Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells.
Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells.
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DOI：
10.1021/tx900301r
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发表时间：
2010-01
影响因子：
4.1
通讯作者：
Sun, Daekyu
中科院分区：
文献类型：
--
作者：
Viswesh, Velliyur;Gates, Kent;Sun, Daekyu
莱那霉素是一种结构新颖的链霉菌衍生天然产物，对各种人类癌细胞系显示出非常有效的活性（IC 50值在低纳摩尔范围内）。先前的体外生物化学研究表明，来那霉素烷基化DNA，产生脱嘌呤（AP）位点和活性氧（ROS），并导致DNA链断裂。但是，目前还不清楚这些事件是否发生在细胞内。在本研究中，我们已经确定了内源性的AP位点和DNA链断裂的基因组DNA和氧化应激的量在人胰腺癌细胞系，MiaPaCa，用莱那霉素处理，通过利用酶反应探针（阿普）测定，彗星试验，和荧光探针，分别。我们证明了AP位点在暴露于莱那霉素后迅速形成，并且AP位点的数量以剂量依赖性方式增加高达7倍。然而，在吸出含有药物分子的培养基并用新鲜培养基替换后2小时，这些位点中只有25-50%保留。我们还观察到来那霉素诱导的ROS产生和伴随的MiaPaCa细胞凋亡的增加。由于AP位点和ROS都有可能在细胞DNA中产生链断裂，因此彗星试验用于检测来那霉素处理的MiaPaCa细胞培养物中对核DNA的损伤。碱性和中性电泳分析表明，莱那霉素产生单，双链DNA损伤的药物处理的细胞在剂量依赖性的方式。两者合计，结果表明，快速转化的来那霉素-鸟嘌呤（N7）加合物到AP位点，产生DNA链断裂，协同来那霉素衍生的ROS，占这种天然产物的非常有效的生物活性。
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 (ARP) 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 was 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, account for the exceedingly potent biological activity of this natural product.
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影响因子：
5.6
作者：
Berquist, Brian R.;McNeill, Daniel R.;Wilson, David M., III
通讯作者：
Wilson, David M., III
DOI：
10.1080/07328319808005194
发表时间：
1998-01-01
期刊：
NUCLEOSIDES & NUCLEOTIDES
影响因子：
--
作者：
Asaeda, A;Ide, H;Kubo, K
通讯作者：
Kubo, K
影响因子：
4.1
作者：
BAILLY, V;VERLY, WG
通讯作者：
VERLY, WG
影响因子：
15
作者：
Asai, A;Hara, M;Saitoh, Y
通讯作者：
Saitoh, Y
影响因子：
3.6
作者：
Breydo, L;Gates, KS
通讯作者：
Gates, KS