Potentiation of the cytostatic effect of melphalan on colorectal cancer hepatic metastases by infusion of buthionine sulfoximine (BSO) in the rat Enhanced tumor glutathione depletion by infusion of BSO in the hepatic artery

Purpose: Glutathione (GSH) plays an important role in the resistance of tumors to cytostatics. Therefore, depletion of GSH by the GSH synthesis inhibitor buthionine sulfoximine (BSO) has been proposed to enhance the efficacy of certain anticancer agents. We studied the effect of BSO in rats bearing intrahepatically implanted tumors of the CC531 colorectal cancer cell line on the antitumor activity of melphalan (L-PAM). Since these liver tumors tend to derive most of their blood supply from the hepatic artery, we evaluated whether delivery of BSO into the hepatic artery would more selectively decrease GSH levels in the implanted tumor tissue as compared with normal liver and extrahepatic tissues. Methods: Tumor-bearing rats were treated with a 24-h continuous infusion of 0.375 mmol/kg BSO via the jugular vein, immediately followed by a bolus L-PAM (15 μmol/kg; 4.5 mg/kg) infusion via the hepatic artery. Laparotomy was performed on day 14 and 28 after treatment for measurement of the liver tumors. For the evaluation of locoregional administration of BSO, a 24-h continuous infusion of 0.375 mmol/kg BSO was delivered into either the hepatic artery, the portal vein, or the jugular vein in freely moving rats and GSH levels in the tumor, liver, kidney, lung, heart, bone marrow, and blood were measured. Results: BSO infusion via the jugular vein increased the antitumor efficacy of L-PAM injected into the hepatic artery 2-fold as determined at 14 days after treatment. Although infusion of BSO via the hepatic artery depleted GSH more severely in the tumor as compared with jugular vein or portal vein administration, the additional effect was only slight (10%). No difference was observed in any other tissue. Conclusion: GSH depletion increased the cytostatic efficacy of L-PAM 2-fold in vivo as determined at 14 days after treatment. Hepatic artery infusion of BSO translated into a statistically significant, but probably not therapeutically relevant, increase in tumor GSH depletion as compared with the other routes of BSO administration. Received: 21 September 1998 / Accepted: 4 January 1999     

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.     

Establishment of l1210 leukemia cells resistant to the distamycin-a derivative (FCE 24517): Characterization and cross-resistance studies

N-deformyl-N-[4-N,N-bis(2-chloroethylamino)benzoyl] distamycin-A (FCE 24517) is a new cytotoxic anti-tumor agent in phase-I clinical trials. We have isolated stable FCE-24517-resistant cell sublines from murine leukemia L1210 cells by in vitro exposure to the drug. FCE 24517 selects a mixed population of resistant cells: the L1210/24517₁ cell line in vitro was in fact resistant to the selecting agent (RI 48.3), as well as to L-PAM (RI 5.4) and DX (RI 8.6) and over-expressed the mdr-I gene. When L1210/24517₁ cells were implanted in vivo and evaluated for sensitivity to the same agents, resistance was observed only to FCE 24517 and partially to L-PAM. whereas DX had the same anti-tumor efficacy as on the sensitive line. The clone derived from the above subline (L1210/24517₂) was resistant to FCE 24517, distamycin-A and other cytotoxic compounds bearing the distamycin-A skeleton, and fully sensitive to DX and other anti-tumor compounds involved in the multi-drug resistance mechanisms, with a complete disappearance of the mdr phenotype. L1210/24517₂ cell line is partially cross-resistant to L-PAM, this resistance being accounted for by higher GSH intracellular levels, which however do not influence the resistance to FCE 24517. In fact, BSO treatment was capable of significantly modifying only the cytotoxicity of L-PAM. Our data suggest that L1210/24517₂ cells present a mechanism of resistance specific for FCE 24517 and related molecules.     

In vivo therapeutic potential of combination thiol depletion and alkylating chemotherapy.

The effect of administering the thiol modulating agent buthionine sulfoximine (BSO) in conjunction with alkylating chemotherapy was investigated in vivo in the mouse KHT sarcomas and bone marrow stem cells. Tumour response to treatment was assessed by an in vivo to in vitro excision assay and bone marrow survival was determined in vitro by CFU-GM. Glutathione (GSH) depletion and recovery kinetics were determined at various times after treatment using high performance liquid chromatography (HPLC) techniques. Following a single 2.5 mmol kg-1 dose of BSO, tumour GSH reached a nadir of approximately 40% of control 12-16 h after treatment. Bone marrow GSH was depleted to approximately 45% of control 4-8 h after treatment but recovered to normal by 16 h. When a range of doses of CCNU, mitomycin C, cyclophosphamide or melphalan (MEL) were given 16 h after mice were exposed to a 2.5 mmol kg-1 dose of BSO, only the antitumour efficacy of MEL was effectively enhanced (by a factor of approximately 1.4). This BSO-MEL combination appeared to be selective for the tumour as the bone marrow toxicity was not increased beyond that seen for MEL alone. Since increasing the administered dose of BSO neither increased the extent of thiol depletion in the tumour nor enhanced the antitumour efficacy of MEL, three other protocols for delivering the thiol depletor were explored. BSO was given either as multiple 2.5 mmol kg-1 doses administered at 6 or 16 h intervals or continuously at a concentration of 30 mM supplied in the animals'' drinking water. Both multi-dose BSO pretreatments were found to increase both the antitumour efficacy and normal tissue toxicity of MEL such that no advantage compared to the single dose combination was achieved. In contrast, maintaining the thiol depletor in the drinking water led to an approximately 1.7-fold increase in the antitumour efficacy of MEL without any corresponding increase in bone marrow stem cell toxicity. For the various pretreatment strategies it was possible, in all cases, to account for the presence or absence of a net therapeutic benefit on the basis of the tumour and bone marrow GSH depletion and recovery kinetics.     

Selective enhancement of hypoxic cell killing by melphalan via thiol depletion: in vitro studies with hypoxic cell sensitizers and buthionine sulfoximine

The relationship between thiol depletion and its enhancement of melphalan (L-PAM) cytotoxicity was studied with the use of V-79-379A Chinese hamster cells in vitro. Selective killing of hypoxic cells by use of a specific and nonspecific reducer of endogenous cellular thiols was the approach used in combining drugs with disparate mechanisms of action. Noncytotoxic concentrations of agents were employed in those experiments designed to mimic a practical scheme for their implementation in vivo. Cells made hypoxic by gassing in suspension with 95% nitrogen and 5% CO2 were treated with buthionine S-R-sulfoximine (BSO), a specific inhibitor of glutathione synthesis and a hypoxic cell sensitizer (i.e., either misonidazole or SR-2508) before their exposure to the alkylating agent. Cellular loss of nonprotein thiols by treatment with BSO correlated with enhanced L-PAM toxicity; however, a far greater effect was achieved when this enzymatic inhibitor was used in combination with a hypoxic cell sensitizer. This chemopotentiation of hypoxic cell killing by L-PAM, along with little potentiation of toxic cell killing, indicated the practical and potential benefit of this sort of drug therapy in vivo.     

Effect of DNA-repair-enzyme modulators on cytotoxicity ofl-phenylalanine mustard andcis-diamminedichloroplatinum (II) in mammary carcinoma cells resistant to alkylating drugs

We investigated the effect of DNA-repair-enzyme inhibitors onl-phenylalanine mustard (L-PAM) andcis-diamminedichloroplatinum (II) (CDDP) cytotoxicity in rat mammary-carcinoma MatB cells sensitive (WT) and resistant (MLNr) to bifunctional alkylating drugs. Among the modulators tested, the combination of arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) significantly increased the sensitivity of the cells to CDDP and, to a lesser extent, L-PAM as compared with cells treated with drug alone. The modulation effect of HU+Ara-C on CDDP and L-PAM cytotoxicity was more effective when intracellular glutathione (GSH) was depleted byl-buthionine-(S,R)-sulfoximine (BSO). This was also associated with a significant increase in DNA-DNA interstrand crosslinks. Caffeine also sensitized both WT and MLNr cells to the cytotoxic effect of L-PAM and CDDP, and this effect was potentiated in GSH-depleted cells. No significant effect was observed with other repair modulators such as aphidicolin, 3-aminobenzamide, novobiocin, or etoposide. These results show (a) that inhibition of DNA repair by HU+Ara-C or caffeine could be a target for modulation of bifunctional alkylating-drug resistance and (b) that GSH depletion renders resistant cells more susceptible to the repair-enzyme modulators, suggesting that intracellular GSH may be involved in the regulation of some of these enzymes. Our results also indicate that a combination of a number of modulators may offer an advantage over the use of a single modulator in tumor resistance that may be associated with multifactorial mechanisms.Abbreviations 3-AB 3-Aminobenzamide - ADP adenosine diphosphate - APD aphidicolin - Ara-C arabinofuranosylcytosine - BSO 1--d-l-buthionine-(S,R)-sulfoximine - CDDP cis-diamminedichloroplatinum (II) (cisplatin) - GSH glutathione - GST glutathione-S-transferase - HU hydroxyurea - L-PAM l-phenylalanine mustard (melphalan) This work was supported in part by the Quebec Lung Association and the National Cancer Institute of Canada     

Augmentation of cisplatin (DDP) cytotoxicity in vivo by DL-buthionine sulfoximine (BSO) in DDP-sensitive and-resistant rat ovarian tumors and its relation to DNA interstrand cross links.

DDP treatment (1.2 mg/kg x 5) prolonged the mean survival time (MST) of rats bearing an experimental ovarian tumor (0-342) from 16.4 to 51.1 days, with one of ten rats surviving more than 90 days. Administration of D,L - buthionine sulfoximine (BSO) (24 and 2 h prior to DDP, respectively) before the last two doses of DDP had no significant effect on DDP therapeutic activity, while daily combination of DDP with BSO (BSO 2 h prior to DDP) throughout the treatment significantly increased MST to 69 days (p less than 0.05, vs. DDP alone), with three of ten rats surviving more than 90 days. In the DDP resistant counterpart (0-342/DDP), on the other hand, DDP alone showed only a slight increase of MST (11.6 days in DDP group vs. 10.7 days in control group), addition of BSO to DDP treatment further prolonged MST to 13.3 days (p less than 0.01 vs. DDP alone). The formation of DNA interstrand cross links (DNA-ISCL) was found to be higher in 0-342 than in 0-342/DDP cells in vitro with a maximum at 24 h following 1 h exposure to DDP. BSO depleted the intracellular GSH level in a dose - and time - dependent manner in the two cell lines. Pretreatment with BSO resulted in a 7.4% increase in DNA-ISCL by DDP in 0-342 cells but a 39% increase in 0-342/DDP cells, which may partially account for chemosensitization of BSO to DDP in vivo. Our result that the chemosensitizing effect of BSO, through depletion of cellular GSH, is more significant in the DDP sensitive O-342 tumor than in its DDP resistant subline in vivo underlines that BSO should be used as a chemosensitizer in combination with DDP at the beginning of chemotherapy for clinical trial.     

Depletion of glutathione in vivo as a method of improving the therapeutic ratio of misonidazole and SR 2508

Depletion of intracellular glutathione (GSH) can enhance misonidazole (MISO) radiosensitizing efficacy both in vivo and in vitro. However, such treatments may also enhance the systemic toxicity in animals. The purpose of the present study was to test various ways of depleting GSH levels in a variety of experimental mouse tumors, to measure the improvement in the efficacy of MISO and its less toxic analog SR 2508 by this depletion, and to determine the effect of daily GSH depletion on the toxicity of MISO and SR 2508. GSH levels were measured daily for 5 days in tumors, livers and brains of mice injected daily with buthionine sulfoximine (BSO), with or without diethylmaleate (DEM). To investigate tumor variability we studied 5 different tumors: EMT-6, RIF-1, KHT, SCC VII, and B16 melanoma. The efficacy of MISO and SR 2508 was evaluated using the KHT and SCC VII tumors either by the regrowth delay assay or by the in vivo/in vitro clonogenic assay. The drug toxicity was evaluated by weight loss and by death. Daily doses of 3 mmole/kg BSO depleted tumor levels of GSH to 20 to 40% of controls by 6 hr after each injection. Injection of DEM (300 mg/kg) 6 hr after BSO further enhanced the depletion. Administration of MISO or SR 2508 at the time of maximum GSH depletion enhanced the MISO efficacy by factors of 2.5 to 8 for depletion to 8% of controls by BSO + DEM, but no enhancement of SR 2508 was seen with tumors at 20% GSH levels achieved with BSO alone in the preliminary experiment. The chronic toxicity of MISO was enhanced not at all or by a factor of up to 2 for BSO and BSO + DEM respectively. Further studies are needed before it can be concluded that GSH depletion by BSO alone may be a useful adjunct to the clinical use of radiosensitizers.     

Effect of glutathione depletion by L-buthionine sulfoximine on the cytotoxicity of cyclophosphamide in single and fractionated doses to EMT6/SF mouse tumors and bone marrow

The combined cytotoxicity of cyclophosphamide [(CYC) CAS: 50-18-0] and glutathione [(GSH) CAS: 70-18-8] depletion by buthionine sulfoximine (BSO) toward EMT6/SF tumors and mouse bone marrow was studied in male BALB/c mice. Tumor GSH was depleted to 28.7 and 7.8% of control by 1 or 2 doses of BSO (5 mmol/kg), respectively, administered ip at 12-hour intervals prior to assay. Tumor GSH could be maintained at a level 8% of control by daily dosing with BSO for 3 days. The same BSO administration schedule lowered bone marrow cell GSH to 31% of control for the 3-day period studied. Tumor growth and bone marrow cell counts were unaffected by all BSO treatments studied. However, BSO pretreatment enhanced the cytotoxicity of CYC to tumor cells, as measured by an in vitro colony-forming assay, but it did not enhance the depletion of bone marrow cells in CYC-treated mice. The magnitude of the enhancement of CYC cytotoxicity by BSO depended on the extent of GSH depletion, tumor size, and the CYC dosing schedule: Single and double doses of BSO enhanced the cytotoxicity of a single dose of CYC by factors of 1.5 and 2.0, respectively, in 7-day-old tumors, based on the dose of CYC required to produce a surviving fraction of 4 X 10(-3). The response of 9-day-old tumors to CYC suggested the presence of a subpopulation of cells that were relatively resistant to CYC. There was no evidence for this subpopulation in BSO-pretreated tumors. Multiple doses of BSO and CYC also combined to give enhanced (1.65-fold) tumor cell killing compared to tumors treated with CYC alone. The data suggest a possible role for BSO as a clinical chemosensitizer, which could be combined with fractionated doses of CYC and may be effective on small cancerous lesions.     

The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma

Melphalan (L-PAM) has been an integral part of multiple myeloma (MM) treatment as a conditioning regimen before stem cell transplant (SCT). After initial response, most treated patients experience relapse with an aggressive phenotype. Increased glutathione (GSH) in MM may mediate resistance to L-PAM. We demonstrated that the GSH synthesis inhibitor buthionine sulfoximine (BSO) synergistically enhanced L-PAM activity (inducing 2–4 logs of cell kill) against nine MM cell lines (also in the presence of marrow stroma or cytokines) and in seven primary MM samples (combination indices <1.0). In MM cell lines, BSO significantly (P<0.05) depleted GSH, increased L-PAM-induced single-strand DNA breaks, mitochondrial depolarization, caspase cleavage and apoptosis. L-PAM depleted GSH, but GSH rapidly recovered in a L-PAM-resistant MM cell line unless also treated with BSO. Treatment with N-acetylcysteine antagonized BSO+L-PAM cytotoxicity without increasing GSH. In human MM xenografted into beige-nude-xid mice, BSO significantly depleted MM intracellular GSH and significantly increased apoptosis compared with L-PAM alone. BSO+L-PAM achieved complete responses (CRs) in three MM xenograft models including maintained CRs >100 days, and significantly increased the median event-free survival relative to L-PAM alone. Combining BSO with L-PAM warrants clinical testing in advanced MM.     

Role of glutathione in the in vitro synergism between 4-hydroperoxy-cyclophosphamide and cisplatin in leukemia cell lines

To explain the sequence-dependent in vitro cytotoxic synergism between 4-hydroperoxycyclophosphamide (4-HC) and cisplatin in the K-562 human leukemia cell line, we have hypothesized that 4-HC decreases cellular glutathione (GSH) levels and that the resulting diminution of the cellular protective effect of GSH leads to the increased cytotoxicity of cisplatin. Exposure of K-562 cells to 4-HC resulted in a concentration- and time-dependent depletion of cellular GSH. To determine the effect of modulation of GSH levels on the toxicity of cisplatin, K-562 cells were exposed to buthionine sulfoximine (BSO) and/or GSH ethyl esters. Depletion of GSH to approximately 10% of control values by BSO potentiated the cytotoxicity of cisplatin, while rapid replenishment of GSH to within normal levels by GSH esters abolished the potentiation of BSO. Doubling cellular GSH by incubation with GSH esters protected against cisplatin cytotoxicity. Of importance, pretreatment of K-562 cells with BSO, in addition to increasing the cytotoxicity of 4-HC and cisplatin, abolished the synergism between the two drugs. The working hypothesis was also tested in two other cell lines in which the cytotoxic synergism between 4-HC and cisplatin was exhibited: the Raji cell line, a human lymphoblastic cell line, and the L1210-CPA cell line, a subclone of the murine L1210 leukemia with resistance to 4-HC. GSH levels in these two cell lines were not altered by incubation with concentrations of 4-HC at which the synergism was observed. In conclusion, the data for the K-562 cell line, indicating that (a) 4-HC depletes cellular GSH levels, (b) the lowering of cellular GSH levels enhances the toxicity of cisplatin, and (c) intact GSH stores are required for the synergism, strongly support the postulate that the cytotoxic synergism between 4-HC and cisplatin is modulated by GSH levels in this cell line. However, the lack of 4-HC-mediated depletion of GSH at concentrations of 4-HC resulting in cytotoxic synergism in the Raji and L1210-CPA cell line indicates that mechanisms other than modulation of GSH levels by 4-HC are responsible for the synergism in these cells.     

Chemosensitization by buthionine sulfoximine in vivo

The in vivo effects of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, on the cytotoxicity of cyclophosphamide (CYM), cisplatin (CDDP) and bleomycin (BLM), were examined by monitoring the changes of non-protein thiols (NPSH) in normal tissues and in the NFSa fibrosarcoma. We used the lung colony assay as a measure of tumor response and the spleen colony assay as a measure of normal tissue response to CYM. In this study, 5 mmol/kg of BSO was subcutaneously injected four times every 12 hr before administration of the above anti-neoplastic drugs. GSH levels in subcutaneous NFSa tumors decreased to 2% of the control 12 hr after the last administration of BSO, but in the bone marrow, had recovered to 41%. In the colony assays, BSO increased the anti-cancer effects of the three chemotherapeutic agents, but did not modify the bone marrow suppression by CYM. This finding was a result of the differential response of GSH depletion in the tumor and in the bone marrow. Our study demonstrates that BSO is an effective chemosensitizer of these drugs and may be of therapeutic value when used at an optimal interval.     

Extracellular: intracellular and subcellular concentration gradients of thiols.

Chinese hamster V79 cells in Eagle's minimum essential medium in vitro at room temperature were incubated with the aminothiol, WR-1065, or glutathione (GSH) at extracellular concentrations of approximately 1 mmol dm-3. Average intracellular concentrations of GSH, cysteine, and WR-1065 were measured by high performance liquid chromatography, and the effective reducing environment near DNA probed by staining the cells with acridine orange (AO) and measuring the delayed fluorescence. Exposure to either thiol resulted in a rapid, 10-fold increase in average intracellular cysteine concentrations (to about 1 mmol dm-3). Adding extracellular GSH after prior depletion of GSH by treatment with L-buthionine sulfoximine (BSO) did not restore intracellular GSH, but intracellular cysteine was elevated 10-fold. These results are ascribed to thiol/disulfide exchange with cystine in the medium. WR-1065 slowly concentrated intracellularly to approximately 160% of the extracellular concentration. Chemical conjugation of GSH in cells decreased the reducing environment near DNA, but BSO treatment altered the uptake of AO. The electrostatic attraction of WR-1065 toward isolated DNA was markedly affected by ionic strength.     

Intracellular glutathione heterogeneity in L1210 murine leukemia sublines made resistant to dna-interacting anti-neoplastic agents

Intracellular glutathione (GSH) content was measured by flow cytometry using monochlorobimane (mBCI) and by the enzymatic assay in a set of 6 sublines of murine L1210 leukemia cells made resistant to DNA-interacting agents having distinct mechanisms of action: L-phenylalanine mustard (L-PAM), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin (DDP), N-de-formyl-N-(4-N, N-bis(2-chloroethylamino) benzoyl) distamycin A (FCE 24517), doxorubicin (DX) and 3′ -deamino-3′ (2-methoxy-4-morpholinyl)-doxorubicin (FCE 23762). A significant correlation was demonstrated between the mean intracellular mBCI fluorescence values measured by flow cytometry and levels of GSH measured by the classical enzymatic assay, despite the possible influence of glutathione-S-transferases and of other thiols on the mBCI fluorescence. Although less specific, the flow cytometric method is more informative than the enzymatic assay, allowing detection of fluorescence distributions, which we proved to be characteristic of each subline. In order to assess a procedure enabling a quantitative analysis to be made of intercellular GSH heterogeneity, we propose the use of appropriate thresholds and parameters of the mBCI flow cytometric distribution. By use of this analysis procedure, distinct types of alterations, with respect to the heterogeneity distribution of the parental L1210 cell line, have been evidenced in resistant cells. A uniform increase in mBCI fluorescence was observed among cells of the sublines resistant to L-PAM and FCE-24517. The mean mBCI fluorescence increase in sublines resistant to DX and DDp was due to a higher number of cells with fairly high mBCI fluorescence, but still within the range spanned by the parental cell line. A less heterogeneous mBCI fluorescence distribution was found in the L1210 subline resistant to FCE 23762, which was, however, similar to a cloned sensitive line. Though GSH was linked to the principal cause of drug resistance only in the L-PAM-resistant cell line, alterations in heterogeneity, as detected by mBCI fluorescence distributions, were found in 5 out of 6 resistant lines.     

Effect of thiol manipulation on chemopotentiation by nitroimidazoles

Highly electron affinic compounds such as the nitroimidazole misonidazole (MISO) have been shown both in vitro and in vivo to be effective potentiators of certain conventional chemotherapeutic agents. Mechanistically, the observation that nitroheterocyclics reduce intra-cellular thiols by enhancing the oxidation of glutathione (GSH), has suggested that thiol depletion by MISO may be a key factor in this enhancement. The present investigations were undertaken to determine whether the use of buthionine sulfoximine (BSO) to affect GSH metabolism may lead to more effective potentiation of chemotherapeutic agents by sensitizers. KHT/iv cells were treated in exponential phase under hypoxic conditions with variable doses of the activated form of cyclophosphamide (4-hydroxy-cyclo-phosphamide, 4OH-CY) administered concomitantly with or without MISO (2.5 mM) for an exposure time of 4 hr. Inclusion of the sensitizer in the treatment protocol resulted in a dose modifying factor of approximately 2.4. Exposing cells to 1.0 mM BSO for 2 hr prior to treatment reduced intracellular GSH levels to 70-80% of control and increased the efficacy of 40H-CY approximately 1.2-fold. If BSO was administered prior to the 4OH-CY + MISO combination, severe tumor cell toxicity resulted. For example, when combined with 4OH-CY, similar cell kill could be achieved with 5 to 6-fold lower MISO doses in the presence of BSO as in the absence of BSO. Ultrastructural evaluations revealed that in the three agent combination, membrane damage, as reflected by the formation of surface blebs, may play a key role in the mechanism of the observed enhanced cytotoxicity.     

Radiosensitizing effect of misonidazole in combination with an inhibitor of glutathione synthesis in murine tumors

The radiosensitizing effects of misonidazole (MISO) in combination with D,L-buthionine-S, R-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, were studied in NFSa tumors of C3H/He mice. The radiation response of tumors was assayed by the tumor growth delay time. The GSH contents in tissues were assayed by high performance liquid chromatography (HPLC). GSH content in the tumors decreased to the minimum level (45% of the control), and then gradually recovered to 75% of the control, respectively, 12 and 24 hr after the intraperitoneal injection of 5 mmole/kg BSO. On the other hand, the maximum non-protein sulfhydryl (NPSH) depletion (29% of the control) in the liver of tumor bearing mice was achieved 6 hr after the administration of the same dose of BSO, but fully recovered 24 hr later. When 5 mmole/kg BSO was injected repeatedly 4 times at an interval of 6 hr, GSH content in the tumors decreased to 19% of the control 24 hr after the first injection of BSO. The radiosensitizing effect of 0.5 mmole/kg MISO was markedly increased by this BSO treatment. The enhancement ratio (ER) of this combined treatment was 1.93. On the other hand, ERs of 1.44 and 1.16 were obtained for MISO (0.5 mmole/kg) and for 4 injections of BSO (5 mmole/kg) in combination with radiation, respectively. Although a considerable increase in the radiosensitizing efficiency of MISO in vivo by the treatment with BSO was found without any notable side effects of the combination, more studies on toxicities are needed to get a definite conclusion on the clinical applicability of the combination.     

Glutathione depletion by buthionine sulfoximine induces DNA deletions in mice

Oxidative stress and genomic rearrangements play a role in cancer development. l-Buthionine-sulfoximine (BSO) induces oxidative stress in a cell by irreversibly inhibiting gamma-glutamylcysteine synthetase, an essential enzyme for the synthesis of glutathione (GSH). We postulated that oxidative stress induced by GSH depletion might lead to genomic rearrangements, such as DNA deletions, and that counteracting such pro-oxidant conditions by the exogenous antioxidant N-acetyl-L-cysteine (NAC), might suppress DNA deletions. Therefore, we determined the frequency of 70 kb DNA deletions and thiol levels in mouse fetuses exposed to BSO (alone or in combination with NAC) via drinking water given to female mice during gestation. BSO treatment resulted in a significantly increased frequency of DNA deletions and decreased concentrations of GSH and cysteine. An amount of 2 mM BSO treatment resulted in a 30% higher DNA deletion frequency, 45% lower GSH and 27% lower cysteine levels, when compared with the untreated control and 20 mM BSO treatment caused a 40% higher DNA deletion frequency, 70% lower GSH and 55% lower cysteine levels. In combination BSO and NAC resulted in reduced levels of GSH consistent with the effect of BSO; however, cysteine levels increased and the frequency of DNA deletions was within the normal range. Thus, NAC protected against genome rearrangements caused by GSH depletion. This study showed that lowering the concentrations of thiol antioxidants results in DNA deletions that may play a role in carcinogenesis.     

Glutathione levels and cytotoxicity of a thiol activated alkylating agent in human and mouse cells

The effects of cellular GSH levels on the cytotoxicity of MNNG and mitomycin C were examined in normal and BSO treated mouse C3H10T1/2 cells. MNNG was less cytotoxic in the GSH depleted cells (less than 10% of normal) whereas the cytotoxicity of mitomycin C was not influenced by thiol status. This is compatible with the alkylating agent MNNG requiring thiols for activation to the methylating electrophile. Conversely, thiols have little if any effect in modulating the activity of mitomycin C. When naturally thiol deficient human fibroblasts were compared with BSO treated fibroblasts depleted to a similar GSH level (less than 10% of normal), only the BSO depleted cells were less effected by MNNG. The GSH deficient cells showed the same MNNG dose response as normal human fibroblasts. These studies indicate that a naturally acquired thiol status and an equivalent induced thiol status need not behave the same and this needs consideration when evaluating the role of thiols in influencing cellular response to chemotherapeutic agents.     

Potentiation of benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis in mice by D,L-buthionine-S,R-sulfoximine-mediated tissue glutathione depletion

In vitro studies have suggested that the glutathione (GSH) S-transferase (GST)-catalyzed GSH conjugation is an important mechanism for the detoxification of (+)-anti-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated form of the widespread environmental pollutant benzo[a]pyrene (BP). However, in vivo experimental evidence for the importance of GSH/GST system in defense against carcinogenic effects of BP is lacking. We hypothesized that if GSH/GST were to play an important role in the detoxification of (+)-anti-BPDE, the tumorigenic activity of BP would be increased by depleting the levels of GSH, which is the required nucleophilic substrate for GST-catalyzed conjugation reactions. In the present study, we have tested the above hypothesis by determining the effect of D, L-buthionine-S,R-sulfoximine (BSO)-mediated tissue GSH depletion on BP-induced tumorigenesis of the lung and forestomach in female A/J mice. Treatment of mice with three i.p. injections of 2.5 mmol BSO/kg (12 h apart) plus 20 mM BSO in drinking water, resulted in a statistically significant reduction in hepatic, pulmonary and forestomach GSH levels. At the same time, BSO-administration caused a statistically significant increase in BP-induced pulmonary and forestomach tumor multiplicity. To the best of our knowledge, the present study is the first report that provides in vivo experimental evidence for the importance of GSH/GST system in cellular protection against carcinogenic effects of BP.     

Glutathione as a determinant of cellular response to doxorubicin

We have studied in detail the relationship between glutathione (GSH) depletion and sensitivity of HEp3 human carcinoma cells to doxorubicin [Adriamycin (ADR)]. Exponentially growing HEp3 cells were incubated with L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, for different periods so that a range of GSH depletion could be obtained. These GSH-depleted cells were then treated with a combination of BSO and ADR (1 microgram/ml) for various durations. Under these conditions, the cytotoxicity of ADR was significantly enhanced by GSH depletion. The extent of ADR dose enhancement was found to be inversely proportional to cellular GSH level at the time of ADR treatment. Furthermore, it was shown that the dose-enhancement factors (DEF) also correlated with the duration of combined BSO and ADR treatment. For example, at a GSH level of 45% of untreated control, 18.5 +/- 3 fmol/cell or 4.8 +/- 0.3 X 10(-3) fmol/mum3 (+/- SD), DEF of 8.0, 6.4, and 5.0 were obtained for treatment periods of 3 hours, 2 hours, and 1 hour, respectively. Further study showed that the GSH kinetics differed significantly for the different treatment times, which indicates that GSH kinetics may be an important factor in determining the intrinsic sensitivity of HEp3 cells to ADR. Furthermore, the kinetics of GSH response to ADR varied significantly between cell lines. In the study of the effect of such differences, the GSH kinetics of 3 human ovarian tumor cell lines with different intrinsic sensitivities to ADR were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)