Caffeic acid phenethyl ester preferentially sensitizes CT26 colorectal adenocarcinoma to ionizing radiation without affecting bone marrow radioresponse

PURPOSE: Caffeic acid phenethyl ester (CAPE), a component of propolis, was reported capable of depleting glutathione (GSH). We subsequently examined the radiosensitizing effect of CAPE and its toxicity. METHODS AND MATERIALS: The effects of CAPE on GSH level, GSH metabolism enzyme activities, NF-kappaB activity, and radiosensitivity in mouse CT26 colorectal adenocarcinoma cells were determined. BALB/c mouse with CT26 cells implantation was used as a syngeneic in vivo model for evaluation of treatment and toxicity end points. RESULTS: CAPE entered CT26 cells rapidly and depleted intracellular GSH in CT26 cells, but not in bone marrow cells. Pretreatment with nontoxic doses of CAPE significantly enhanced cell killing by ionizing radiation (IR) with sensitizer enhancement ratios up to 2.2. Pretreatment of CT26 cells with N-acetyl-L-cysteine reversed the GSH depletion activity and partially blocked the radiosensitizing effect of CAPE. CAPE treatment in CT26 cells increased glutathione peroxidase, decreased glutathione reductase, and did not affect glutathione S-transferase or gamma-glutamyl transpeptidase activity. Radiation activated NF-kappaB was reversed by CAPE pretreatment. In vivo study revealed that pretreatment with CAPE before IR resulted in greater inhibition of tumor growth and prolongation of survival in comparison with IR alone. Pretreatment with CAPE neither affected body weights nor produced hepatic, renal, or hematopoietic toxicity. CONCLUSIONS: CAPE sensitizes CT26 colorectal adenocarcinoma to IR, which may be via depleting GSH and inhibiting NF-kappaB activity, without toxicity to bone marrow, liver, and kidney.     

Effects of glutathione depletion by buthionine sulfoximine on the sensitivity of EMT6/SF cells to chemotherapy agents or X radiation

The effects of depletion of cellular glutathione (GSH) on the sensitivity of cultured EMT6/SF cells to chemotherapy agents or x rays under hypoxic and aerated conditions were investigated. Buthionine sulfoximine (BSO), a potent inhibitor of the enzyme gamma-glutamyl-cysteine synthetase, was used to deplete cellular GSH. Addition of BSO (50 microM) to EMT6/SF cultures depleted cellular GSH with a half-time of approximately 2 hr. Cellular GSH reached very low levels within hours of addition of BSO. After removal of BSO, cellular GSH recovered with approximately the same kinetics as was seen for depletion. Incubation of EMT6/SF cells with BSO concentrations of up to 1 mM did not reduce the viability or inhibit growth when exposure was limited to times less than 24 hr. However, for longer exposure times, toxicity and growth inhibition were demonstrated in a dose dependent fashion. EMT6/SF cells were treated with chemotherapy agents under either aerated or extremely hypoxic conditions. Cells were more sensitive to cis-dichlorodiammino Pt(II) (DDP), mitomycin C (MitC), L-phenylalanine mustard (L-PAM), and nitrogen mustard (HN2) when treatment was under hypoxic conditions. The magnitude of this sensitization under hypoxic conditions ranged from a dose modifying factor (DMF) of 1.4 (HN2) to 4.1 (MitC), measured at the 0.1 level of cell survival. Hypoxic EMT6/SF cells were more resistant to the cytotoxic effects of actinomycin D (ActD) under hypoxic conditions (DMF = 10 at SF = 0.3). When cellular GSH was depleted to less than 5% of control by treatment with 50 microM BSO for 12-14 hr, cells were sensitized to DDP, L-PAM and HN2 under both aerated and hypoxic conditions. DMF's ranged from 1.4-6.5, depending on the agent. Hypoxic cell sensitization was never significantly greater than that seen in aerated cells, as was the case for X radiation (DMF = 1.3 for hypoxic cells only). GSH depletion also sensitized to MitC, but only under aerated conditions (DMF = 2.1). Hypoxic EMT6/SF cells were not sensitized to MitC by depletion of GSH. GSH depletion afforded slight protection against ActD toxicity under both aerated and hypoxic conditions. These studies suggest that cellular GSH plays an important role in modifying cellular response to cytotoxic drugs. GSH depletion may sensitize tumor cells to some chemotherapy agents, but differential sensitization of tumors compared to normal tissues, based on hypoxic tumor cells as targets, would not be expected based on these in vitro experiments.     

{gamma}-Glutamyltranspeptidase-conferred resistance to hydroquinone induced GSH depletion and toxicity in isolated hepatocytes

Hepatocyte resistance against glutathione (GSH) depleting xenobioticswas studied in an in vitro model. Hepatocytes were isolatedfrom carcinogen treated rats that had received phenobarbitalfor three weeks. Isolated cells were incubated in GSH containingbuffer with hydroquinone, which depleted GSH. Cells were thenseeded on collagen coated plates and cultured overnight in completemedium. Attached cells were stained and the proportion of -glutamyltranspeptidase(GGT)-positive cells was counted. It was found that toxicityrelated to GSH depletion increased the proportion of GGTpositivecells from 10–15% up to 40–60%, indicating thatthe toxicity mainly affected GGT-negative cells. GSH added tothe buffer was essential for this effect. It is concluded thatGGT may protect GGT-positive hepatocytes from GSH depletionand toxicity early during liver carcinogenesis.     

Modification of antitumor disulfide cytotoxicity by glutathione depletion in murine cells

Decyl and phenyl disulfide derivatives of 6-mercaptopurine and 6-thioguanine (6-TG) were examined for antineoplastic activity under aerobic and hypoxic conditions toward EMT6 cells in culture. Although these derivatives did not display selective toxicity toward the hypoxic cells, they were significantly more toxic than 6-TC to this cell line at 500 microM after a 2-h exposure. In conjunction with this cytotoxicity, these agents were found to deplete the cellular glutathione (GSH) levels to varying degrees at this same concentration after a 1-h period. Therefore, the effect of modulating the cellular GSH on the cytotoxicity of these agents was investigated. When the GSH was depleted to less than 5 or 41% of control levels, the cytotoxicity exhibited by these agents was significantly increased while that of 6-TG remained unchanged. The cytotoxicity of these agents was similar to that of decanethiol and thiophenol, the thiol portion of the molecules, both under normal treatment conditions or after depletion of GSH. The lack of selective toxicity toward hypoxic cells was correlated to the finding that the disulfides were broken down to the parents, 6-mercaptopurine, and 6-TG, by cells under aerobic conditions. However, these studies demonstrate that manipulation of GSH levels might yield a therapeutic gain for these disulfide derivatives of antitumor agents.     

Selected flavonoids potentiate the toxicity of cisplatin in human lung adenocarcinoma cells: a role for glutathione depletion

Adjuvant therapies that enhance the anti-tumor effects of cis-diammineplatinum(II) dichloride (cisplatin, CDDP) are actively being pursued. Growing evidence supports the involvement of mitochondrial dysfunction in the anti-cancer effect of cisplatin. We examined the potential of using selective flavonoids that are effective in depleting tumor cells of glutathione (GSH) to potentiate cisplatin-mediated cytotoxicity in human lung adenocarcinoma (A549) cells. We found that cisplatin (40 microM, 48-h treatment) disrupts the steady-state levels of mitochondrial respiratory complex I, which correlates with elevated mitochondrial reactive oxygen species (ROS) production and cytochrome c release. The flavonoids, 2',5'-dihydroxychalcone (2',5'-DHC, 20 microM) and chrysin (20 microM) potentiated the cytotoxicity of cisplatin (20 microM), which could be blocked by supplementation of the media with exogenous GSH (500 microM). Both 2',5'-DHC and chrysin were more effective than the specific inhibitor of GSH synthesis, L-buthionine sulfoximine (BSO, 20 microM), in inducing GSH depletion and potentiating the cytotoxic effect of cisplatin. These data suggest that the flavonoid-induced potentiation of cisplatin's toxicity is due, in part, to synergetic pro-oxidant effects of cisplatin by inducing mitochondrial dysfunction, and the flavonoids by depleting cellular GSH, an important antioxidant defense.     

Depletion of intracellular glutathione by 1-methyl-2-nitrosoimidazole.

In addition to their ability to radiosensitize, nitroimidazoles are selectively toxic toward hypoxic cells. Reduction of the nitro group is required to observe cytotoxicity. One of the reduction products believed to play a role in this cytotoxicity is the nitroso-derivative. One-methyl-2-nitrosoimidazole (INO), chemically synthesized from one-methyl-2-nitroimidazole (INO2), has been used as a model to study the reactivity of 2-nitrosoimidazoles. The ability of INO to react rapidly with glutathione in Chinese hamster ovary cells treated with a sub-toxic and toxic level of the drug has been measured. The kinetics of GSH loss as well as oxidized GSH (GSSG) formation and loss were assessed at short times (0-15 min) after INO exposure using a high pressure liquid chromatography (HPLC) assay for GSH and GSSG. The results obtained were consistent with a model, based on previous chemical studies of the reaction of INO with GSH, whereby GSH reduces INO forming GSSG and as well reacts with a reduced form of INO to form an adduct (I-SG). These results suggest possible strategies for modifying the toxicity of reduction products of one-substituted-2-nitroimidazoles.     

Biochemical mechanism of acetylsalicylic acid (Aspirin) selective toxicity toward melanoma cell lines

In the current work, we investigated the biochemical toxicity of acetylsalicylic acid (ASA; Aspirin) in human melanoma cell lines using tyrosinase enzyme as a molecular cancer therapeutic target. At 2 h, ASA was oxidized 88% by tyrosinase. Ascorbic acid and NADH, quinone reducing agents, were significantly depleted during the enzymatic oxidation of ASA by tyrosinase to quinone. The 50% inhibitory concentration (48 h) of ASA and salicylic acid toward SK-MEL-28 cells were 100 micromol/l and 5.2 mmol/l, respectively. ASA at 100 micromol/l was selectively toxic toward human melanocytic SK-MEL-28, MeWo, and SK-MEL-5 and murine melanocytic B16-F0 and B16-F10 melanoma cell lines. However, ASA was not significantly toxic to human amelanotic C32 melanoma cell line, which does not express tyrosinase enzyme, and human nonmelanoma BJ, SW-620, Saos, and PC-3 cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased ASA toxicity toward SK-MEL-28 cells indicating quinone formation and intracellular GSH depletion played important mechanistic roles in ASA-induced melanoma toxicity. Ascorbic acid, a quinone reducing agent, and GSH, an antioxidant and quinone trap substrate, prevented ASA cell toxicity. Trifluoperazine, inhibitor of permeability transition pore in mitochondria, prevented ASA toxicity. ASA led to significant intracellular GSH depletion in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ASA also led to significant reactive oxygen species (ROS) formation in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ROS formation was exacerbated by dicoumarol and 1-bromoheptane in SK-MEL-28. Our investigation suggests that quinone species, intracellular GSH depletion, ROS formation, and mitochondrial toxicity significantly contributed toward ASA selective toxicity in melanocytic SK-MEL-28 melanoma cells.     

Combination of cyclosporin A and buthionine sulfoximine (BSO) as a pharmacological strategy for circumvention of multidrug resistance in small cell lung cancer cell lines selected for resistance to doxorubicin.

The small cell lung cancer (SCLC) cell lines U-1285 and U-1690 were adapted to growth in continuous presence of doxorubicin (Dox). The resulting cell lines U-1285R and U-1690R were investigated with respect to sensitivity to the glutathione (GSH) depleting agent buthionine sulfoximine (BSO) and the immunosuppressant cyclosporin A (CsA) as well as the Dox resistance modifying ability of these agents. The parental U-1285 cells were more sensitive to BSO compared to parental U-1690 and the multidrug resistant (MDR) sublines, whereas no difference in sensitivity to CsA was observed between parental and MDR lines. BSO (10 microM) or CsA (1 microgram/ml) alone were able to partially reverse Dox resistance in the MDR cell lines, CsA being only marginally active in U-1285R cells. However, the combination of these two drugs at the same concentrations completely reversed Dox resistance in the MDR U-1690R cells whereas the combination was less effective in the U-1285R cells. The results demonstrate that a combination of low concentrations of BSO and CsA, only partially active by themselves in modifying Dox resistance, may be used as a pharmacological strategy to increase Dox sensitivity in some MDR SCLC cells.     

Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide

The role of glutathione (GSH) as a determinant of cellular sensitivity to the cytotoxic and DNA-damaging effects of cyclophosphamide (CP) was studied in a dual culture system of rat hepatocytes and K562 human chronic myeloid leukemia cells, which have elevated aldehyde dehydrogenase activity with a corresponding insensitivity to activated CP. Exposure of K562 cells to 50 microM DL-buthionine-S,R-sulfoximine for 24 h resulted in a depletion of cellular GSH content to 10% of control values without toxicity. Subsequent 1-h exposure of GSH-depleted cells to activated cyclophosphamide, obtained by incubation of CP with suspension cultures of rat hepatocytes, resulted in a 5-fold potentiation of the cytotoxicity of CP. Alkaline elution analysis of cellular DNA demonstrated that the level of apparent interstrand cross-linking was 3 to 4 times higher in GSH-depleted cells than in nondepleted cells. GSH-depleted cells were, in addition, more sensitive to induction of DNA single strand breaks than nondepleted cells. Depletion of GSH content did not increase cellular sensitivity to the cytotoxicity of phosphoramide mustard. Preincubation of K562 cells with 1 mM cysteine for 4 h resulted in an approximately 60% increase in cellular GSH content, which was accompanied by decreased sensitivity to the cytotoxicity of hepatocyte-activated CP. Exposure of nondepleted cells to clinically relevant concentrations of hepatocyte-activated CP resulted in depletion of cellular GSH content. Replenishment of GSH content in these cells was relatively slow following CP exposure. Acrolein was highly effective at depleting cellular GSH content, whereas phosphoramide mustard had no effect on cellular GSH content. The depletion of GSH by intracellularly released acrolein may be important in the mechanism of cytotoxicity of CP.     

Glutathione depletion and cytotoxicity of buthionine sulphoximine and SR2508 in rodent and human cells

SR2508 (1 mM) increases the rate of glutathione (GSH) depletion by L-buthionine-S-R-sulphoximine (BSO) in hypoxic V79 rodent and A549 human cells. Specifically, the GSH content for V79 and A549 cells, after incubating for about 6 hr with 50 and 100 microM BSO, respectively, was lower by at least 10-fold when 1 mM SR2508 was present. In addition, 1 mM SR2508 is extremely toxic to hypoxic cells with lower GSH content. Survival probabilities of GSH-depleted V79 and A549 cells are about 10(-3) after 10 hr incubation with 1 mM SR2508. By itself, 1 mM SR2508 or 50-100 microM BSO decreased cellular viability by about 50% with a 10 hr treatment period. Both the phenomena described above are preferential towards hypoxic cells with minimal effect on aerobic cells.     

Hypoxanthine concentrations in normal subjects and patients with solid tumors and leukemia

Mean plasma hypoxanthine (Hyp) concentrations determined by high-pressure liquid chromatography were 0.56 microM (range, 0.2 to 1.9 microM) in 16 normal subjects, 0.68 microM (range, 0.1 to 1.1 microM) in 10 untreated acute leukemic subjects, and 0.89 microM (range, 0.3 to 2.6 microM) in 14 solid tumor patients. Despite large differences in Hyp concentration between patients, every 4-hr sampling, indicated that diurnal variation in individual patients was small (maximum, 2.3-fold). While the mean plasma and malignant effusion Hyp concentrations did not differ significantly, bone marrow plasma Hyp concentration averaged 4.0-fold greater than that of simultaneously drawn venous plasma. Allopurinol 300 mg p.o. caused a mean 1.5-fold increase in plasma Hyp within 3 hr. In 17 patients with acute leukemia, treatment with allopurinol at 300 mg daily plus initiation of chemotherapy caused a mean 7-fold increase in plasma Hyp to 4.6 microM (range, 1 to 12 microM). The ability of Hyp to modulate the toxicity of antimetabolites affecting purine synthesis (6- diazao -5- oxonorleucine , 6-methylmercaptopurine riboside, 6-mercaptopurine, and 6-thioguanine) was determined in vitro using human B-lymphoblast (WI-L2) and promyelocytic leukemia (HL-60) cell lines. Hyp permitted growth of both cell lines in the presence of clinically achievable concentrations of all 4 drugs, but the initial culture concentrations of Hyp required were above those found in patients. Since Hyp was consumed rapidly during the culture period, the average Hyp concentrations required for the protection of cells were actually much lower. We conclude that, in patients with acute leukemia receiving allopurinol during chemotherapy, plasma Hyp concentrations are significantly elevated; the potential for antagonism of antimetabolite activity is uncertain.     

Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism

Ultraviolet (UV) light-induced activation of activator protein-1 (AP-1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP-1 activation. We have, therefore, examined the effects of two well-known inducers of phase II enzymes, sulforaphane (SF) and tert-butylhydroquinone (tBHQ), on UVB-induced AP-1 activation, with an AP-1-luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase-1 (QR-1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1-10 microM SF or 11-45 microM tBHQ for 24 h resulted in up to 1.4-fold and 1.7-fold increase of QR-1 activity, respectively, and up to 1.5-fold and 1.6-fold increases in cellular GSH levels, respectively. AP-1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m(2) of UVB. While the above SF treatment dose-dependently reduced the UVB-induced AP-1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB-induced AP-1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB-induced AP-1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP-1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB-induced AP-1 activation. The inhibitory effect of SF on UVB-induced AP-1 activation appears to be at least partly due to the direct inhibition of AP-1 DNA binding activity. This direct effect of SF on AP-1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP-1 activation.     

The effect of GSH depletion on thermal radiosensitization

Depletion of intracellular glutathione (GSH) increased aerobic thermal radiosensitization in Chinese hamster ovary (CHO) cells gamma irradiated and heated at 42 degrees C. The GSH concentration was decreased to various stable levels by exposure to increasing concentrations of diethylmaleate (DEM). Analysis of dose-response curves indicated that GSH depletion affected thermal sensitization and thermal radiosensitization at 42 degrees when greater than 95% of the GSH had been depleted. GSH depletion also increased the fixation of radiation damage. For example, survival after 10 Gy decreased from 0.012 to 0.006 if CHO cells were incubated in 100 microM DEM at 37 degrees for 2 hrs after irradiation. The results show that GSH might be an important agent for the protection of cells against thermal enhancement of radiation damage.     

Differential specificity of monochlorobimane for isozymes of human and rodent glutathione S-transferases

Monochlorobimane (MCB) has been used as a glutathione (GSH) specific fluorescent probe capable of delineating GSH heterogeneity in cellular systems. Generally, low concentrations of MCB (less than 50 microM) have been used to quantitatively label GSH in rodent cell lines. Incubation of the hamster cell lines, CHO AB1 and V79, with 10 microM MCB labeled 75 and 39% of the reduced GSH pool, respectively. In contrast, incubation of 7 different human cell lines with 10 microM MCB labeled less than 4% of the total reduced GSH pool. The human cell lines required 1000 microM MCB to label an average of 73% of the GSH pool (range, 60-88%). When using 1000 microM MCB to label GSH, flow cytometry results from 7 different cell lines (human and rodent) were in good agreement with high performance liquid chromatography and standard spectrophotometric analysis with regards to a rank ordering of the GSH content determined for each cell line. The human glutathione S-transferases B2B2, B1B2, psi, pi, and the rat transferases 1-2, 3-3, and 3-4 were isolated and purified for steady state kinetic analysis with MCB and GSH as the primary substrates. The human basic transferases, B1B2 and B2B2, had Km values for MCB of 354 and 283 microM and Vmax values of 33.3 and 34.6 mumol bimane-GSH/min/mg protein, respectively. The rat basic transferase 1-2 showed similar kinetic results with a Km of 199 microM and a Vmax of 35.5 mumol bimane-GSH/min/mg protein. The human neutral transferase (psi) had a Km for MCB of 204 microM with a Vmax of 6.5 mumol bimane-GSH/min/mg protein. In contrast, MCB has a high affinity for the rat neutral transferase with a Km of 2.6 microM and a Vmax of 35.1 mumol bimane-GSH/min/mg protein. The human acidic transferase (pi), the predominate transferase found in most human tumor cell lines, has a Km of 264 microM for MCB and a Vmax of 1.99 mumol bimane-GSH/min/mg protein. The kcat/Km values indicated that MCB is an excellent substrate for the rat neutral transferases while the human pi glutathione S-transferase showed the least reactivity. Collectively the data indicate that MCB fails to label GSH at lower concentrations (less than 50 microM) in human cell lines because of the reduced affinity of MCB for the human transferases and possibly also due to differences in glutathione S-transferase isozyme expression between rodent and human cell lines.     

Glutathione dependence of neocarzinostatin cytotoxicity and mutagenicity in Chinese hamster V-79 cells

Neocarzinostatin (NCS) is mutagenic in bacteria, yeast, fungi, and mammalian cells. In cell-free systems, DNA strand breakage induced by NCS requires a reducing agent like 2-mercaptoethanol, unless very high (greater than 100 micrograms/ml) concentrations of NCS are used. In this study, we have investigated the role of the sulfhydryl compound glutathione (GSH), which is usually the most common intracellular thiol, in the bioactivation of NCS to a toxic and mutagenic species. Chinese hamster V79 cells were pretreated with one of two GSH depleting agents, buthionine sulfoximine or diethyl maleate. These agents deplete GSH via different mechanisms, but both will lower GSH levels within the cell to less than 5% of control (untreated) values. GSH-depleted cells and control cells were then exposed to NCS concentrations of 0.5-2.5 micrograms/ml for 1 h, assayed for survival, and plated for expression of hypoxanthine-guanine phosphoribosyltransferase-negative (HGPRT-) mutants. After an expression period of 7 days, during which the cultures were subcultured twice, HGPRT- mutants were selected by plating in hypoxanthine-free medium containing 5 micrograms of 6-thioguanine per ml, at a density of 2 X 10(5) cells per 100 mm dish. NCS alone decreased the surviving fraction to about 1% at 2.5 micrograms/ml and produced dose-related increases in HGPRT-mutants that reached greater than 10 times the spontaneous mutation frequency at 2.5 micrograms NCS per ml. In GSH-depleted cells, however, NCS was only mildly cytotoxic (60-80% surviving fraction) and did not produce dose-related increases in HGPRT- mutants over cells treated only with diethyl maleate or buthionine sulfoximine. Thus, GSH appears to be the main reducing agent for the bioactivation of NCS to a toxic and mutagenic species in Chinese hamster V79 cells.     

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.     

Sensitivity of human melanoma cells to L-dopa and DL-buthionine (S,R)-sulfoximine

Four of seven human melanoma cell lines were sensitive to killing by L-dopa (D37 1.0-4.7 microM) compared with fibroblasts, Hela, and three ovarian tumor cell lines (D37 12-59 microM). All seven melanoma lines, however, were sensitive to DL-buthionine(S,R)sulfoximine (BSO) (D37 0.73-8.5 microM) compared with the nonmelanoma cells (D37 25-68 microM). The melanoma line most sensitive to BSO (MM418) was highly melanized, proliferated slowly and was resistant to other agents [dopa, 5-(3-methyl-1-triazeno)5-imidazole-4-carboxamide, melphalan, methotrexate, hydroxyurea, etoposide, Adriamycin]. In most cell lines, L-dopa and BSO blocked cell proliferation in all phases of the cell cycle. Cellular sensitivity to dopa or BSO did not correlate with levels of total soluble SH, glutathione (GSH), GSH reductase, GSH peroxidase or GSH transferase, or with the extent of GSH depletion induced by the drug. No GSH transferase activity could be detected in the dopa-resistant HeLa line, indicating that detoxification of quinones is not an important mechanism of resistance. Within the group of melanoma cell lines, sensitivity to dopa correlated with decreased level of gamma-glutamyl transpeptidase (r = 0.81). However, the gamma-glutamyl transpeptidase inhibitor azaserine was less effective than BSO in enhancing the toxicity of dopa. It can be inferred that (a) there is no simple relationship between GSH metabolism and sensitivity to dopa or BSO in human melanoma cells, and (b) BSO may be an effective agent for melanoma.     

Caspase inhibitor decreases apoptosis in pyrogallol-treated lung cancer Calu-6 cells via the prevention of GSH depletion

Pyrogallol (PG) is a polyphenol compound and is known to be an O2.- generator. In the present study, we evaluated the anti-apoptotic effects of caspase inhibitors in relation to changes in reactive oxygen species (ROS) and glutathione (GSH) levels in PG-treated human pulmonary adenocarcinoma Calu-6 cells. Treatment with 50 microM PG inhibited the growth of Calu-6 cells approximately 60% and induced apoptosis approximately 17% at 24 h, accompanied by mitochondrial membrane potential loss (DeltaPsim). Treatment with pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) significantly prevented apoptosis in PG-treated Calu-6 cells at 24 h. PG increased the ROS and depleted GSH contents in Calu-6 cells. Treatment with each caspase inhibitor did not significantly change the ROS and GSH levels in PG-treated Calu-6 cells at 24 h. However, Z-VAD significantly prevented GSH depletion in PG-treated Calu-6 cells at the late time phase of 72 h. Conclusively, the anti-apoptotic effect of caspase inhibitor on PG-induced Calu-6 cell death was closely related to changes in GSH content rather than ROS levels.     

Glutathione and related enzymes in rat brain tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea and nitrogen mustard

Reduced glutathione (GSH) and activities of several glutathione-related enzymes were measured in two 9L rat brain tumor cell lines with differing sensitivities to both 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and nitrogen mustard. GSH, measured by a specific high-performance liquid chromatographic method, was found to be approximately twice as high in 9L cells sensitive to BCNU but resistant to nitrogen mustard. The nitrogen mustard resistant cell line was also found to have 2.5-fold more bulk glutathione transferase activity and approximately 3-fold more gamma-glutamyl transpeptidase activity. Glutathione reductase activity, protein thiol, and total protein content were similar in the two cell lines. Pretreatment of 9L cells with 50 microM buthionine sulfoximine for 24 h to deplete GSH only slightly potentiated BCNU cytotoxicity in a clonogenic assay whereas that of nitrogen mustard was markedly potentiated in both cell lines. Similarly, buthionine sulfoximine pretreatment had little effect on the induction of sister chromatid exchanges by BCNU, but significantly increased the number of sister chromatid exchanges induced by nitrogen mustard in both cell lines. Depleting GSH also had no significant effect on the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea to 9L cells. Pretreatment of 9L cells with 1 mM GSH significantly protected against nitrogen mustard cytotoxicity. Moreover, nitrogen mustard incubated with GSH and glutathione transferase was 4-fold less cytotoxic than nitrogen mustard incubated with GSH alone. Incubation of BCNU with GSH alone or with glutathione transferase had no effect on BCNU cytotoxicity. These results indicate that elevated GSH and glutathione transferase activity is one mechanism of cellular resistance to nitrogen mustard in the 9L cell line, but it does not correlate with resistance to BCNU or other clinically important nitrosoureas.     

Relationship between intracellular glutathione level and the mode of cell death induced by pingyangmycin

The effects of intracellular glutathione (GSH) concentration on the toxicity of pingyangmycin in human squamous cell carcinoma cell line were evaluated. By using the GSH synthesis inhibitor D,L-buthionine-S,R-sulfoximine and the precursor of cysteine 2-oxothiazolidine-4-carboxylate (OTZ), intracellular glutathione levels were artificially changed. After exposed to different GSH concentrations cultured tumor cells were treated with pingyangmycin and the resultant mode of cell death was analyzed using morphological and biochemical criteria. It was found that the toxicity of pingyangmycin was obviously increased to cultured tumor cells on lowering GSH levels, with the mode of cell death switching from necrosis to apoptosis. In contract, treatment with OTZ increased GSH level compared with that of control cells, inhibited cell death induced by pingyangmycin via a necrotic rather than apoptotic process. These observations suggest that modulation of GSH levels effects the toxicity of pingyangmycin and that GSH influences the mode of cell death induced by pingyangmycin.