Optimizing radiotherapy of brain tumours by a combination of temozolomide & lonidamine.

BACKGROUND & OBJECTIVE: Temozolomide (TMZ), a second generation alkylating drug, an effective cytotoxic agent as well as radiosensitizer for malignant brain tumours, has side effects like myelosuppression. Lonidamine (LND) increases the effectiveness of several experimental multiple chemotherapy protocols, without increasing bone marrow toxicities and is effective in brain tumour patients. The objective of the present studies was to investigate whether combining clinically relevant doses of LND and TMZ could increase the proliferation and radiation response of malignant human brain tumour cells in vitro. METHODS: A malignant human glioma (U373MG) cell line was used in these studies. TMZ (20, 40 or 60 microM) or LND (100, 150 or 200 microM), or the combination of both (20 and 100 microM, respectively) in 0.1 per cent dimethyl sulphoxide (DMSO) were added three days after setting up cultures, in six well plates (5 x 10(4) cells/ well). The effects of continuous treatment for two days on proliferation response and cytotoxicity were studied after trypsinization; by cell counts and the uptake of trypan blue dye (0.5%). For the study of radiation (60Co-Gamma-rays, 2 Gy) response, drugs were removed 4 h after irradiation and cultures were grown further in drug free, normal growth medium for another 20 h or 44 h. RESULTS: Continuous presence of TMZ or LND for two days significantly inhibited cell proliferation in a concentration dependent manner. The frequencies of non viable cells increased significantly only at higher concentrations of LND. Combination of 20 microM TMZ with 100 microM LND had additive effects on proliferation response, without affecting cell viability. Short-term drug treatments without irradiation did not induce micronuclei formation. Cell proliferation and viability were also not affected. However, post-irradiation presence of either of these drugs for 4 h significantly reduced the proliferation response, 24 and 48 h after treatments. It was further inhibited by the combination treatment. On the contrary, radiation induced micronuclei formation was enhanced by either of the drugs; which was significantly increased by the combined treatment, 24 h as well as 48 h after irradiation. No effects on cell viability were observed, immediately after these treatments as well as at later time points. INTERPRETATION & CONCLUSION: Our findings showed that combination of TMZ and LND at clinically achievable, low plasma concentrations could inhibit tumour growth, and lonidamine could reduce the dose of temozolomide required for radiosensitization of brain tumours.

Optimizing radiation therapy of brain tumours by combination of 5-bromo-2-deoxy-uridine & 2-deoxy-D-glucose.

The effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) on 60Co-gamma ray induced damage were studied in a human glioma cell line grown as monolayer. Radiation induced micronuclei formation was used as an index of cytogenetic damage. Exponentially growing cells (doubling time 16-20 h) were incubated in the presence of BrdU (0.8 microM, in dark) for 24 h. After removing BrdU, cells were irradiated (1-4 Gy), incubated with or without 2-DG (2-3 h), and grown further (for 18, 24, 30 or 45 h) for assay of damage. It was observed that (i) BrdU and 2-DG treatments did not induce micronuclei formation in unirradiated cultures; (ii) pre-irradiation presence of BrdU increased the gamma-ray induced micronuclei formation; (iii) incubation of irradiated cells under sub-optimal growth conditions [Dulbecco's modified minimal essential medium (DMEM) + 1% serum, or DMEM alone] instead of growth medium (DMEM + 5% serum) progressively decreased micronuclei formation; and (iv) post-irradiation presence of 2-DG (1.25, 2.5, 5 mM, 2-3 h in DMEM + 1% serum) enhanced the radiation damage with and without BrdU treatment at all the time points studied. These observations suggest that (i) radiation induced lesions leading to micronuclei formation in proliferating cells are, at least, partly repairable; (ii) the presence of 2-DG (2DG/glucose > or = 0.25) for short intervals (approximately 2 h), could enhance radiation damage in proliferating brain tumour cells, in the absence as well as presence of BrdU incorporation; and (iii) the combination of 2-DG could reduce BrdU doses required for radiosensitization of brain tumours, reducing, thereby, its toxic side effects.

Differential modification of radiation damage in 5-bromo-2-deoxy-uridine sensitized human glioma cells and PHA-stimulated peripheral leukocytes by 2-deoxy-D-glucose.

Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) on 60-Co-gamma-ray induced damage were studied in monolayer cultures of glioma (BMG-1) cells, and PHA-stimulated peripheral leukocytes from normal donors. Micronuclei formation was used as an index of cytogenetic damage. BrdU and 2-DG treatments did not induce micronuclei formation in unirradiated cultures. Presence of BrdU (0.8 microM) for more than one cell cycle (24 hr) significantly increased gamma-ray (1-4 Gy) induced micronuclei formation in exponentially growing BMG-1 cells. Incubation of irradiated cells under sub-optimal growth conditions (DMEM with 1% serum) for 3 hr, instead of growth medium, significantly decreased micronuclei formation. Post-irradiation presence of 2-DG (5 mM; 3 hr, in DMEM + 1% serum) significantly increased radiation damage. In BrdU sensitized cells also, 2-DG significantly increased radiation damage further. In PHA-stimulated leukocytes from normal donors, 2-DG (5mM, equimolar with glucose; for 2 hr) did not increase gamma-ray (2-Gy, 42 hr after PHA-stimulation) induced micronuclei formation. Pre-irradiation presence of BrdU (1.6 microM) significantly increased micronuclei. On the contrary, 2-DG treatment reduced radiation induced micronuclei formation in BrdU sensitized leukocyte cultures. These results suggest that (i) radiation induced lesions leading to micronuclei formation in proliferating tumour cells, are, at least, partly repairable; (ii) combination of 2-DG could reduce BrdU doses required for radiosensitization of proliferating tumour cells; and (iii) 2-DG could differentially increase radiation damage in BrdU sensitized proliferating tumour cells, while reducing manifestation of damage in normal proliferating cells.

Potentiation of radiation effects in plateau phase human glioma cells by combination of metabolic inhibitors.

Effects of glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on radiation damage were studied in a human glioma cell line (BMG-1), grown to confluence in monolayer. After irradiation (60Co-gamma-rays, 2 Gy) and incubation with low concentrations of 2-DG (0.5, 1.25 mM; 2-DG/glucose = 0.1, 0.25; 2 hr), in the absence or presence of respiratory inhibitor KCN (0.5-2 mM), cells were trypsinized and plated to assay radiation induced cytogenetic damage (micronuclei formation). The observations made were: (1) 2-DG and/or KCN treatments did not induce damage in unirradiated cells. (2) Either of these treatments did not increase radiation induced micronuclei formation. (3) Presence of 2-DG along with KCN (1,2 mM) significantly enhanced the radiation induced micronuclei formation. (4) Preliminary experiments by macrocolony assay showed that radiation induced cell death was also significantly increased by the combined treatment. These observations suggest that presence of clinically feasible, low concentrations of 2-DG (2-DG/glucose < 0.5) for short intervals of time after radiation could increase radiation damage in non-cycling, hypoxic tumour cells with impaired oxidative and increased glycolytic energy metabolism.

Modification of radiation damage in transformed mammalian cells by 5-bromo-2-deoxy-uridine and 2-deoxy-D-glucose.

The effects of 2-deoxy-D-glucose (2-DG) and 5-bromo-2-deoxy-uridine (BrdU) on gamma ray (60Co) induced damage were studied in monolayer cultures of transformed mammalian (BHK-21) cells. Micronuclei formation and changes in DNA content dispersion were used as indices of cytogenetic damage. Exposure of cells to BrdU (0.8 microM) for nearly two cell cycles before irradiation significantly increased micronuclei formation in exponentially growing cells. Incubation of irradiated cells under suboptimal growth conditions (in HBSS) for 4 hr, instead of growth medium, decreased the manifestation of damage. However, post-irradiation presence of 2-DG (5 mM, equimolar with glucose; 4 hr) in growth medium or HBSS significantly increased radiation damage. The effects of 2-DG treatment following irradiation in plateau phase were quantitatively less. These results suggest that: (i) radiation induced DNA lesions leading to micronuclei formation in BrdU incorporated cells are partly repairable; (ii) 2-DG could increase radiation induced cytogenetic damage in transformed mammalian cells, possibly by inhibiting the cellular repair processes; and (iii) combination of 2-DG treatment may decrease the BrdU doses required for radiosensitization of proliferating tumour cell populations.

Effects of 5-bromo-2-deoxy-uridine and 2-deoxy-D-glucose on the radiosensitivity of proliferating transformed mammalian cells.

Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) were studied in exponentially growing transformed mammalian (BHK-21) cells, grown as monolayer. Micronuclei formation as an index of radiation damage was studied by i) cytokinesis block technique from cytochalasin-B induced binucleated cells, and ii) conventional technique. Presence of BrdU (0.8 microM) for nearly 2 cell cycles before gamma-irradiation (2.5 Gy) significantly increased frequencies of cells with micronuclei. Post-irradiation incubation of cultures in liquid holding medium (HBSS) however, reduced micronuclei formation, especially in the BrdU treated cells. Presence of 2-DG (4 hr, equimolar with glucose) in growth as well as liquid holding medium further increased micronuclei frequencies. These observations suggest that radiation induced DNA lesions in BrdU substituted cells, leading to chromosome fragmentation are partly repairable. 2-DG increased cytogenetic damage, possibly by inhibiting the repair of such repairable lesions. Present studies suggest that combination of 2-DG could optimize BrdU-radiation therapy of brain tumors, by reducing the BrdU doses required for tumor radiosensitization.

2-Deoxy-D-glucose induced modification of chromosomal damage in UV-irradiated peripheral human leukocytes.

UV-irradiation (0.6 J/m2) of peripheral human leukocytes 27 hr after PHA-stimulation induced a considerable mitotic delay in the cultures. Approximately two thirds of the chromosomal aberrations induced by UV were gaps of the chromatid and isochromatid types. Treatment with glucose antimetabolite 2-deoxy-D-glucose (2-DG) alone did not induce any chromosomal damage. Presence of 2-DG (5 mM, equimolar with glucose) for 2 hr after UV-irradiation resulted in a significant reduction in the frequency of cells with aberrations. Decrease in the total aberrations per cell was also observed. The data are consistent with earlier observations that 2-DG reduces the manifestation of radiation damage in normal proliferating cells.