Vorinostat (suberoylanilide hydroxamic acid), a histone deacetylase inhibitor, is currently undergoing clinical evaluation as therapy for cancer. We investigated the effects of vorinostat on tumor cell radiosensitivity in a breast cancer brain metastasis model using MDA-MB-231-BR cells. In vitro radiosensitivity was evaluated using clonogenic assay. Cell cycle distribution and apoptosis was measured using flow cytometry. DNA damage and repair was evaluated using γH2AX. Mitotic catastrophe was measured by immunostaining. Growth delay and intracranial xenograft models were used to evaluate the in vivo tumor radiosensitivity. Cells exposed to vorinostat for 16 hours before and maintained in the medium after irradiation had an increase in radiosensitivity with a dose enhancement factor of 1.57. γH2AX, as an indicator of double-strand breaks, had significantly more foci per cell in the vorinostat plus irradiation group. Mitotic catastrophe, measured at 72 hours, was significantly increased in cells receiving vorinostat plus irradiation. Irradiation of s.c. MDA-MB-231-BR tumors in mice treated with vorinostat resulted in an increase in radiation-induced tumor growth delay. Most importantly, animals with intracranial tumor implants lived the longest after combination treatment. These results indicate that vorinostat enhances tumor cell radiosensitivity in vitro and in vivo. There was a greater than additive improvement in survival in our intracranial model. Combining vorinostat with radiation may be a potential treatment option for patients with breast cancer who develop brain metastases. [Mol Cancer Ther 2009;8(6):1589–95]

Overexpression or activation of Src family kinases (SFKs) has been implicated in a number of different cancers. Because SFKs are involved in multiple cellular signaling pathways, including cell division, survival, motility, and invasion, development of SFKs inhibitors is ongoing. Here, we examine the effect of one such agent AZD0530 on response to radiation (IR) in a glioblastoma multiforme cell line. U251 human GBM cell line was treated for 16 hours with AZD0530 (3μmol/L) pre-irradiation in all in vitro experiments. The effects of AZD0530 on the in vitro radiosensitivity of U251 was evaluated using clonogenic assay. DNA damage and repair were evaluated using γH2AX at 1, 6 and 24 hours after 2 Gy IR. Neutral comet assay was performed at 0, 3, 6, 16, 24 hours after 10 Gy IR for further evaluate DNA damages and repair. Mechanism of cell death after DNA damage was determined by immunostaining for mitotic catastrophe and flow cytometry for apoptosis. Drug induced cell cycle changes were evaluated by staining of phospho-H3 and flow cytometry. Exposure of U251 to AZD0530 for 16 hour prior to radiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 of 1.31. AZD0530 had no effect on the number of cells in each phase of the cell cycle nor on the activation of the G2 cell cycle checkpoint after IR. As a measure of DNA double strand breaks, γH2AX foci were determined as a function of time after the AZD0530 + IR combination. The number of γH2AX foci per cell was significantly greater at 24 hours after the combined modality as compared to the individual treatments. Mitotic catastrophe, measured at 72 hours, was also significantly increased in cells receiving the AZD0530 + IR combination as compared to the single treatments. These results indicate that AZD0530 can enhance tumor cell radiosensitivity in vitro and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.

Purpose/Objective(s)Thalidomide has anti-inflammatory and antiangiogenic effects, and it is widely used to treat both autoimmune diseases and cancer. However, it has toxic side effects, thus, novel thalidomide analogs have been developed to both enhance activity and reduce toxicity. We have investigated the effects of one such analog (CPS45) on the radiosensitivity of two human tumor cell lines (MiaPaCa pancreatic cancer and U251 glioma).Materials/MethodsClonogenic survival assays were used to evaluate cell survival after exposure to CPS45 followed by radiation. DNA damage and repair were evaluated using phosphorylated histone H2AX (γH2AX) and the neutral comet assay. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-H3 and flow cytometry. In vivo activity was measured using a tumor growth delay assay.ResultsExposure of each cell line to CPS45 for 16 h before irradiation (IR) resulted in an increase in radiosensitivity with dose enhancement factors of 1.8 and 1.5 for U251 and MiaPaCa cells, respectively (PF = 0.43, 0.31 respectively in CPS45 treated cells). CPS45 had no effect on IR-induced apoptosis or on the activation of the G2 cell cycle checkpoint. However, CPS45 did modify the time course of γH2AX expression in IR cells. There was significant difference in IR-induced γH2AX foci count at both 6 h and at 24 h after IR in the CPS45 + IR group than either individual treatment. In the neutral comet assay, there was a significant increase in the percentage of DNA damage remaining in cells exposed to CPS45 before IR compared to IR alone. Mitotic catastrophe, measured at 48 and 72 hour, was also significantly increased in cells receiving the CPS45 + IR combination compared with the individual treatments. Tumor growth delay demonstrated a greater than additive effect of the combination of CPS45 and IR than either treatment alone.ConclusionsThese results indicate that CPS45 can enhance tumor cell radiosensitivity and suggest that this effect involves an inhibition of double strand breaks repair leading to an increase in mitotic catastrophe. Purpose/Objective(s)Thalidomide has anti-inflammatory and antiangiogenic effects, and it is widely used to treat both autoimmune diseases and cancer. However, it has toxic side effects, thus, novel thalidomide analogs have been developed to both enhance activity and reduce toxicity. We have investigated the effects of one such analog (CPS45) on the radiosensitivity of two human tumor cell lines (MiaPaCa pancreatic cancer and U251 glioma). Thalidomide has anti-inflammatory and antiangiogenic effects, and it is widely used to treat both autoimmune diseases and cancer. However, it has toxic side effects, thus, novel thalidomide analogs have been developed to both enhance activity and reduce toxicity. We have investigated the effects of one such analog (CPS45) on the radiosensitivity of two human tumor cell lines (MiaPaCa pancreatic cancer and U251 glioma). Materials/MethodsClonogenic survival assays were used to evaluate cell survival after exposure to CPS45 followed by radiation. DNA damage and repair were evaluated using phosphorylated histone H2AX (γH2AX) and the neutral comet assay. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-H3 and flow cytometry. In vivo activity was measured using a tumor growth delay assay. Clonogenic survival assays were used to evaluate cell survival after exposure to CPS45 followed by radiation. DNA damage and repair were evaluated using phosphorylated histone H2AX (γH2AX) and the neutral comet assay. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-H3 and flow cytometry. In vivo activity was measured using a tumor growth delay assay. ResultsExposure of each cell line to CPS45 for 16 h before irradiation (IR) resulted in an increase in radiosensitivity with dose enhancement factors of 1.8 and 1.5 for U251 and MiaPaCa cells, respectively (PF = 0.43, 0.31 respectively in CPS45 treated cells). CPS45 had no effect on IR-induced apoptosis or on the activation of the G2 cell cycle checkpoint. However, CPS45 did modify the time course of γH2AX expression in IR cells. There was significant difference in IR-induced γH2AX foci count at both 6 h and at 24 h after IR in the CPS45 + IR group than either individual treatment. In the neutral comet assay, there was a significant increase in the percentage of DNA damage remaining in cells exposed to CPS45 before IR compared to IR alone. Mitotic catastrophe, measured at 48 and 72 hour, was also significantly increased in cells receiving the CPS45 + IR combination compared with the individual treatments. Tumor growth delay demonstrated a greater than additive effect of the combination of CPS45 and IR than either treatment alone. Exposure of each cell line to CPS45 for 16 h before irradiation (IR) resulted in an increase in radiosensitivity with dose enhancement factors of 1.8 and 1.5 for U251 and MiaPaCa cells, respectively (PF = 0.43, 0.31 respectively in CPS45 treated cells). CPS45 had no effect on IR-induced apoptosis or on the activation of the G2 cell cycle checkpoint. However, CPS45 did modify the time course of γH2AX expression in IR cells. There was significant difference in IR-induced γH2AX foci count at both 6 h and at 24 h after IR in the CPS45 + IR group than either individual treatment. In the neutral comet assay, there was a significant increase in the percentage of DNA damage remaining in cells exposed to CPS45 before IR compared to IR alone. Mitotic catastrophe, measured at 48 and 72 hour, was also significantly increased in cells receiving the CPS45 + IR combination compared with the individual treatments. Tumor growth delay demonstrated a greater than additive effect of the combination of CPS45 and IR than either treatment alone. ConclusionsThese results indicate that CPS45 can enhance tumor cell radiosensitivity and suggest that this effect involves an inhibition of double strand breaks repair leading to an increase in mitotic catastrophe. These results indicate that CPS45 can enhance tumor cell radiosensitivity and suggest that this effect involves an inhibition of double strand breaks repair leading to an increase in mitotic catastrophe.

Purpose/Objective(s)Glioblastoma multiforme (GBM) continues to have a poor survival despite advances in surgical resection and the addition of temozolomide to radiation. Radiotherapy, one of the mainstays of treatment for GBM, is known to induce tumor cell killing through DNA double strand breaks (DSB). Poly (ADP-ribose) polymerase (PARP) is an enzyme involved in the repair of DSBs. Here we investigate the in vitro and in vivo radiosensitizing effects of the novel PARP inhibitor, GPI 21016, on a GBM cell line.Materials/MethodsThe U251 human GBM cell line was treated for 6 h with GPI 21016 (3 uM) pre-irradiation in all in vitro experiments. Inhibition of PARP-1 by GPI 21016 was evaluated using a PARP chemiluminescent assay. Clonogenic survival assays were performed according to standard protocol. To assess DSBs, γH2AX foci were assessed at 1, 6 and 24 h after 2 Gy. To further evaluate DSBs, neutral comet assay was performed at 0, 1, 3, 6 and 24 h after exposure to 10 Gy. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-histone H3 and flow cytometry. For in vivo studies, U251 cells were implanted in both SC and IC models.ResultsA 6h exposure of GPI 21016 (3uM) inhibited PARP activity by 73%. Clonogenic survival resulted in a dose enhancement factor (DEF) of 1.6 at a surviving fraction of 10% (SF = 0.49 in drug alone cells) in cells treated with GPI 21016 and IR vs. IR alone. To assess DSB repair, γH2AX measured at 24 h post-IR had significantly more foci/cell in the combination group vs. the IR alone group. Neutral comet assay further suggested unrepaired DSBs with significantly greater DNA damage at 6 h post-IR in the combination group vs. IR alone, however this damage resolved by 24 h. Mitotic catastrophe (MC) staining revealed significantly greater cells in MC at 72 h post-IR in the combination group vs. the IR alone group. Drug, IR and combination groups showed little apoptosis at 24 and 72 h post-treatment. Cell cycle analysis revealed no changes with exposure to drug pre-IR. In both SC and IC in vivo models, the combination therapy had a greater than additive anti-tumor effect than either single modality.ConclusionsGPI 21016 effectively inhibits PARP leading to decreased DSB repair and enhanced mitotic catastrophe. Thereby, GPI 21016 radiosensitizes U251 cells in vitro and results in decreased tumor growth in vivo, suggesting a potential role for this drug in the treatment of GBM. Purpose/Objective(s)Glioblastoma multiforme (GBM) continues to have a poor survival despite advances in surgical resection and the addition of temozolomide to radiation. Radiotherapy, one of the mainstays of treatment for GBM, is known to induce tumor cell killing through DNA double strand breaks (DSB). Poly (ADP-ribose) polymerase (PARP) is an enzyme involved in the repair of DSBs. Here we investigate the in vitro and in vivo radiosensitizing effects of the novel PARP inhibitor, GPI 21016, on a GBM cell line. Glioblastoma multiforme (GBM) continues to have a poor survival despite advances in surgical resection and the addition of temozolomide to radiation. Radiotherapy, one of the mainstays of treatment for GBM, is known to induce tumor cell killing through DNA double strand breaks (DSB). Poly (ADP-ribose) polymerase (PARP) is an enzyme involved in the repair of DSBs. Here we investigate the in vitro and in vivo radiosensitizing effects of the novel PARP inhibitor, GPI 21016, on a GBM cell line. Materials/MethodsThe U251 human GBM cell line was treated for 6 h with GPI 21016 (3 uM) pre-irradiation in all in vitro experiments. Inhibition of PARP-1 by GPI 21016 was evaluated using a PARP chemiluminescent assay. Clonogenic survival assays were performed according to standard protocol. To assess DSBs, γH2AX foci were assessed at 1, 6 and 24 h after 2 Gy. To further evaluate DSBs, neutral comet assay was performed at 0, 1, 3, 6 and 24 h after exposure to 10 Gy. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-histone H3 and flow cytometry. For in vivo studies, U251 cells were implanted in both SC and IC models. The U251 human GBM cell line was treated for 6 h with GPI 21016 (3 uM) pre-irradiation in all in vitro experiments. Inhibition of PARP-1 by GPI 21016 was evaluated using a PARP chemiluminescent assay. Clonogenic survival assays were performed according to standard protocol. To assess DSBs, γH2AX foci were assessed at 1, 6 and 24 h after 2 Gy. To further evaluate DSBs, neutral comet assay was performed at 0, 1, 3, 6 and 24 h after exposure to 10 Gy. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-histone H3 and flow cytometry. For in vivo studies, U251 cells were implanted in both SC and IC models. ResultsA 6h exposure of GPI 21016 (3uM) inhibited PARP activity by 73%. Clonogenic survival resulted in a dose enhancement factor (DEF) of 1.6 at a surviving fraction of 10% (SF = 0.49 in drug alone cells) in cells treated with GPI 21016 and IR vs. IR alone. To assess DSB repair, γH2AX measured at 24 h post-IR had significantly more foci/cell in the combination group vs. the IR alone group. Neutral comet assay further suggested unrepaired DSBs with significantly greater DNA damage at 6 h post-IR in the combination group vs. IR alone, however this damage resolved by 24 h. Mitotic catastrophe (MC) staining revealed significantly greater cells in MC at 72 h post-IR in the combination group vs. the IR alone group. Drug, IR and combination groups showed little apoptosis at 24 and 72 h post-treatment. Cell cycle analysis revealed no changes with exposure to drug pre-IR. In both SC and IC in vivo models, the combination therapy had a greater than additive anti-tumor effect than either single modality. A 6h exposure of GPI 21016 (3uM) inhibited PARP activity by 73%. Clonogenic survival resulted in a dose enhancement factor (DEF) of 1.6 at a surviving fraction of 10% (SF = 0.49 in drug alone cells) in cells treated with GPI 21016 and IR vs. IR alone. To assess DSB repair, γH2AX measured at 24 h post-IR had significantly more foci/cell in the combination group vs. the IR alone group. Neutral comet assay further suggested unrepaired DSBs with significantly greater DNA damage at 6 h post-IR in the combination group vs. IR alone, however this damage resolved by 24 h. Mitotic catastrophe (MC) staining revealed significantly greater cells in MC at 72 h post-IR in the combination group vs. the IR alone group. Drug, IR and combination groups showed little apoptosis at 24 and 72 h post-treatment. Cell cycle analysis revealed no changes with exposure to drug pre-IR. In both SC and IC in vivo models, the combination therapy had a greater than additive anti-tumor effect than either single modality. ConclusionsGPI 21016 effectively inhibits PARP leading to decreased DSB repair and enhanced mitotic catastrophe. Thereby, GPI 21016 radiosensitizes U251 cells in vitro and results in decreased tumor growth in vivo, suggesting a potential role for this drug in the treatment of GBM. GPI 21016 effectively inhibits PARP leading to decreased DSB repair and enhanced mitotic catastrophe. Thereby, GPI 21016 radiosensitizes U251 cells in vitro and results in decreased tumor growth in vivo, suggesting a potential role for this drug in the treatment of GBM.

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.