Evaluation of thymoquinone (TQ) as an antiproliferative, proapoptotic, and immunomodulatory agent in a non-small cell lung cancer cell (NSCLC) line

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14644-e14644
Author(s):  
S. H. Jafri ◽  
J. Glass ◽  
R. Shi ◽  
H. Kleiner
Keyword(s):  
Flow Cytometry ◽  
Cell Growth ◽  
Cell Line ◽  
Mtt Assay ◽  
Statistical Significance ◽  
Control Cell ◽  
Annexin V ◽  
Nsclc Cell ◽  
Antibody Array ◽  
Nsclc Cell Line

e14644 Background: NSCLC is the most common cause of cancer death worldwide. We performed in vitro testing of Thymoquinone (TQ), a derivative of black caraway seed against NSCLC cell line NCI-H460 Methods: Cells were grown in RPMI and were plated at a density of 5,000 cells per well in a 96 well plate and cell growth determined in the presence of 80 and 100μM of TQ dissolved in DMSO with appropriate solvent only as control. Cell proliferation was determined at 24, 48 and 72 hrs intervals using 3-(4,5-dimethyltiazol 2-yl)-2,5-diphenyltetraolium bromide (MTT) assay. Factorial analyses of variance (ANOVA) were used to determine the effect of TQ and control with the time. Student-Newman-Keuls test was used to determine statistical significance with P value <0.05 considered significant. Apoptosis was detected using Annexin V-FITC Aptosis Detection Kit analyzing the effects of TQ at 24 hrs after treatment by flow cytometry. The immunomodulatory effects of TQ were tested using RayBio Human Cytokine Antibody Array C series 200. NCI-H460 cells (50,000 cells per well in duplicate 6 well plates) were grown in serum free RPMI media. 24 hrs after treatment with TQ or DMSO media was collected and analyzed for expression of various cytokines. Results: The MTT assay showed that TQ at 80 and 100 μM significantly inhibited cell growth as compared to control and at 24 hrs for example, 100 μM TQ inhibited cell growth by 78%. Apoptosis occurred rapidly after treatment with TQ with 73.5% of cells being positive for expression of Annexin-V as detected by flow cytometry after 24 hrs of exposure to TQ as compared to 2.6% of controls. The cytokine array revealed that TQ significantly decreased expression of ENA-78(Epithelial neutrophil activating peptide), and GRO (Growth related oncogene).ENA-78 is correlated with vascularity and tumor growth in NSCLC tumor, whereas GRO is associated with neoangiogenesis. Conclusions: TQ is shown to be a powerful anti-proliferative, pro-apoptotic and anti- angiogenic agent in a NSCLC cell line. No significant financial relationships to disclose.

scholarly journals Preclinical Validation of Ubiquitin Receptor PSMD4 As Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4403-4403
Author(s):  
Yan Song ◽  
Ting DU ◽  
Arghya Ray ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Line ◽  
Therapeutic Potential ◽  
Statistical Significance ◽  
Annexin V ◽  
Endoplasmic Reticulum Stress Response ◽  
In Vivo Models

Background and Rationale Our preclinical studies have focused on Identification and validation of components within the ubiquitin proteasome system which can be targeted with novel therapies to overcome proteasome-inhibitor (PI)-resistance in multiple myeloma (MM). Our siRNA screening studies identified ubiquitin Receptor (UbR) PSMD4/Rpn10 as a mediator of MM cell growth and survival. Rpn10 is localized on the 19S regulatory lid of the proteasome, and plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. Here, we show that inhibition of Rpn10 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. Materials and Methods Rpn10 knockout 293 cell line was generated using CRISPR/Cas9. Rpn10-inducible knockdown (KD) MM.1S cell line was generated using shRNA. Drug sensitivity, cell viability, and apoptosis assays were performed using WST/CellTiter-Glo assay, and Annexin V staining, respectively. Cell signaling and caspase activity were determined by western blotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2012, 22:345-358). A xenograft human MM model was used to characterize the role of Rpn10 on tumor progression. Statistical significance was assessed with Student's t test. Results 1) Analysis of MM patient gene expression profiling database showed that Rpn10 expression inversely correlates with overall survival (n=175; p= 0.00064). 2)Real-time PCR, immunoblotting, and immunohistochemistry of MM patient bone marrow showed higher Rpn10 levels in patient MM cells and MM cell lines versus normal cells. 3)Transient transfection of MM cells with Rpn10-siRNA decreased their viability; conversely, transfection with Rpn10-WT specifically rescued cells from growth-inhibitory activity of Rpn10-siRNA. Immunoblot analysis confirmed significant knockdown of Rpn10 by Rpn10-siRNA versus scrambled (scr)-siRNA, and restoration of Rpn10 levels in cells transfected with Rpn10-WT versus Rpn10-siRNA. 4)Genetic blockade of Rpn10 decreased viability in bortezomib-resistant MM cells. 5)CRISPR/Cas9-mediated knockout (KO) of Rpn10 decreased cell growth. 5)Both Rpn10-KO and inducible Rpn10-KD cells showed elevated levels of polyubiquitylated proteins. 6)Inhibition of Rpn10 induced apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling. 7)Reduced tumor growth was noted in mice xenografted with Rpn10-KD MM.1S cells versus mice engrafted with WT-MM.1S cells. Conclusion Our data demonstrate the therapeutic potential of targeting ubiquitin receptor Rpn10/PSMD4, and provide the preclinical basis for development of Rpn10 inhibitors to overcome PI-resistance in MM. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Hepatotoxicity During Eltrombopag Administration Might Be Due to Necrosis of Hepatocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2228-2228
Author(s):  
Kazunori Murai ◽  
Shugo Kowata ◽  
Akiko Abo ◽  
Tatsuo Oyake ◽  
Shigeki Ito ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Growth Inhibition ◽  
Cell Line ◽  
Mtt Assay ◽  
Annexin V ◽  
Similar Data ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Annexin V Assay

Abstract Abstract 2228 Background: Eltrombopag is an oral thrombopoietin receptor agonist for the treatment of immune thrombocytopenic patients who are refractory to the medications including corticosteroid. In RAISE study (The Lancet 2011: 377; 393–402), 79% patients in the eltrombopag group responded to treatment at least once during the study. However, 7% of eltrombopag-treated patients had increase of alanine aminotransferase (ALT) concentration and 4% of total bilirubin. The mechanism of eltrombopag-induced hepatobiliary toxicity remains unknown. In this study, we evaluated the effects of eltrombopag on hepatocytes using HepG2, human hepatocellular carcinoma cell line. Method: Cell proliferation was analyzed by MTT assay. Analysis of apoptosis/necrosis was analyzed by flow cytometry assay using Annexin V and propium iodide (PI) (Annexin-/PI-, viable cells; Annexin+/PI-, early apoptosis cells; Annexin+/PI+, late apoptosis and necrosis cells; Annexin-/PI+, late necrosis cells). Reduced glutathione was measured by DTNB colorimetric method. Murine hepatoma cell line Hepa 1–6 and murine normal liver derived cell line NCTC clone 1469 were also used in this study. Results: HepG2 was incubated with eltrombopag for 72 hrs and then MTT assay was performed. Figure 1a showed the growth inhibition curve of HepG2. HepG2 growth was suppressed by eltrombopag in a dose dependent manner (Figure 1a: without NAC, 12.5 μM 44.3 ± 8.7 %). In the addition of N-acetylcysteine (NAC) canceled the inhibitory effect (Figure 1a: with NAC 10mM: 60.4 ± 22.3 % at 12.5μM eltrombopag, p<0.05). To investigate whether this suppression was due to apoptosis or necrosis, we used PI/ Annexin V assay using flow cytometry. Figure 1b showed that each cell fraction after 72 hrs incubation with eltrombopag. PI positive and PI negative fraction was markedly increased (0μM: 0.7 ± 0.4%, 12.5 μM: 8.0 ± 6.0% p<0.01, 25μM: 39.6 ± 12.2% p<0.001). To confirm that the inhibitory effects of eltrombopag might be due to necrosis, we used IM-54, which is inhibitory molecule against oxidative stress induced necrosis, in MTT assay and PI/ Annexin V assay. As shown in Figure 2 a and 2 b, IM-54 canceled the inhibitory effects of eltrombopag in MTT assay (IM-54 0μM: 18.3 ± 7.5 %, IM-54 10 μM: 33.9 ± 10.6 % at eltrombopag 25μM, p<0.01) and PI/Annexin V assay (IM-54 0μM: 48.3 ± 12.4 %, IM-54: 10 μM 74.2 ± 8.3 % in PI/Annexin V double negative fraction at eltrombopag 25μM, p<0.01). The addition of Caspase-Inhibitor III (Boc-D-FMK) did not cancel the growth inhibition by eltrombopag in MTT assey. We measured GSH concentration of HepG2 cells. After the treatment of eltrombopag for 72 hrs, GSH decreased at 12.5μM of eltrpmbopag, however IM-54 restored the GSH concentration (83 mM at IM-54 0μM and eltrombopag 0 μM, IM-54 0 μM: 42.0 mM and IM-54 10μM: 83.3 mM at eltrombopag 12.5μM). Similar data were obtained in the study using Hepa 1–6 and NCTC clone 1469. Conclusion: Basic structure of eltrombopag is similar to that of 3-bipheyl-carboxylic acid, which is a strong oxidizing agent. Taken together, eltrombopag have an oxidative stress on hepatocytes to result in hepatotoxicity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tin Mesoporphyrin Selectively Reduces Non-Small-Cell Lung Cancer Cell Line A549 Proliferation by Interfering with Heme Oxygenase and Glutathione Systems

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 917
Author(s):  
Valeria Sorrenti ◽  
Agata Grazia D’Amico ◽  
Ignazio Barbagallo ◽  
Valeria Consoli ◽  
Salvo Grosso ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Line ◽  
Heme Oxygenase ◽  
Cell Lung Cancer ◽  
Small Cell ◽  
Nsclc Cell ◽  
Antioxidant Systems ◽  
Nsclc Cell Line ◽  
Small Cell Lung

In order to maintain redox homeostasis, non-small-cell lung cancer (NSCLC) increases the activation of many antioxidant systems, including the heme-oxygenase (HO) system. The overexpression of HO-1 has been often associated with chemoresistance and tumor aggressiveness. Our results clearly showed an overexpression of the HO-1 protein in A549 NSCLC cell lines compared to that in non-cancerous cells. Thus, we hypothesized that “off-label” use of tin mesoporphyrin, a well-known HO activity inhibitor clinically used for neonatal hyperbilirubinemia, has potential use as an anti-cancer agent. The pharmacological inhibition of HO activity caused a reduction in cell proliferation and migration of A549. SnMP treatment caused an increase in oxidative stress, as demonstrated by the upregulation of reactive oxygen species (ROS) and the depletion of glutathione (GSH) content. To support these data, Western blot analysis was performed to analyze glucose-6-phosphate dehydrogenase (G6PD), TP53-induced glycolysis and the apoptosis regulator (TIGAR), and the glutamate cysteine ligase catalytic (GCLC) subunit, as they represent the main regulators of the pentose phosphate pathway (PPP) and glutathione synthesis, respectively. NCI-H292, a subtype of the NSCLC cell line, did not respond to SnMP treatment, possibly due to low basal levels of HO-1, suggesting a cellular-dependent antitumorigenic effect. Altogether, our results suggest HO activity inhibition may represent a potential target for selective chemotherapy in lung cancer subtypes.

scholarly journals AURKA upregulation plays a role in fibroblast-reduced gefitinib sensitivity in the NSCLC cell line HCC827

2015 ◽  
Vol 33 (4) ◽  
pp. 1860-1866 ◽  
Author(s):  
JIA CHEN ◽  
HUIQI LU ◽  
WANG ZHOU ◽  
HUABIN YIN ◽  
LISHUANG ZHU ◽  
...  
Keyword(s):  
Cell Line ◽  
Nsclc Cell ◽  
Nsclc Cell Line

scholarly journals Constitutive Cell Proliferation Regulating Inhibitor of Protein Phosphatase 2A (CIP2A) Mediates Drug Resistance to Erlotinib in an EGFR Activating Mutated NSCLC Cell Line

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 716
Author(s):  
Hisham Saafan ◽  
Ahmad Alahdab ◽  
Robin Michelet ◽  
Linus Gohlke ◽  
Janine Ziemann ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Drug Resistance ◽  
Cell Line ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Nsclc Cell ◽  
Egfr Mutations ◽  
Nsclc Cell Line ◽  
Acquired Drug Resistance ◽  
Phosphatase 2A

Exploring mechanisms of drug resistance to targeted small molecule drugs is critical for an extended clinical benefit in the treatment of non-small cell lung cancer (NSCLC) patients carrying activating epidermal growth factor receptor (EGFR) mutations. Here, we identified constitutive cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A) in the HCC4006rErlo0.5 NSCLC cell line adapted to erlotinib as a model of acquired drug resistance. Constitutive CIP2A resulted in a constitutive activation of Akt signaling. The proteasome inhibitor bortezomib was able to reduce CIP2A levels, which resulted in an activation of protein phosphatase 2A and deactivation of Akt. Combination experiments with erlotinib and bortezomib revealed a lack of interaction between the two drugs. However, the effect size of bortezomib was higher in HCC4006rErlo0.5, compared to the erlotinib-sensitive HCC4006 cells, as indicated by an increase in Emax (0.911 (95%CI 0.867–0.954) vs. 0.585 (95%CI 0.568–0.622), respectively) and decrease in EC50 (52.4 µM (95%CI 46.1–58.8 µM) vs. 73.0 µM (95%CI 60.4–111 µM), respectively) in the concentration–effect model, an earlier onset of cell death induction, and a reduced colony surviving fraction (0.38 ± 0.18 vs. 0.95 ± 0.25, respectively, n = 3, p < 0.05). Therefore, modulation of CIP2A with bortezomib could be an interesting approach to overcome drug resistance to erlotinib treatment in NSCLC.

scholarly journals Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Christo J. Botha ◽  
Sarah J. Clift ◽  
Gezina C.H. Ferreira ◽  
Mxolisi G. Masango
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Ethical Issues ◽  
Sesquiterpene Lactones ◽  
Annexin V ◽  
Metabolic Activation ◽  
Suitable Model ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12) using methyl-thiazol-tetrazolium (MTT) and lactate dehydrogenase (LDH) assays, annexin V and propidium iodide (PI) flow cytometry and transmission electron microscopy (TEM). Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12) is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

Generation of Osimertinib-Resistant Cells from EGFR L858R/T790M Mutant NSCLC Cell Line

2020 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
Nalini Devi Verusingam ◽  
Yi-Chen Chen ◽  
Heng-Fu Lin ◽  
Chao-Yu Liu ◽  
Ming-Cheng Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Nsclc Cell ◽  
Nsclc Cell Line ◽  
Resistant Cells
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