Melatonin Loading Chitosan-Tripolyphosphate Nanoparticles: Application in Attenuating Etoposide-Induced Genotoxicity in HepG2 Cells.

Etoposide is one of the most effective chemotherapeutic agents used in the treatment of various types of cancers. However, as a Topoisomerase II inhibitor, during clinical use, several side effects may occur. In addition, in several in vivo and in vitro studies, etoposide has been shown to have a range of genotoxic effects including single and double strand breaks. Melatonin is an anti-aging and antioxidant hormone synthesized from the pineal gland. The genoprotective, antioxidant, and free radical scavenger properties of melatonin have been well explained in various studies. The aim of this study was to explore whether melatonin nanoparticles protects against etoposide-induced genotoxicity in the HepG2 cell line. HepG2 cells (25 × 104 cells/well) were cultured in 24-well plates: a control group and 3 melatonin and its nanoparticles + etoposide groups (pre- and cotreatment conditions). Our results show that etoposide induced a noticeable genotoxic effect in HepG2 cells. Melatonin reduced the effects of etoposide significantly in both types of experiment conditions, through the reduction of the level of DNA damage measured via comet assay. Furthermore, melatonin decreased the intracellular reactive oxygen species generation. It also increased the intracellular glutathione levels in HepG2 cells. Nano melatonin is more effective than regular melatonin. The most protective effect was observed with melatonin when it was administrated 24 h before etoposide treatment.

Antimutagenicity and antigenotoxicity of Aloe arborescens Miller and Aloe barbadensis Miller in Aspergillus nidulans and Wistar rats.

Medicinal plants such as Aloe arborescens Miller and Aloe barbadensis Miller are used by the general population to treat various diseases. Therefore, the aim of this study was to evaluate the antimutagenicity of these two species using a methG1 system in Aspergillus nidulans and the comet assay in rats. The animals were treated with the plants at concentrations of 360 and 720 mg/kg body weight (1 and 2, respectively) by gavage for 14 days, followed by the administration of etoposide on treatment day 8. Blood samples were prepared for analysis of DNA damage. For the test in A. nidulans, the biA1methG1 lineage conidia were treated for 4 h with both plant species at concentrations of 4 and 8% (w/v). Then, they were washed and plated on a selective medium for frequency analysis of survival and mutation. The results of the comet assay showed that both plants were antigenotoxic compared to etoposide, which was not a typical response of methG1 systems, where only the highest concentration of plant extracts usually exhibit beneficial effects. This study demonstrates the potential antigenotoxicity and antimutagenicity of the Aloe plants tested and, therefore, supports their use as a form of preventive therapy and for health maintenance by the population.

Nifedipine prevents etoposide-induced caspase-3 activation, prenyl transferase degradation and loss in cell viability in pancreatic ß-cells.

Emerging evidence implicates novel roles for post-translational prenylation (i.e., farnesylation and geranylgeranylation) of various signaling proteins in a variety of cellular functions including hormone secretion, survival and apoptosis. In the context of cellular apoptosis, it has been shown previously that caspase-3 activation, a hallmark of mitochondrial dysregulation, promotes hydrolysis of several key cellular proteins. We report herein that exposure of insulin-secreting INS 832/13 cells or normal rat islets to etoposide leads to significant activation of caspase-3 and subsequent degradation of the common α-subunit of farnesyl/geranylgeranyl transferases (FTase/GGTase). Furthermore, the above stated signaling steps were prevented by Z-DEVD-FMK, a known inhibitor of caspase-3. In addition, treatment of cell lysates with recombinant caspase-3 also caused FTase/GGTase α-subunit degradation. Moreover, nifedipine, a calcium channel blocker, markedly attenuated etoposide-induced caspase-3 activation, FTase/GGTase α-subunit degradation in INS 832/13 cells and normal rat islets. Further, nifedipine significantly restored etoposide-induced loss in metabolic cell viability in INS 832/13 cells. Based on these findings, we conclude that etoposide induces loss in cell viability by inducing mitochondrial dysfunction, caspase-3 activation and degradation of FTase/GGTase α-subunit. Potential significance of these findings in the context of protein prenylation and ß-cell survival are discussed.

Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.

Delphinidin modulates the DNA-damaging properties of topoisomerase II poisons.

The anthocyanidin delphinidin (DEL) has recently been shown to inhibit human topoisomerase I and II, without stabilizing the covalent DNA/topoisomerase intermediate [Habermeyer, M., Fritz, J., Barthelmes, H. U., Christensen, M. O., Larsen, M. K., Boege, F., and Marko, D. (2005) Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. Chem. Res. Toxicol. 18, 1395-404]. In the present study, we demonstrated that DEL affects the catalytic activity of topoisomerase IIalpha in a redox-independent manner. Furthermore, this potent inhibitory effect is not limited to a cell-free system, but is also of relevance within intact cells. DEL at micromolar concentrations was found to significantly decrease the level of topoisomerase IIalpha/DNA intermediates stabilized by the topoisomerase II poison doxorubicin in the human colon carcinoma cell line (HT29). In addition, DEL diminished the DNA-damaging properties of topoisomerase II poisons in HT29 cells without affecting the level of sites sensitive to formamidopyrimidine-DNA-glycosylase. However, the preventive effect on DNA damage exhibited an apparent maximum at a concentration of 10 microM DEL, followed by a recurrence of DNA damage at higher DEL concentrations. Furthermore, the incubation of HT29 cells with 10 microM DEL resulted in a decrease of etoposide (ETO)-induced DNA strand breaks. However, the level of ETO-stabilized covalent topoisomerase/DNA intermediates did not affect DEL, indicating an additional mechanism of action. An impact of DEL on genes involved in the repair of DNA double-strand breaks and the onset of apoptosis has to be considered. In conclusion, the natural food constituent DEL represents, depending on the concentration range, a protective factor against the DNA-damaging effects of topoisomerase II poisons in vitro. Further studies are needed to clarify whether in vivo a high DEL intake might compromise the therapeutic outcome of these anticancer agents.

Ornithine decarboxylase attenuates leukemic chemotherapy drugs-induced cell apoptosis and arrest in human promyelocytic HL-60 cells.

Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in cell cycle, tumor promotion and anti-apoptosis. In our previous studies, overexpression of ODC prevented apoptosis induced by tumor necrosis factor-alpha and methotrexate. We further investigated the apoptotic mechanisms of the cancer chemotherapeutic drugs, including etoposide (VP-16), paclitaxel (TAX) and cisplatin (CDDP), and the influences of ODC on apoptosis and cell cycle. Our results showed that the investigated drugs induced caspase-dependent apoptosis, the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (Deltapsi m) in HL-60 cells, all of which were reversed by putrescine, glutathione or N-acetyl-l-cysteine. Overexpression of ODC prevented the cancer chemotherapeutic drugs-induced apoptosis, ROS generation and the disruption of Deltapsi m. After drug administrations, the decline of Bcl-2, cytochrome c release and caspases' activation were inhibited by ODC overexpression. In cell cycle, ODC overexpressed cells seemed to overcome the G1 arrest and G2/M arrest, caused by VP-16 and TAX, respectively, and kept on the cell cycle rolling. Overexpression of ODC increased the expression of Cyclin A, D, E and Cdk4 and the enzyme activity of Cdk1 and Cdk2 after the treatment of VP-16 and TAX, respectively. In conclusions, the cancer chemotherapeutic drugs-induced apoptosis is through ROS-related, mitochondria-mediated and caspase-dependent pathways. With higher ODC activity, cells are resistant to the cancer chemotherapeutic drugs-induced apoptosis and keep on the cell cycle rolling with the significant interference in G1/S arrest caused by VP-16 and G2/M arrest by TAX.

Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation.

In this study, we investigated the influence of platelet-activating factor (PAF) on the induction of apoptosis-regulating factors in B16F10 melanoma cells. PAF increased the expression of mRNA and the protein synthesis of antiapoptotic factors, such as Bcl-2 and Bcl-xL, but did not increase the expression of the proapoptotic factor, Bax. A selective nuclear factor-kappaB (NF-kappaB) inhibitor, parthenolide, inhibited the effects of PAF. Furthermore, PAF inhibited etoposide-induced increases in caspase-3, caspase-8, and caspase-9 activities, as well as cell death. p50/p65 heterodimer increased the mRNA expression of Bcl-2 and Bcl-xL and decreased etoposide-induced caspase activities and cell death. In an in vivo model in which Matrigel was injected s.c., PAF augmented the growth of B16F10 cells and attenuated etoposide-induced inhibition of B16F10 cells growth. These data indicate that PAF induces up-regulation of antiapoptotic factors in a NF-kappaB-dependent manner in a melanoma cell line, therefore suggesting that PAF may diminish the cytotoxic effect of chemotherapeutic agents.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells.

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.

Herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 expression.

PURPOSE: Monoclonal antibodies, such as herceptin and trastuzumab, against the epidermal growth factor receptor ErbB2 (also known as HER2/neu) are an effective therapy for breast cancer patients with overexpression of ErbB2. Herceptin, in combination with standard chemotherapy, such as taxol or etoposide, gives a synergistically apoptotic response in breast tumors. EXPERIMENTAL DESIGN: The mechanism underlying this synergy between chemotherapy and herceptin treatment is not well understood. Herein, we have determined that addition of herceptin, sensitized breast cancer cell lines MDA-MB-231 and MCF-7 to etoposide- or taxol-induced apoptosis. RESULTS: This treatment resulted in reduced expression of ErbB2 and the antiapoptotic Bcl-2 family member Mcl-1 in MDA-MB-231 cells. Using antisense oligonucleotides against Mcl-1, MDA-MB-231 cells were rendered sensitive to etoposide-induced apoptosis similar to herceptin, but combined treatment of antisense against Mcl-1 and herceptin failed to give a significant increase in apoptosis. In 29 human breast tumors immunostained for ErbB2 and Mcl-1, we found that when ErbB2 was overexpressed, there was a corresponding increase in Mcl-1 expression. DISCUSSION: Using murine fibroblasts that express human ErbB2, but no other ErbB family member (NE2), these cells showed resistance to both taxol- and etoposide-induced apoptosis compared with parental cells. In addition, NE2 cells preferentially express the antiapoptotic Bcl-2 family member Mcl-1 compared with parental cells, and treatment with herceptin reduces Mcl-1 expression. Taken together, these results suggest that herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 protein levels.

Rho GDP dissociation inhibitor protects cancer cells against drug-induced apoptosis.

Rho GDP dissociation inhibitor (RhoGDI) plays an essential role in control of a variety of cellular functions through interactions with Rho family GTPases, including Rac1, Cdc42, and RhoA. RhoGDI is frequently overexpressed in human tumors and chemo-resistant cancer cell lines, raising the possibility that RhoGDI might play a role in the development of drug resistance in cancer cells. We found that overexpression of RhoGDI increased resistance of cancer cells (MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells) to the induction of apoptosis by two chemotherapeutic agents: etoposide and doxorubicin. Conversely, silencing of RhoGDI expression by DNA vector-mediated RNA interference (small interfering RNA) sensitized MDA-MB-231 cells to drug-induced apoptosis. Resistance to apoptosis was restored by reintroduction of RhoGDI protein expression. The mechanism for the anti-apoptotic activity of RhoGDI may derive from its ability to inhibit caspase-mediated cleavage of Rac1 GTPase, which is required for maximal apoptosis to occur in response to cytotoxic drugs. Taken together, the data show that RhoGDI is an anti-apoptotic molecule that mediates cellular resistance to these chemotherapy agents.

Aspirin reduces the outcome of anticancer therapy in Meth A-bearing mice through activation of AKT-glycogen synthase kinase signaling.

Aspirin displays, at millimolar concentrations, several mechanisms independent from its ability to inhibit cyclooxygenases. Occasionally, the mechanisms displayed in vitro have been clearly related to an effect of clinical relevance in vivo. An expanding literature has been focusing on the cytoprotective effect of aspirin in neurodegenerative disorders and the activation of AKT pathway in neuroprotection and induction of resistance to anticancer drugs. In this work, we tested the ability of aspirin to activate the AKT survival pathway in methylcholanthrene-induced fibrosarcoma cells (Meth A) transplanted into BALB/c nude mice and the clinical effect of aspirin cotreatment during etoposide (VP-16)-based anticancer therapy. We found that cotreatment with aspirin reduced VP-16-induced apoptosis and activated AKT in vitro and in vivo. In Meth A-bearing mice, aspirin administration also activated glycogen synthase kinase-3 and reduced the activity and the efficacy of anticancer therapy in VP-16 cotreated animals. Our data suggest that the antiapoptotic effect of aspirin operates in vivo through the activation of AKT-glycogen synthase kinase pathway causing a decrease in the outcome of VP-16-based therapy. These findings could have clinical relevance in treatment of human malignancies.

Lovastatin protects human endothelial cells from the genotoxic and cytotoxic effects of the anticancer drugs doxorubicin and etoposide.

BACKGROUND AND PURPOSE: 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are frequently used lipid-lowering drugs. Moreover, they exert pleiotropic effects on cellular stress responses and death. Here, we analysed whether lovastatin affects the sensitivity of primary human endothelial cells (HUVEC) to the anticancer drug doxorubicin. EXPERIMENTAL APPROACH: We investigated whether pretreatment of HUVEC with low dose of lovastatin influences the cellular sensitivity to doxorubicin. To this end, cell viability, proliferation and apoptosis as well as DNA damage-triggered stress response were analysed. KEY RESULTS: Lovastatin reduced the cytotoxic potency of doxorubicin in HUVEC. Lovastatin attenuated the doxorubicin-induced increase in p53 as well as activation of checkpoint kinase (Chk-1) and stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK). Acquired doxorubicin resistance was independent of alterations in doxorubicin efflux and cell cycle progression. Also, doxorubicin-triggered production of reactive oxygen species (ROS) and formation of oxidative DNA lesions remained unaffected by lovastatin. However, lovastatin impaired DNA strand break formation induced by doxorubicin. Notably, lovastatin also conferred cross-resistance to the cytotoxic and genotoxic effects of etoposide, indicating that lovastatin shields topoisomerase II against poisons. CONCLUSIONS AND IMPLICATIONS: Based on these data, we suggest that lovastatin-mediated resistance to topoisomerase II inhibitors is due to a reduction in DNA damage and, hence, it attenuates stress responses leading to cell death that are triggered by DNA damage. Therefore, lovastatin might be useful clinically for alleviating side-effects of anticancer therapies that include topoisomerase II inhibitors.

Digitoxin, at concentrations commonly found in the plasma of cardiac patients, antagonizes etoposide and idarubicin activity in K562 leukemia cells.

Digitoxin is used in the treatment of cardiac congestion and some types of cardiac arrhythmias. The mechanism of action of this cardiac glycoside suggested that it might antagonize the anticancer activity of topoisomerase II poisons. The present report shows that digitoxin, at concentrations commonly found in the plasma of cardiac patients, significantly reduced etoposide and idarubicin-induced topoisomerase II cleavable complexes in K562 leukemia cells. This may lead to a reduction in the anticancer effect of these two topoisomerase II poisons when they are used in the clinic concurrently with digitoxin.

Protection from chemotherapy-induced alopecia by 1,25-dihydroxyvitamin D3.

We have previously reported that several biological agents, when given simultaneously with cytosine arabinoside or cytoxan, will protect from cytosine arabinoside-induced but not from cytoxan-induced alopecia. In the present study we used the secosteroid 1,25-dihydroxyvitamin D3 in a different timing schedule to protect from chemotherapy-induced alopecia. In three separate experiments, 0.2 microgram of topical, 1,25-dihydroxyvitamin D3 protected rats from alopecia induced by etoposide, cytoxan, and an Adriamycin-cytoxan combination. In another experiment, 0.1 microgram protected rats from etoposide-induced alopecia at the site of application. 1,25-Dihydroxyvitamin D3 may offer a new and exciting approach to the prevention of chemotherapy-induced alopecia.

Insulin-like growth factor I (IGF-I) and the IGF-I receptor prevent etoposide-induced apoptosis.

The interaction of insulin-like growth factors (IGF) with the IGF-I receptor promotes cell proliferation and survival. We examined the role of the IGF-I receptor as a possible direct inhibitor of apoptosis induced by the topoisomerase I inhibitor etoposide. When exposed to this agent, BALB/c 3T3 cells that constitutively overexpress the human IGF-I receptor (p6 cells) arrested in S phase and subsequently underwent apoptosis as determined by the appearance of a pre-G1 apoptotic peak when studied by flow cytometry and the characteristic internucleosomal fragmentation of DNA. The addition of IGF-I markedly inhibited etoposide-induced apoptosis in a concentration-dependent manner. IGF-I was not mitogenic in the presence of etoposide. IGF-I was less effective in preventing apoptosis in parental BALB/c 3T3 cells and had no effect on etoposide-induced cell killing of mouse embryo fibroblasts that have a targeted disruption of the IGF-I receptor gene. These results demonstrate an important role for the IGF-I receptor as an inhibitor of apoptosis, independent of its mitogenic actions.

Transient protection of cultured human cells against antitumor agents by 12-O-tetradecanoylphorbol-13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown in cell cultures to enhance the frequency of resistance to methotrexate. However, we found that TPA could also partially protect human KB cells over a short time (72 h) from the cytotoxicity of several antitumor agents, including 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene)-beta-D-glucopyranoside (VP-16), vincristine, mitoxantrone, and methotrexate, but not 1-beta-D-arabinofuranosylcytosine or 5-fluorouracil. The modes of protection were different for methotrexate and VP-16. Protection by TPA was concentration dependent up to 40 nM and could be accomplished by a 2-h incubation of cells with TPA alone prior to drug treatment. This protection disappeared after a 24-h drug-free incubation. TPA-induced protection could not be mimicked by treatment of cells with 1-oleoyl-2-acetyl-glycerol (a stimulator of protein kinase C) or phospholipase C, which increased the cellular content of diacylglycerols. Thus the action of TPA on protein kinase C may not be sufficient to exert protection. Verapamil, a calcium-channel blocker which has been found to circumvent resistance of multiple drug-resistant cells, also circumvented the protective effect of TPA when used with VP-16. The presence of TPA during a 24-h exposure to radiolabeled VP-16 reduced the cellular drug content by about 30%, whereas verapamil enhanced drug content by at least 50% in TPA-treated and untreated cultures. Since substances with some TPA-like activity have been found in foods and in human circulation, the observation of clinical resistance to some compounds may partly be due to a related mechanism.

Hematopoietic rescue via T-cell-dependent, endogenous granulocyte-macrophage colony-stimulating factor induced by the pineal neurohormone melatonin in tumor-bearing mice.

We investigated whether melatonin can affect tumor growth and/or hematopoiesis in mice transplanted with Lewis lung carcinoma and treated with cyclophosphamide or etoposide. These agents were injected i.p. for 5 days at two different cumulative doses (cyclophosphamide, 40 and 160 mg/kg body weight; etoposide, 20 and 40 mg/kg body weight) from day 8 through day 12 after tumor transplantation. Melatonin was injected s.c. at a dose of 1 mg/kg body weight/day, from day 8 throughout the experiments and from days 8 through 12 or from day 13 onwards. Melatonin did not influence tumor growth but selectively counteracted bone marrow toxicity when administered together with the cancer chemotherapy compounds without interfering with their anticancer action. In vitro, melatonin proved to counteract apoptosis in bone marrow cells incubated with etoposide. Such protection was reflected by an increased frequency of granulocyte/macrophage-colony forming units but not of the pluripotent spleen-colony forming units. The effect of melatonin was neutralized by anti-granulocyte/macrophage-colony-stimulating factor monoclonal antibodies. When athymic, T-cell-deficient mice were used as bone marrow donors, melatonin did not exert any protective effect. This suggested that melatonin is able to stimulate the endogenous production of granulocyte/macrophage-colony-stimulating factor via bone marrow T-cells. Due to the well known lack of toxic and undesirable side effects of melatonin, these findings might have a straightforward clinical application.

Targeting the cytotoxicity of topoisomerase II-directed epipodophyllotoxins to tumor cells in acidic environments.

The epipodophyllotoxins etoposide and teniposide are probably the most important drugs in the treatment of small cell lung cancer. The drugs are used in maximally tolerated doses, and the toxicity of the drugs precludes significant dose increments. The cellular target is the nuclear enzyme topoisomerase II which, in the presence of these drugs, causes an extensive fragmentation of DNA. The cell kill can be antagonized by distinct drug types. We have demonstrated previously that the intercalating drug aclarubicin and the cardioprotecting agent ICRF-187 antagonize the cytotoxicity of etoposide in vitro. We have studied possible ways of using this antagonism as a means of differentially protecting normal tissue. Here we demonstrate that the intercalating agent chloroquine prevents the introduction of topoisomerase II-mediated DNA breaks and thereby antagonizes the cytotoxicity of etoposide. This interaction depends on the extracellular pH. Chloroquine, in contrast to etoposide, is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. We propose that such a pH-dependent drug interaction may be useful in directing topoisomerase II drug effects toward solid tumors. Thus, by lowering the extracellular pH (pHe) from neutral (pHe = 7.4) to acidic (pHe = 6.0), [3H]chloroquine accumulation was decreased 5-fold in a human small cell lung cancer cell line, OC-NYH, and in murine leukemia L1210 cells. In parallel, the antagonism exhibited by chloroquine on etoposide cytotoxicity was pHe dependent. Thus, no protection by chloroquine was observed at pHe = 6.5, whereas at pHe = 7.4, etoposide cytotoxicity was almost completely antagonized with a 460-fold protection or more than eight doublings of the cell population. This exploitation of antagonist extracellular trapping by acidic pH is a novel model for regulation of anticancer drug effects.

Inhibition of etoposide-induced DNA damage and cytotoxicity in L1210 cells by dehydrogenase inhibitors and other agents.

The mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), an important antitumor agent, is unclear. There is evidence that DNA may be the target of action because VP-16 causes single-strand and double-strand breaks in DNA and produces cytotoxicity over a similar dose range. We have hypothesized that an enzyme system, such as dehydrogenase, catalyzes an oxidation-reduction reaction involving the pendant phenolic group which forms an active metabolite that causes the DNA damage and cytotoxicity. To test our hypothesis, we investigated the effect of disulfiram, an aldehyde dehydrogenase inhibitor, and its metabolite, diethyldithiocarbamate, on VP-16-induced DNA damage in L1210 cells. Using the alkaline elution technique to assay DNA damage, we found that disulfiram and diethyldithiocerbamate inhibited VP-16-induced single-strand breaks. Both compounds were also capable of significantly reducing VP-16-induced cytotoxicity. Oxalic acid, pyrophosphate, and malonic acid, competitive inhibitors of succinate dehydrogenase, and the naturally occurring dehydrogenase substrates, succinic acid, beta-glycerophosphate, and isocitric acid, also blocked the effects of VP-16. Free-radical scavengers were also studied. While sodium benzoate was particularly effective in preventing drug-induced DNA damage and cytotoxicity, a number of other scavengers were not. Our data are consistent with the hypothesis that VP-16 is activated by an enzyme such as a dehydrogenase which transforms it into an active intermediate resulting in DNA damage and, consequently, cell death.