Targeting the COX2/MET/TOPK signaling axis induces apoptosis in gefitinib-resistant NSCLC cells

Abstract MET overactivation is one of the crucial reasons for tyrosine kinase inhibitor (TKI) resistance, but the mechanisms are not wholly clear. Here, COX2, TOPK, and MET expression were examined in EGFR-activating mutated NSCLC by immunohistochemical (IHC) analysis. The relationship between COX2, TOPK, and MET was explored in vitro and ex vivo. In addition, the inhibition of HCC827GR cell growth by combining COX2 inhibitor (celecoxib), TOPK inhibitor (pantoprazole), and gefitinib was verified ex vivo and in vivo. We found that COX2 and TOPK were highly expressed in EGFR-activating mutated NSCLC and the progression-free survival (PFS) of triple-positive (COX2, MET, and TOPK) patients was shorter than that of triple-negative patients. Then, we observed that the COX2-TXA2 signaling pathway modulated MET through AP-1, resulting in an inhibition of apoptosis in gefitinib-resistant cells. Moreover, we demonstrated that MET could phosphorylate TOPK at Tyr74 and then prevent apoptosis in gefitinib-resistant cells. In line with these findings, the combination of celecoxib, pantoprazole, and gefitinib could induce apoptosis in gefitinib-resistant cells and inhibit tumor growth ex vivo and in vivo. Our work reveals a novel COX2/MET/TOPK signaling axis that can prevent apoptosis in gefitinib-resistant cells and suggests that a triple combination of FDA-approved drugs would provide a low-cost and practical strategy to overcome gefitinib resistance.

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Real-Time Wireless Platform for In Vivo Monitoring of Bone Regeneration

Sensors ◽

10.3390/s20164591 ◽

2020 ◽

Vol 20 (16) ◽

pp. 4591 ◽

Cited By ~ 1

Author(s):

Pablo Blázquez-Carmona ◽

Manuel Sanchez-Raya ◽

Juan Mora-Macías ◽

Juan Antonio Gómez-Galán ◽

Jaime Domínguez ◽

...

Keyword(s):

Bone Regeneration ◽

Real Time ◽

Ex Vivo ◽

Low Cost ◽

Bone Lengthening ◽

Bone Callus ◽

Reliable Solution ◽

Regeneration Processes

For the monitoring of bone regeneration processes, the instrumentation of the fixation is an increasingly common technique to indirectly measure the evolution of bone formation instead of ex vivo measurements or traditional in vivo techniques, such as X-ray or visual review. A versatile instrumented external fixator capable of adapting to multiple bone regeneration processes was designed, as well as a wireless acquisition system for the data collection. The design and implementation of the overall architecture of such a system is described in this work, including the hardware, firmware, and mechanical components. The measurements are conditioned and subsequently sent to a PC via wireless communication to be in vivo displayed and analyzed using a developed real-time monitoring application. Moreover, a model for the in vivo estimation of the bone callus stiffness from collected data was defined. This model was validated in vitro using elastic springs, reporting promising results with respect to previous equipment, with average errors and uncertainties below 6.7% and 14.04%. The devices were also validated in vivo performing a bone lengthening treatment on a sheep metatarsus. The resulting system allowed the in vivo mechanical characterization of the bone callus during experimentation, providing a low-cost, simple, and highly reliable solution.

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Entrectinib, a TRK/ROS1 inhibitor with anti-CNS tumor activity: differentiation from other inhibitors in its class due to weak interaction with P-glycoprotein

Neuro-Oncology ◽

10.1093/neuonc/noaa052 ◽

2020 ◽

Vol 22 (6) ◽

pp. 819-829 ◽

Cited By ~ 3

Author(s):

Holger Fischer ◽

Mohammed Ullah ◽

Cecile C de la Cruz ◽

Thomas Hunsaker ◽

Claudia Senn ◽

...

Keyword(s):

Steady State ◽

Lipid Membrane ◽

Kinase Inhibitor ◽

Ex Vivo ◽

Intracranial Tumor ◽

Brain Penetration ◽

In Vivo Experiments ◽

P Glycoprotein

Abstract Background Studies evaluating the CNS penetration of a novel tyrosine kinase inhibitor, entrectinib, proved challenging, particularly due to discrepancies across earlier experiments regarding P-glycoprotein (P-gp) interaction and brain distribution. To address this question, we used a novel “apical efflux ratio” (AP-ER) model to assess P-gp interaction with entrectinib, crizotinib, and larotrectinib, and compared their brain-penetration properties. Methods AP-ER was designed to calculate P-gp interaction with the 3 drugs in vitro using P-gp–overexpressing cells. Brain penetration was studied in rat plasma, brain, and cerebrospinal fluid (CSF) samples after intravenous drug infusion. Unbound brain concentrations were estimated through kinetic lipid membrane binding assays and ex vivo experiments, while the antitumor activity of entrectinib was evaluated in a clinically relevant setting using an intracranial tumor mouse model. Results Entrectinib showed lower AP-ER (1.1–1.15) than crizotinib and larotrectinib (≥2.8). Despite not reaching steady-state brain exposures in rats after 6 hours, entrectinib presented a more favorable CSF-to-unbound concentration in plasma (CSF/Cu,p) ratio (>0.2) than crizotinib and larotrectinib at steady state (both: CSF/Cu,p ~0.03). In vivo experiments validated the AP-ER approach. Entrectinib treatment resulted in strong tumor inhibition and full survival benefit in the intracranial tumor model at clinically relevant systemic exposures. Conclusions Entrectinib, unlike crizotinib and larotrectinib, is a weak P-gp substrate that can sustain CNS exposure based on our novel in vitro and in vivo experiments. This is consistent with the observed preclinical and clinical efficacy of entrectinib in neurotrophic tropomyosin receptor kinase (NTRK) and ROS1 fusion-positive CNS tumors and secondary CNS metastases.

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The E3 Ubiquitin Ligase NEDD4-1 Mediates Temozolomide-Resistant Glioblastoma through PTEN Attenuation and Redox Imbalance in Nrf2–HO-1 Axis

International Journal of Molecular Sciences ◽

10.3390/ijms221910247 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10247

Author(s):

Hao-Yu Chuang ◽

Li-Yun Hsu ◽

Chih-Ming Pan ◽

Narpati Wesa Pikatan ◽

Vijesh Kumar Yadav ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Lines ◽

Biological Effects ◽

Critical Role ◽

Clinical Samples ◽

Redox Imbalance ◽

Signaling Axis ◽

Resistant Cells

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to chemotherapy, and tumor recurrence is common. Neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ligase that controls embryonic development and animal growth. NEDD4-1 regulates the tumor suppressor phosphatase and tensin homolog (PTEN), one of the major regulators of the PI3K/AKT/mTOR signaling axis, as well as the response to oxidative stress. Methods: The expression levels of NEDD4-1 in GBM tissues and different cell lines were determined by quantitative real-time polymerase chain reaction and immunohistochemistry. In vitro and in vivo assays were performed to explore the biological effects of NEDD4-1 on GBM cells. Temozolomide (TMZ)-resistant U87MG and U251 cell lines were specifically established to determine NEDD4-1 upregulation and its effects on the tumorigenicity of GBM cells. Subsequently, miRNA expression in TMZ-resistant cell lines was investigated to determine the dysregulated miRNA underlying the overexpression of NEDD4-1. Indole-3-carbinol (I3C) was used to inhibit NEDD4-1 activity, and its effect on chemoresistance to TMZ was verified. Results: NEDD4-1 was significantly overexpressed in the GBM and TMZ-resistant cells and clinical samples. NEDD4-1 was demonstrated to be a key oncoprotein associated with TMZ resistance, inducing oncogenicity and tumorigenesis of TMZ-resistant GBM cells compared with TMZ-responsive cells. Mechanistically, TMZ-resistant cells exhibited dysregulated expression of miR-3129-5p and miR-199b-3p, resulting in the induced NEDD4-1 mRNA-expression level. The upregulation of NEDD4-1 attenuated PTEN expression and promoted the AKT/NRF2/HO-1 oxidative stress signaling axis, which in turn conferred amplified defense against reactive oxygen species (ROS) and eventually higher resistance against TMZ treatment. The combination treatment of I3C, a known inhibitor of NEDD4-1, with TMZ resulted in a synergistic effect and re-sensitized TMZ-resistant tumor cells both in vitro and in vivo. Conclusions: These findings demonstrate the critical role of NEDD4-1 in regulating the redox imbalance in TMZ-resistant GBM cells via the degradation of PTEN and the upregulation of the AKT/NRF2/HO-1 signaling pathway. Targeting this regulatory axis may help eliminate TMZ-resistant glioblastoma.

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A platform utilizing Drosophila ovulation for nonhormonal contraceptive screening

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2026403118 ◽

2021 ◽

Vol 118 (28) ◽

pp. e2026403118

Author(s):

Kewa Jiang ◽

Jiyang Zhang ◽

Yuping Huang ◽

Yingzheng Wang ◽

Shuo Xiao ◽

...

Keyword(s):

Ex Vivo ◽

Unmet Need ◽

Dependent Manner ◽

Female Reproduction ◽

Contraceptive Agents ◽

Follicle Rupture ◽

Approved Drugs ◽

Fda Approved Drugs

A significant unmet need for new contraceptive options for both women and men remains due to side-effect profiles, medical concerns, and the inconvenience of many currently available contraceptive products. Unfortunately, the development of novel nonsteroidal female contraceptive medicine has been stalled in the last couple of decades due to the lack of effective screening platforms. Drosophila utilizes conserved signaling pathways for follicle rupture, a final step in ovulation that is essential for female reproduction. Therefore, we explored the potential to use Drosophila as a model to screen compounds that could inhibit follicle rupture and be nonsteroidal contraceptive candidates. Using our ex vivo follicle rupture assay, we screened 1,172 Food and Drug Administration (FDA)–approved drugs and identified six drugs that could inhibit Drosophila follicle rupture in a dose-dependent manner. In addition, we characterized the molecular actions of these drugs in the inhibition of adrenergic signaling and follicle rupture. Furthermore, we validated that three of the four drugs consistently inhibited mouse follicle rupture in vitro and that two of them did not affect progesterone production. Finally, we showed that chlorpromazine, one of the candidate drugs, can significantly inhibit mouse follicle rupture in vivo. Our work suggests that Drosophila ovulation is a valuable platform for identifying lead compounds for nonsteroidal contraceptive development and highlights the potential of these FDA-approved drugs as novel nonsteroidal contraceptive agents.

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Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in In Vitro, In Vivo and Ex Vivo Models of Pulmonary Fibrosis

10.1101/2022.01.04.474955 ◽

2022 ◽

Author(s):

Farida Ahangari ◽

Christine Becker ◽

Daniel G Foster ◽

Maurizio Chioccioli ◽

Meghan Nelson ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Recombinant Adenovirus ◽

Ex Vivo ◽

Src Kinase ◽

Lung Fibroblasts

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two FDA approved anti-fibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Using an in-silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor, originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. We investigated the anti-fibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: (i) in vitro in normal human lung fibroblasts (NHLFs); (ii) in vivo in bleomycin and recombinant adenovirus transforming growth factor-beta (Ad-TGF-β) murine models of pulmonary fibrosis; and (iii) ex vivo in precision cut lung slices from these mouse models. In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone.

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Cochlear Dummy Electrodes for Insertion Training and Research Purposes: Fabrication, Mechanical Characterization, and Experimental Validation

BioMed Research International ◽

10.1155/2015/574209 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Jan-Philipp Kobler ◽

Anandhan Dhanasingh ◽

Raphael Kiran ◽

Claude Jolly ◽

Tobias Ortmaier

Keyword(s):

Mechanical Properties ◽

Force Measurement ◽

Ex Vivo ◽

Low Cost ◽

Hearing Preservation ◽

Insertion Force ◽

A New Technique ◽

Temporal Bones

To develop skills sufficient for hearing preservation cochlear implant surgery, surgeons need to perform several electrode insertion trials inex vivotemporal bones, thereby consuming relatively expensive electrode carriers. The objectives of this study were to evaluate the insertion characteristics of cochlear electrodes in a plastic scala tympani model and to fabricate radio opaque polymer filament dummy electrodes of equivalent mechanical properties. In addition, this study should aid the design and development of new cochlear electrodes. Automated insertion force measurement is a new technique to reproducibly analyze and evaluate the insertion dynamics and mechanical characteristics of an electrode. Mechanical properties of MED-EL’s FLEX28, FLEX24, and FLEX20electrodes were assessed with the help of an automated insertion tool. Statistical analysis of the overall mechanical behavior of the electrodes and factors influencing the insertion force are discussed. Radio opaque dummy electrodes of comparable characteristics were fabricated based on insertion force measurements. The platinum-iridium wires were replaced by polymer filament to provide sufficient stiffness to the electrodes and to eradicate the metallic artifacts in X-ray and computed tomography (CT) images. These low-cost dummy electrodes are cheap alternatives for surgical training and forin vitro, ex vivo, andin vivoresearch purposes.

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Identification and Characterization of a Series of Novel Jak2 Small Molecule Inhibitor Compounds.

Blood ◽

10.1182/blood.v110.11.539.539 ◽

2007 ◽

Vol 110 (11) ◽

pp. 539-539

Author(s):

Jacqueline Sayyah ◽

Robert W. Allen ◽

Raul C. Braylan ◽

Gyorgy M. Keseru ◽

Peter P. Sayeski

Keyword(s):

Small Molecule ◽

Kinase Inhibitor ◽

Ex Vivo ◽

Molecular Model ◽

Jak2 V617f ◽

Vehicle Control ◽

Jak2 V617f Mutation ◽

V617f Mutation

Abstract Recent work from our lab has been aimed at identifying novel Jak2 tyrosine kinase inhibitor molecules. Here, we refined our previous Jak2 molecular model using information obtained from the crystal structures of the Jak2 and Jak3 kinase domains. A total of 223,481 compounds within the NIH small molecule database were then screened in silico using FlexX 1.13.2 in order to identify compounds that specifically bind and inhibit Jak2. In what is now our third generation of Jak2 inhibitors, analysis of the highest scoring compounds identified a set of structurally diverse molecules that potently inhibited Jak2 autophosphorylation not only at Tyr 1007, but at all 49 tyrosine residues. These compounds significantly inhibited proliferation of the human erythroleukemia (HEL) cells, which express the Jak2-V617F mutation on both alleles (Fig. 1). One compound in particular, herein designated as G6, was further examined in greater detail. The mechanism by which G6 inhibits Jak2-V617F dependent cell growth is via a marked increase in cellular apoptosis (Fig. 2). We found that G6 does not inhibit c-Src and Tyk2 autophosphorylation at doses that completely inhibit Jak2, therefore suggesting a high degree of specificity. We found that both the G6 and the G13 compounds significantly reduced ex vivo megakaryocyte colony formation of cells taken from a patient diagnosed with essential thrombocythemia and harboring the Jak2-V617F mutation. Finally, we examined the ability of G6 to inhibit red blood cell formation in C57BL/6 mice following acute hemolytic anemia. We found that treatment of animals with G6 (10 mg/kg/day for five days, IP) resulted in a marked inability of these animals to increase their hematocrits following acute anemia when compared to vehicle control animals. Additionally, histological analysis of the bone marrow of G6 injected animals exhibited erythroid hypoplasia resulting in a skewed M:E ratio when compared to vehicle control animals. Collectively, our data demonstrate that these small molecule compounds inhibit Jak2 function in vitro, ex vivo, and in vivo. As such, they may have therapeutic value in treating diseases that are caused by aberrant Jak2 kinase function. Figure 1 Figure 1. Figure 2 Figure 2.

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Abstract 364: TRPV4 Channel Acts as a Negative Regulator of Angiogenesis by Modulation of Rho/Rho Kinase Pathway

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.364 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Holly C Cappelli ◽

Roslin J Thoppil ◽

Ravi K Adapala ◽

Sailaja Paruchuri ◽

Charles K Thodeti

Keyword(s):

Tumor Growth ◽

Kinase Inhibitor ◽

Ex Vivo ◽

Rho Kinase ◽

Aortic Ring ◽

Negative Regulator ◽

Cancer Drug ◽

Kinase Pathway

Angiogenesis, the formation new blood vessels from pre-existing ones, is critical for maintenance of normal cardiovascular physiology. However, excessive or insufficient angiogenesis can contribute to various diseases including cancer, atherosclerosis, and retinopathy. While the mechanism by which angiogenesis occurs is well established, little is known about the mechanisms that negatively regulate this process. Therefore, we investigated the role of mechanosensitive ion channel, TRPV4, in the regulation of angiogenesis by employing in vitro, ex vivo, and in vivo techniques. In the present study, we first cultured aortic ring explants isolated from wild-type (WT) and TRPV4KO mice and found a significant increase in the sprouting from TRPV4KO aortic rings after 5 days. Next, we found that endothelial cells (EC) isolated from TRPV4KO mice (TRPV4KO EC) exhibited increased proliferation, migration, as well as abnormal angiogenesis in vitro, compared to their WT counterparts. Further, in vivo Matrigel plug assays revealed abnormal vascular growth in TRPV4KO mice. Mechanistically, we found that absence of TRPV4 results in a significant increase in basal Rho activity and that pharmacological inhibition of the Rho/Rho kinase pathway was able to normalize the abnormal tube formation exhibited by TRPV4KO EC in vitro . To confirm these findings, we examined tumor growth in TRPV4KO mice treated with Rho kinase inhibitor, Y-27632, and anti-cancer drug Cisplatin, alone and in combination. We found that Y-27632 treatment, in conjunction with Cisplatin but not alone, was able to significantly reduce the abnormal tumor growth seen in TRPV4KO mice, suggesting that Rho kinase inhibition may have normalized the tumor vasculature and improved the delivery of Cisplatin. Taken together, these data suggest that TRPV4 is a negative regulator of angiogenesis and potentially a novel target for pathological and/or therapeutic angiogenesis.

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Applications of novel bioreactor technology to enhance the viability and function of cultured cells and tissues

Interface Focus ◽

10.1098/rsfs.2019.0090 ◽

2020 ◽

Vol 10 (2) ◽

pp. 20190090 ◽

Cited By ~ 2

Author(s):

H. W. Hoyle ◽

L. A. Smith ◽

R. J. Williams ◽

S. A. Przyborski

Keyword(s):

Cultured Cells ◽

Ex Vivo ◽

Low Cost ◽

Tissue Slices ◽

Cell Culture Techniques ◽

Culture Techniques ◽

Tissue Constructs ◽

And Function

As the field of tissue engineering continues to advance rapidly, so too does the complexity of cell culture techniques used to generate in vitro tissue constructs, with the overall aim of mimicking the in vivo microenvironment. This complexity typically comes at a cost with regards to the size of the equipment required and associated expenses. We have developed a small, low-cost bioreactor system which overcomes some of the issues of typical bioreactor systems while retaining a suitable scale for the formation of complex tissues. Herein, we have tested this system with three cell populations/tissues: the culture of hepatocellular carcinoma cells, where an improved structure and basic metabolic function is seen; the culture of human pluripotent stem cells, in which the cultures can form more heterogeneous tissues resembling the in vivo teratoma and ex vivo liver tissue slices, in which improved maintenance of cellular viability is seen over the 3 days tested. This system has the flexibility to be used for a variety of further uses and has the potential to provide a more accessible alternative to current bioreactor technologies.

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miR-365a-5p suppresses gefitinib resistance in non-small-cell lung cancer through targeting PELI3

Pharmacogenomics ◽

10.2217/pgs-2020-0006 ◽

2020 ◽

Vol 21 (11) ◽

pp. 771-783

Author(s):

Fannian Li ◽

Haitao Li ◽

Shuai Li ◽

Baolei Lv ◽

Junjie Shi ◽

...

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Kinase Inhibitor ◽

Small Cell ◽

Small Cell Lung ◽

Signaling Axis ◽

Nsclc Patients ◽

Gefitinib Resistance

Aim: Demonstrate the function of dysregulated miR-365a-5p–PELI3 signaling axis in the generation of gefitinib resistance during treatment for non-small-cell lung cancer (NSCLC). Patients & methods: All the NSCLC patients who participated in this research were recruited from the Second Hospital of Hebei Medical University. PC9 cells and PC9GR cells were cultured for in vitro experiments. Results: Patients who were primary resistant to EGFR-tyrosine kinase inhibitor had lower miR-365a-5p levels. MiR-365a-5p directly targeted PELI3 mRNA. MiR-365a-5p overexpression enhanced the function of gefitinib in inhibiting cell viability. Tumor growth was suppressed through miR-365a-5p in nude mice. Conclusion: Dysregulated miR-365a-5p–PELI3 signaling axis triggered the generation of gefitinib resistance in NSCLC.

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