Abstract
Background2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy.ResultsWe found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it.ConclusionsThese results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.
Highlights
poly (ADP-ribose)-polymerase (PARP)-1 is a nuclear enzyme present in eukaryotes in a high copy number
HepG2 cells were synchronized by overnight serum deprivation, treated with 0.5 or 1 μM IK11 for 24 hrs, DNA content of the cells was determined by flow cytometry following propidium iodide staining
By using two different methods, we demonstrated that IK11 induced depolarization of the mitochondrial membrane as early as 30 min after its application, suggesting that both apoptotic and necrotic cell death induced by the drug could be mitochondria-mediated
Summary
PARP-1 is a nuclear enzyme present in eukaryotes in a high copy number. One of its major physiological functions is responding to single- and double-strand DNA breaks and facilitating DNA repair. Inhibition of PARP-1 sensitizes cells to DNA-damaging agents [1] indicating its potentiality in facilitating tumor therapy. It was shown that cells deficient in breast cancer associated gene-1 and −2 (BRCA1/2) are extremely sensitive to PARP-1 inhibition because of the defective double-strand DNA break repair [2] in these cells. PARP inhibition is considered as a useful therapeutic strategy for the treatment of BRCA mutation-associated tumors, and for the treatment of a wider range of tumors bearing a variety of deficiencies in the homologous recombination DNA repair pathway [3]. PARP inhibitors were found to protect cells and tissues in different pathophysiological conditions [4] by various mechanisms including activation of the cytoprotective phosphatidylinositol-3 kinase (PI3K)-Akt pathway [5] that could even impair the efficacy of tumor therapy and mediate drug-resistance [6,7]
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