Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer.

BACKGROUND: Pro-survival Bcl-2 family members can promote cancer development and contribute to treatment resistance. Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. Inhibition of anti-apoptotic Bcl-2 family members therefore represents an appealing strategy to overcome resistance to anti-cancer therapies. The aim of this study was to evaluate combined effects of radiation and the pan-Bcl-2 inhibitor AT-101 in HNSCC in vitro. In addition, we determined human plasma levels of AT-101 obtained from a phase I/II trial, and compared these with the effective in vitro concentrations to substantiate therapeutic opportunities. METHODS: We examined the effect of AT-101, radiation and the combination on apoptosis induction and clonogenic survival in two HNSCC cell lines that express the target proteins. Apoptosis was assessed by bis-benzimide staining to detect morphological nuclear changes and/or by propidium iodide staining and flow-cytometry analysis to quantify sub-diploid apoptotic nuclei. The type of interaction between AT-101 and radiation was evaluated by calculating the Combination Index (CI) and by performing isobolographic analysis. For the pharmacokinetic analysis, plasma AT-101 levels were measured by HPLC in blood samples collected from patients enrolled in our clinical phase I/II study. These patients with locally advanced HNSCC were treated with standard cisplatin-based chemoradiotherapy and received dose-escalating oral AT-101 in a 2-weeks daily schedule every 3 weeks. RESULTS: In vitro results showed that AT-101 enhances radiation-induced apoptosis with CI's below 1.0, indicating synergy. This effect was sequence-dependent. Clonogenic survival assays demonstrated a radiosensitizing effect with a DEF37 of 1.3 at sub-apoptotic concentrations of AT-101. Pharmacokinetic analysis of patient blood samples taken between 30 min and 24 h after intake of AT-101 showed a dose-dependent increase in plasma concentration with peak levels up to 300-700 ng/ml between 1.5 and 2.5 h after intake. CONCLUSION: AT-101 is a competent enhancer of radiation-induced apoptosis in HNSCC in vitro. In addition, in vitro radiosensitization was observed at clinically attainable plasma levels. These finding support further evaluation of the combination of AT-101 with radiation in Bcl-2-overexpressing tumors.

Radiosensitization of human glioma cells by cyclooxygenase-2 (COX-2) inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration.

PURPOSE: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines. MATERIALS AND METHODS: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 - 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis. RESULTS: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 microM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 microM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor administration after radiotherapy. Treatment of D384 with NS-398 (200 microM) or Celecoxib (50 microM) and U87 with NS-398 (200 microM) after radiation resulted even in radioprotection. CONCLUSIONS: Effectiveness of COX-2 inhibitors on cell proliferation and radio-enhancement was independent of COX-2 protein expression. The sequence of COX-2 inhibitor addition and irradiation is very important.

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0-6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24-72 h exposure to 250-750 microM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 microM meloxicam for 24 h increased the fraction of cells in the radiosensitive G(2)/M cell cycle phase in D384 (18-27%) and U251 (17-41%) cells. 750 microM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

Expression of cyclooxygenase-2 and epidermal growth factor receptor in primary and recurrent glioblastoma multiforme.

PURPOSE: To investigate the pattern and level of cyclooxygenase-2 (COX-2) expression in a series of high grade primary and recurrent glioblastoma multiforme (GBM) and correlation with time to recurrence and patients' survival following therapy. The relationship between COX-2 and epidermal growth factor receptor (EGFR) immunoreactivities was evaluated. MATERIALS AND METHODS: Specimens of 14 primary and 14 recurrent GBMs (eight pairs) following surgery and full course radiation therapy were processed for immunostaining on COX-2 and EGFR. Tumor cell positivity was semi-quantitatively scored. COX-2 scores of the primary tumor and recurrence were correlated with the time to radiological tumor progression and patients' survival. RESULTS: COX-2 positive tumor cells were disseminated throughout the tumor parenchyma. The intensity and pattern of COX-2 expression were heterogeneous, with predominant expression in areas surrounding tumor necrosis. Scoring of COX-2 positivity revealed values between 1 and 80% of the cells. Primary GBMs with COX-2 expression levels between 25% and 70% of the tumor cells showed a shorter time to radiological recurrence than GBMs with <10% COX-2 positive tumor cells (respectively, 219 +/- 50 and 382 +/- 77 days). No correlation was found between the COX-2 expression in the primary tumor and patients' survival (r (s) = -0.073) following therapy. No correlation was found either between COX-2 and EGFR immunoreactivity. CONCLUSIONS: Immunohistochemical expression of COX-2 in GBM showed large variation. Hence, determination of COX-2 expression in tumor specimen for each individual might be relevant for selection of those patients, who could benefit from adjuvant therapy with selective COX-2 inhibitors.

Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution.

When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.