Flow Cytometric Cell Cycle Analysis Research Articles

The Characteristic Function of Blood-Derived Exosomes and Exosomal circRNAs Isolated from Dairy Cattle during the Dry Period and Mid-Lactation.

Exosomes are key mediators of intercellular communication. They are secreted by most cells and contain a cargo of protein-coding genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), which modulate recipient cell behavior. Herein, we collected blood samples from Holstein cows at days 30 (mid-lactation) and 250 (dry period) of pregnancy. Prolactin, follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone levels showed an obvious increase during D250. We then extracted exosomes from bovine blood samples and found that their sizes generally ranged from 100 to 200 nm. Further, Western blotting validated that they contained CD9, CD63, and TSG101, but not calnexin. Blood-derived exosomes significantly promoted the proliferation of mammary epithelial cells, particularly from D250. This change was accompanied by increased expression levels of proliferation marker proteins PCNA, cyclin D, and cyclin E, as detected by EdU assay, cell counting kit-8 assay, and flow cytometric cell cycle analysis. Moreover, we treated mammary epithelial cells with blood-derived exosomes that were isolated from the D30 and D250 periods. And RNA-seq of two groups of cells led to the identification of 839 differentially expressed genes that were significantly enriched in KEGG signaling pathways associated with apoptosis, cell cycle and proliferation. In bovine blood-derived exosomes, we found 12,747 protein-coding genes, 31,181 lncRNAs, 9374 transcripts of uncertain coding potential (TUCP) candidates, and 460 circRNAs, and 32 protein-coding genes, 806 lncRNAs, 515 TUCP candidates, and 45 circRNAs that were differentially expressed between the D30 and D250 groups. We selected six highly expressed and four differentially expressed circRNAs to verify their head-to-tail splicing using PCR and Sanger sequencing. To summarize, our findings improve our understanding of the key roles of blood-derived exosomes and the characterization of exosomal circRNAs in mammary gland development.

STUDY EFFECT OF TAMOXIFEN ON CELL CYCLE PHASE DISTRIBUTION, IN BREAST CANCER CELLS

Hormone therapy also known as estrogen suppression therapy is the first targeted therapy and is frequently used as a secondi therapeutic to lower the risk of cancer returning after surgery or spread. Tamoxifen is a drug that is commonly used to treat breast cancer by mediating estrogen receptor signaling pathways. Breast cancer is becoming a spread disease that needs a specific therapy. The main focusi of this study is the anti-cancer drug Tamoxifen (TAM)i and it’s effected on celli cycle (Culture) of breast cancer cell line measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. This study include the determination the effects of tamoxifen on MCF-7i on breast cancer celli line and investigate of the mechanism by which the affected of it on the cell cycle phases using Flowcytometric cell cycle analysis assay. As well study the Flowy cytometric cell cycle analysis of tamoxifen-treated cultures showed an increase in the proportion of cells in the GI phase of the cell cycle.

MicroRNA-145-5p inhibits prostate cancer bone metastatic by modulating the epithelial-mesenchymal transition.

ObjectiveTo investigate the effects of miRNA-145-5p on the tumor development and progression of prostate cancer (Pca) bone metastasis.MethodsLevels of miRNA-145-5p were assessed by real-time quantitative PCR in PC3 (bone metastatic Pca cells), 22RV1 (non-metastatic Pca cells), RWPE-1 (non-cancerous prostate epithelial cells) and Pca tissues collected from patients with and without bone metastases. The impact of miRNA-145-5p on cell proliferation was tested by CCK8 assay, colony formation assay and flow cytometric cell cycle analysis. Effects on invasion and migration of PC3 cells were determined by Transwell and wound healing assays. Western blotting, enzyme-linked immunosorbent assay, and flow cytometry apoptosis analyses were also performed to assess roles in metastasis.ResultsLevels of miRNA-145-5p were decreased in Pca bone metastases and miRNA-145-5p inhibited cell proliferation, migration and invasion. miRNA-145-5p inhibited the expression of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) and transforming growth factor-β (TGF-β) in PC3 cells. miR-145-5p increased the expression of the epithelial marker E-cadherin and reduced the expression of matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9). It was found that miRNA-145-5p mediated the epithelial-mesenchymal transition (EMT) and induced apoptosis.ConclusionsmiRNA-145-5p negatively regulated the EMT, inhibited Pca bone metastasis and promoted apoptosis in Pca bone metastasis. Mimicry of miRNA-145-5p action raises the possibility of a novel target for treating Pca with bone metastases.

Pleiotropic Effects of Icariside II on the Cardiovascular System: Novel Applications of Ethnopharmacology in Targeting Vascular Remodeling.

Pleiotropic Effects of Icariside II on the Cardiovascular System: Novel Applications of Ethnopharmacology in Targeting Vascular Remodeling.

Development and Optimization of Osimertinib-loaded Biodegradable Polymeric Nanoparticles Enhance In-vitro Cytotoxicity in Mutant EGFR NSCLC Cell Models and In-vivo Tumor Reduction in H1975 Xenograft Mice Models.

Osimertinib (OMB), a third-generation EGFR inhibitor, specifically and irreversibly inhibits EGFRT790M mutant form. Nevertheless, its clinical use is limited due to poor solubility, low absorption, and oral bioavailability. To overcome the low therapeutic capabilities of the free drug, we developed OMB-loaded PCL or CHS nanoparticles and characterized them. Among fifteen devised nanoparticle formulations (Npfs), OMB-PCL-f3, f9, and OMB-CHS-f3 showed great characteristics such as particle size (ranges from 101.3 ± 8.2 to 119.7 ± 10.4 nm), zeta potential (-36.4 ± 3.2 to -31.7 ± 3.9 mV), and polydispersity index (0.227 ± 0.037 to 0.261 ± 0.025). The % entrapment (91.25 ± 5.84 to 95.25 ± 5.88) and drug loading (29.64 ± 2.38 to 33.59 ± 2.36) indicated the formulation optimization. OMB-CHS-f3 demonstrated long-term in-vitro release, with a % cumulative OMB release of 99.99 ± 2.67 within 24 h, and the cytotoxicity of OMB-CHS-f3 showed 2.6- and 2.4-fold superior activity in mutant EGFR harboring H1975 and PC-9 cells, respectively, compared to plain OMB. Quantitative assessment of OMB cellular uptake from OMB-CHS-f3 showed superior drug accumulation of 81.59 ± 5.8% and 77.31 ± 4.6% in H1975 and PC-9 cells which was more than OMB-CHS-f9 and plain OMB. Flow cytometric cell cycle analysis revealed that OMB-CHS-f3 triggered G2/M phase arrest greater than OMB-PCL-f9 and plain OMB. In vivo, OMB-CHS-f3 Npf treatment reduced tumor size and body weight gain compared to Tagrisso treatment (p < 0.05). These findings showed that chitosan-coated OMB Npfs might improve outcomes by overcoming complications, including resistance and disease recurrence in NSCLC patients.

2, 4, 5-Trideoxyhexopyranosides Derivatives of 4'-Demethylepipodophyllotoxin: De novo Synthesis and Anticancer Activity.

Podophyllotoxin is a natural lignan which possesses anticancer and antiviral activities. Etoposide and teniposide are semisynthetic glycoside derivatives of podophyllotoxin and are increasingly used in cancer medicine. The present work aimed to design and synthesize a series of 2, 4, 5-trideoxyhexopyranosides derivatives of 4'-demethylepipodophyllotoxin as novel anticancer agents. A divergent de novo synthesis of 2, 4, 5-trideoxyhexopyranosides derivatives of 4'- demethylepipodophyllotoxin has been established via palladium-catalyzed glycosylation. The abilities of synthesized glycosides to inhibit the growth of A549, HepG2, SH-SY5Y, KB/VCR and HeLa cancer cells were investigated by MTT assay. Flow cytometric analysis of cell cycle with propidium iodide DNA staining was employed to observe the effect of compound 5b on cancer cell cycle. Twelve D and L monosaccharide derivatives 5a-5l have been efficiently synthesized in three steps from various pyranone building blocks employing de novo glycosylation strategy. Dmonosaccharide 5b showed the highest cytotoxicity on five cancer cell lines with the IC50 values ranging from 0.9 to 6.7 μM. It caused HepG2 cycle arrest at G2/M phase in a concentrationdependent manner. The present work leads to the development of novel 2, 4, 5-trideoxyhexopyranosides derivatives of 4'-demethylepipodophyllotoxin. The biological results suggest that the replacement of the glucosyl moiety of etoposide with 2, 4, 5-trideoxyhexopyranosyl is favorable to their cytotoxicity. D-monosaccharide 5b was observed to cause HepG2 cycle arrest at the G2/M phase in a concentration- dependent manner.

A flow cytometric journey into cell cycle analysis.

Cell cycle involves a series of changes that lead to cell growth and division. Cell cycle analysis is crucial to understand cellular responses to changing environmental conditions. Since its inception, flow cytometry has been particularly useful for cell cycle analysis at single cell level due to its speed and precision. Previously, flow cytometric cell cycle analysis relied solely on the measurement of cellular DNA content. Later, methods were developed for multiparametric analysis. This review explains the journey of flow cytometry to understand different molecular and cellular events underlying cell cycle using various protocols. Recent advances in the field that overcome the shortcomings of traditional flow cytometry and expand its scope for cell cycle studies are also discussed.

Pyrrolizidine alkaloids cause cell cycle and DNA damage repair defects as analyzed by transcriptomics in cytochrome P450 3A4-overexpressing HepG2 clone 9 cells

Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.Graphical abstract

Cisplatin Cytotoxicity in Human Testicular Germ Cell Tumor Cell Lines Is Enhanced by the CDK4/6 Inhibitor Palbociclib.

Cisplatin-based chemotherapy is the mainstay of pharmacological treatment of testicular germ cell tumors (TGCTs) that, together with early diagnosis, surgery, and/or radiotherapy, has dramatically improved the prognosis. However, under the pressure of such pharmacological therapy (both classical cytotoxic drugs and targeted therapy), cancer cells may develop resistance. Thus, combination therapy that may include cytotoxic drugs and targeted therapy could offer an advantage to curing cancers. Here, we investigated the in vitro and in vivo antitumor activity of cisplatin, as a single-agent or in combination with palbociclib. The cell viability of Ntera-2/cl.D1 (NT2/D1) and 833K after exposure to palbociclib and/or cisplatin was evaluated by MTT dye reduction assay and by ATPLite Luminescence Assay. Gene and protein expression was evaluated by quantitative reverse transcription polymerase chain reaction and by western blot. Flow cytometric cell-cycle analysis was performed, as well. The in vivo experiments were conducted on NT2/D1 xenografts in AB zebrafish embryos exposed to the drugs. Palbociclib and cisplatin decreased TGCT cell viability both in vitro and in vivo. This effect was additive when cells were exposed to the drug combination. In the NT2/D1 cell lines, the drug combination also exerted a positive effect with regard to delaying cell recovery after the toxic insult. In the combination experiments, cisplatin-induced cell accumulation in G2/M was predominant compared with the palbociclib effect. These results could provide the rationale for developing further studies to improve the pharmacological treatment of TGCTs, but they must be demonstrated in a dedicated clinical trial.

A novel ligand of the translationally controlled tumor protein (TCTP) identified by virtual drug screening for cancer differentiation therapy

SummaryIntroduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology.

Controlled synthesis of (CuO-Cu2O)Cu/ZnO multi oxide nanocomposites by facile combustion route: A potential photocatalytic, antimicrobial and anticancer activity

Photocatalytic activity of (CuO-Cu2O)Cu/ZnO hetero-junction nanocomposites along with their luminescent, biological applications in the progress of anticancer and antibacterial agents is investigated. The Cu and Zn bi-components modified (CuO-Cu2O)Cu/ZnO nanocomposites were synthesized via facile combustion route in the presence of controlled fuel to oxidizer ratio and were characterized by X-Ray Diffraction (XRD) patterns, Transmission electron microscopy (TEM), High resolution Transmission electron microscopy (HRTEM), Scanning Electron Microscopy (SEM), X-ray photoelectron Spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), photoluminescence (PL) and energy dispersive X-ray (EDX) analysis. The PL and UV–Visible diffused reflectance spectral (UV–Vis-DRS) techniques were used to measure the optical sensitivity and tuning of band gap in the samples. The excellent photocatalytic degradation of Methylene Blue and industrial waste water under Sunlight irradiation depends on the mass ratios of Cu/Zn. The findings show that the addition of a certain proportion of CuO, Cu2O, ZnO, and Cu can promote efficiency in Sunlight harvesting and separation of charge carriers. Process parameters namely catalyst quantity, dye concentration and a proposal for the mechanism of degradation pathway, experiments for trapping and enhancer are investigated. The study of photoluminescence, CIE and CCT calculations suggests that the present nanocomposite may find applications as phosphor material in warm white LEDs. The second segment of this study deals with the investigation of antibacterial performance of composites upon Gram-negative and Gram-positive bacteria. The results indicate that nanocomposites can be used in antibacterial control systems and as an important growth inhibitor in various microorganisms. The cytotoxic effect of the (CuO-Cu2O)Cu/ZnO (CCCZ11) nanocomposite was determined by colorimetric and flow cytometric cell cycle analysis. Our experimental results show that the nanocomposite can induce apoptosis and suppress the proliferation of HeLa cells. The applications of nanocomposites based on Cu, an abundant and inexpensive metal has created much interest in various multifunctional applications.

In vitro screening of plant extracts traditionally used as cancer remedies in Ghana – 15-Hydroxyangustilobine A as the active principle in Alstonia boonei leaves

Ethnopharmacological relevanceCancer represents a major health burden and drain on the global healthcare systems. Traditional African medicine widely use a variety of plant species for treatment of different kinds of cancer. A previous systematic survey by traditional healers in the Ashanti region of Ghana revealed a good overview on the plant species and herbal materials used for the different types of cancer. Aims of the studyThe following study aimed to investigate 18 herbal materials from 10 plant species based on the cancer survey in Ghana regarding potential cytotoxicity against different cancer cell lines under in vitro conditions followed by subsequent bioassay-guided fractionation towards the active principle. Materials and methodsEthanol-water (1:1) extracts were tested (1–100 μg/mL) against a panel of cancer cell lines according to their respective traditional use. Selected extracts with relevant cytotoxicity in this screening were also tested against common pediatric malignancies (leukemias (HL-60, REH) and Ewing sarcoma (RD-ES and CADO-ES1)). Bioassay-guided fractionation of the hydroalcoholic extract from Alstonia boonei was performed by liquid-liquid chromatography and preparative HPLC. Preliminary mechanistic studies on the mode of action were performed by flow cytometric cell cycle analysis as well as apoptosis and necrosis staining. ResultsScreening of plant extracts revealed relevant cytotoxicity against all tested cancer cell lines for Alstonia boonei leaves and stem of Paulinia pinnata. The A. boonei extract was additionally found to be active against common pediatric tumor types (leukemias and Ewing sarcoma). Bioassay-guided fractionation of the A. boonei extract revealed the presence of 15-hydroxyangustilobine A 1 as the active principle (IC50 26 μM against MCF-7 cells). This is the first report of this compound in A. boonei. 1 was shown to lead to cell cycle arrest in the G2/M-phase (MCF-7 cells), triggering cells at least partially into apoptosis. ConclusionIn summary, an appreciable in vitro activity was revealed for the leaf extract from A. boonei and the isolated vallesamine type indole alkaloid 1, which has to be investigated in future studies towards a potential clinical use.

Abstract 1727: Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin

Abstract Background: Temozolomide (TMZ) is a DNA alkylating prodrug used for the treatment of malignant, glioblastoma multiforme (GBM) tumors. Yet, TMZ therapy is blighted by resistance systems in GBM that thwart eradication of all cancer cells remaining after surgery and radiotherapy. Resistance mechanisms include drug efflux by P-glycoprotein 1 (Pgp), present on the blood brain barrier (BBB) and on GBM cells, overexpression of O6-methylguanine DNA-methyltransferase (MGMT), which removes cytotoxic O6-methylated guanine (O6-MeG) lesions caused by TMZ, and deficiency in mismatch repair (MMR), which leads to tolerance of O6-MeG lesions. Nanoscale delivery systems are proposed as a strategy to enhance drug targeting and accumulation under systemically tolerable conditions. Apoferritin (AFt), a hollow protein capsule, has been used to deliver anticancer agents. Transferrin receptor 1 (TfR1) binding sites on the AFt cage has reportedly been shown to allow AFt to cross the BBB and accumulate in GBM cells, as both BBB endothelial and GBM (but not glial) cells express TfR1. Thus, we use AFt for targeted delivery, specific uptake and controlled release of TMZ in GBM. Methods: TMZ was encapsulated into AFt via molecular diffusion through the channels in the AFt cage. Encapsulation efficiency and stability for this formulation were assessed by dynamic light scattering (DLS) and UV-vis spectroscopy. In vitro assays were conducted (MTT, live cell count, clonogenic, flow cytometry and ELISA) against isogenic GBM cell lines, U373V (vector control) and U373M (MGMT overexpressing), as well as MMR deficient and Pgp overexpressing HCT116 (colorectal carcinoma) and healthy MRC-5 (lung fibroblasts) cells. Environmental scanning electron microscopy (ESEM) and confocal microscopy were further employed to assess morphological changes to the cells after treatment. Results: Encapsulation of &amp;gt; 500 molecules of TMZ per AFt cage was achieved, with encapsulation efficiency &amp;gt; 70%. MTT assays demonstrated significantly increased activity of AFt-TMZ compared to TMZ alone in all cancer cell lines, with 50% growth inhibition (GI50) in resistant cancer cell lines at &amp;lt; 15 μM, compared to &amp;gt; 200 μM for TMZ alone. Clonogenic and flow cytometric cell cycle and DNA damage analyses further corroborate these findings. Additionally, ELISA quantification demonstrated significantly (P &amp;lt; 0.001) elevated O6-MeG levels following treatment of U373V and U373M cells with AFt-TMZ compared to naked TMZ, correlating with enhanced γ-H2AX burden in cells following exposure to AFt-TMZ. Against MRC-5, lower activity was observed, with GI50 for AFt-TMZ &amp;gt; 70 μM. Furthermore, cell shrinkage and blebbing were observed in GBM cells following 24 h treatment with AFt-TMZ. Conclusions: TMZ delivered by AFt demonstrated enhanced potency. Overcoming resistance by enhancing TMZ intracellular accumulation, may prove to be a turning point in enhancing drug efficacy in GBM. Citation Format: Kaouthar Bouzinab, Neil R. Thomas, Lyudmila Turyanska, Pavel Gershkovich, Nicola Weston, Marianne B. Ashford, Tracey D. Bradshaw. Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin [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 1727.

Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors in vitro: Antitumoral Effects.

Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target.

Piperlongumine induces apoptosis and G2/M phase arrest in human osteosarcoma cells by regulating ROS/PI3K/Akt pathway

Previous research has reported that piperlongumine exerts antitumor properties on several types of tumor cells. However, its effect on osteosarcoma cells remains unknown. This study aimed to investigate the antitumor effects of piperlongumine on osteosarcoma cells (MG63 and U2OS cells) in vitro and determined the underlying mechanism. Cell viability was measured using MTT assay. Cell apoptosis was assessed via AO/EB staining and flow cytometry apoptosis as well as western blot analysis. Cell cycle distribution was detected by flow cytometric cell cycle and western blot analysis. In our research, we found that piperlongumine induced apoptosis and G2/M phase arrest of MG63 cells. Western blot analysis not only confirmed the above results, but also demonstrated that piperlongumine induced apoptosis of osteosarcoma cells by activating Caspase-9-dependent apoptotic pathway. Furthermore, we also found that piperlongumine significantly induced apoptosis and cell cycle arrest of osteosarcoma cells by regulating ROS/PI3K/Akt signaling pathway. In summary, our findings suggested that piperlongumine inhibited osteosarcoma progression by promoting apoptosis of osteosarcoma cells. In addition, the underlying mechanism demonstrated that piperlongumine produced potent antitumor properties in osteosarcoma cells by regulating ROS/PI3K/Akt signaling pathway

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms

Ethnopharmacological relevanceRhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer.Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. Material and methodCytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. ResultsCytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ± 0.005 μg/ml and 0.18 ± 0.007 μg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ± 0.084 μg/ml and 0.32 ± 0.077 μg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. ConclusionOur study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs.

Using cell cycle measurements with flow cytometry to predict the growth rate of Walleye Pollock Gadus chalcogrammus larvae

Growth rate during early life stages can be an important factor in the recruitment process of marine fishes, and in this study we develop a methodology based on cell cycle measurements to predict the growth rate of Walleye Pollock (Gadus chalcogrammus) larvae. Results from cell-cycle analysis of muscle cell nuclei of laboratory-reared Walleye Pollock larvae measured with flow cytometry were used as covariates in a generalized additive model to predict the growth rate (growth in length per day, mm d−1) of individual larvae from the day exogenous feeding began (first feeding) to the time of capture (r2 = 0.79). Additional covariates used were temperature and standard length. A generalized additive model to classify a larva as fast- or slow-growing was also formulated using the same covariates. Validation testing with an independent set of 45 laboratory-reared larvae showed that 33% of the laboratory growth rates (i.e., growth rate based on age and size) fell within the 95% confidence interval of the predicted growth rates, and predicted growth rates were significantly less than otolith-derived growth rates. The growth classification model was more accurate than the growth rate model, correctly classifying the growth rate type (fast or slow growing) of 71% of the same set of larvae showing that flow cytometric cell cycle analysis may be better suited for classifying larvae as fast- or slow-growing rather than for predicting absolute growth rate. Predicted growth rates for larvae ≤11 mm in length collected from the Gulf of Alaska were within the range of published values for that area, and were not significantly different than corresponding otolith-derived growth rates. The growth rates of Gulf of Alaska larvae >11 mm were overestimated when compared to both otolith-derived growth rates and published values, and that may be due in part because those larvae were outside of the size range used to formulate the model. Predicted growth rate and growth classification of late-stage, field-collected Walleye Pollock larvae would be improved by adding larvae as large as 15 mm to the model to encompass the size range of larvae typically collected during spring ichthyoplankton surveys conducted in the Bering Sea or Gulf of Alaska. Flow cytometric cell cycle analysis offers promise as an alternative method for determining growth rate of fish larvae when otolith daily increments cannot be reliably counted.

Salidroside suppresses the growth and invasion of human osteosarcoma cell lines MG63 and U2OS in vitro by inhibiting the JAK2/STAT3 signaling pathway.

Previous research has reported that salidroside exerts antitumor properties on numerous types of tumor cells; however, its effect on osteosarcoma cells remains unknown. The present study aimed to investigate the effects of salidroside on the viability, apoptosis and invasion of osteosarcoma cells in vitro, and determine the underlying mechanism of action. The results of an MTT revealed that salidroside suppressed the viability of osteosarcoma cells (MG63 and U2OS cells) in a time- and concentration-dependent manner. The results of cell morphological analysis (profile observations and Hoechst 33258 staining) and the detection of apoptosis by flow cytometry further indicated that the decrease in osteosarcoma cell viability induced by salidroside was associated with cell apoptosis. Western blot analysis not only confirmed these results but also suggested that salidroside induced the apoptosis of osteosarcoma cells by activating the caspase-9-dependent apoptotic pathway. In addition, we reported that salidroside induced G0/G1 phase arrest and suppressed the invasion of osteosarcoma cells, as measured by flow cytometric cell cycle analysis and a Transwell invasion assay, respectively. Western blot analysis confirmed the aforementioned results. Furthermore, our findings demonstrated that salidroside induced the apoptosis, G0/G1 phase arrest and suppressed the invasion of osteosarcoma cells by inhibiting the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, as determined by western blot analysis. In summary, the findings of the present study suggested that salidroside may inhibit the progression of osteosarcoma by suppressing the growth and invasion of osteosarcoma cells. Furthermore, the investigations into the underlying mechanism demonstrated that salidroside exerted notable antitumor activity in osteosarcoma cells by inhibiting the JAK2/STAT3 signaling pathway.

Next-Generation Sequencing Analysis of mRNA Profile in Cisplatin-Resistant Gastric Cancer Cell Line SGC7901.

BackgroundCisplatin-resistant gastric cancer (GC) occurs in patients with GC treated with cisplatin-based chemotherapy, which results in disease progression and early recurrence during the treatment.Material/MethodsTo understand the initiation and developmental mechanism underlying cisplatin-resistant GC, we developed cisplatin-resistant SGC7901 cells (SGC7901/DDP) from the parental cells (SGC7901/S) by continuous exposure to increasing concentrations of cisplatin and subjected these 2 cell lines to RNA sequencing analysis. The data were verified by quantitative polymerase chain reaction and their functional role was evaluated by cell counting kit 8 assay and cell apoptosis and cell cycle flow cytometric analysis. Bioinformatics analysis was performed to classify the differentially-expressed genes (DEGs) involved in the development of cisplatin resistance.ResultsIn comparison with SGC7901/S cells, SGC7901/DDP cells showed a total of 3165 DEGs (2014 upregulated and 1151 downregulated, fold change ≥2, and adjusted P value <0.001). qRT-PCR confirmed the reliability of the RNA sequencing results. Depletion of the top 5 upregulated mRNAs reversed the resistant index, increased apoptotic SGC7901/DDP cells, and arrested the cells at G2/M phase. Gene ontology analysis revealed that the DEGs mainly regulate metabolic process, immune system, locomotion, cell adhesion, cell growth, cell death, cytoskeleton organization, cell binding, signal transducing activity, and antioxidant activity. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs were mainly involved in the PI3K-Akt signaling pathway, Rap1 signaling pathway, proteoglycans in cancer, regulation of actin cytoskeleton, and pathways in cancer.ConcluisonsThe present study is the first to interrogate mRNAs profiles in human GC cells with cisplatin resistance using RNA sequencing, which may assist in discovering potential therapeutic targets for cisplatin-resistant GC patients.

Repurposing of idebenone as a potential anti-cancer agent.

Glioblastoma (GB) represents the most common and aggressive form of malignant primary brain tumour associated with high rates of morbidity and mortality. In the present study, we considered the potential use of idebenone (IDE), a Coenzyme Q10 analogue, as a novel chemotherapeutic agent for GB. On two GB cell lines, U373MG and U87MG, IDE decreased the viable cell number and enhanced the cytotoxic effects of two known anti-proliferative agents: temozolomide and oxaliplatin. IDE also affected the clonogenic and migratory capacity of both GB cell lines, at 25 and 50 µM, a concentration equivalent to that transiently reached in plasma after oral intake that is deemed safe for humans. p21 protein expression was decreased in both cell lines, indicating that IDE likely exerts its effects through cell cycle dysregulation, and this was confirmed in U373MG cells only by flow cytometric cell cycle analysis which showed S-phase arrest. Caspase-3 protein expression was also significantly decreased in U373MG cells indicating IDE-induced apoptosis that was confirmed by flow cytometric Annexin V/propidium iodide staining. No major decrease in caspase-3 expression was observed in U87MG cells nor apoptosis as observed by flow cytometry analysis. Overall, the present study demonstrates that IDE has potential as an anti-proliferative agent for GB by interfering with several features of glioma pathogenesis such as proliferation and migration, and hence might be a drug that could be repurposed for aiding cancer treatments. Furthermore, the synergistic combinations of IDE with other agents aimed at different pathways involved in this type of cancer are promising.