| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 103 === I. Combination therapy of SAHA and PARP inhibitors in HCC treatment
Genomic instability is one of the cancer hallmarks. Because severe DNA damage results in cell death, inhibitors of the DNA repair system can be used as anticancer drugs. An inhibitor of histone deacetylases (HDACs) - suberoylanilide hydroxamic acid (SAHA), is being evaluated for phase2/3 clinical trial to treat several types of cancers. Mechanism of SAHA-induced cell death is not totally clear, but reactive oxygen species (ROS) release and double strand break (DSB) may play a role in apoptosis of SAHA-treated cells. Upon DNA damage, p53 activates its downstream target genes and triggers DNA repair or apoptosis. In our previous study, mutation of TP53 and down-regulation of PARP4 were found to be associated with chromosome 4q loss of heterozygosity in hepatocellular carcinoma (HCC), implicating that sensitivity to DNA-damaging agents in HCC cells might depend on the TP53 status and PARP4 expression level. A pan-PARP inhibitor 3-aminobenzamide (3-ABA), known to inhibit the single strand break repairing system by targeting PARP1/2, was chosen as a candidate to be combined with SAHA for treating HCC cells with TP53 mutation. As a result of impaired DNA repair, DSB and apoptosis were enhanced, as measured by γ-H2AX staining and caspase 3/PARP1 cleavage, respectively. Sensitivity to SAHA and 3-ABA was reduced in TP53 knocked down or mutant TP53 stably transfected HepG2 cells. Depending on genetic background, SAHA/3-ABA-induced cytotoxicity was variable and could be attributed to cell cycle arrest and cell death. In HepG2 cells, SAHA-induced up-regulation of CDKN1A was enhanced by 3-ABA. In Hep3B cells, TXNIP level was further elevated by 3-ABA, contributing to ROS release and DSB. Consistently, N-acetylcysteine, a ROS inhibitor, reduced the expression of the targeted genes, GRP94 and CHOP, of unfolded protein response, and blunted SAHA/3-ABA induced cell death. Another PARP inhibitor rucaparib, when combining with SAHA, induced prominent cell death, and it was accompanied by up-regulation of p21 (WAF1/CIP). Unlike 3-ABA, rucaparib did not enhance SAHA-induced DSB. Thus our results support that multiple pathways and effectors are involved in SAHA-induced cancer cell death, and, contingent on TP53, a new strategy of therapeutic development can be designed by adding PARP inhibitors, to achieve personalized medicine for HCC.
II. Cellular characterization of PARP4
Poly(ADP-ribose) polymerase family, member 4 (PARP4) catalyzes poly(ADP-ribosyl)ation PARsylation of numerous proteins localized in cytosol and nuclei. In previous studies, PARsylation of targeted proteins were known to be involved in regulating DNA damage detection, DNA repair, and cell death pathways. To investigate whether PARP4 also participates in the maintenance of genome integrity, subcellular localization of PARP4 was analyzed. Nuclear translocation of PARP4 was observed with DSBs induced by various DNA-damaging agents. In H2O2-treated HeLa cells, PARP4 localized with γ-H2AX, a DSB marker, while in etoposide-treated HEK293T cells, co-localization of PARP4 and PAR was observed. Notably, PARP4 nuclear localization is an early event and it became detectable by fluorescence microscopy two hours after addition of 4 μM SAHA. PARP4 knockdown enhanced DSBs and cell death in SAHA/3-ABA-treated cancer cells. Finally, SAHA targeted on PARP4 by down-regulation of mRNA and protein expression, suggesting that enhancement of cell death by combining SAHA with PARP inhibitors might work through their effects on the PARP4 level and DNA repair. Taken together, our data support that PARP4 may function in the maintenance of genome integrity, and it could be served as a target to design anticancer drugs.
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