Background
Poly(ADP-ribose) polymerases (PARPs) are activated by DNA single- and double-strand breaks and promote repair of DNA damage through the relaxation of chromatin and recruitment of other repair proteins.1,2,3 Inhibition of PARP activity has been linked to synthetic lethality in cells with mutations in BRCA1 or BRCA2 and is used as a therapeutic strategy to selectively target cancers.4,5 Rucaparib is a potent, cell-permeable inhibitor of PARP1 (Ki = <5 nM) that is used in clinical therapy to sensitize cancer cells to chemotherapy.6,7 Rucaparib inactivates PARP activity in cells with homologous recombination DNA repair pathway mutations at LC50 values ranging from 1.3-5.5 μM.7 At 25 mg/kg, rucaparib arrests tumor growth in mice bearing epigenetically silenced BRCA1 UACC3199 xenograft tumors.7 It has been shown to increase efficacy of temozolomide in medulloblastoma cells and xenografts.6
1.Yuan, Y., Liao, Y.M., Hsueh, C.T., et al.Novel targeted therapeutics: Inhibitors of MDM2, ALK and PARPJ. Hematol. Oncol.4(16)1-14(2011)
2.Javle, M., and Curtin, N.J.The potential for poly (ADP-ribose) polymerase inhibitors in cancer therapyTher. Adv. Med. Oncol.3(6)257-267(2011)
3.Plummer, R.Poly(ADP-ribose) polymerase inhibition: A new direction for BRCA and triple-negative breast cancer?Breast Cancer Res.13(4)1-6(2011)
4.Johnson, N., Li, Y.C., Walton, Z.E., et al.Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibitionNat. Med.17(7)875-882(2011)
5.Rowe, B.P., and Glazer, P.M.Emergence of rationally designed therapeutic strategies for breast cancer targeting DNA repair mechanismsBreast Cancer Res.12(2)1-11(2010)
6.Daniel, R.A., Rozanska, A.L., Mulligan, E.A., et al.Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699Br. J. Cancer103(10)1588-1596(2010)
7.Drew, Y., Mulligan, E.A., Vong, W.T., et al.Therapeutic potential of poly(ADP-ribose) polymerase inhibitor AG014699 in human cancers with mutated or methylated BRCA1 or BRCA2JNCI103(4)334-346(2011)
Protocol
Kinase experiment: | Inhibition of PARP activity in 5×103 D283Med cells is measured using various concentrations of Rucaparib (0-1 μM), compared with DMSO-only. Maximally stimulated PARP activity is measured in samples of permeabilised cells by immunologica[1]. |
Cell experiment: | Medulloblastoma cell lines are seeded in 96-well plates at a density of 1×103, 3×103 and 3×103, respectively. At 24 hours (D384Med) or 48 hours (D283Med and D425Med) after seeding, the cells are exposed to various concentrations of temozolomide in the presence or absence of 0.4 μM Rucaparib. After 3 days (D425Med and D384Med) or 5 days (D283Med) of culture, cell viability is evaluated by a XTT cell proliferation kit assay. Cell growth is expressed as a percentage in relation to DMSO or 0.4 μM Rucaparib-alone controls. The concentration of temozolomide, alone or in combination with Rucaparib that inhibited growth by 50% (GI50) is calculated. The potentiation factor 50 (PF50) is defined as the ratio of the GI50 of temozolomide in the presence of Rucaparib to the GI50 of temozolomide alone[1]. |
Animal experiment: | A single dose of temozolomide is administrated p.o. as a suspension in saline at 200 mg/kg either alone or in combination with a single i.p. administration of PARP inhibitor administered at 0.1 [Rucaparib and MS-AG14644 (equivalent to 0.078 mg/kg free AG14644 only)], 1.0, and 10 mg/kg (for the mesylate salts equivalent to 0.79 and 7.9 mg/kg free AG14451 and AG14452 and 0.78 and 7.8 free AG14531 and AG14644). Control animals are treated with either normal saline p.o. and i.p or normal saline p.o and PARP inhibitor 10 mg/kg i.p[1]. |
参考文献: [1]. Thomas HD, et al. Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial. Mol Cancer Ther, 2007, 6(3), 945-956.
[2]. Hunter JE, et al. NF-κB mediates radio-sensitization by the PARP-1 inhibitor, AG-014699. Oncogene, 2012, 31(2), 251-264.
[3]. Daniel RA, et al. Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699. Br J Cancer, 2010, 103(10), 1588-1596.
[4]. Daniel RA, et al. Inhibition of poly(ADP-ribose) polymerase-1 enhances temozolomide and topotecan activity against childhood neuroblastoma. Clin Cancer Res, 2009, 15(4), 1241-1249. |
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