scholarly journals Excessive Autophagy Activation and Increased Apoptosis Are Associated with Palmitic Acid-Induced Cardiomyocyte Insulin Resistance

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Shanxin Li ◽  
Hui Li ◽  
Di Yang ◽  
Xiuyan Yu ◽  
David M. Irwin ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Western Blot ◽  
Palmitic Acid ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
H9c2 Cells ◽  
Dynamic Changes ◽  
Lc3 Puncta ◽  
Excessive Activation

Diabetic cardiomyopathy (DCM) remains the major cause of death associated with diabetes. Researchers have demonstrated the importance of impaired cardiac insulin signaling in this process. Insulin resistance (IR) is an important predictor of DCM. Previous studies examining the dynamic changes in autophagy during IR have yielded inconsistent results. This study aimed to investigate the dynamic changes in autophagy and apoptosis in the rat H9c2 cardiomyocyte IR model. H9c2 cells were treated with 500 μM palmitic acid (PA) for 24 hours, resulting in the induction of IR. To examine autophagy, monodansylcadaverine staining, GFP-LC3 puncta confocal observation, and Western blot analysis of LC3I-to-LC3II conversion were used. Results of these studies showed that autophagic acid vesicles increased in numbers during the first 24 hours and then decreased by 36 hours after PA treatment. Western blot analysis showed that treatment of H9c2 cells with 500 μM PA for 24 hours decreased the expression of Atg12-Atg5, Atg16L1, Atg3, and PI3Kp85. Annexin V/PI flow cytometry revealed that PA exposure for 24 hours increased the rate of apoptosis. Together, this study demonstrates that PA induces IR in H9c2 cells and that this process is accompanied by excessive activation of autophagy and increases in apoptosis.

SIRT1 Activator 3 Potently Induces Apoptosis in Multiple Myeloma Cell Lines and in Primary Human Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3845-3845
Author(s):  
Tommasina Guglielmelli ◽  
Emilia Giugliano ◽  
Ilaria Defilippi ◽  
Marisa Pautasso ◽  
Roberta Merlini ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
Fixed Dose ◽  
Cell Leukaemia ◽  
Myeloma Cells ◽  
Sirt1 Activator

Abstract Abstract 3845 Poster Board III-781 Introduction Multiple myeloma (MM) is a malignant plasma cells disorder and as far as now remains an incurable disease so that new target therapies are necessary to improve survival of MM patients. Histone deacetylases (HDACs) are enzymes that deacetylate acetyl lysines in histones and various non-histone proteins. Class I and II HDACs have been identified as valid anticancer targets so that clinical studies on their inhibitors as new anticancer agents are ongoing. Much less is known about the consequences of inhibition and activation of the seven members of class III HDACs (sirtuins, SIRT1-7). Recent studies highlight the role of SIRT1 in stress response and cell survival. Increased levels of SIRT1 were observed in normal colon mucosa but not in advanced colon carcinomas. Moreover, SIRT1 activators have been shown to have anti-inflammatory property mediated by TNF-alpha. We have investigated the anti-myeloma activity of SIRT1 activator 3 in RPMI8226 and U226 MM cell lines and in 4 primary human MM cells. Methods In this study RPMI8226 and U226 MM cell lines were used. Bone marrow samples of 3 MM patients were subjected to CD138 immunomagnetic purification for plasmacells enrichment. Peripheral plasmacells from a patients with plasmacell leukaemia dexametasone-melphalan resistant were also collected. SIRT1 activator 3 was used at increasing dose of 50, 100 and 500 μM alone and in combination with 1 μM of dexametasone in MM cell lines and at the fixed dose of 500 μM in primary human plasmacells. Apoptosis has been assayed at 12h, 24h and 48h after treatment in RPMI8226 and U226 cells and at 24h in human plasmacells by flow cytometry evaluating annexin V marker. Western blot analysis was performed to assess the effect of SIRT1 activator 3 agent on NFkB activity (localization of p65 subunit), AKT, p-AKT, mTOR, p-mTOR, Src and p-Src. Results The highest level of apoptosis was observed in RPMI8226 cells with SIRT1 activator 3 agent at the dosage of 500 μM at 24 h. (annexin V positivity 53%, P<0.05). U226 cells resulted sensitive in the same conditions ((annexin V positivity 41%, P<0.05). SIRT1 activator 3 (500 μM) induced significant apoptosis at 24h (range 29-47%) in MM cells of all four patients. The highest level of apoptosis was observed in plasmacells of the patient with plasma cell leukaemia dexametasone-melphalan resistant (47% annexin V positivity) Dexametasone do not significantly enhances SIRT1 activator 3 induced apoptosis in both MM cell lines and primary human MM cells. Western blot analysis demonstrated strong reduction of AKT, mTOR and Src phosphorylation in RPMI8226 and U226 cells as early as 24h after exposure. Conclusion SIRT1 activator 3 induces significant cell death in MM cell lines and primary human myeloma cells in a dose-dependent manner. The mechanisms of SIRT1 activator 3 cytotoxicity are related to down-regulation of AKT, SRC and TOR phosphorylation. Together, these findings may give useful insights into a novel anti-myeloma therapy. Disclosures: Guglielmelli: Celgene: Honoraria; Janssen Cilag: Honoraria. Saglio:Celgene: Honoraria; Novartis: Honoraria; Bristol Myers: Honoraria.

Rab7b suppresses autophagy and induces apoptosis of K562 leukemic cells treated with homoharringtonine

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4884-4884 ◽  
Author(s):  
Peilin Lu ◽  
Donghua He ◽  
Yang Yang ◽  
Pengfei Hu ◽  
Yi Zhao ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Rab Proteins ◽  
Wild Type ◽  
Akt Phosphorylation

Abstract Abstract 4884 Homoharringtonine is an effective anti-leukemia medicine developed by Chinese. It has been found to induce differentiation and apoptosis of leukemia cells. However, the mechanism of its anti-leukemia function has not been fully understood. Rab is a kind of G protein of Ras superfamily and participates in endocytosis and exocytosis of protein transport process. In recent studies, some Rab proteins such as Rab7 are found to be associated with cellular autophagy and apoptosis. We previously identified a new small GTPase homologous to Rab7, named Rab7b, which is selectively expressed in promyeloid and monocytic cells and is localized to lysosome-associated compartments. To investigate the roles of Rab7b in acute myeloid leukemia, we used leukemia cell line K562 as a model in the present study. After treatment of K562 cells with various doses of HHT, cell viability and apoptosis were measured by MTT assay and Annexin V/PI staining respectively. Protein expression of LC3, a marker of autophagy, and caspase3, 9, ERK1/2,Akt were determined by Western blot analysis. Using stable gene transfection, several Rab7b variants, including Rab7b wild-type, active mutant Rab7b-Q67L and localization-deficient mutant Rab7b-ΔCC as well as Rab7b RNAi were transfected in to K562 cells and their roles in regulation of apoptosis in K562 leukemia cells induced by HHT were further evaluated. Our data showed that the viability of the K562 cells was greatly reduced by HHT treatment in a dose- and time- dependant manner. Treatment of the K562 with HHT significantly increases apoptosis in the cells as measured by Annexin V/PI staining. Using Western blot analysis, we further determined that the expression of caspase3, 9 was increased, and ERK1/2 augmented with Akt was suppressed in the cells treated with HHT. After suppressing autophagy with 3-MA, apoptosis was enhanced in the K562 cells treated with HHT. (p<0.05). By constitutively expression of Rab7B and variants in K562 cells, we found that the rate of apoptotic cells are much higher in the K562 cells transfected with Rab7b wild-type and Rab7b-Q67L variants, along with increased expression of caspase3, 9, ERK1/2 and decreased expression of Akt in the transfectants with Rab7b wild-type and active mutant Rab7b-Q67L. Our study suggests that HHT is able to suppress autophagy and enhance apoptosis in K562 leukemia cells in a caspase-dependent way, which is associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Over-expression of Rab7b can enhance HHT induced apoptosis in K562 cells, which may also be associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Taken together, our study elucidates a new recognition for the mechanism of HHT in anti-leukemia therapy and provides a new insight into understanding the relationship between autophagy and apoptosis in leukemia cells induced chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals Protocol for determining protein cysteine thiol redox status using western blot analysis

2021 ◽  
Vol 2 (2) ◽  
pp. 100566
Author(s):  
Bikram Datt Pant ◽  
Sunhee Oh ◽  
Kirankumar S. Mysore
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Redox Status ◽  
Thiol Redox

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

scholarly journals OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Alessandro Bressan ◽  
Francesca Bozzo ◽  
Carlo Alberto Maggi ◽  
Monica Binaschi
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Tandem Repeat ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Ovarian Cancer Cells ◽  
Repeat Region ◽  
Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

scholarly journals DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jiechao Yang ◽  
Liang Zhou ◽  
Yanping Zhang ◽  
Juan Zheng ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Laryngeal Squamous Cell Carcinoma ◽  
Poor Overall Survival

Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.

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