Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells

Velliyur Viswesh, Kent Gates, Daekyu Sun

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC50 values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.

Original languageEnglish (US)
Pages (from-to)99-107
Number of pages9
JournalChemical Research in Toxicology
Volume23
Issue number1
DOIs
StatePublished - Jan 18 2010

Fingerprint

Biological Products
DNA Damage
Cells
Anti-Bacterial Agents
DNA
Neoplasms
DNA Breaks
Reactive Oxygen Species
Assays
Comet Assay
leinamycin
Cell Line
Oxidative stress
Single-Stranded DNA
Guanine
Streptomyces
Bioactivity
Fluorescent Dyes
Cell culture
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Toxicology

Cite this

Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells. / Viswesh, Velliyur; Gates, Kent; Sun, Daekyu.

In: Chemical Research in Toxicology, Vol. 23, No. 1, 18.01.2010, p. 99-107.

Research output: Contribution to journalArticle

@article{0d2dd042316543eda1304e87d30e00f2,
title = "Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells",
abstract = "Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC50 values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50{\%} of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.",
author = "Velliyur Viswesh and Kent Gates and Daekyu Sun",
year = "2010",
month = "1",
day = "18",
doi = "10.1021/tx900301r",
language = "English (US)",
volume = "23",
pages = "99--107",
journal = "Chemical Research in Toxicology",
issn = "0893-228X",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells

AU - Viswesh, Velliyur

AU - Gates, Kent

AU - Sun, Daekyu

PY - 2010/1/18

Y1 - 2010/1/18

N2 - Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC50 values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.

AB - Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC50 values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.

UR - http://www.scopus.com/inward/record.url?scp=75149196885&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=75149196885&partnerID=8YFLogxK

U2 - 10.1021/tx900301r

DO - 10.1021/tx900301r

M3 - Article

C2 - 20017514

AN - SCOPUS:75149196885

VL - 23

SP - 99

EP - 107

JO - Chemical Research in Toxicology

JF - Chemical Research in Toxicology

SN - 0893-228X

IS - 1

ER -