scholarly journals Estrogen Induces Metastatic Potential of Papillary Thyroid Cancer Cells through Estrogen Receptorαandβ

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Wenwu Dong ◽  
Hao Zhang ◽  
Jing Li ◽  
Haixia Guan ◽  
Liang He ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Metastatic Potential ◽  
Migration And Invasion ◽  
Selective Agonist ◽  
E Cadherin

Estradiol (E2) promotes metastatic propensity. However, the detailed mechanism remains largely unknown. E-cadherin, vimentin, and MMP-9 play a dominant role in the metastatic process. We aimed to investigate the effects of E2 on metastatic potential of PTC cell line BCPAP and on E-cadherin, vimentin, and MMP-9 protein expression. PTC cell line BCPAP was evaluated for the presence of estrogen receptor (ER) by western blot analysis. The effects of E2, PPT (a potent ERα-selective agonist), and DPN (a potent ERβ-selective agonist) on modulation of metastatic phenotype were determined by usingin vitroscratch wound assay and invasion assay. In addition, the effects on E-cadherin, vimentin, and matrix metalloproteinase-9 (MMP-9) protein expression were evaluated by Western blot analysis. We found that BCPAP cells expressed ERαand ERβ. E2 and PPT enhanced, but DPN inhibited, the migration and invasion of BCPAP cells in anin vitroexperimental model system that is modulated by E-cadherin, vimentin, and MMP-9. These findings indicate that E2 induces the metastatic potential of BCPAP cells through ERαand ERβ. The two ER subtypes play differential roles in modulation of BCPAP cell metastasis and the related molecule expressions including E-cadherin, vimentin, and MMP-9.

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

scholarly journals Moderate Autophagy Inhibits Vascular Smooth Muscle Cell Senescence to Stabilize Progressed Atherosclerotic Plaque via the mTORC1/ULK1/ATG13 Signal Pathway

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Zhenli Luo ◽  
Wenhuan Xu ◽  
Sai Ma ◽  
Hongyu Qiao ◽  
Lei Gao ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Foam Cell ◽  
Cell Model ◽  
P53 Protein ◽  
Atherosclerosis Progression

In order to investigate the effects of autophagy induced by rapamycin in the development of atherosclerosis plaque we established murine atherosclerosis model which was induced in ApoE−/− mice by high fat and cholesterol diet (HFD) for 16 weeks. Rapamycin and 3-Methyladenine (MA) were used as autophagy inducer and inhibitor respectively. The plaque areas in aortic artery were detected with HE and Oil Red O staining. Immunohistochemical staining were applied to investigate content of plaque respectively. In contrast to control and 3-MA groups, rapamycin could inhibit atherosclerosis progression. Rapamycin was able to increase collagen content and a-SMA distribution relatively, as well as decrease necrotic core area. Then we used MOVAS and culture with ox-LDL for 72 h to induce smooth muscle-derived foam cell model in vitro. Rapamycin and 3-MA were cultured together respectively. Flow cytometry assay and SA-β-Gal staining experiments were performed to detect survival and senescence of VSMCs. Western blot analysis were utilized to analyze the levels of protein expression. We found that rapamycin could promote ox-LDL-induced VSMCs autophagy survival and alleviate cellular senescence, in comparison to control and 3-MA groups. Western blot analysis showed that rapamycin could upregulate ULK1, ATG13 and downregulate mTORC1 and p53 protein expression.

Synthesis, In Vitro Antitumor Activity and Molecular Mechanism of Novel Furan Derivatives and their Precursors

2020 ◽  
Vol 20 (12) ◽  
pp. 1475-1486
Author(s):  
Dan Lu ◽  
Ya Zhou ◽  
Qin Li ◽  
Juan Luo ◽  
Qihua Jiang ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Antiproliferative Activity ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Proliferative Activity ◽  
Furan Derivatives ◽  
Dicarbonyl Compounds ◽  
Sw620 Cell

Background: Compounds featuring furan nucleus exhibit diverse biological properties. Lots of furan derivatives have been explored as pharmaceutical compounds. Hence it is of great interest to explore furan derivatives and their precursors as antitumor agents. Objective: A series of novel furan derivatives and their precursors (1-36) were synthesized from α-haloketones and β-dicarbonyl compounds. Methods: The reactions between β-dicarbonyl compounds and α-haloketones under basic conditions produced tricarbonyls or dihydrofurans, which were then condensed into their corresponding furan products. Their potential antiproliferative activity in vitro against two human tumor cell lines-cervical (HeLa) and colorectal (SW620) was evaluated using CCK-8 assay. Compounds 1 and 24 were selected for Western blot analysis. Results: Pronounced anti-proliferative effect in the micromolar level was observed for compounds (1, 4, 17, 20, 21, 24, 27, 31 and 32) in HeLa cells, with their IC50 values ranging from 0.08 to 8.79μM. Additionally, furan compounds (24, 26, 32 and 35) had moderate to potent anti-proliferative activity against the SW620 cell line. Furthermore, the possible targets of these compounds were explored by Western blot analysis. The results indicated that the candidates (compounds 1 and 24) exhibited excellent antiproliferative activity, which may be mediated by promoting the activity of PTEN to suppress PI3K/Akt and Wnt/β-catenin signaling. Conclusion: Most of the furan derivatives and their precursors reported herein exhibited moderate to excellent anti-proliferative activity against HeLa cell line and/or SW620 cell line. Compounds 1 and 24, as well as their analogues may be developed as promising anti-cancer agents.

HER4 promotes the growth and metastasis of osteosarcoma via the PI3K/AKT pathway

2020 ◽  
Vol 52 (4) ◽  
pp. 345-362 ◽  
Author(s):  
Xiaodong Li ◽  
Qingshan Huang ◽  
Shenglin Wang ◽  
Zhen Huang ◽  
Fengqiang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Osteosarcoma Cells ◽  
Her4 Protein

Abstract Osteosarcoma is the most common primary malignant bone tumor, which occurs in adolescents. As reported by our previous studies, HER4 indicates a poor prognosis of primary osteosarcoma. However, its mechanisms in the pathogenesis of osteosarcoma have not yet been studied. The purpose of this study was to investigate the role of HER4 in osteosarcoma and whether the PI3K/AKT pathway is involved. In this study, western blot analysis was used to investigate the expression of HER4 protein in osteosarcoma tissues and cell lines. CCK8 and transwell assays were used to detect the effects of HER4 on the proliferation, migration, and invasion of osteosarcoma cells in vitro. The effects of HER4 on the growth and metastasis of osteosarcoma in vivo were detected by tumor formation and immunofluorescence in nude mice. The role of the PI3K/AKT pathway in HER4 regulation of the growth and metastasis of osteosarcoma was examined by western blot analysis and immunofluorescence assay. We found that HER4 protein was highly expressed in clinical osteosarcoma specimens and osteosarcoma cells. HER4 markedly promoted the proliferation, migration, and invasion of osteosarcoma cells in vitro as well as the growth and metastasis of osteosarcoma in vivo. HER4 overexpression upregulated the expression of phosphorylated protein kinase B (pAKT), proliferation marker antigen Ki67, and metastasis cell marker matrix metalloproteinase 9 (MMP9). Notably, PI3K/AKT inhibitor LY294002 significantly inhibited the effects of HER4 via the downregulation of pAKT, Ki67, and MMP9. Moreover, LY294002 markedly blocked the effects of HER4-induced upregulation of tumor malignancy. The present study suggests that HER4 may promote the growth and metastasis of osteosarcoma via the PI3K/AKT pathway. The HER4/PI3K/AKT pathway could serve as a potential target for the treatment of osteosarcoma.

scholarly journals Effect of cigarette smoke on placental antioxidant enzyme expression

2007 ◽  
Vol 293 (2) ◽  
pp. R754-R758 ◽  
Author(s):  
Elizabeth H. Sidle ◽  
Richard Casselman ◽  
Graeme N. Smith
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Cigarette Smoke ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cigarette Smoke Exposure ◽  
Vitro Experiment ◽  
In Vitro Experiment

Cigarette smoking is associated with systemic oxidative stress leading to an upregulation of antioxidant systems [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and heme oxygenase (HO)] in some tissues, but the response in the human placenta is unknown. The aim of this study was to determine the effect of cigarette smoke exposure on placental antioxidant expression in vivo, as well as the effect on antioxidant expression in the human trophoblast choriocarcinoma (HTR)-8SVNeo cell line. In the in vivo experiment, normal-term placentas were obtained following elective caesarean section. The chorionic villi (CV), anchoring villi (AV), and basal plate (BP) were dissected, and Western blot analysis was carried out for HO-1, HO-2, SOD, CAT, and GPx. In vitro experiment, a cigarette smoke extract (CSE) was prepared by bubbling the smoke form three cigarettes through 15 ml of RPMI. This 100% CSE was syringe filtered and diluted to 0.1, 0.5, 1, 2, 5, and 10% concentrations. HTR-8SVNeo cells were cultured with the CSE for 48 h. The cells were harvested, protein was extracted, and run on SDS-PAGE gels, and Western blot analysis was carried out for HO-1, HO-2, SOD, and CAT. Immunofluorescence for HTR-8SVNeo cells HO-1 was carried out following increasing concentrations of CSE. In the in vivo experiment, HO-1 and HO-2 expression was increased in the BP of placentas from smokers compared with nonsmokers. CAT, GPx, and SOD levels in all placental regions, as well as HO-1 and HO-2 expression in the AV and CV were unchanged. In the in vitro experiment, The 5%, 10%, and 20% dilutions were toxic to the cells. The 0.1% CSE solution did not significantly alter HO-1 expression. Treatment with the 0.5%, 1% and 2% CSE solutions resulted in a dose-dependent increase in HO-1 expression. None of the CSE treatments resulted in a significant alteration in HO-2, SOD, GPx, or CAT expression. HO-1 immunoflourescence confirmed the HO-1 expression studies. Cigarette smoke exposure increases HO-1 and HO-2 expression in the placental basal plate and increases HO-1 expression in the HTR-8SVNeo cell line. Increased HO-1 and HO-2 protein expression may increase the production of the antioxidants biliverdin and bilirubin, which are products of heme metabolism. This could function to reduce the oxidative load that is released into the maternal plasma from the preeclamptic placenta and may contribute to the observed decreased incidence of preeclampsia in smokers.

scholarly journals RNF216 Promotes Occurrence and Progression of Cholangiocarcinoma via Regulation of the DIAPH3 Ubiquitination

2020 ◽  
Author(s):  
Jianfei Tu ◽  
Weiqian Chen ◽  
Miaomiao Meng ◽  
Siyu Zhao ◽  
Fazong Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Migration And Invasion ◽  
Metastatic Progression ◽  
Loss Of Function ◽  
Invasion And Migration ◽  
Potential Biomarker

Abstract BackgroundCholangiocarcinoma is a relatively uncommon malignant tumor with high mortality. However, the molecular underpinnings behind malignant progression of cholangiocarcinoma are incompletely understood. Here we demonstrate that RNF216 plays a suppressive role in cholangiocarcinoma occurrence and metastasis.MethodsIHC and Western blot analysis were performed to examine the expression pattern of RNF216 and DIAPH3 in the clinical CRC cholangiocarcinoma. The relationship between RNF216 and DIAPH3 was then validated using in western blot analysis. The mechanism of RNF216-mediated ubiquitination modification of DIAPH3 was analyzed via Co-IP analysis. Gain- or loss-of-function approaches were manipulated to evaluate the modulatory effects of RNF216 and DIAPH3 on cell growth and metastasis. The mediatory effects of RNF216 and DIAPH3 on cancerogenesis were validated in vivo.ResultsClinical data indicated that expression levels of RNF216 were associated with favorable clinical outcomes. RNF216 was downregulated in cholangiocarcinoma and inhibited cell proliferation and colony formation in vitro and xenograft tumorigenicity in vivo. Moreover, RNF216 suppressed Invasion and migration of cholangiocarcinoma. Mechanistic investigations further showed that RNF216 was involved in the ubiquitination of DIAPH3, a member of formin family related to assembly of actin cytoskeleton. RNF216 elicits tumor suppressor role by promoting degradation of DIAPH3. Importantly, expression of DIAPH3 rescued RNF216-mediated suppression of proliferation, cell migration, and invasion. ConclusionOur findings uncover a suppressive role for RNF216 in cholangiocarcinoma proliferation and metastatic progression and provide novel insight into that RNF216 is a potential biomarker or serves as a therapeutic target for cholangiocarcinoma.

scholarly journals E- and N-cadherin expression and distribution during luteinization in the rat ovary

Reproduction ◽  
2003 ◽  
pp. 791-800 ◽  
Author(s):  
NH Machell ◽  
R Farookhi
Keyword(s):  
Western Blot ◽  
Corpus Luteum ◽  
Expression Pattern ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Adult Rats ◽  
Preovulatory Follicles ◽  
E Cadherin

Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control.

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.

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