy and support GWAS findings reporting a difference in CDI susceptibility based on β-catenin genotype.

Mechanistic Study of Triazole Based Aminodiol Derivatives in Leukemic Cells-Crosstalk between Mitochondrial Stress-Involved Apoptosis and Autophagy.

Various derivatives that mimic ceramide structures by introducing a triazole to connect the aminodiol moiety and long alkyl chain have been synthesized and screened for their anti-leukemia activity. SPS8 stood out among the derivatives, showing cytotoxic selectivity between leukemic cell lines and human peripheral blood mononuclear cells (about ten times). DAPI nuclear staining and H&E staining revealed DNA fragmentation under the action of SPS8. SPS8 induced an increase in intracellular Ca2+ levels and mitochondrial stress in HL-60 cells identified by the loss of mitochondrial membrane potential, transmission electron microscopy (TEM) examination, and altered expressions of Bcl-2 family proteins. SPS8 also induced autophagy through the detection of Atg5, beclin-1, and LC3 II protein expression, as well as TEM examination. Chloroquine, an autophagy inhibitor, promoted SPS8-induced apoptosis, suggesting the cytoprotective role of autophagy in hindering SPS8 from apoptosis. Furthermore, SPS8 was shown to alter the expressions of a variety of genes using a microarray analysis and volcano plot filtering. A further cellular signaling pathways analysis suggested that SPS8 induced several cellular processes in HL-60, including the sterol biosynthesis process and cholesterol biosynthesis process, and inhibited some cellular pathways, in which STAT3 was the most critical nuclear factor. Further identification revealed that SPS8 inhibited the phosphorylation of STAT3, representing the loss of cytoprotective activity. In conclusion, the data suggest that SPS8 induces both apoptosis and autophagy in leukemic cells, in which autophagy plays a cytoprotective role in impeding apoptosis. Moreover, the inhibition of STAT3 phosphorylation may support SPS8-induced anti-leukemic activity.

Effect of Different Concentration of Cellulose Nanocrystals Comprising Hydroxyethyl Cellulose / Poly(Vinyl Alcohol) as a Bone Tissue Engineering Scaffold

In this study, biodegradable scaffolds based on hydroxyethyl cellulose (HEC) (5 wt%) and poly (vinyl alcohol) (PVA) (15 wt%) with different percentages of celullose nanocrystal (CNC) (1 and 7 wt%) were fabricated by lyophilization method to get highly porous scaffolds. These scaffolds were made water insoluble by cross-linking via heat treatment. The morphology and thermal properties of HEC/PVA/CNCs scaffolds were characterized by using Scanning Electron Microscope (SEM) and Thermogravimetric Analysis (TGA). The morphological study showed that both prepared scaffold have highly porous structures with good pore interconnected structure. It was observed that thermal properties of scaffolds increased significantly as the concentration of CNCs increased. Cytotoxicity studies on scaffolds were carried out by utilizing human fetal osteoblast (hFOB) cells using DAPI nuclear stain and then confirmed using SEM. hFOB cells were able to attach and spread on all scaffolds. Incorporated CNCs as reinforcing nanofiller on scaffolds promising a superior functionality in bone tissue engineering.

Synthesis of Substituted Cinnamido Linked Quinazolinone Congeners as Potential Anticancer Agents via Mitochondrial Dependent Intrinsic Apoptotic Pathway.

The synthesis of novel heterocyclic scaffolds with amide functionality is a key research area due to their plethora of medicinal applications. The present study aims to explore the synthesis of new cinnamido linked quinazolinone congeners and their potential as anticancer agents. Cytotoxicity evaluation, Cell cycle analysis, JC-1 staining, ROS, Annexin V assays, AO/EB, DAPI nuclear staining, Colony-forming assay and Western blot analysis. Among the synthesized compounds, 5eb and 5fc have shown promising cytotoxic activity with an IC50 value of 3.89±1.01µM and 4.05±0.62µM against HeLa cell lines. The flow-cytometry analysis demonstrated that the compound 5eb arrested the sub-G1 phase of the cell cycle and induced apoptosis. Furthermore, the compound 5eb triggered the collapse of mitochondrial membrane potential (ΔΨm), which was assessed by JC-1 staining and also induced the generation of Reactive Oxygen Species. An increase in the expression of proapoptotic proteins such as Bax, p53, cleaved PARP and cleaved caspase-3 by 5eb confirmed the activation of the mitochondrial-dependent intrinsic apoptosis pathway. Our results suggest that compound 5eb and 5fc of cinnamido linked quinazolinone derivatives could serve as potential leads in the development of novel chemotherapeutic agents.

Evaluation of Cytotoxicity of Different Part Extracts of Ipomoea turpethum against Breast Cancer Cell Lines.

The aim of the present study was to explore the anti-breast cancer activity of the plant I. turpethum together with the comparative analysis of the cytotoxicity of the extracts from different parts of the plant. The gas chromatography-mass spectrometry (GC-MS) analysis of the plant revealed the identification of 71 compounds. The cytotoxicity of the extracts from different parts (whole, aerial, and root) of the plant was evaluated against the breast cancer cell lines (MCF-7 and MDA MB-231) by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The findings of these assays were further confirmed by the carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation assay, DAPI (4',6-diamidino-2-phenylindole) nuclear staining, and simple microscopy images. The results of the MTT assay revealed that the root extract showed the lowest IC50 values, i.e., 516 μg/mL for MCF-7 and 396 μg/mL for MDA MB-231 cell lines. The LDH release in the medium was high in the root extract treated cells, i.e., 28.87% for MCF-7 and 23.62% for MDA MB-231 cell lines. Moreover, the CFSE proliferation assay also showed the decrease in the proliferation of the cells up to 48.7% and 46.4% in case of MCF-7 and MDA MB-231 cells, respectively. The fragmented and condensed nuclear material in both the cell lines was observed in the DAPI nuclear staining. In conclusion, results showed that the root extract possess the highest cytotoxicity among all other tested extracts toward both breast cancer cell lines (MCF-7 and MDA MB-231).

SB365 induces apoptosis and suppresses proliferation of glioblastoma cells.

CONTEXT:Glioblastoma is a malignant brain tumor with limited treatment modalities due to its nature. SB365, Pulsatilla saponin D, is known to induce apoptosis and inhibit the growth of many cancer cells.AIM:We elucidated the anticancer effects of SB365 in glioblastoma cells.METHODS:We examined the antiproliferative activity of SB365 in human glioblastoma cell lines. Apoptosis was evaluated using the Hoechst assay, TUNEL assay, DAPI nuclear staining, and Western blotting analysis. To test the antimetastatic capacity of SB365, cell migration assay was conducted, and hypoxia-inducible factor-1 alpha (HIF-1α) expression and vascular endothelial growth factor (VEGF) level were determined under hypoxic conditions.STATICAL ANALYSIS:Significance of the results was confirmed by a one-way analysis of variance analysis.RESULTS:SB365 treatment suppressed the growth of glioblastoma cells and resulted in apoptotic morphological features such as nuclear condensation and fragmentation, enhancing the expression of cleaved poly (ADP-ribose) polymerase and caspase-3. It also significantly delayed cell migration and decreased the HIF-1α expression and VEGF secretion.CONCLUSION:Our findings thus demonstrate that SB365 induced apoptosis and delayed the growth and migration of human glioblastoma cells. It is considered that SB365 would be a promising therapeutic option for glioblastoma.

Glutathione capped core/shell CdSeS/ZnS quantum dots as a medical imaging tool for cancer cells

Nanomaterials have secured an enduring position as a promising contender for various biological applications. Quantum dot (QD) is one such example of nano sized semiconductor material having unique optical characteristics that makes it an ideal candidate to be used as imaging tool for today’s medical industry. In the present study, the L-Glutathione (L-GSH) capped L-GSH-CdSeS/ZnS core/shell QDs were applied to visualize MCF-7 human breast cancer cells. Time and dose dependent studies were done, to study the cell penetration and visualization of QDs within the cancer cells. MTT assay was done to derive the percent cell viability of cancer cells at different QDs dose concentrations. Cell cytotoxicity of the capped CdSeS/ZnS QDs were also studied on non-cancerous healthy cells. Human cancerous cells were treated with the selected doses of QDs for different time durations followed by counter staining of the cells with fluorescent DAPI nuclear stain to observe the reflected QD mediated effects. The small sized QDs were localized predominantly in the cytoplasm of MCF-7 cells and displayed no significant cytotoxicity on healthy cells. Our findings suggest that our new synthesized L-GSH-CdSeS/ZnS core/shell QDs could be highly biocompatible and can have a high probability of applications in bio-labeling and imaging for cancer diagnostics.

Artemisia absinthium nanoparticles induce apoptosis in breast cancer cells via inhibiting vesicular trafficking related proteins

Abstract Background The limitations of the currently available anti-breast cancer agents like they exert side effects can be overcome by combining two approaches together i.e. loading of herbal drugs into the nanoparticles. Methods Keeping in mind the tumor micro environment, double triggered (temperature and pH responsive) polymeric nanoparticles (NPs) were prepared and characterized by TEM, DLS and IR. The cytotoxicity of the different part extracts of A. absinthium was evaluated on the breast cancer cell lines (MCF-7 & MDA MB-231) by MTT and LDH assay. The extract with least IC50 value (whole plant extract) was loaded into the NPs and various cytotoxic parameters like MTT assay, CFSE cell proliferation assay, apoptosis assay, cell cycle study and DAPI nuclear staining was performed to evaluate cytotoxicity against breast cancer cell lines by. To further delineate the mechanism of action of extract loaded NPs, nano-LC-MS/MS analysis was performed. Finally, the probable mechanism behind the caused cytotoxicity was explored by the interactome analysis of highly differentially expressed proteins via STRING online tool. Results The cytotoxic studies together with proteome analysis suggest that extract loaded NPs effectively inhibits cell proliferation and induce apoptosis and cell cycle arrest in G0/G1 phase in both the breast cancer cell lines via significantly modulating the expression of the key proteins related to the proliferation, vesicular trafficking, apoptosis, and tumor suppression. Moreover, interactome analysis uncovered the key apoptotic proteins responsible for the caused cytotoxicity. Conclusions In conclusion, encapsulation enhances the cytotoxicity of the whole extract of A. absinthium and causes apoptosis in breast cancer cells. Furthermore, nano LCMS/MS and STRING analysis uncover the probable mechanism behind the caused cytotoxicity. Legal entity responsible for the study Saima Wajid. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.