Cancer therapy strategies that selectively target tumors while sparing normal tissues would provide significant clinical benefit to cancer patients. GMX1777/1778 is a potent and specific inhibitor of the nicotinamide adenine dinucleotide (NAD+) biosynthesis enzyme, phosphoribosyl transferase (NAMPT). Since cancer cells have a high rate of NAD(+) turnover, modulation of NAD(+) biosynthesis would be an attractive target for GMX 1777/1778. Addition of exogenous nicotinic acid (NA) restored NAD+ level via nicotinic acid phosphoribosyltransferase 1 (NAPRT1). During the course of this study we observed that in addition to the depletion of NAD(+) level, GMX 1777/1778 also induced oxidative stresses by specifically increasing the superoxide level in certain cancer cells. Since NAD(+) is also involved in reducing oxidative stresses, we reasoned that the lack of NAPRT1 enzyme in certain cancers would provide cancer specific oxidative stresses. Furthermore, since p53 plays a critical role in mitigating cellular oxidative stresses for the survival of normal and certain cancer cells, we also investigated the effect of p53 status for the survival of cancer cells defective in NAPRT1. Steady-state levels of ROS were measured using DHE after cells were treated with GMX1777/1778 with or without niacin. Glutathione [reduced (GSH) plus oxidized (GSSG)] content was measured by GSH Detection Kit. Apoptosis was measured by Guava EasyCyte Plus as per the manufacturer's protocol. Status of NAPRT1 and p53 expression was determined by western-blot analysis. Intracranial orthotopic brain model was used to determine the efficacy of GMX1777/1778 on glioblastomas lacking NAPRT1 and p53. The oxidative stresses induced by GMX1777/1778 were maintained even in the presence of nicotinic acid (NA) when the NAPRT1 was lacking. Of critical importance, the induction of oxidative stresses in normal tissues are lacking, suggesting improved therapeutic index by GMX 1777/1778. The absence of functional p53 further increased the effect of GMX1777/1778 on cancer cells with minimal effect on normal cells. Since most of the glioblastomas lack the NAPRT1, the potential of GMX1777/1778 was investigated using an intracranial orthotopic brain tumor model. The result presented here suggests clear clinical benefit of prolonged survival in mice treated with GMX1777/1778 and ionizing radiation with minimal toxicities to normal tissues. Taken together, these results clearly showed that GMX1777/1778 has a strong potential as a molecular targeted agent specifically targeting non-oncogenic addiction of certain tumors with minimal collateral damage to normal tissues.

A comparison was made between two K vitamin analogs. Growth in vitro of Hep G2 hepatoma cells was inhibited both by Compound 5 (Cpd 5), a recently synthesized thioalkyl analog of vitamin K or 2-(2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone, as well as by synthetic vitamin K3 (menadione). Using synchronized Hep G2 hepatoma cells, the actions of both Cpd 5 and vitamin K3 on cell cycle regulating proteins were examined. Cpd 5 decreased the levels of cyclin D1, Cdk4, p16, p21 and cyclin B1. By contrast, VK3 only decreased the level of cyclin D1, but had no effect on the levels of Cdk4, p16 or p21. Interestingly, both VK3 and VK2 increased the levels of p21. The naturally occurring K vitamins had little effect on cell growth and none on the cyclins or Cdks. Amounts and activity of the G1/S phase controlling Cdc25A were measured. We found that Cpd 5 directly inhibited both Cdc25A activity and its protein expression, whereas VK3 did not. Thus, the main effects of Cpd 5 were on G1 and S phase proteins, especially Cdk4 and Cdc25A amounts in contrast to VK3. Computer docking studies of Cpd 5 and VK3 to Cdc25A phosphatase showed three binding sites. In the best conformation, Cpd 5 was found to be closer to the enzyme active site than VK3. These findings show that Cpd 5 represents a new class of anticancer agent, being a protein tyrosine phosphatase (PTP) antagonist, that binds to Cdc25A with suppression of its activity. Tumors expressing high levels of oncogenic Cdc25A phosphatase may thus be susceptible to the growth inhibitory activities of this class of compound.

Purpose: Because ribonucleotide reductase (RR) plays a role in DNA repair, it may serve as a molecular target for radiosensitization. Unlike previously investigated RR inhibitors, Triapine potently inhibits both RR holoenzymes. Therefore, the effects of Triapine on tumor cell radiosensitivity were investigated.Experimental Design: The effects of Triapine on the in vitro radiosensitivity of three human tumor cell lines and one normal cell line were evaluated using a clonogenic assay. Growth delay was used to evaluate the effects of Triapine on in vivo tumor radiosensitivity. The levels of the RR subunits were determined using immunoblot analysis and DNA damage and repair were evaluated using gamma H2AX foci.Results: Exposure of the tumor cell lines to Triapine before or immediately after irradiation resulted in an increase in radiosensitivity. In contrast, Triapine enhanced the radiosensitivity of the normal fibroblast cell line only when the exposure was before irradiation. There were no consistent differences between cell lines with respect to the expression of the RR subunits. Whereas Triapine had no effect on radiation-induced gamma H2AX foci at 1 hour, the number of gamma H2AX foci per cell was significantly greater in the Triapine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Triapine administration to mice bearing tumor xenografts immediately after irradiation resulted in a greater than additive increase in radiation-induced tumor growth delay.Conclusions: These results indicate that Triapine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.

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 14-3-3 sigma) 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, gamma H2AX expression was determined. Whereas zebularine had no effect on radiation-induced gamma H2AX foci at 1 hour, the number of gamma H2AX 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 micetreated 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.

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.

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 gammaH2AX 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 gammaH2AX. 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.