Abstract 1252: A Mouse Model of a Familial Dilated Cardiomyopathy Mutation in Titin Recapitulates the Human Phenotype

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Michael Gramlich ◽  
Beate Michely ◽  
Brenda Gerull ◽  
Christian Krohne ◽  
Ingo Morano ◽  
...  
Keyword(s):  
Mouse Model ◽  
Western Blot ◽  
Dilated Cardiomyopathy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Autosomal Dominant ◽  
Premature Stop Codon ◽  
Left Ventricular ◽  
Insertion Mutation ◽  
Human Phenotype

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial diseases and the third most common cause of heart failure. Familial occurrence, mostly as an autosomal dominant trait, is responsible for 20 –30% of all DCM cases. We have previously shown that mutations in the giant muscle filament titin ( TTN ) cause dilated cardiomyopathy. In a large DCM kindred (A1) with autosomal dominant inherited DCM, we could identify a 2 bp insertion mutation in exon 326 of TTN . This heterozygous nonsense mutation leads to a framshift generating a premature stop codon after the addition of 4 novel amino acid residues. We have recently evaluated a cardiac biopsy sample from an affected family member of kindred A1 showing that no truncated protein is observed in a western blot analysis. To further investigate the functional consequences of the identified human TTN mutation, we now generated a mouse model that includes the 2bp insertion at the corresponding site in the mouse genome. Heterozygous mice are viable and fertile. As in the human situation, the truncated titin is not detectable in western blot analysis of cardiac tissue indicating haploinsufficiency. The ventricles of the heterozygous animals show a decrease in ventricular stiffness as seen in isolated working heart pressure measurements and transmitral Doppler echocardiography (E:A 1.34 vs. 1.075, p<0.01; IVRT 13.57ms vs. 17.01ms, p<0.05). When exposed to angiotensin II (1.4 mg/kg/d for 14d) as a cardiac stressor, heterozygous animals develop dilatation of the left ventricles (4.45mm vs. 3.77 mm, p<0.05) with impaired fractional shortening (25.12% vs. 32.86%, p<0.01) and a diffuse myocardial fibrosis. Homozygous mice die in utero before E8.0. Whether a defect in the formation of sarcomeres or, alternatively, a defect in yet unknown non-muscle functions of titin account for this early embryonic lethality remains to be determined. Conclusion: Our mouse model shows that a mutation in TTN leads to impaired biomechanical properties of the heart, resulting in left ventricular dilatation and decreased systolic function, thereby recapitulating the human phenotype.

Overcoming resistance to antiandrogens with a TGF-β RI inhibitor in preclinical mouse model of PCa.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 288-288
Author(s):  
Channing Judith Paller ◽  
Hong Pu ◽  
Diane Begemann ◽  
Mary Nakazawa ◽  
Natasha Kyprianou
Keyword(s):  
Cell Proliferation ◽  
Mouse Model ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Combined Treatment ◽  
Prostate Tumor ◽  
Metastatic Progression ◽  
Mesenchymal Transition

288 Background: Epithelial-mesenchymal transition (EMT) is a significant contributor to PCa metastatic progression and therapeutic resistance in patients treated with the androgen receptor (AR) directed therapies. We previously demonstrated that aberrant TGF-β signaling accelerates prostate tumor progression in the TRAMP mouse model of tumorigenesis via selective effects on EMT. Methods: We hypothesize that the combination of the TGF-β receptor inhibitor, galunisertib (G), and enzalutamide (E) will perturb the interactive signaling between TGF-β and AR signaling affecting the phenotypic landscape of EMT. This perturbation may be exploited in our mouse model, towards enhanced anti-tumor efficacy in advanced castration-resistant PCa (CRPC). We treated 2-week old mice for two weeks with the G (75mg/kg) and/or E (30mg/kg) in combination and as single agents. Results: Treatment with G alone or in combination with E resulted in a significant reduction in prostate tumor weight without affecting total body weight. Immunohistochemical (IHC) and Western blot analysis showed that, while treatment with the G alone led to increased apoptosis and decreased cell proliferation, combination of G and E had significantly higher efficacy in inducing apoptosis and inhibiting cell proliferation than either E or G alone. As expected treatment with the G decreased the levels of nuclear Smad4 protein; the combination of G and E further decreased nuclear Smad4 expression. Furthermore the combination of G and E reversed phenotypic EMT to MET (mesenchymal-epithelial-transition), as assessed by the increase in E-cadherin among the prostate tumor cell populations. IHC and Western blot analysis also revealed that the combined treatment of G and E led to a significant decrease in nuclear AR levels compared to E-only-treated or vehicle-control tumors. Conclusions: These results provide significant insights as to the therapeutic impact of G to effectively impair the TGF-β signaling and overcome resistance of PCa patients to E by reversing EMT to potentially sensitize tumors to the antiandrogen effect. This study has major translational relevance; the combination of G and E may lead to synergistic anti-tumor impact in patients with CRPC.

scholarly journals Mild hypothermia preserves myocardial conduction during ischemia by maintaining gap junction intracellular communication and Na+ channel function

2017 ◽  
Vol 312 (5) ◽  
pp. H886-H895 ◽  
Author(s):  
Michelle M. J. Nassal ◽  
Xiaoping Wan ◽  
Zack Dale ◽  
Isabelle Deschênes ◽  
Lance D. Wilson ◽  
...  
Keyword(s):  
Cardiac Arrest ◽  
Western Blot ◽  
Gap Junction ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mild Hypothermia ◽  
Conduction Block ◽  
Left Ventricular ◽  
Cardiac Ischemia ◽  
Cellular Excitability

Acute cardiac ischemia induces conduction velocity (CV) slowing and conduction block, promoting reentrant arrhythmias leading to sudden cardiac arrest. Previously, we found that mild hypothermia (MH; 32°C) attenuates ischemia-induced conduction block and CV slowing in a canine model of early global ischemia. Acute ischemia impairs cellular excitability and the gap junction (GJ) protein connexin (Cx)43. We hypothesized that MH prevented ischemia-induced conduction block and CV slowing by preserving GJ expression and localization. Canine left ventricular preparations at control (36°C) or MH (32°C) were subjected to no-flow prolonged (30 min) ischemia. Optical action potentials were recorded from the transmural left ventricular wall, and CV was measured throughout ischemia. Cx43 and Na+ channel (NaCh) remodeling was assessed using both confocal immunofluorescence (IF) and/or Western blot analysis. Cellular excitability was determined by microelectrode recordings of action potential upstroke velocity (d V/d tmax) and resting membrane potential (RMP). NaCh current was measured in isolated canine myocytes at 36 and 32°C. As expected, MH prevented conduction block and mitigated ischemia-induced CV slowing during 30 min of ischemia. MH maintained Cx43 at the intercalated disk (ID) and attenuated ischemia-induced Cx43 degradation by both IF and Western blot analysis. MH also preserved d V/d tmax and NaCh function without affecting RMP. No difference in NaCh expression was seen at the ID by IF or Western blot analysis. In conclusion, MH preserves myocardial conduction during prolonged ischemia by maintaining Cx43 expression at the ID and maintaining NaCh function. Hypothermic preservation of GJ coupling and NaCh may be novel antiarrhythmic strategies during resuscitation. NEW & NOTEWORTHY Therapeutic hypothermia is now a class I recommendation for resuscitation from cardiac arrest. This study determined that hypothermia preserves gap junction coupling as well as Na+ channel function during acute cardiac ischemia, attenuating conduction slowing and preventing conduction block, suggesting that induced hypothermia may be a novel antiarrhythmic strategy in resuscitation.

scholarly journals Verbascoside Inhibits Glioblastoma Cell Proliferation, Migration and Invasion While Promoting Apoptosis Through Upregulation of Protein Tyrosine Phosphatase SHP-1 and Inhibition of STAT3 Phosphorylation

2018 ◽  
Vol 47 (5) ◽  
pp. 1871-1882 ◽  
Author(s):  
Wei-Qiang Jia ◽  
Zhao-Tao Wang ◽  
Ming-Ming Zou ◽  
Jian-Hao Lin ◽  
Ye-Hai Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mouse Model ◽  
Western Blot ◽  
Protein Tyrosine Phosphatase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tyrosine Phosphatase ◽  
Migration And Invasion ◽  
Glioblastoma Cell ◽  
Stat3 Phosphorylation

Background/Aims: As a natural antioxidant, verbascoside (VB) is proved to be a promising method for the treatment of oxidative-stress-related neurodegenerative diseases. Thus, this study aimed to investigate the effects of VB on glioblastoma cell proliferation, apoptosis, migration, and invasion as well as the mechanism involving signal transducer and activator of transcription 3 (STAT3) and Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). Methods: U87 cells were assigned to different treatments. The MTT assay was used to test cell proliferation, flow cytometry was used to detect cell apoptosis, and a Transwell assay was used for cell migration and invasion. We analyzed the glioblastoma tumor growth in a xenograft mouse model. Western blot analysis was employed to determine the protein expression of related genes. Results: Glioblastoma cells exhibited decreased cell proliferation, migration, invasion, and increased apoptosis when treated with VB or TMZ. Western blot analysis revealed elevated SHP-1 expression and reduced phosphorylated (p)-STAT3 expression in glioblastoma cells treated with VB compared with controls. Correspondingly, in a xenograft mouse model treated with VB, glioblastoma tumor volume and growth were decreased. Glioblastoma xenograft tumors treated with VB showed elevated SHP-1, Bax, cleaved caspase-3, and cleaved PARP expression and reduced p-STAT3, Bcl-2, survivin, MMP-2, and MMP-9 expression. siRNA-SHP-1 inhibited the VB effects on glioblastoma. Conclusion: This study demonstrates that VB inhibits glioblastoma cell proliferation, migration, and invasion while promoting apoptosis via SHP-1 activation and inhibition of STAT3 phosphorylation.

scholarly journals ARHGEF12 Is Essential for Human Megakaryocyte Differentiation and Plays Critical Roles in Platelet Function

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 341-341
Author(s):  
Siying Zou ◽  
Alexandra M Teixeira ◽  
Chad D Sanada ◽  
Ping-xia Zhang ◽  
Diane Krause
Keyword(s):  
Platelet Aggregation ◽  
Mouse Model ◽  
Western Blot ◽  
Platelet Activation ◽  
Platelet Function ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Thrombin Receptors ◽  
Platelet Functions

Abstract Megakaryocytopoiesis, the process by which hematopoietic stem cells develop into mature megakaryocytes (MK), and thrombopoiesis, platelet production/release, are critical for blood homeostasis. We tested the hypothesis that the Rho guanine exchange factor, ARHGEF12 (also known as LARG), is critical for MK differentiation and platelet functions based on the following: 1) ARHGEF12 is part of a recurrent translocation with MLL in acute myeloid leukemia. 2) Both published microarray datasets and deep-sequencing data from our lab on primary human CD34+ cells differentiating into MKs show that ARHGEF12 expression goes up dramatically during MK differentiation. 3) ARHGEF12 is one of the most highly expressed guanine exchange factors in platelets. 4) ARHGEF12 forms a complex with G proteins and stimulates Rho-dependent signals. It is known that platelet activation can be initiated by extracellular stimuli working through G protein-coupled receptors and Rho signaling, suggesting that ARHGEF12 may function in platelet activation. 5) Mice with KO of RhoA (a known ARHGEF12 substrate) in the MK-lineage have macrothrombocytopenia and defective platelet activation. To test this hypothesis, we used ARHGEF12 shRNA mediated KD and an ARHGEF12 specific pharmacological inhibitor (Y16) in both murine and human primary cells, and characterized a LARG KO mouse model for MK and platelet phenotypes, and found: ARHGEF12 is differentially upregulated during MK differentiation and is enriched in platelets Using quantitative RT-PCR and western blot analysis at different timepoints of primary FACSorted Mk progenitors induced to differentiate into mature MK in vitro, ARHGEF12 RNA and protein expression increases during MK differentiation in both the murine and human systems. Also western blot analysis of murine platelet rich plasma shows that ARHGEF12 protein is highly expressed in platelets. ARHGEF12 is essential for human MK differentiation To test the function of ARHGEF12 in Mk differentiation, we used lentiviral shRNA to knockdown ARHGEF12 in FACSorted primary human Mk progenitors from mobilized peripheral blood differentiated in vitro to MK. The results show that ARHGEF12 knockdown blocks MK polyploidization (not shown) and maturation (Fig. A). This was confirmed using a published ARHGEF12 inhibitor (Y16) in the differentiation culture of human MK progenitors, in which there was a dose-dependent block in MK differentiation (Fig. B). These data suggested that ARHGEF12 is essential for human MK differentiation. We researched the function of ARHGEF12 in the murine system using a constitutive ARHGEF12 knockout mouse model. The mice have enlarged platelets (p=0.07) and a decreased platelet count (p=0.01). However, the knockout mice have normal BM cellularity with no change in megakaryocyte number or ploidy, suggesting that ARHGEF12 is dispensable for murine MK differentiation in vivo. ARHGEF12 is essential for platelet function in both the murine and human systems: To test whether ARGEF12 functions in platelet activation, we compared WT versus KO platelet activation in vitro. We tested activation in response to ADP, U46619 (Thromboxane), ADP+U46619, and Thrombin. KO plateelts have significantly reduced activation in response to U46619 and thrombin, with no effects on ADP-induced activation. Analogous studies using the ARHGEF12 inhibitor (Y16) on WT platelets revealed supportive evidence. Lastly, we tested ARHGEF12 function in human platelet aggregation using the Y16 compound. Consistent with the murine data, Y16 blocked platelet aggregation in response to both U46619 and Thrombin. Taken together, these data strongly suggest that ARHGEF12 is essential for platelet function and acts downstream of the Thromboxane and Thrombin receptors. In summary, we found that ARHGEF12 is differentially up-regulated in MK differentiation both in human and in mouse system,. It plays a critical role in human Mk differentiation but is dispensable in murine MK differentiation, and ARHGEF12 is critical for platelet functions in both human and mouse systems, potentially acting downstream of Thromboxane and Thrombin receptors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Role of microtubules in myocyte contractile dysfunction during cardiac hypertrophy in the rat

1996 ◽  
Vol 271 (5) ◽  
pp. H1978-H1987 ◽  
Author(s):  
Y. Ishibashi ◽  
H. Tsutsui ◽  
S. Yamamoto ◽  
M. Takahashi ◽  
K. Imanaka-Yoshida ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Contractile Function ◽  
Pressure Overload ◽  
Progressive Increase ◽  
Left Ventricular ◽  
Contractile Dysfunction

We have shown that increased microtubules cause myocyte contractile dysfunction in feline right ventricular pressure-overload hypertrophy. To investigate the association between the progression of cardiac hypertrophy and microtubules and to delineate the role of microtubules in contractile defects in hypertrophied myocytes, we assessed the amounts of free and polymerized tubulin proteins, using Western blot analysis and immunofluorescence micrograph, and evaluated the sarcomere mechanics of myocytes isolated from rats with pressure-overload left ventricular (LV) hypertrophy. Total and polymerized tubulins were progressively and persistently increased in LV after the imposition of pressure overload. The increase in microtubules was associated with the development and progression of hypertrophy and not the immediate response to the stress loading to the myocardium. The contractile function of hypertrophied myocytes was depressed in parallel with the increase in microtubules. Depolymerization of microtubules normalized the initially depressed LV myocyte contractile function. Thus the progressive increase of microtubule density during LV hypertrophy due to persistent pressure overloading to the myocardium may cause the consequent myocyte contractile dysfunction.

Abstract 18442: Cardiac-specific SOCS3 Deficiency Leads to Left Ventricular Dysfunction With Advanced Age Through Impaired Autophagy

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Shoichiro Nohara ◽  
Hideo Yasukawa ◽  
Toyoharu Oba ◽  
Takanobu Nagata ◽  
Jinya Takahashi ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Cardiac Dysfunction ◽  
Western Blot Analysis ◽  
Cardiac Fibrosis ◽  
Left Ventricular ◽  
Negative Regulator ◽  
Advanced Age ◽  
Cytokine Signaling ◽  
Age Related

Introduction: Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible negative regulator of JAK-STAT signaling pathway. Cardiac-specific SOCS3 deficient mice (SOCS3-CKO) spontaneously develop cardiac dysfunction with advanced age. Hypothesis: We thus evaluated the underlying mechanism of age-related cardiac dysfunction in SOCS3-cKO mice. Methods: To create SOCS3-CKO mice, SOCS3-flox mice were bred with mice harboring a transgene encoding Cre recombinase driven by the α-myosin heavy chain promoter. Cardiac fibrosis was measured by Sirius-red staining. We evaluated autophagy induction by western blot using an antibody raised against microtubule-associated protein light chain 3 (LC3). Results: SOCS3-CKO mice were born at the expected Mendelian ratio, but developed cardiac dysfunction estimated by echocardiogram from 25 weeks of age. Cardiac fibrosis was greater in SOCS3-CKO mice compared to control mice (p<0.01). Western blot analysis revealed that increased activation of STAT3 from 25 weeks of age in SOCS3-CKO mice (p<0.01). TUNEL-positive cells were not observed in both control and SOCS3-CKO mice at 35 weeks of age. Expressions of Bax and Bad were comparable between control and SOCS3-CKO mice, indicating that apoptosis is not a main mechanism in the cardiac dysfunction with advanced age in SOCS3-CKO mice. In SOCS3-CKO mice hearts from 25 weeks of age, LC3II/LC3I ratio was significantly decreased (p<0.01). Western blot analysis revealed that expression of parkin and PINK1 but not p62 and Bnip3 were decreased in SOCS3-CKO mice compared with control mice from 25 weeks of age (p<0.01). We found that fasting-induced changes of LC3II/LC3I ratio were correlated with activation of STAT3 in WT mice hearts (p<0.01). Conclusions: These results suggest that age-related cardiac dysfunction in SOCS3-CKO mice may be due to impaired autophagy through sustained STAT3 activation.

Abstract P464: Cardiac Dysfunction In A Mouse Model For Autosomal Dominant Polycystic Kidney Disease

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
William Perez ◽  
Troy Hendrickson ◽  
Gabriele G Schiattarella ◽  
Joseph A Hill ◽  
Francisco J Altamirano
Keyword(s):  
Kidney Disease ◽  
Mouse Model ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Western Blot ◽  
Blot Analysis ◽  
Cardiac Dysfunction ◽  
Autosomal Dominant ◽  
Polycystic Kidney ◽  
Autosomal Dominant Polycystic Kidney

Mutations in polycystin-1 (PC1) cause autosomal dominant polycystic kidney disease (ADPKD), a disorder that manifests with cardiac hypertrophy and dysfunction. We recently showed that cardiomyocyte-specific PC1 KO mice exhibit both systolic and diastolic dysfunction without signs of cardiac hypertrophy. The purpose of this study was to determine the effects of ADPKD-causing PC1 mutations on cardiac function using a mouse model for ADPKD harboring a mutation in PC1 R3277C (RC/RC). We used echocardiography to determine cardiac function and Western blot analysis to explore signaling pathways in WT and RC/RC mice (2-4 months of age).We observed a slight but significant decrease in ejection fraction (77.2±0.7 vs 86.2±2.4 %, N=5, 4, P=0.015) without signs of left ventricular hypertrophy (LV mass 78.4±5.5 vs 93.5±4.2 mg, N=5, 4, P>0.05) in RC/RC compared to WT mice. Western blot analysis from total heart lysates (WT and RC/RC; N=5) revealed no changes in protein levels of hypertrophic markers: beta myosin heavy chain (β-MHC) and regulator of calcineurin 1 (RCAN1). In addition, we studied multiple signaling pathways involved in cardiac hypertrophy by analyzing their phosphorylation status by Western blot (phosphorylated/total protein). We observed no changes in mTOR, S6K1 and S6 phosphorylation. However, a decrease in p-4EBP1 and p-eIF4B was observed in RC/RC compared to WT. Moreover, we observed a significant increase in p-ERK and p-CaMKII. Our data suggest that alterations in PC1 signaling promote cardiac dysfunction but do not promote hypertrophy in young mice (2-4 months of age). Published evidence (PMID: 32730856) suggest that RC/RC hearts become hypertrophic at 6 months of age. However, our data suggest there may be dysfunction prior to cardiac hypertrophy. This warrants further investigation into the more primary role of ADPKD-associated co-morbidities. More studies, with a larger animal cohort, are necessary to unveil the effects of mutant PC1 on cardiac function.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

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