Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.     

Arsenic trioxide-induced apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma cell lines

We treated four hepatocellular carcinoma cell lines, HLE, HLF, HuH7, and HepG2 with ATO and demonstrated that arsenic trioxide (ATO) at low doses (1--3 muM) induced a concentration-dependent suppression of cell growth in HLE, HLF, and HuH7. HLE cells underwent apoptosis at 2 microM ATO, which was executed by the activation of caspase-3 through the mitochondrial pathway mediated by caspase-8 activation and Bid truncation. When these cell lines were exposed to ATO in combination with l-S,R-buthionine sulfoximine (BSO) which inhibits GSH synthesis, a synergistic growth suppression was induced, even in HepG2 showing a lower sensitivity to ATO than other cell lines tested. The intracellular GSH levels after the treatment with ATO plus BSO were considerably decreased in HLE cells compared with those after the treatment with ATO or BSO alone. The production of reactive oxygen species (ROS) which was examined by 2' ,7' -dichlorodihydrofluorescein diacetate, increased significantly after the treatment with ATO plus BSO in HLE cells. These findings indicate that ATO at low concentrations induces growth inhibition and apoptosis, and furthermore that the ATO-BSO combination treatment enhances apoptosis through increased production of ROS in hepatocellular carcinoma cells.     

Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK

A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and an increasing amount of evidence suggests that kaempferol may have cancer chemopreventative properties. However, the precise underlying protective mechanisms are poorly understood. To elucidate these mechanisms, we challenged human lung cancer cell line A549 with kaempferol and investigated its effects upon cellular growth and signal transduction pathways. Treatment of A549 cells with kaempferol resulted in a dose- and time-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 0.9+/-0.5, 5.2+/-1.5, 16.8+/-2.0, 25.4+/-2.6, and 37.8+/-4.5% on treatment with 0, 17.5, 35.0, 52.5, and 70.0 microM kaempferol, respectively. Concomitantly, kaempferol treatments led to a 1.2-, 2.7-, 3.3-, and 3.4-fold increase in Bax. Similar elevations were also observed in Bad which increased 1.2-, 3.3-, 3.7-, and 4.7-fold, respectively, as compared to control. Bcl-2 and Bcl-xL expression were inhibited in a dose-dependent fashion. While the Akt-1 and phosphorylated Akt-1 were inhibited, the mitogen-activated protein kinase (MAPK) was activated upon kaempferol treatment. Kaempferol induced apoptosis was associated with the cleavage of caspase-7 and poly ADP-ribose polymerase (PARP). Inhibition of MEK1/2 but not PI-3 kinase blocked kaempferol-induced cleavage of caspase-7, PARP cleavage, and apoptosis. The results suggest that inactivation of Akt-1 and alteration of Bcl-2 family of proteins are not sufficient for kaempferol to induce apoptosis and activation of MEK-MAPK is a requirement for kaempferol-induced cell death machinery in A549 cells.     

Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica

Tremella mesenterica (TM) is a common food and folk medicine widely used in several Asian countries as a tonic for the lungs. In the present study, we compared the effects of extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and ethanol extract (EE) of Tremella mesenterica on the induction of apoptosis into human lung carcinoma A549 epithelial cells. The EE, but not the EPS or the IPS, almost completely inhibited the growth of A549 cells. The results of Annexin V-FITC/PI staining and flow cytometric analysis indicated that the percentage of Annexin V(+)/PI(-) cells in EE-treated cells increased to 32.8%. The results of further investigation showed a disruption of mitochondrial transmembrane potential (DeltaPsi(m)), the production of reactive oxygen species (ROS), and the activation of caspase-3 protein in EE-treated cells. These findings suggest that EE can decrease cell viability and induce apoptosis in A549 cell lines by activating a mitochondrial pathway.     

Ruthenium methylimidazole complexes induced apoptosis in lung cancer A549 cells through intrinsic mitochondrial pathway

Ruthenium(II) methylimidazole complexes, with the general formula [Ru(MeIm)₄(N?N)]²⁺ (N?N = tip (RMC1), iip (RMC2), dppz (RMC3), dpq (RMC4); MeIm = 1-methylimidazole, tip = 2-(thiophene-2-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, iip = 2-(1H-imidazol-4-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, dppz = dipyrido[3,2-a:2′,3′-c]phenazine, dpq = pyrazino [2,3-f] [1,10]phenanthroline), were synthesized and characterized. As determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, these complexes displayed potent anti-proliferation activity against various cancer cells. RMC1 inhibited the growth of A549 (human lung adenocarcinoma) lung cells through induction of apoptotic cell death, as evidenced by the accumulation of cell population in sub-G1 phase. RMC1 also induced the depletion of mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Another experiment showed that Bid protein was also activated by RMC1, which implied that RMC1 could existed two pathways crosstalk, namely, have exogenous death receptor signaling pathway. These results demonstrated that RMC1 induced cancer cell death by acting on both mitochondrial and death receptor apoptotic pathways, suggesting that RMC1 could be a candidate for further evaluation as a chemotherapeutic agent against human cancers.     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine).

Buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), the most potent of a series of analogs of methionine sulfoximine thus far studied (Griffith, O.W., Anderson, M.E., and Meister, A. (1979) J. Biol. Chem. 254, 1205-1210), inhibited gamma-glutamylcysteine synthetase about 20 times more effectively than did prothionine sulfoximine and at least 100 times more effectively than methionine sulfoximine. The findings support the conclusion that the S-alkyl moiety of the sulfoximine binds at the enzyme site that normally binds the acceptor amino acid. Thus, the affinity of the enzyme for the S-ethyl, S-n-propyl, and S-n-butyl sulfoximines increases in a manner which is parallel to those of the corresponding isosteric acceptor amino acid substrates, i.e. glycine, alanine, and alpha-aminobutyrate. Buthionine sulfoximine did not inhibit glutamine synthetase detectably, nor did it produce convulsions when injected into mice. Injection of buthionine sulfoximine into mice decreased the level of glutathione in the kidney to a greater extent (less than 20% of the control level) than found previously after giving prothionine sulfoximine. alpha-Methyl buthionine sulfoximine was also prepared and found to be almost as effective as buthionine sulfoximine; this compound would not be expected to undergo substantial degradative metabolism. Buthionine sulfoximine and alpha-methyl buthionine sulfoximine may be useful agents for inhibition of glutathione synthesis in various experimental systems.     

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-l-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.     

The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells

Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC₅₀ of 16.4 ± 1.6 μM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G₂/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.     

Induction of endoreduplication by a JNK inhibitor SP600125 in human lung carcinoma A 549 cells

The effect of the pan c-Jun N-terminal kinase (JNK) inhibitor SP600125 on the proliferation of human lung carcinoma A549 cells has been evaluated. We have shown that SP600125 completely inhibited the proliferation of A549 cells, the cycle arrest being in G2/M phase. When cells were treated with SP600125 for >12h, a cell population with DNA content of 4n to 8n was detected. Moreover, the effect of SP600125 on the expression of cell cycle related proteins was an upregulation of p53 protein accompanied by an increase in its molecular mass. Prolonged SP600125 treatment downregulated p21, Bax and Mdm2 expression, but increased the level of the cellular p53-Mdm2 complex. Taken together, we show that SP600125 could induce G2/M cell cycle arrest and endoreduplication in a p21 independent manner, and that SP600125 could also post-translationally modify p53 to modify its function. Our data show that basic JNK activity plays an important role in the progression of the cell cycle at G2/M cell phase.     

Smad7 sensitizes A549 lung cancer cells to cisplatin-induced apoptosis through heme oxygenase-1 inhibition

Smad7, an inhibitory Smad, acts as a key regulator forming autoinhibitory feedback loop in transforming growth factor-beta (TGF-β) signaling. However, a growing body of evidences suggests that Smad7 is capable of apoptotic function. In the present study, we have demonstrated a proapoptotic function of Smad7 as a negative regulator of survival protein heme oxygenase-1 (HO-1). The HO-1 protein level was elevated in cisplatin-resistant A549 human lung cancer cells and blockade of HO-1 activation sensitized the cells to apoptosis. Interestingly, overexpression of Smad7 decreased HO-1 gene expression and its enzymatic activity. Notably, Smad7 reduced Akt activity and infection with adenovirus expressing a constitutively active form of the Akt reversed the inhibitory effects of Smad7 to HO-1, indicating a negative action mechanism of Smad7 to Akt-HO-1-linked survival pathway. Consistently, Smad7 sensitized A549 cells to cisplatin-induced apoptosis and these effects were dependent on HO-1 and Akt inhibition. Based on these findings, we suggest that targeting Smad7 may be an efficient strategy for overcoming drug-resistance in cancer therapy.     

Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells

Previously, we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells by activating caspase-3, -8, and -9. In this study, we further investigated which upstream pathways were activated by safrole oxide during the apoptosis. Immunofluorescence assay combined with laser scanning confocal microscopy revealed that both Fas and Fas ligand (FasL) were up-regulated by the small molecule. In addition, Fas protein distribution was altered, showing a clustering distribution instead of a homogeneous one. Subsequently, Western blot analysis confirmed the up-regulations of Fas and its membrane-binding form of FasL (m-FasL), as well as P53 protein. Conversely, safrole oxide hardly affected integrin beta4 subunit expression or distribution, which was reflected from the data obtained by immunofluorescence assay combined with laser scanning confocal microscopy. The results suggested that Fas/FasL pathway might be involved in safrole oxide-induced apoptosis of A549 cells, while integrin beta4 might be irrelevant to the apoptosis. Nevertheless, we first found the strong expression of integrin beta4 in A549 cells. The study first suggested that safrole oxide might be used as a small molecular promoter of Fas/FasL pathway to elicit apoptosis in A549 cells, which would lay the foundation for us to insight into the new strategies for lung cancer therapy.     

Protection from hyperbaric oxidant stress by administration of buthionine sulfoximine.

To explore the role of glutathione in protecting rats from hyperbaric hyperoxia, we administered buthionine sulfoximine (BSO) to block gamma-glutamyl cysteine synthase activity and decrease tissue glutathione synthesis. We then exposed these animals and their vehicle-treated matched controls to 100% oxygen at 4 ATA or room air at 1 ATA. After BSO treatment, glutathione concentrations in air-exposed controls decreased 62% in lung, 76% in liver, 28% in brain, and 62% in plasma. Paradoxically, BSO-treated rats were protected from hyperbaric hyperoxia. The BSO-treated animals seized significantly later and had a markedly prolonged time of survival compared with the vehicle-treated controls. We conclude that BSO treatment protects rats from hyperbaric hyperoxia, despite its effects of lowering plasma and tissue glutathione concentrations. This protection may be related to a direct effect of the compound in decreasing free radical-mediated tissue injury, increasing tissue antioxidant defenses, or increasing seizure threshold.     

Induction of apoptosis by eugenol in human breast cancer cells

In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol.     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

葛根素对人非小细胞肺癌A549细胞凋亡的作用

目的：观察葛根素对人非小细胞肺癌A549细胞生长的抑制作用及其机制。方法：体外培养人非小细胞肺癌细胞（A549）,不同浓度（60 μg/ml,120 μg/ml,240 μg/ml）葛根素处理24 h后;采用CCK-8法观察葛根素对细胞的增值抑制作用;吖啶橙（AO）/溴化乙锭（EB）双染法及AnnexinⅤ-PI双染流式细胞术检测药物作用前后A549细胞的形态学变化及凋亡状况;Western blot法检测Apelin/APJ蛋白水平的变化。结果：CCK-8法检测结果说明葛根素能抑制A549细胞的增值,具有浓度和时间依赖关系;流式细胞术进一步证实葛根素具有诱导细胞凋亡的作用,与A549细胞组比较,葛根素各处理组Apelin/APJ蛋白水平均有不同程度下调。结论：葛根素可能通过调节Apelin/APJ蛋白的表达诱导A549细胞凋亡。     

DPMA,a deoxypodophyllotoxin derivative,induces apoptosis and anti-angiogenesis in non-small cell lung cancer A549 cells

We found that the deoxypodophyllotoxin derivative, 2,6-dimethoxy-4-(6-oxo-(5R,5aR,6,8,8aR,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)phenyl ((R)-1-amino-4-(methylthio)-1-oxobutan-2-yl)carbamate (DPMA), exhibited superior cytotoxicity compared with etoposide. In this study, we investigated the mechanism of action of DPMA. DPMA exhibited anti-proliferative activity and induced apoptosis in A549 cells in a dose- and time-dependant manner. DPMA inhibited microtubule formation and induced expression of Bax, cleaved caspase-3, p53 and ROS, and inhibited Bcl-2 expression. DPMA also affected cyclinB1, cdc2 and p-cdc2 expression, inducing cell cycle arrest. DPMA also inhibited tube formation of VEGF-induced human umbilical vein endothelial cells. These studies demonstrate that DPMA inhibits p53/cdc2/Bax signaling, thereby inhibiting cell growth/angiogenesis and inducing apoptosis.     

Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells

Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.