Rapamycin Reduces Podocyte Apoptosis and is Involved in Autophagy and mTOR/ P70S6K/4EBP1 Signaling

Background/Aims: The purpose of this study was to investigate the impact of rapamycin (RAP) on autophagy in podocytes and the therapeutic effects of RAP on idiopathic membranous nephropathy (IMN). Methods: We established an in vitro model of IMN by preconditioning mouse podocytes with puromycin aminonucleoside (PAN). A Cell Counting Kit-8 was used to detect the proliferation of each group of podocytes. Podocyte apoptosis was analyzed by flow cytometry via annexin V/propidium iodide dual staining. Subsequently, we observed the number of autophagosomes by transmission electron microscopy. Western blotting was used to detect the levels of LC3, mTOR, p-mTOR, 4EBP1, p-4EBP1, P70S6K, and p-P70S6K in each group. Results: The number of podocytes in the PAN + 100 ng/mL RAP group, PAN + 200 ng/mL RAP group, and PAN + 300 ng/mL RAP group was significantly increased (P < 0.01). The apoptotic rate of podocytes was significantly different between the PAN group and the PAN + RAP group (P < 0.001). There were fewer autophagic corpuscles in the PAN group and more autophagosomes were observed in the PAN + RAP group. LC3 protein expression was down-regulated in the PAN group, while its expression was up-regulated in the PAN + RAP group. In the PAN group, the levels of phosphorylated mTOR, 4EBP1, and P70S6K were increased, while in the PAN + RAP group, protein phosphorylation was reduced. Conclusions: RAP can effectively inhibit the mTOR/P70S6K/4EBP1 signaling pathway, and activate podocyte autophagy, consequently reducing podocyte apoptosis. Therefore, RAP could be used for the treatment of idiopathic membranous nephropathy.

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A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

Cancer Cell International ◽

10.1186/s12935-021-01973-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Xuejie Gao ◽

Bo Li ◽

Anqi Ye ◽

Houcai Wang ◽

Yongsheng Xie ◽

...

Keyword(s):

Mouse Model ◽

Tumor Burden ◽

High Rate ◽

Cell Counting ◽

Tumor Xenograft ◽

Luciferase Reporter ◽

Therapeutic Effects ◽

Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

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Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study

Biomedicine Hub ◽

10.1159/000491074 ◽

2018 ◽

Vol 3 (3) ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Madhuravasal Krishnan Janani ◽

Venkatakrishnan Jaichandran ◽

Hajib Narahari Rao Madhavan ◽

Lingam Vijaya ◽

Ronnie Jacob George ◽

...

Keyword(s):

Culture Medium ◽

In Vitro Model ◽

Morphological Changes ◽

Human Fibroblasts ◽

Annexin V ◽

In Vitro Study ◽

Tenon’S Capsule ◽

Tenon's Capsule ◽

Vitro Model

Purpose: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon’s capsule fibroblast (HTFs) in an in vitro model. Patients and Methods: Tenon’s capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. Results: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5–2.0%), the viability of cells decreased from 1.0% lignocaine. Conclusion: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

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P13.08 Novel Thioridazine derivates: Antiproliferative and apoptosis-inducing activity on glioblastoma cells in vitro

Neuro-Oncology ◽

10.1093/neuonc/noab180.116 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii34-ii34

Author(s):

S G Schwab ◽

K Sarnow ◽

E Alme ◽

R Goldbrunner ◽

H Bjørsvik ◽

...

Keyword(s):

Imaging System ◽

Therapeutic Potential ◽

Flow Cytometric Analysis ◽

Annexin V ◽

Therapeutic Effects ◽

Cell Viability Assay ◽

Selective Cytotoxicity ◽

Viability Assay

Abstract BACKGROUND Although withdrawn from the market due to cardiotoxicity, we have shown that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of the presented project was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. MATERIAL AND METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay as well as a real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p<0,0001, U251 p<0,0001, LN18 p=0,0004) as well as on glioma stem cells (GSC) (P3 p<0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in the GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We have developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

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Downregulation of CCKBR Expression Inhibits the Proliferation of Gastric Cancer Cells, Revealing a Potential Target for Immunotoxin Therapy

Current Cancer Drug Targets ◽

10.2174/1568009622666220106113616 ◽

2022 ◽

Vol 22 ◽

Author(s):

Meng Li ◽

Jiang Chang ◽

Honglin Ren ◽

Defeng Song ◽

Jian Guo ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Cells ◽

Cell Counting ◽

Migration And Invasion ◽

Gastric Cancer Cells ◽

Aberrant Expression ◽

Promising Target ◽

The Impact

Background Increased CCKBR expression density or frequency has been reported in many neoplasms. Objective We aimed to investigate whether CCKBR drives the growth of gastric cancer (GC) and its potential as a therapeutic target of immunotoxins. Methods A lentiviral interference system was used to generate CCKBR-knockdown gastric cancer cells. Cell Counting Kit-8 and clonogenic assays were used to evaluate cell proliferation. Wound-healing and cell invasion assays were performed to evaluate cell mobility. Cell cycle was analyzed by flow cytometry. Tumor growth in vivo was investigated using a heterologous tumor transplantation model in nude mice. In addition, we generated the immunotoxin FQ17P and evaluated the combining capacity and tumor cytotoxicity of FQ17P in vitro. Results Stable downregulation of CCKBR expression resulted in reduced proliferation, migration and invasion of BGC-823 and SGC-7901 cells. The impact of CCKBR on gastric cancer cells was further verified through CCKBR overexpression studies. Downregulation of CCKBR expression also inhibited the growth of gastric tumors in vivo. Furthermore, FQ17P killed CCKBR-overexpressing GC cells by specifically binding to CCKBR on the tumor cell surface. Conclusion The CCKBR protein drives the growth, migration, and invasion of gastric cancer cells, and it might be a promising target for immunotoxin therapy based on its aberrant expression, functional binding interactions with gastrin, and subsequent internalization.

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Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death

Alternatives to Laboratory Animals ◽

10.1177/026119290903700210 ◽

2009 ◽

Vol 37 (2) ◽

pp. 209-218 ◽

Cited By ~ 10

Author(s):

Mathieu Vinken ◽

Elke Decrock ◽

Elke De Vuyst ◽

Luc Leybaert ◽

Tamara Vanhaecke ◽

...

Keyword(s):

Cell Death ◽

In Vitro Model ◽

Caspase 3 ◽

Fas Ligand ◽

Apoptotic Cell ◽

Annexin V ◽

Apoptotic Cell Death ◽

Biochemical Characterisation ◽

Vitro Model

This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.

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CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model

Cancer Cell International ◽

10.1186/s12935-020-01718-6 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Mustafa Magan ◽

Emilia Wiechec ◽

Karin Roberg

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Head And Neck ◽

Treatment Response ◽

Tumor Cells ◽

In Vitro Model ◽

Tumor Spheroids ◽

Vitro Model ◽

The Impact

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. Methods Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. Results The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. Conclusion Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

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Impact of Bacterial Biofilm Formation on In Vitro and In Vivo Activities of Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.4.895 ◽

1998 ◽

Vol 42 (4) ◽

pp. 895-898 ◽

Cited By ~ 112

Author(s):

Silvia Schwank ◽

Zarko Rajacic ◽

Werner Zimmerli ◽

Jürg Blaser

Keyword(s):

Animal Model ◽

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Phenotypic Resistance ◽

Vitro Model ◽

The Impact ◽

Isogenic Strains

ABSTRACT The impact of bacterial adherence on antibiotic activity was analyzed with two isogenic strains of Staphylococcus epidermidis that differ in the features of their in vitro biofilm formation. The eradication of bacteria adhering to glass beads by amikacin, levofloxacin, rifampin, or teicoplanin was studied in an animal model and in a pharmacokinetically matched in vitro model. The features of S. epidermidis RP62A that allowed it to grow on surfaces in multiple layers promoted phenotypic resistance to antibiotic treatment, whereas strain M7 failed to accumulate, despite initial adherence on surfaces and growth in suspension similar to those for RP62A. Biofilms of S. epidermidis M7 were better eradicated than those of strain RP62A in vitro (46 versus 31%;P < 0.05) as well as in the animal model (39 versus 9%; P < 0.01).

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T-2 Toxin Exposure Induces Apoptosis in TM3 Cells by Inhibiting Mammalian Target of Rapamycin/Serine/Threonine Protein Kinase(mTORC2/AKT) to Promote Ca2+Production

International Journal of Molecular Sciences ◽

10.3390/ijms19113360 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3360 ◽

Cited By ~ 4

Author(s):

Ji Wang ◽

Chenglin Yang ◽

Zhihang Yuan ◽

Jine Yi ◽

Jing Wu

Keyword(s):

Cell Injury ◽

Mammalian Target Of Rapamycin ◽

Annexin V ◽

Target Of Rapamycin ◽

Dependent Manner ◽

Toxin Exposure ◽

Concentration Changes ◽

Vitro Model ◽

Induced Apoptosis

Although mTOR (the mammalian target of rapamycin) can regulate intracellular free Ca2+concentration in normal cultured podocytes, it remains elusive as to how mTORC2/AKT-mediated Ca2+participates in the process of T-2 toxin-induced apoptosis. The potential signaling responsible for intracellular Ca2+ concentration changes was investigated using immunoblot assays in an in vitro model of TM3 cell injury induced by T-2 toxin. Changes in Ca2+ were assessed using the Ca2+-sensitive fluorescent indictor dye Fura 2-AM. The cytotoxicity of TM3 cells was assessed with an MTT bioassay, and apoptosis was measured using Annexin V-FITC staining. Following T-2 toxin treatment, the growth of cells, phospho-mTORSer2481, phospho-mTORSer2448, and phospho-AktSer473 were significantly decreased in a time-dependent manner, whereas Ca2+ and apoptosis were increased. T-2 toxin-induced apoptosis was prevented by BAPTA-AM (a Ca2+chelator) and MHY1485 (an mTOR activator), and the application of mTOR activator MHY1485 also prevented the increase of intracellular free Ca2+concentration in TM3 cells. Our results strongly suggest that T-2 toxin exposure induces apoptosis in TM3 cells by inhibiting mTORC2/AKT to promote Ca2+ production.

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Apoptosis-Promoting Effects on A375 Human Melanoma Cells Induced by Exposure to 35.2-GHz Millimeter Wave

Technology in Cancer Research & Treatment ◽

10.1177/1533033820934131 ◽

2020 ◽

Vol 19 ◽

pp. 153303382093413

Author(s):

Ruiting Zhao ◽

Yonghong Liu ◽

Sida Liu ◽

Tong Luo ◽

Guang Yuan Zhong ◽

...

Keyword(s):

Millimeter Wave ◽

Caspase 3 ◽

Malignant Tumors ◽

Annexin V ◽

Human Melanoma ◽

Wave Exposure ◽

Cell Counting ◽

Melanoma Tumor ◽

A375 Cells

Malignant tumors pose a major problem in the medical field. Millimeter wave (MMW) exposure have potential apoptosis-promoting effects on several types of tumors. Considering that the penetration depth of millimeter wave is usually several millimeters, we study the apoptosis-promoting effects of millimeter wave exposure on A375 human melanoma tumor cells in vitro, and this topic has not been explored in the previous literature. In this study, we use the A375 human melanoma cell line as an experimental model exposed to 35.2 GHz millimeter wave in vitro to determine any positive effect and further explore the underlying mechanisms. In this study, 2 groups namely, exposed and sham groups, were set. The exposed groups included 4 exposure time periods of 15, 30, 60, and 90 minutes. The cells in the sham group did not receive millimeter wave exposure. After millimeter wave exposure, the A375 cells in the exposed and sham groups were collected for further experimental procedures. The cell viability after exposure was determined using a cell counting kit, and the apoptosis of A375 cells was assessed by Annexin V/propidium iodide. Changes in the expression of apoptosis-related proteins, including cleaved-caspase-3, and -8, were examined by Western blot. We observed that the millimeter wave exposure could inhibit the viability and induce apoptosis in A375 cells, and the expression of cleaved caspase-3 and -8 were upregulated ( P < .05). The results indicated that the millimeter wave at 35.2 GHz exerted apoptosis-promoting effects on the A375 cells via a pathway by activating of caspase-8 and -3.

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