scholarly journals Cytotoxicity evaluation of chloroquine and hydroxychloroquine in multiple cell lines and tissues by dynamic imaging system and PBPK model

2020 ◽  
Author(s):  
Jianling Yang ◽  
Meng Wu ◽  
Xu Liu ◽  
Qi Liu ◽  
Zhengyang Guo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Imaging System ◽  
Cell Types ◽  
Dynamic Imaging ◽  
Time Dependent ◽  
Dependent Manner ◽  
Physiologically Based Pharmacokinetic ◽  
Relative Safety ◽  
Short Period ◽  
Multiple Cell

AbstractChloroquine (CQ) and hydroxychloroquine (HCQ) have been used in treating COVID-19 patients recently. However, both drugs have some contradictions and rare but severe side effects, such as hypoglycemia, retina and cardiac toxicity. To further uncover the toxicity profile of CQ and HCQ in different tissues, we evaluated the cytotoxicity of them in 8 cell lines, and further adopted the physiologically-based pharmacokinetic models (PBPK) to predict the tissue risk respectively. Retina, myocardium, lung, liver, kidney, vascular endothelium and intestinal epithelium originated cells were included in the toxicity evaluation of CQ and HCQ respectively. The proliferation pattern was monitored in 0-72 hours by IncuCyte S3, which could perform long-term continuous image and video of cells upon CQ or HCQ treatment. CC50 and the ratio of tissue trough concentrations to CC50 (RTTCC) were brought into predicted toxicity profiles. The CC50 at 24 h, 48 h, 72 h of CQ and HCQ decreased in the time-dependent manner, which indicates the accumulative cytotoxic effect. HCQ was found to be less toxic in 7 cell types except cardiomyocytes H9C2 cells (CC50-48 h=29.55 μM; CC50-72 h=15.26 μM). In addition, RTTCC is significant higher in CQ treatment group compared to HCQ group, which indicates that relative safety of HCQ. Both CQ and HCQ have certain cytotoxicity in time dependent manner which indicates the necessity of short period administration clinically. HCQ has the less impact in 7 cell lines proliferation and less toxicity compared to CQ in heart, liver, kidney and lung.

scholarly journals Cytotoxicity Evaluation of Chloroquine and Hydroxychloroquine in Multiple Cell Lines and Tissues by Dynamic Imaging System and Physiologically Based Pharmacokinetic Model

2020 ◽  
Vol 11 ◽  
Author(s):  
Jianling Yang ◽  
Zhengyang Guo ◽  
Xu Liu ◽  
Qi Liu ◽  
Meng Wu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Imaging System ◽  
Cell Types ◽  
Vero Cells ◽  
Dynamic Imaging ◽  
Dependent Manner ◽  
Physiologically Based Pharmacokinetic ◽  
Relative Safety ◽  
Short Period ◽  
Physiologically Based

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been challenged in treating COVID-19 patients and still under debate due to the uncertainty regarding the effectiveness and safety, and there is still lack of the systematic study on the toxicity of these two drugs. To further uncover the toxicity profile of CQ and HCQ in different tissues, we evaluated the cytotoxicity of them in eight cell lines and further adopted the physiologically based pharmacokinetic models to predict the tissue risk, respectively. Retina, myocardium, lung, liver, kidney, vascular endothelium, and intestinal epithelium originated cells were included in the toxicity evaluation of CQ and HCQ, respectively. The proliferation pattern was monitored in 0–72 h by IncuCyte S3. CC50 and the ratio of tissue trough concentrations to CC50 (RTTCC) were brought into predicted toxicity profiles. Compared to CQ, HCQ was found to be less toxic in six cell types except Hep3B and Vero cells. In addition, RTTCC was significantly higher in CQ treatment group compared to HCQ group, which indicates relative safety of HCQ. To further simulate the situation of the COVID-19 patients who suffered the dyspnea and hypoxemia, we also tested the cytotoxicity upon hypoxia and normoxia (1, 5 vs. 21% O2). It was found that the cytotoxicity of CQ was more sensitive to hypoxia compared with that of HCQ, particularly in liver originated cells. Both CQ and HCQ showed cytotoxicity in time-dependent manner which indicates the necessity of short period administration clinically.

scholarly journals Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 787
Author(s):  
Enrique García-Pérez ◽  
Dojin Ryu ◽  
Hwa-Young Kim ◽  
Hae Dun Kim ◽  
Hyun Jung Lee
Keyword(s):  
Oxidative Stress ◽  
Ochratoxin A ◽  
Cell Lines ◽  
Time Dependent ◽  
Mrna Levels ◽  
Dependent Manner ◽  
In Vitro System ◽  
Glutathione Peroxidase 1 ◽  
Glucose 6 Phosphate Dehydrogenase

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

scholarly journals A versatile automated high-throughput drug screening platform for zebrafish embryos

2020 ◽  
Author(s):  
Alexandra Lubin ◽  
Jason Otterstrom ◽  
Yvette Hoade ◽  
Ivana Bjedov ◽  
Eleanor Stead ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Imaging System ◽  
Cell Types ◽  
Wide Range ◽  
Stem And Progenitor Cells ◽  
Multiple Cell ◽  
Flexible Imaging ◽  
Automated Screening ◽  
Screening Platform

AbstractZebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed a easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan®Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft®Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions, and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and x-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high content screening in zebrafish.

scholarly journals Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis

2006 ◽  
Vol 203 (1) ◽  
pp. 189-201 ◽  
Author(s):  
Ronit Vogt Sionov ◽  
Orly Cohen ◽  
Shlomit Kfir ◽  
Yael Zilberman ◽  
Eitan Yefenof
Keyword(s):  
T Cell ◽  
Glucocorticoid Receptor ◽  
Cell Lines ◽  
Cell Types ◽  
Thymic Epithelial Cells ◽  
Dependent Manner ◽  
Localization Signal ◽  
Induced Apoptosis ◽  
T Cell Lines

The mechanisms by which glucocorticoid receptor (GR) mediates glucocorticoid (GC)-induced apoptosis are unknown. We studied the role of mitochondrial GR in this process. Dexamethasone induces GR translocation to the mitochondria in GC-sensitive, but not in GC-resistant, T cell lines. In contrast, nuclear GR translocation occurs in all cell types. Thymic epithelial cells, which cause apoptosis of the PD1.6 T cell line in a GR-dependent manner, induce GR translocation to the mitochondria, but not to the nucleus, suggesting a role for mitochondrial GR in eliciting apoptosis. This hypothesis is corroborated by the finding that a GR variant exclusively expressed in the mitochondria elicits apoptosis of several cancer cell lines. A putative mitochondrial localization signal was defined to amino acids 558–580 of human GR, which lies within the NH2-terminal part of the ligand-binding domain. Altogether, our data show that mitochondrial and nuclear translocations of GR are differentially regulated, and that mitochondrial GR translocation correlates with susceptibility to GC-induced apoptosis.

scholarly journals Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation

2020 ◽  
Vol 21 (4) ◽  
pp. 1274
Author(s):  
Hideka Saotome ◽  
Atsumi Ito ◽  
Atsushi Kubo ◽  
Masafumi Inui
Keyword(s):  
Cell Types ◽  
Live Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Post Translational Modifications ◽  
Extracellular Signal ◽  
Multiple Cell ◽  
Sox9 Activity ◽  
Reporter Signal

Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.

scholarly journals Inhibition of Wnt/β-Catenin Signaling in Neuroendocrine Tumors In Vitro: Antitumoral Effects

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 345
Author(s):  
Xi-Feng Jin ◽  
Gerald Spöttl ◽  
Julian Maurer ◽  
Svenja Nölting ◽  
Christoph Josef Auernhammer
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroendocrine Tumors ◽  
Density Lipoprotein ◽  
Time Dependent ◽  
Dependent Manner ◽  
Antitumor Properties

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.

The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5120-5120
Author(s):  
Hatice Demet Kiper ◽  
Burcin Tezcanli Kaymaz ◽  
Ozlem Purclutepe ◽  
Ceyda Tunakan Dalgic ◽  
Nur Selvi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Drug Exposure ◽  
Conflicts Of Interest ◽  
Time Dependent ◽  
Blue Dye ◽  
Dependent Manner ◽  
Down Regulation ◽  
Expression Levels ◽  
Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

Biologic Sequelae of CDC2 Inhibition in MM.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4797-4797
Author(s):  
Mariateresa Fulciniti ◽  
Pierfrancesco Tassone ◽  
Teru Hideshima ◽  
Kenneth C. Anderson ◽  
Nikhil C. Munshi
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Cell Cycle Checkpoint ◽  
Time Dependent ◽  
Thymidine Uptake ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Apoptotic Pathway ◽  
M Phase

Abstract Multiple Myeloma (MM) is a malignant proliferation of plasma cells characterized by disruption of cell cycle checkpoint controls which maintain G2M transition and/or mitosis. CDC2 is the cyclin-dependent kinase that normally drives cells into mitosis and is universally expressed in MM. To examine the biologic role of CDC2 in MM, we evaluated cellular and molecular effects of Terameprocol (M4N, tetra-O-methyl nordihydroguaiaretic acid) that has been shown to inhibit cell cycle progression at the G2/M phase by inhibiting the transcription of sp-1 dependent expression of CDC2. We observed that Terameprocol downregulated the expression of cdc2 in a time-dependent manner, with a maximal effect at 24h. This was associated with induction of G2/M growth arrest in a panel of MM cell lines (INA6, OPM1, OPM2, MM1S, RPMI-8226, U266), as determined by PI staining. Interestingly, Terameprocol treatment led to increase in p21waf1 protein levels. Importantly, we observed inhibition of DNA synthesis by Terameprocol in a dose- and time-dependent manner, with IC50 range from 1–20 uM for a 24 hours period of treatment, as assessed by 3H-thymidine uptake. Longer exposure of MM cells to Terameprocol resulted in cytoxicity, as assessed by MTT assay, via induction of apoptosis, evidenced by Annexin V+ /PI staining, in all the MM cell lines tested. Terameprocol -induced apoptosis is predominantly associated with caspase-9 and caspase-3, but not caspase-8 activation, suggesting that Terameprocol triggers intrinsic apoptotic pathway in MM cells. Our results show that genes that control entry and progression of G2/M phase, especially cdc2, may be an attractive target for MM therapy and Terameprocol represents a prototypic agent for the control of unregulated cellular proliferation in MM.

scholarly journals Inhibition of Phosphodiesterase 5 and Increasing the Level of Cyclic Guanosine 3′,5′ Monophosphate by Hydroalcoholic Achillea wilhelmsii C. Koch Extract in Human Breast Cancer Cell Lines MCF-7 and MDA-Mb-468

2017 ◽  
Vol 11 ◽  
pp. 117822341769017 ◽  
Author(s):  
Ramin Saravani ◽  
Hamid Reza Galavi ◽  
Ali Shahraki
Keyword(s):  
Cell Lines ◽  
Messenger Rna ◽  
Colorimetric Assay ◽  
Time Dependent ◽  
Breast Cancer Cell Lines ◽  
Dependent Manner ◽  
Cgmp Signaling ◽  
Achillea Wilhelmsii ◽  
Phosphodiesterase 5 ◽  
Mcf 7

This study aimed to investigate the effect of hydroalcoholic Achillea wilhelmsii C. Koch extract (HAWE) on phosphodiesterase 5 (PDE5) gene expression and cyclic guanosine 3′,5′ monophosphate (cGMP) signaling in the MCF-7 and MDA-Mb-468 cell lines. The effective dose (ED50) of HAWE was examined in both cell lines using a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide viability test, and the type of cell death was detected by flow cytometry. The expression of PDE5 and the concentration of cGMP were measured in a time-dependent manner in the ED50 by real-time polymerase chain reaction and a colorimetric assay, respectively. Treatment with HAWE showed 25 µg/mL to be the ED50 for both cell lines, and HAWE led to a reduction in the PDE5 messenger RNA expression. The intracellular cGMP increased in a time-dependent manner. The results showed that HAWE has an antiproliferative property in MCF-7 and MDA-Mb-468 cell lines through the cGMP pathway. Therefore, HAWE is a potential source to effectively isolate inhibitory PDE5.

WP1066: A Novel PI-3K Inhibitor with Antileukemic Activity in Philadelphia Chromosome-Positive ALL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 850-850
Author(s):  
Alessandra Ferrajoli ◽  
Stefan Faderl ◽  
Tony Wang ◽  
Waldemar Priebe ◽  
Hagop Kantarjian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tyrosine Kinases ◽  
Philadelphia Chromosome ◽  
Annexin V ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dose Dependent

Abstract Prognosis for patients with Philadelphia chromosome (Ph) positive ALL remains dismal. Ph+ ALL is characterized by the activation of several tyrosine kinases that provide the neoplastic clone with its proliferative capacity and survival advantage. We found that WP1066, a novel derivative of the tyrphostin AG490, inhibits the JAK-STAT pathway and cytokine-dependent and independent signaling pathways. Therefore, we sought to investigate the activity of WP1066 in Ph+ ALL. To do this, we first studied the effect of WP1066 on the Ph+ ALL cell lines Z-119 and Z-181 (Estrov Z et al. J. Cell Physiol.166(3):18, 1996). Using the MTT assay we found that WP1066 inhibited the growth of both Z-119 and Z-181 cells in a concentration-dependent manner with only 8% and 4% of the cells surviving at a concentration of 4 mM, respectively. Similarly, the clonogenic growth of both Z-119 and Z-181 cells was effectively inhibited by WP1066 with more than 90% reduction in colony numbers at concentration of 4 mM. Using Western Immunoblott analysis of cell lysates, we found that 4 mM of WP1066 induced caspase-3 cleavage in a time- and dose-dependent manner in both Z-119 and Z-181 cells. In addition, WP-1066 downregulated uncleaved poly (adenosine diphosphate-ribose) polymerase (PARP) and upregulated cleaved PARP protein levels in a time-dependent manner after 2 hours of exposure to 4 mM. We further evaluated induction of apoptosis using the annexin V-FITC assay and showed a dose dependent increase of the fraction of apoptotic cells in both Z-119 and Z-181 cells. After 24 hour of exposure to 4 mM of WP1066 the fraction of apoptotic cells increased by 23% and 43%, respectively. To elucidate the mechanisms by which WP1066 induces growth inhibition and apoptosis in Ph+ ALL cells, we investigated the effect of this agent on the phosphatidylinositol 3-kinase (PI-3K) pathway because the PI-3K pathway is constitutively activated in Ph+ leukemias. We found that WP1066 inhibited the phosphorylation of AKT in a time-dependent fashion in both cell lines and that this inhibitory effect lasted for 24 hours. In conclusion, our data suggest that exposure to WP1066 induces caspase-dependent apoptosis, is associated with PI3-K inhibition and reduces the growth of the Ph+ cell lines Z-119 and Z-181. The activity of WP1066 in Ph+ ALL should be further studied.

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