Quantification of mRNA Using Real-Time PCR and Western Blot Analysis of MAPK Events in Chondrocyte/Agarose Constructs

Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level. However, whether miRNAs regulate EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b rescues impaired EPC angiogenesis in vitro and in vivo via suppressing anti-angiogenic molecule thrombospondin-1 (TSP-1) in type 2 diabetes. Bone marrow-derived EPCs from adult male (C57BLKS/J, 9 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 371.8±37.8 vs. 167.5±21.3 mg/dL, n=38, p<0.05) were used. miRNA processing enzyme Dicer in EPCs was decreased by >40% in db/db vs. db/+ mice (Western blot analysis, n=4 p<0.01), paralleled with >66% reduction of mir-27b expression (real-time PCR, n=4, p<0.05). Both TSP-1 mRNA and protein in EPCs were significantly higher in db/db vs. db/+ mice (real-time PCR, 130.1%, n=4, p<0.05, Western blot analysis, 127.4%, n=4 p<0.05), which were suppressed upon mir-27b mimic transfection (by 75%, real-time PCR and 69%, Western blot analysis, n=4 – 6, p<0.01). EPC-induced angiogenesis was decreased by >70% in db/db vs. db/+ mice (Matrigel tube formation assay, n=4, p<0.05), which was rescued upon mir-27b mimic transfection or silencing TSP-1 expression by its siRNA (both n=4, p<0.05). Furthermore, inhibition of mir-27b in normal EPCs increased their TSP-1 protein by 117.5% (n=6, p<0.05) and impaired their angiogenesis by 81.5% (n=4, p<0.01), both were reversed by silencing TSP-1 expression by its siRNA. Finally, excisional wound closure was markedly delayed in db/db vs. db/+ mice (4-mm punch biopsy, n=4, p<0.05), accompanied by impaired wound angiogenesis (perfusion index by Laser Doppler, n=4, p<0.05). Cell therapy of diabetic EPCs (3×10 5 cells) transfected with mir-27b mimic onto diabetic wounds significantly accelerated their closure rates (n=4, p<0.05 vs. diabetic EPCs alone), with a concomitant augmentation of in vivo wound angiogenesis (n=4, p<0.05). Mir-27b rescues impaired EPC angiogenesis and accelerates wound healing in type 2 diabetic mice, at least in part, via suppressing TSP-1 expression. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

Download Full-text

233 EXPRESSION AND LOCALIZATION OF DNMT1 DURING EARLY BOVINE DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab233 ◽

2005 ◽

Vol 17 (2) ◽

pp. 267 ◽

Cited By ~ 1

Author(s):

D.F. Russell ◽

D.H. Betts

Keyword(s):

Dna Methylation ◽

Embryonic Development ◽

Western Blot ◽

Real Time ◽

Blot Analysis ◽

Real Time Pcr ◽

Western Blot Analysis ◽

Cell Stage ◽

Rna Levels

DNA methylation of CG motifs is an important mechanism of transcriptional regulation. During embryonic development DNA methyltransferase 1 (DNMT1) has been implicated in the maintainance of gametic and embryonic epigenetic patterns. Here we report the characterization of DNMT1 expression patterns within in vitro-produced (IVP) bovine embryos. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and either denuded (germinal vesicle, GV) or matured in vitro for 24 h (metaphase II, MII). Embryos at the 1-, 2-, 4-, 8-, 16-cell stage, and morula (Mo, Day 6 post-insemination, p.i.) and blastocyst (Days 7, 8, and 9 p.i.) stages were produced by in vitro fertilization and cultured in SOFm for appropriate times under a 5% O2, 5% CO2, and 90% N2 atmosphere. Oocytes/embryos were either snap frozen (−80°C) for DNMT1 mRNA and protein expression profiles analysis or wholemount fixed and permeabilized for DNMT1 immunostaining followed by laser confocal microscopy. DNMT1 RNA, determined by real-time PCR analysis, was present throughout bovine embryonic development (replications = 3, n = 10 embryos/pool and normalized by Histone H2A expression). Growing oocytes accumulated DNMT1 transcripts until the MII stage (20-fold increase), whereas after fertilization DNMT1 RNA levels decreased and remained constant until the 16-cell stage when DNMT1 RNA levels decreased further and then remains constant until the blastocyst (Day 8 p.i.) stage. Confocal analysis of DNMT1 immunostained oocytes/embryos (n = 20/stage) revealed that DNMT1 is localized in the cytoplasm of the oocyte and pre-implantation embryo with the exception of the 16-cell stage, when the enzyme is translocated to nucleus (confirmed by Hoechst co-localization). Moreover, a punctuate staining pattern was observed for DNMT1, which could be due to its association with the mitochondria or endoplasmic reticulum. Interestingly, in GV oocytes DNMT1 was present in localized areas of the nucleus, suggestive of nucleoli association. DNMT1 was observed in the majority of the nuclei in early blastocysts, while after expansion, DNMT1 accumulated in the cytoplasm of the trophectoderm and was localized in both the cytoplasm and the nucleus of the majority of cells within the inner cell mass. Western blot analysis revealed low levels of DNMT1 protein in oocytes and pre-implantation embryos with the exception of the 16-cell embryos and Mo stages during which a significant (P < 0.05) increase in the levels of DNMT1 protein was observed. Specificity of primers and conditions for the real-time PCR assay were confirmed by cDNA sequencing whereas specificity of the antibody used for immunofluorescence and western blot analysis was confirmed by amino acid sequencing. These results suggest the participation of DNMT1 in the bovine embryonic genome activation process, supporting a passive DNA demethylation process during the early cleavage stages in the bovine. The low expression profile of DNMT1 after morula stage indicates that other methylases are required for the maintenance of DNA methylation during blastocyst formation and expansion. This work was funded by CFIA, NSERC, CIHR, and OMAF.

Download Full-text

Qualitative and Quantitative Analysis of IDH1 Mutation in Progressive Gliomas by Allele-Specific qPCR and Western Blot Analysis

Technology in Cancer Research & Treatment ◽

10.1177/1533033819828396 ◽

2019 ◽

Vol 18 ◽

pp. 153303381982839 ◽

Cited By ~ 1

Author(s):

Moritz Perrech ◽

Lena Dreher ◽

Gabriele Röhn ◽

Pantelis Stavrinou ◽

Boris Krischek ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Western Blot ◽

Real Time ◽

Blot Analysis ◽

Western Blot Analysis ◽

Idh1 Mutation ◽

World Health ◽

Chain Reaction ◽

Polymerase Chain ◽

Health Organization

To date, diagnosis of IDH1 mutation is based on DNA sequencing and immunohistochemistry, methods limited in terms of sensitivity and ease of use. Recently, the diagnosis of IDH1 mutation by real-time polymerase chain reaction was introduced as an alternative method. In this study, real-time polymerase chain reaction was validated as a tool for detection of IDH1 mutation, and expression levels were analyzed for correlation with course of the disease. A total of 113 tumor samples were obtained intraoperatively from 84 patients with glioma having a diagnosis of diffuse glioma (World Health Organization II), anaplastic glioma (World Health Organization III), secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy (World Health Organization IV). Tumor samples were snap frozen and processed for sectioning and RNA and protein isolation. Presence of IDH1 mutation was determined by DNA sequencing. Hereafter, quantitative expression of IDH1 messenger RNA was assessed using real-time polymerase chain reaction with specific primers for IDH1 mutation and –wt; protein expression was verified by Western Blot analysis and immunohistochemistry. Additionally, 19 samples of low-grade glioma and their consecutive high-grade glioma were analyzed at different time points of the disease. IDH1 mutation was identified in 63% of samples by DNA sequencing. In correlation with the real-time polymerase chain reaction results, a cutoff value was determined. Above this threshold, sensitivity and specificity of real-time polymerase chain reaction in detecting IDH1 mutation were 98% and 94%, respectively. Quantitative analysis revealed that IDH1 mutation expression is upregulated in secondary glioblastoma (mean ± standard error of mean: 3.52 ± 0.55) compared to lower grade glioma (II = 1.54 ± 0.22; III = 1.67 ± 0.23). In contrast, IDH1 wt expression is upregulated in all glioma grades (concentration >0.1) compared to control brain tissue (0.007 ± 0.0016). Western Blot analysis showed a high concordance to both sequencing and real-time polymerase chain reaction results in qualitative analysis of IDH1 mutation status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in secondary glioblastoma. In our study, real-time polymerase chain reaction and Western Blot analysis were found to be highly efficient methods in detecting IDH1 mutation in glioma samples. As cost-effective and time-saving methods, real-time polymerase chain reaction and Western Blot analysis may therefore play an important role in IDH1 mutation analysis in the future. IDH1 mutation expression level was found to correlate with the course of disease to a certain extent. Yet, clinical factors as recurrent disease or prior radiochemotherapy did not alter IDH1 mutation expression level.

Download Full-text

Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells

Blood ◽

10.1182/blood.v116.21.1695.1695 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1695-1695

Author(s):

Norina Tanaka ◽

Yan-Hua Wang ◽

Masayuki Shiseki ◽

Minoko Takanashi ◽

Toshiko Motoji

Keyword(s):

Cell Cycle ◽

Acute Leukemia ◽

Western Blot ◽

Real Time ◽

Blot Analysis ◽

Western Blot Analysis ◽

Caspase 3 ◽

S Phase ◽

Leukemic Cells ◽

Rt Pcr

Abstract Abstract 1695 Introduction: The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods: The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results: A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions: Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽

10.1182/blood.v126.23.4213.4213 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4213-4213

Author(s):

Priya Khoral ◽

Robert J Guo ◽

Jahangir Abdi ◽

Hong Chang

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Western Blot ◽

Real Time ◽

Cell Lines ◽

Blot Analysis ◽

Drug Combination ◽

Western Blot Analysis ◽

Rt Pcr ◽

Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Effects of Electroacupuncture on Ovarian Expression of the Androgen Receptor and Connexin 43 in Rats with Letrozole-Induced Polycystic Ovaries

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/3608062 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Ge Xu ◽

Andong Zhang ◽

Jiandang Liu ◽

Xi Wang ◽

Jiwei Feng ◽

...

Keyword(s):

Androgen Receptor ◽

Western Blot ◽

Real Time ◽

Blot Analysis ◽

Western Blot Analysis ◽

Connexin 43 ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Endocrine Dysfunction ◽

Rt Pcr

Background. Polycystic ovarian syndrome (PCOS) occurs in women of reproductive age and is often characterized by reproductive and endocrine dysfunction. Androgens play a major role in PCOS, and previous studies reported abnormal expression of Connexin 43 (Cx43) in animal models of PCOS, suggesting an association of Cx43 with PCOS pathogenesis. Experimental and clinical evidence indicated that acupuncture may be a safe and effective approach for treating reproductive and endocrine disorders in women with PCOS. This study aimed to determine the effects of electroacupuncture (EA) on PCOS and its relationship with the expression of the androgen receptor (AR) and Cx43. Methods. In total, 30 female Sprague Dawley rats (6 weeks old) were randomly divided into three groups: control group, letrozole (LE) group, and LE + EA group. Rats were administered LE solution (1.0 mg/kg) for 21 consecutive days to induce PCOS. For the LE + EA group, additional EA treatment was conducted (2 Hz, 20 min/d) with “Guanyuan” (CV3) for 14 consecutive days. After hematoxylin-eosin staining, the ovarian structure was observed with an optical microscope, and serum levels of the following hormones were examined via enzyme-linked immunosorbent assay (ELISA): testosterone (T), estradiol (E2), sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH); luteinizing hormone (LH), insulin (INS), anti-Müllerian hormone (AMH), and inhibin B (INHB). Fasting blood glucose (FBG) levels were evaluated using glucose oxidase-peroxidase. Ovarian mRNA and protein expressions of AR and Cx43 were determined by real-time RT-PCR and Western blot analysis. Results. EA was found to restore the cyclicity and ovarian morphology in the PCOS rat model. Serum derived from the LE + EA group showed significant decreases in the levels of T, free androgen index (FAI), LH, LH/FSH ratio, AMH, INHB, and fasting serum insulin (FINS), and significant increases in the levels of E2, FSH, and SHBG. Western blot analysis showed a decreased protein expression of ovarian AR and Cx43; real-time RT-PCR showed reduced expression of ovarian mRNA levels of AR and Cx43. Conclusions. In conclusion, our results showed that EA can ease hyperandrogenism and polycystic ovary morphology in PCOS rats. Furthermore, EA counteracted the letrozole-induced upregulation of AR and Cx43. These results suggested that acupuncture can break the vicious cycle initiated by excessive androgen secretion and may be an effective treatment method for improving the reproductive and endocrine dysfunction caused by PCOS.

Download Full-text

Substance P Promotes the Progression of Endometrial Adenocarcinoma

International Journal of Gynecological Cancer ◽

10.1097/igc.0000000000000683 ◽

2016 ◽

Vol 26 (5) ◽

pp. 845-850 ◽

Cited By ~ 12

Author(s):

Jing Ma ◽

Shifa Yuan ◽

Jianxin Cheng ◽

Shan Kang ◽

Wenhong Zhao ◽

...

Keyword(s):

Substance P ◽

Western Blot ◽

Endometrial Carcinoma ◽

Real Time ◽

Blot Analysis ◽

Western Blot Analysis ◽

Endometrial Adenocarcinoma ◽

Expression Levels ◽

Ishikawa Cells ◽

Proliferation And Invasion

ObjectivesIt has been demonstrated that substance P (SP) promotes while neurokinin-1 receptor (NK-1R) antagonist inhibits the proliferation of several human cancer cells. Currently, it is still unknown whether such actions exist in human endometrial carcinoma. This study aimed to explore the role of SP/NK-1R signaling in the progression of endometrial adenocarcinoma.Materials and MethodsThe expression levels of SP and NK-1R in endometrial adenocarcinoma tissues and Ishikawa cell line were detected by real-time quantitative PCR and Western blot analysis. The effects of SP on Ishikawa cells proliferation and invasion were analyzed using MTT assay and transwell matrigel invasion assay, respectively. The expression levels of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) in Ishikawa cells after administration of SP were detected by real-time quantitative RCR and Western blot analysis.ResultsThe expression levels of SP and NK-1R were significantly higher in endometrial adenocarcinoma tissues and Ishikawa cells than in normal endometrium. Substance P significantly enhanced the proliferation and invasion of Ishikawa cells. In addition, SP induced the expression of MMP-9 and VEGF-C in Ishikawa cells, whereas NK-1R antagonist inhibited these effects.ConclusionsSubstance P plays an important role in the development of endometrial carcinoma by inducing the expression of MMP-9 and VEGF-C and promoting cancer cell proliferation and metastasis, which can be blocked by NK-1R antagonist.

Download Full-text

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

Journal of Neurosurgery ◽

10.3171/2010.7.gks10972 ◽

2010 ◽

Vol 113 (Special_Supplement) ◽

pp. 228-235 ◽

Cited By ~ 4

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.

Download Full-text

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

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200731174216 ◽

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.

Download Full-text

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

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200227092623 ◽

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.

Download Full-text