diethyl-maleate and Lung-Neoplasms

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor kappaB (NF-kappaB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-kappaB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-kappaB. Cells treated for 1 h with 1 mM diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-kappaB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-kappaB function approximately 50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-kappaB binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor kappaB-alpha (IkappaB-alpha). Furthermore, acrolein decreased NF-kappaB activation in cells depleted of IkappaB-alpha by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-kappaB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-kappaB in the cytosol prior to chemical dissociation from IkappaB with detergent. Together, these data support the conclusion that the inhibition of NF-kappaB activation by acrolein and DEM is IkappaB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-kappaB subunits.

Topics: Acrolein; Adenocarcinoma; Cell Division; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Humans; I-kappa B Proteins; Lung Neoplasms; Maleates; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The relationship between intracellular sulfhydryl(SH) compounds and the kinetics of the inhibitory effect of selenite on cellular nucleic acid synthesis has been examined. In A549 cells, with a relatively high SH level, exposure to low concentrations of selenite caused inhibition even after short exposure times. In contrast, in VA cells, with a relatively low level of SH compounds, selenite had no significant effect at short exposure times, but inhibited significantly with longer exposures. Selenodicysteine, the product of the reaction of selenite with cysteine (an important intracellular SH compound), inhibited synthesis in both cell types at short exposure times. Exposure of cells to diethylmaleate, which decreased the level of intracellular SH compounds, reduced the inhibitory effect of a short exposure to selenite but did not affect a long exposure. These results indicate that the reaction of selenite with intracellular SH compounds may be a determining factor in the kinetics of its inhibitory effect on cellular DNA and RNA synthesis.

Topics: Adenocarcinoma; Cell Line, Transformed; DNA; Embryo, Mammalian; Embryo, Nonmammalian; Kinetics; Lung; Lung Neoplasms; Maleates; RNA; Sodium Selenite; Sulfhydryl Compounds; Tumor Cells, Cultured

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-T27) cells to the cytotoxic effects of Cd++. The effects of GSH depletion on Cd++ accumulation and Cd++-induced metallothionein (MT) content were investigated to determine the possible role of these Cd++ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd++-induced accumulation of exogenous 35s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd++ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd++ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd++, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd++-induced MT accumulation rates.

Topics: Buthionine Sulfoximine; Cadmium; Glutathione; Humans; Lung Neoplasms; Maleates; Metallothionein; Methionine Sulfoximine; Sulfur Radioisotopes; Tumor Cells, Cultured

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd++-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd++ cytotoxic effect and decreased the LD50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd++. Thus the results demonstrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd++ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd++ cytotoxic response. Comparison of reduced glutathione levels and of Cd++ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd++ resistance.

Topics: Buthionine Sulfoximine; Cadmium; Cell Survival; Glutathione; Humans; Lung Neoplasms; Maleates; Methionine Sulfoximine; Tumor Cells, Cultured

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased.

Topics: Buthionine Sulfoximine; Cell Line; Cell Survival; Dose-Response Relationship, Radiation; Glutathione; Humans; Lung Neoplasms; Maleates; Methionine Sulfoximine; Radiation-Sensitizing Agents; Time Factors

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently we discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH because this agent irreversibly inhibits gamma-glutamyl synthetase. For A549 monolayer cultures, there is approximately 50% regeneration 6 hr after removal of 0.01 mM BSO, 20% 6 hr after 0.1 mM BSO, and only 5% 6 hr after 0.5 mM BSO. We found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH. After removal of DEM, A549 cells immediately begin GSH resynthesis, and return to control levels occurs within 2 hr. Exogenous 5 mM GSH increases the rate of resynthesis of GSH in DEM-treated cells, but not in BSO-treated cells.

Topics: Aerobiosis; Buthionine Sulfoximine; Cell Line; Cell Survival; Dose-Response Relationship, Radiation; Glutathione; Humans; Lung Neoplasms; Maleates; Methionine Sulfoximine; Radiation-Sensitizing Agents; Sulfhydryl Compounds; Time Factors

Glutathione (GSH)-depletion by buthionine sulphoximine (BSO) altered both the aerobic and anaerobic radiation response of A549 human lung cancer cells grown in vitro. The oxygen enhancement ratio (o.e.r) was increased slightly from 3.0-3.3. The lack of an effect of GSH-depletion on o.e.r. reduction, provides a system whereby the mechanism of action of the thiol reactive reagent diethylmaleate (DEM) can be investigated. Pretreatment of cells with DEM, under non-toxic concentrations, removed 13 per cent of the intracellular NPSH and resulted in an o.e.r. of 2. When BSO followed by DEM was used, so that both GSH and NPSH were reduced to zero, an o.e.r. of 1.5 was obtained. Cells treated with 1 mM BSO for 24 hours contained 10 per cent NPSH and no GSH. When these cells were exposed to 0.5 or 1 mM DEM briefly, during irradiation, the o.e.r. was 2.4 and 1.7 respectively. In some cases altered o.e.r.s occurred in combination with increased aerobic responses. This was especially true for aerobic irradiations of BSO-treated cells in the presence or absence of DEM. However, the increased aerobic response was offset by a more dramatic increase in the hypoxic response. These results indicate (a) that GSH plays a significant role in aerobic radiation response but is not a principal factor in o.e.r.-reduction, and (b) that reduction of the o.e.r. by DEM is not due primarily to GSH-removal. The preferential radiosensitization of hypoxic cells by DEM may involve reactions of this compound with NPSH or protein SH, or may be related to the ability of DEM to mimic oxygen as a hypoxic cell radiosensitizer.

Topics: Aerobiosis; Anaerobiosis; Cell Line; Cell Survival; Dose-Response Relationship, Radiation; Glutathione; Humans; Lung Neoplasms; Maleates

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.

Topics: Animals; Buthionine Sulfoximine; Carcinoma; Cell Line; Cells, Cultured; Cricetinae; Dose-Response Relationship, Radiation; Glutathione; Humans; Hypoxia; Lung; Lung Neoplasms; Maleates; Methionine Sulfoximine; Oxygen Consumption; Radiation-Sensitizing Agents; Time Factors

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.

Topics: Animals; Azo Compounds; Buthionine Sulfoximine; Cell Survival; Cells, Cultured; Chemical Phenomena; Chemistry; Cricetinae; Diamide; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Female; Glutathione; Humans; Lung Neoplasms; Maleates; Methionine Sulfoximine; Ovary; Oxygen; Radiation-Sensitizing Agents; Sulfhydryl Compounds