Novel Molecular Mechanism for Actinomycin D Activity as an Oncogenic Promoter G-Quadruplex Binder

Abstract

Actinomycin D (ActD) is a natural antibiotic that inhibits the transcription of genes by interacting with a GC-rich duplex, a single-stranded or hairpin form of DNA, and then interfering with the action of RNA polymerase. In this study, we identified a novel molecular mechanism of anticancer activity of ActD as an oncogenic c-Myc promoter G-quadruplex binder. ActD selectively inhibits the elongation of oligonucleotides containing c-Myc promoter G-quadruplex sequence in PCR-stop assays. UV-vis spectroscopic and circular dichroism studies suggest that ActD interacts with c-Myc promoter G-quadruplex via a surface end stacking interaction, inducing a mixed-type conformation of the G-quadruplex. ActD selectively inhibits the cellular growth and synthesis of c-Myc mRNA in Ramos cells having the NHEIII(1) region in the translocated c-Myc gene. In addition, the results of promoter assays using two kinds of NHEIII(1) region mutants and wild-type constructs strongly support the idea that binding of ActD with G-quadruplex formed in the promoter region results in the reporter gene being turned off. Our study reveals a novel mechanism underlying the anticancer activity of ActD, whereby ActD interacts with oncogenic promoter G-quadruplex DNA to repress gene expression.

In silico identification of novel ligands for G-quadruplex in the c-MYC promoter

“…Taq DNA polymerase and dNTP mixtures were purchased from Koma Biotech (Korea). The principles and procedures for this experiment were previously described [37]. PCR was performed in a reaction buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM KCl and 1.5 mM Mg (OAc) 2 with 0.2 lM of each oligonucleotide.…”

“…The method for real-time PCR was previously described [37]. Two B-cell lymphoma cell lines, Ramos and CA46, were treated with ligands for 48 h and total RNA was extracted by Easy-Blue RNA extraction kits (Intron Biotechnology).…”

“…PCR was performed using an initial denaturing at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. Melting curves were obtained by 0.5°C/steps and an incubation time of 30 s. The following primers were used for PCR: 5 0 -TCAAGAGGCGA ACACACAAC-3 0 (forward) and 5 0 -GGCCTTTTCATTG-TTTTCCA-3 0 (reverse) for c-MYC and 5 0 -GGACTTCGAGC AAGAGATGG-3 0 (forward) and 5 0 -AGCACTGTGTTGGC GTACAG-3 0 (reverse) for b-Actin [37].…”

“…pGL3-Pu27 had the wild type NHEIII 1 of c-MYC upstream of firefly luciferase gene and pGL3-Pu27Mut had a one-point mutated sequence in this region. The scheme used for cloning these constructs was described in the previous study [37]. HeLa cells (3-4 9 10 4 ) were precultured with Dulbecco's modified Eagle's medium (DMEM) containing 10 % FBS and 1 % penicillin/streptomycin (Hyclone) for 24 h. Cells were transfected with 0.5 lg of pGL3-constructsusing WelFect transfection kits (Welgene) according to manufacturer's instructions.…”

Actinomycin D Induces Phosphorylation of STAT3 through Down-Regulation of SOCS3 in Renal Cancer Cells

“…세포 독성과 항종양성을 가진다고 알려져 있으며 암세포의 DNA에 작용하 는 항암제로 알킬화제에 포함된다 [12,13]. Actinomycin D는 세포자멸사(apoptosis)를 일으키는 강력한 유도물질로 DNA 에 결합하여 유전 정보 전사를 저해한다 [8,13]. 이들 유사 약물 은 생식세포종양(germ cell tumors), 연조직육종(soft-tissue sarcoma), 흑색종(melanoma), 융모암종(choriocarcinoma) 등 의 치료에 사용되는 항암 화학 치료 약물이다.…”