Valproic acid (VA) is a well-tolerated drug used to treat seizure disorders and has recently been shown to inhibit histone deacetylase (HDAC). Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we investigated the effects of VA on the radiosensitivity of human brain tumor cells grown in vitro and in vivo. The human brain tumor cell lines SF539 and U251 were used in our study. Histone hyperacetylation served as an indicator of HDAC inhibition. The effects of VA on tumor cell radiosensitivity in vitro were assessed using a clonogenic survival assay and ␥H2AX expression was determined as a measure of radiation-induced DNA double strand breaks. The effect of VA on the in vivo radioresponse of brain tumor cells was evaluated according to tumor growth delay analysis carried out on U251 xenografts. Irradiation at the time of maximum VA-induced histone hyperacetylation resulted in significant increases in the radiosensitivity of both SF539 and U251 cells. Key words: valproic acid; radiosensitization; histone deacetylase; in vivo; murine Valproic acid (VA), an 8-carbon branched-chained fatty acid, has well-established efficacy in the treatment of epilepsy and other seizure disorders. 1,2 Its broad-spectrum anticonvulsant activity has been suggested to result from a combination of mechanisms including the increase in ␥-aminobutyric acid, a decrease in ␥-hydroxybutyric acid and a direct interaction with the neuronal membrane blocking voltage dependent sodium channels. 3 Because of its effectiveness, oral bioavailability and generally low toxicity profile VA has been frequently used as a chronic anti-epileptic therapy. 4 Whereas generally free of other major side effects, VA is teratogenic in humans and rodents. In each case administration of VA during pregnancy can result in major malformations, dysmorphic syndromes and an estimated risk of neural tube defects of 1-3%. 5 Structure activity studies using VA and VA-related compounds, however, showed that the teratogenic effects could be separated from the anticonvulsant actions. 6,7 Moreover, the teratogenic activity of VA was attributed to reduced rate of cell proliferation resulting in a disruption in neuroepithelial-mesenchymal interactions. 8 Attempts to define the mechanism responsible for the neural tube defects led to in vitro studies using neuroblastoma cell lines, which showed that VA inhibited cell proliferation and induced morphological differentiation. 9 Subsequent investigations supported a potential anti-cancer action with VA reported to inhibit the proliferation of a variety of human tumor cells grown in vitro and as in vivo xenografts. 10 -12 Valproic acid was shown recently to be an effective inhibitor of histone deacetylase (HDAC). 6,11 Using VA and VA analogs, Phiel et al. 6 showed that the teratogenic but not the anti-epileptic activity of VA is likely due to HDAC inhibition. Moreover, their results along with previous reports 11 indicate that the anti-tumor effects of VA are likely the resu...
Purpose Preclinical studies evaluating histone deacetylase (HDAC) inhibitor-induced radiosensitization have largely focused on the preirradiation setting based on the assumption that enhanced radiosensitivity was mediated by changes in gene expression. Our previous investigations identified maximal radiosensitization when cells were exposed to HDAC inhibitors in both the preradiation and postradiation setting. We now expand on these studies to determine whether postirradiation exposure alone affects radiosensitivity. Experimental Design The effects of the HDAC inhibitor valproic acid (VA) on postirradiation sensitivity in human glioma cell lines were evaluated using a clonogenic assay, exposing cells to VA up to 24 h after irradiation. DNA damage repair was evaluated using γH2AX and 53BP1foci and cell cycle phase distribution was analyzed by flow cytometry. Western blot of acetylated γH2AX was done following histone extraction on AUT gels. Results VA enhanced radiosensitivity when delivered up to 24 h after irradiation. Cells accumulated in G2-M following irradiation, although they returned to baseline at 24 h, mitigating the role of cell cycle redistribution in postirradiation sensitization by VA. At12 h after irradiation, significant γH2AX and 53BP1foci dispersal was shown in the control, although cells exposed to VA after irradiation maintained foci expression. VA alone had no effect on the acetylation or phosphorylation of H2AX, although it did acetylate radiation-induced γH2AX. Conclusions These results indicate that VA enhances radiosensitivity at times up to 24 h after irradiation, which has direct clinical application.
Aberrant DNA hypermethylation is a frequent finding in tumor cells, which has suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145, and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1, and14-3-3r) was determined as a function of zebularine exposure. Zebularine resulted in DNA demethylation in a time-dependent manner, with the maximum loss of methylation detected by 48 hours. Treatment of cells with zebularine for 48 hours also resulted in an increase in radiosensitivity with dose enhancement factors of >1.5. As a measure of radiation-induced DNA damage, gH2AX expression was determined.Whereas zebularine had no effect on radiation-induced gH2AX foci at 1hour, the number of gH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Zebularine administration to mice reactivated gene expression in U251xenografts; irradiation of U251tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay.These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.
Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4Ј-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiationinduced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.
We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by micro-array. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity
Purpose:Temozolomide, a DNA methylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomide has been shown to increase survival rates of patients with malignant gliomas when given combined with radiation, and there is conflicting preclinical data concerning the radiosensitizing effects of temozolomide, we further investigated the possible temozolomide-induced enhancement of radiosensitivity. Experimental Design: The effects of temozolomide on the in vitro radiosensitivity of U251 (a human glioma) and MDA-MB231BR (a brain-seeking variant of a human breast tumor) cell lines was evaluated using clonogenic assay. DNA damage and repair were evaluated using phosphorylated histone H2AX (gH2AX), and mitotic catastrophe was measured using nuclear fragmentation. Growth delay was used to evaluate the effects of temozolomide on in vivo (U251) tumor radiosensitivity. Results: Exposure of each cell line to temozolomide for 1 h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G 2 cell cycle checkpoint. As a measure of DNA double strand breaks, gH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of gH2AX foci per cell was significantly greater at 24 h after the combined modality compared with the individual treatments. Mitotic catastrophe, measured at 72 h, was also significantly increased in cells receiving the temozolomide + irradiation combination compared with the single treatments. In vivo studies revealed that temozolomide administration to mice bearing U251 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay with a dose enhancement factor of 2.8. Conclusions: These results indicate that temozolomide can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.Temozolomide has known anticancer effects against a broad range of tumor histologies including gliomas (1, 2). Temozolomide is a lipophilic molecule that can be given p.o. and crosses the blood-brain barrier. At physiologic pH, temozolomide is converted to the active metabolite methyltriazenoimidazole-carboxamide, which forms methyl adducts at O 6 -position of guanine in DNA. The formation of O 6 -methylguanine then results in mismatch pairing with thymine during subsequent cycles of DNA replication, followed by DNA strand-break formation and eventually cell death (2). Critical to its effectiveness as an antitumor agent is the cellular expression of O 6 -methlyguanine-DNA-methyltransferase (MGMT; refs. 3,4), which removes the O 6 -methyl adducts and, thus, acts to repair temozolomide-induced DNA damage (2, 3). In a recently published study by Stupp et al. (5), patients with primary glioblastoma multiforme (GBM) treated...
The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient. (Cancer Res 2005; 65(15): 6967-75)
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