scholarly journals Effect of Epigenetic Drug Candidate Olsalazine on the Expression of CDH1 and uPA Genes in MCF-7 Breast Cancer Cell Line

2020 ◽  
Vol 15 (4) ◽  
Author(s):  
Mojgan Naghitorabi ◽  
Ghasem Saki ◽  
Sedighe Gharishvandi
Keyword(s):  
Mtt Assay ◽  
Cancer Cell Line ◽  
Dna Methyltransferases ◽  
Pcr Analysis ◽  
Drug Candidate ◽  
Rt Pcr ◽  
Epigenetic Drug ◽  
Ic50 Values ◽  
Polymerase Chain ◽  
Mcf 7

Background: A main epigenetic change in cancer is DNA methylation, which leads to the inactivation of tumor suppressor genes. Due to its reversible nature, many studies have focused on how to correct epigenetic imbalances via inhibiting DNA methyltransferases (DNMTs). Recent studies have shown that olsalazine can be a potent candidate for DNMT inhibition. Objectives: The current study aimed to assess the cytotoxic effect of olsalazine on MCF-7 cells and the expression of CDH1 and uPA, as cancer-related genes, compared to decitabine. Methods: The cytotoxicity of olsalazine and decitabine on MCF-7 cells was assessed by MTT assay. To evaluate the effect of drugs on the expression of CDH1 and uPA genes, MCF-7 cells were treated with olsalazine and decitabine in concentrations below their IC50 values. After 24 h, RNA of treated cells was extracted and then subjected to a quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Results: The MTT assay showed that olsalazine was more toxic (IC50 = 1.75 mM) in MCF-7 cells than decitabine (IC50 = 3mM). Q-RT-PCR analysis showed that olsalazine can significantly increase uPA expression along with a non-significant increase in CDH1 expression. Meanwhile, no significant change was found in gene expression after treatment with decitabine. Conclusions: This study demonstrated that olsalazine was more cytotoxic than decitabine on MCF-7 cells. Also, compared to decitabine, olsalazine could increase the expression of CDH1 and uPA genes. It suggests that olsalazine might be more potent than decitabine in inhibiting DNMTs, although further studies are needed.

scholarly journals Expanding the Structural Diversity of DNA Methyltransferase Inhibitors

2020 ◽  
Vol 14 (1) ◽  
pp. 17
Author(s):  
K. Eurídice Juárez-Mercado ◽  
Fernando D. Prieto-Martínez ◽  
Norberto Sánchez-Cruz ◽  
Andrea Peña-Castillo ◽  
Diego Prada-Gracia ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Dna Methyltransferase ◽  
Structural Diversity ◽  
Dna Methyltransferases ◽  
Dna Methyltransferase Inhibitors ◽  
Epigenetic Drug ◽  
Ic50 Values ◽  
Dietary Component ◽  
Approved Drugs ◽  
Epigenetic Processes

Inhibitors of DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug discovery. They are also chemical tools to understand the biochemistry of epigenetic processes. Herein, we report five distinct inhibitors of DNMT1 characterized in enzymatic inhibition assays that did not show activity with DNMT3B. It was concluded that the dietary component theaflavin is an inhibitor of DNMT1. Two additional novel inhibitors of DNMT1 are the approved drugs glyburide and panobinostat. The DNMT1 enzymatic inhibitory activity of panobinostat, a known pan inhibitor of histone deacetylases, agrees with experimental reports of its ability to reduce DNMT1 activity in liver cancer cell lines. Molecular docking of the active compounds with DNMT1, and re-scoring with the recently developed extended connectivity interaction features approach, led to an excellent agreement between the experimental IC50 values and docking scores.

scholarly journals TRIGONELLA FOENUM-GRAECUM L. EXHIBITS ESTROGENIC EFFECT THROUGH PRESENILIN 2 GENE EXPRESSION IN THE BREAST CANCER CELL LINE MCF-7

2019 ◽  
Vol 12 (1) ◽  
pp. 438
Author(s):  
Kurnia Agustini ◽  
Michael Wink ◽  
Wahono Sumaryono ◽  
Frans Suyatna ◽  
Nurjati Chairani Siregar
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Cell Line ◽  
Active Fraction ◽  
Rt Pcr ◽  
Fenugreek Seeds ◽  
Trigonella Foenum Graecum ◽  
Presenilin 2 ◽  
Trigonella Foenum Graecum L ◽  
Mcf 7

Objective: The objective of this study is to investigate the estrogenic and antiestrogenic activity of Fenugreek seeds, Trigonella foenum-graecum L. in the estrogen-dependent breast cancer cell line, MCF-7, including its effect on the expression of estrogen-dependent presenilin 2 (pS2) gene.Methods: An activity guided fractionation was carried out with extracts from fenugreek seeds in MCF-7 cells. Cytotoxic activity assays were conducted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Most fractions were also tested also tested in media with estradiol 10 nM We also analysed the expression of pS2 gene. For the analysis of pS2 gene expression we employed PCR primers for pS2 and for β-actin as a housekeeping gene using real-time polymerase chain reaction (RT-PCR).Results: Based on cytotoxic activity assay in MCF-7, the active fractions are ethyl acetic fraction and its phases ethyl acetic (EA) 2 and EA 2.2. The most active fraction was EA 2.2 (IC50=27.129 ppm), which exhibited a biphasic effect; at low concentrations, it stimulated the growth, and at high concentrations it showed strong cytotoxic effects. EA2.2 fraction in concentration 20 ppm, also could induce pS2 gene expression in media with and without estrogen.Conclusion: The most active fraction was the ethyl acetate phase and further subfractions. The most active fraction also induced the expression of pS2 gene which was studied by RT-PCR.

scholarly journals Regulation of Chloride Intracellular Channel Protein 1 and Caspase -3 mRNA Expression by Hydroethanolic Extract of Aegle marmelos Fruit Human Breast Cancer Cell Line-MCF-7

2021 ◽  
pp. 586-593
Author(s):  
S. Dhivya ◽  
R. Gayatri Devi ◽  
J. Selvaraj ◽  
A. Jothi Priya
Keyword(s):  
Breast Cancer ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Human Breast ◽  
Rt Pcr ◽  
Aegle Marmelos ◽  
Hydroethanolic Extract ◽  
The Given ◽  
Intracellular Channel ◽  
Mcf 7

Introduction: Cancer is the second leading cause of death all over the world where among all types of cancer breast cancer is said to be the leading cancer followed by lung cancer. The aim of this study is to find the regulation of chloride intracellular channel protein 1 and caspase -3 mRNA expression by hydroethanolic extract of Aegle marmelos fruit human breast cancer cell line-MCF-7. Materials and methods: MCF-7 cells were collected from NCCS Pune, India. It is stored in Dubecos Modified Eagle's Medium. The Aegle marmelos fruit was collected from the herbal department and its extract was prepared. The extract of Aegle marmelosis used in treating MCF-7 cells at different dosages in in vitro.  Isolation of total RNA from MCF-7 cells. The cells will be mixed with total RNA isolation reagent, sonicated and RNA will be isolated as per the standard method. c-DNA conversion and real time polymerase chain reaction. The c-DNA will be synthesized using reverse transcription by commercially available (RT-PCR) kit. Two microlitres of c-DNA will be used for amplification of clic-1 and caspase-3 using gene specific primers by commercially available RT-PCR kit (SyBr kit) and comparative CT method will be used to see the expression of genes. Untreated MCF-7 cells were compared with MCF-7 cells treated with various concentrations of the extract (10, 20 and 40ug). The statistical data’s were collected from the SPSS software version 21. Result: The given extract inhibits the proliferation of MCF-7 cells therefore said to have antiproliferative activity. Different doses of extract were tested (200ug-500ug) out of which 400ug of extract were preferred. Conclusion: The given plant extract has anti proliferative properties and hence can be used as a drug to treat breast cancer.

IN-SILICO AND IN VITRO ASSESSMENT OF SYNTHESIZED DIAZENYLSULFONAMIDES AS APOPTOSIS INDUCERS AND RADICAL SCAVENGERS

INDIAN DRUGS ◽  
2020 ◽  
Vol 57 (06) ◽  
pp. 49-59
Author(s):  
Priyambada Kshiroda Nandini Sarangi ◽  
Jyotirmaya Sahoo ◽  
Chita Ranjan Sahoo ◽  
Sudhir Kumar Paidesetty ◽  
Guru Prasad Mohanta
Keyword(s):  
Cell Line ◽  
Cancer Cell ◽  
Radical Scavenging ◽  
Cancer Cell Line ◽  
Scavenging Effect ◽  
Ic50 Values ◽  
Radical Scavenging Effect ◽  
The Individual ◽  
Mcf 7

A series of eight quinoline-thiazole hybrid-bearing diazenylsulfonamides, 4a-4h, were synthesized and characterized by UV-Vis, FT/IR, 1H NMR and lC-MS. These compounds were formed when two prepared intermediate precursors of Schiff-base compounds, (E)-N-((2-chloroquinolin-3-yl)methylene)-4phenylthiazol-2-amine (3a) and (E)-N-((2-chloroquinolin-3-yl)methylene)-4-chlorophenylthiazol-2-amine (3b) were converted to the corresponding diazenyl compounds 4a-4h by treating and coupling with the individual diazonium salts of sulfa-drugs. The results of in vitro cytotoxic activity of the synthesized compounds in two cancer cell lines MCF 7 (human breast cancer cell line) and K562 (myelogenousleukemia cell line) have shown the IC50 values as given: 4b against MCF 7 19.52 and against K562 20.55µM; 4d against MCF 7 15.96 and against K562 13.05µM. Moreover, the compound 4-(((Z)-(2-chloroquinolin-3yl)(4-phenylthiazol-2-ylimino)methyl)diazenyl)benzenesulfonic acid (4d) induced maximum percentage of apoptosis. Furthermore, the in vitro antioxidant activity study revealed that among all the synthesized compounds, compound 4d has an excellent radical scavenging effect. Molecular docking was additionally performed to investigate the binding affinity of H-bonding interaction of synthesized compounds with a targeted enzyme and to compare it with the anticancer drugs, dasatinib, bosutinib and dacarbazine.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Prospective Multi-Institutional Study of Reverse Transcriptase Polymerase Chain Reaction for Molecular Staging of Melanoma

2006 ◽  
Vol 24 (18) ◽  
pp. 2849-2857 ◽  
Author(s):  
Charles R. Scoggins ◽  
Merrick I. Ross ◽  
Douglas S. Reintgen ◽  
R. Dirk Noyes ◽  
James S. Goydos ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Peripheral Blood ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Molecular Staging ◽  
Polymerase Chain

Purpose To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream. Patients and Methods In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma ≥ 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant–DFS (DDFS), and overall survival (OS) were analyzed. Results A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR–positive (n = 620) and RT-PCR–negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. Conclusion In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

scholarly journals REAL TIME POLYMERASE CHAIN REACTION (RT-PCR) FOR DNA PIG CONTAMINATION IDENTIFICATION SAUSAGE IN SIX DISTRICT PANDEGLANG BANTEN

2021 ◽  
Vol 1 (1) ◽  
pp. 81-88
Author(s):  
Hadi Susilo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Product ◽  
Meat Products ◽  
Pcr Analysis ◽  
Processed Meat ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Dna Purity ◽  
Polymerase Chain

Sausage is a meat product processed that is popular food especially in Pandeglang, Banten Province. The importance of halal certificates or the existence of the MUI (Indonesian Ulama Council) halal logo for processed meat products makes Muslim people confident to consume them. The aim this research was to identify pig DNA contamination in sausage products in six  districts in Pandeglang without the MUI halal labels using RT-PCR (Real Time-Polymerase Chain Reaction). RT PCR that can calculate to pig to fill these sample free from pig contamination. This research was divided into two stage, the first stage is extracted or carried out DNA and the second stage is RT PCR analysis. The results of the DNA purity test on sausage samples had DNA purity values ​​of 1.84-1.9 (A260 / A280) and resulted in sample concentrations ranging from 37.8 to 102.5 ng / µl.  The only amplification on the FAM curve was in the positive control pig.  the Cq value ranges from 30 - 31.29. The results of RT PCR on sausage samples in the district area in Pandeglang Banten did not detect the presence of pig DNA.

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