David Cerna

Enhancement of in vitro and in vivo tumor cell radiosensitivity by valproic acid

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 γH2AX 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. The radiosensitization was accompanied by a prolonged expression of γH2AX. VA administration to mice resulted in a clearly detectable level of histone hyperacetylation in U251 xenografts. Irradiation of U251 tumors in mice treated with VA resulted in an increase in radiation‐induced tumor growth delay. Valproic acid enhanced the radiosensitivity of both SF539 and U251 cell lines in vitro and U251 xenografts in vivo, which correlated with the induction of histone hyperacetylation. Moreover, the VA‐mediated increase in radiation‐induced cell killing seemed to involve the inhibition of DNA DSB repair.

In vitro and In vivo Radiosensitization Induced by the DNA Methylating Agent Temozolomide

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 (γH2AX), 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 0.1 ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. As a measure of DNA double strand breaks, γH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of γH2AX 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.

Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine

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, and 14-3-3σ) 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, γH2AX expression was determined. Whereas zebularine had no effect on radiation-induced γH2AX foci at 1 hour, the number of γH2AX 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 U251 xenografts; irradiation of U251 tumors 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.

Postradiation Sensitization of the Histone Deacetylase Inhibitor Valproic Acid

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 53BP1 foci 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. At 12 h after irradiation, significant γH2AX and 53BP1 foci 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.

Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide]

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 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], 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 radiation-induced 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.

Histone deacetylation as a target for radiosensitization.

Due to an increase in the understanding of molecular radiobiology, strategies for enhancing tumor radiosensitivity have begun to focus on targeting the molecules and processes that regulate cellular radioresponse. Toward this end, histone acetylation has begun to receive considerable attention as a potential target for radiosensitization. Histone acetylation, which is determined by the competing actions of histone acetylases (HATs) and histone deacetylases (HDACs), plays a role in regulating chromatin structure and gene expression--two parameters that have long been considered determinants of radioresponse. As a means of modifying histone acetylation status, considerable effort has been put into the development of inhibitors of HDAC activity, which is often aberrant in tumor cells. This has led to the generation of a relatively large number of structurally diverse compounds that inhibit HDAC activity and result in histone hyperacetylation, and importantly, are applicable to patient treatment. Whereas a number of these HDAC inhibitors have antitumor activity in preclinical cancer models when delivered as single agents, recent studies have indicated that these compounds also significantly enhance tumor cell radiosensitivity. A structurally diverse set of HDAC inhibitors have been shown to enhance the in vitro radiosensitivity of human tumor cell lines generated from a spectrum of solid tumors. Moreover, HDAC inhibitors increased the radiosensitivity of human tumor xenografts. Although the mechanism responsible for this radiosensitization has not been definitely elucidated, data suggest that inhibiting the repair of radiation-induced DNA damage may be involved. Whereas HDAC inhibitors are currently in clinical trials as single modalities and in combination with chemotherapeutic agents, recent results suggest that these compounds may also enhance the antitumor effectiveness of radiotherapy.

ErbB3 Expression Predicts Tumor Cell Radiosensitization Induced by Hsp90 Inhibition

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.

PX-478, an inhibitor of hypoxia-inducible factor-1α, enhances radiosensitivity of prostate carcinoma cells

Overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF‐1α is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF‐1α inhibitor PX‐478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX‐478 for 20 hr, and HIF‐1α protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX‐478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX‐478 decreased HIF‐1α protein in PC3 and DU 145 cells. PX‐478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU‐145. PX‐478 (20 μmol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF‐1α and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 μmol/L concentration. PX‐478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of γH2AX expression in the irradiated cells. PX‐478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX‐478 as a clinical radiation enhancer. Published 2008 Wiley‐Liss, Inc.

Fractionated radiation alters oncomir and tumor suppressor miRNAs in human prostate cancer cells.

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 microarray. 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.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner.

Background: GMX1778 is an inhibitor of nicotinamide phosphoribosyltransferase for the regeneration of NAD+ from nicotinamide. Results: GMX1778 increases intracellular ROS in cancer cells but does not induce ROS in normal cells. Conclusion: Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumor cells. Significance: Selectively modulating intracellular ROS in cancers by GMX1778 provides a useful therapeutic opportunity. Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD+ from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD+ level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.