Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: implications in the prevention of etoposide-induced apoptosis

Etoposide (VP-16) is known to promote cell apoptosis either in cancer or in normal cells as a side effect. This fact is preceded by the induction of several mitochondrial events, including increase in Bax/Bcl-2 ratio followed by cytochrome c release and consequent activation of caspase-9 and -3, reduction of ATP levels, depolarization of membrane potential (DeltaPsi) and rupture of the outer membrane. These events are apoptotic factors essentially associated with the induction of the mitochondrial permeability transition (MPT). VP-16 has been shown to stimulate the Ca2+-dependent MPT induction similarly to prooxidants and to promote apoptosis by oxidative stress mechanisms, which is prevented by glutathione (GSH) and N-acetylcysteine (NAC). Therefore, the aim of this work was to study the effects of antioxidants and thiol protecting agents on MPT promoted by VP-16, attempting to identify the underlying mechanisms on VP-16-induced apoptosis. The increased sensitivity of isolated mitochondria to Ca2+-induced swelling, Ca2+ release, depolarization of DeltaPsi and uncoupling of respiration promoted by VP-16, which are prevented by cyclosporine A proving that VP-16 induces the MPT, are also efficiently prevented by ascorbate, the primary reductant of the phenoxyl radicals produced by VP-16. The thiol reagents GSH, dithiothreitol and N-ethylmaleimide, which have been reported to prevent the MPT induction, also protect this event promoted by VP-16. The inhibition of the VP-16-induced MPT by antioxidants agrees with the prevention of etoposide-induced apoptosis by GSH and NAC and suggests the generation of oxidant species as a potential mechanism underlying the MPT that may trigger the release of mitochondrial apoptogenic factors responsible for apoptotic cascade activation.     

Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.     

Synthesis and biological evaluation of novel conjugates of podophyllotoxin and 5-FU as antineoplastic agents

A series of novel conjugates of podophyllotoxin and 5-FU were designed using association strategy and were synthesized by coupling 4′-demethylepipodophyllotoxin with 5-FU-N¹-alkyl amino acid ester. These derivatives have been evaluated for cytotoxicity in vitro against tumour cell lines (HL-60, K562, A-549 and AGS), and their octanol–water partition coefficients (log P) were also determined. As compared with VP-16, most compounds showed superior water solubility, as well as more potent inhibitions against these four tumour cell lines. Compound 21 showed interaction with calf thymus DNA, and it was relatively resistant to metabolism by human plasma.     

TAT Peptide-Conjugated Magnetic PLA-PEG Nanocapsules for the Targeted Delivery of Paclitaxel: <Emphasis Type="Italic">In Vitro</Emphasis> and Cell Studies

Paclitaxel (PTX) and organophilic iron oxide nanocrystals of 7 nm average size were co-encapsulated in the oily core of poly(lactide)-poly(ethyleneglycol) (PLA-PEG) nanocapsules in order to develop magnetically responsive nanocarriers of PTX. The nanocapsules were prepared by a solvent displacement technique and exhibited satisfactory drug and iron oxide loading efficiency, high colloidal stability, and sustained drug release properties. Drug release also proved responsive to an alternating magnetic field. Magnetophoresis experiments showed that the magnetic responsiveness of the nanocapsules depended on their SPION content. The PTX-loaded nanocapsules exhibited comparable to free PTX cytotoxicity against the A549 lung cancer cell line at 24 h of incubation but higher cytotoxicity than free drug at 48 h of incubation. The conjugation of a cysteine-modified TAT peptide (HCys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH₂) on the surface of the nanocapsules resulted to highly increased uptake of nanocapsules by cancer cells, as well as to profound improvement of their cytotoxicity against the cancer cells. The results obtained justify further investigation of the prospects of these multifunctional PLA-PEG nanocapsules as a targeted delivery system of paclitaxel.     

Improving platinum(II)-based anticancer drug delivery using cucurbit[n]urils

Despite the synthesis of hundreds of new platinum(II) and platinum(IV)-based complexes each year as potential anticancer drugs, only three have received world-wide approval: cisplatin, carboplatin and oxaliplatin. The next big advance in platinum-based chemotherapy is not likely to come from the development of new drugs, but from the controlled and targeted delivery of already approved drugs or those in late stage clinical trials. Encapsulation of platinum drugs inside macromolecules has already demonstrated promise, and encapsulation within cucurbit[n]urils has shown particular potential. Partial or full encapsulation within cucurbit[n]urils provides steric hindrance to drug degradation by peptides and proteins, and the use of different sized cucurbit[n]urils allows for the tuning of drug release rates, cytotoxicity and toxicity.     

GL3, a Novel 4β-Anilino-4′-O-Demethyl-4-Desoxypodophyllotoxin Analog,Traps Topoisomerase II Cleavage Complexes and Exerts Anticancer Activities

A novel VP-16 derivative, 4β-[N -(4?-acetyloxyl-phenyl-1?-carbonyl)-4″-aminoanilino]-4′-O-demethyl-4-desoxypodophyllotoxin (GL3), displayed a wide range of cytotoxicity in a panel of human tumor cell lines, with half-maximal inhibitory concentration (IC₅₀) values ranging from 0.82 to 4.88 µM, much less than that of VP-16 (4.18–39.43 µM). Importantly, GL3 induces more significant apoptosis and cell cycle arrest than VP-16. The molecular and cellular machinery studies showed that GL3 functions as a topoisomerase II (Top 2) poison through direct binding to the enzyme, and the advanced cell-killing activities of GL3 were ascribed to its potent effects on trapping Top 2-DNA cleavage complex, Moreover, GL3-triggered DNA double-strand breaks and apoptotic cell death were in a Top 2-dependent manner, because the catalytic inhibitor aclarubicin attenuated these biologic consequences caused by Top 2 poisoning in GL3-treated cells. Taken together, among a series of 4β-anilino-4′-O-demethyl-4-desoxypodophyllotoxin analog, GL3 stood out by its improved anticancer activity and well-defined Top 2 poisoning mechanisms, which merited the potential value of GL3 as an anticancer lead compound/drug candidate deserving further development.     

A protease pathway for the repair of topoisomerase II-DNA covalent complexes

Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. gamma-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2beta knock-out mouse embryonic fibroblasts and Top2beta small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIalpha was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIalpha-DNA and TopIIbeta-DNA adducts. By contrast, hydrogen peroxide-induced gamma-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage.     

Apoptosis-resistant mitochondria in T cells selected for resistance to Fas signaling

Jurkat leukemic T cells are highly sensitive to the extrinsic pathways of apoptosis induced via the death receptor Fas or tumor necrosis factor-related apoptosis-inducing ligand as well as to the intrinsic/mitochondrial pathways of death induced by VP-16 or staurosporin. We report here that clonal Jurkat cell lines selected for resistance to Fas-induced apoptosis were cross-resistant to VP-16 or staurosporin. Each of the apoptotic pathways was blocked at an apical phase, where common regulators of apoptosis have not yet been defined. The Fas pathway was blocked at the level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in resistant cells in response to either Fas-cross-linking or VP-16 treatment. Also, no apoptosis-associated alterations in the mitochondrial inner membrane, outer membrane, or matrix were detected in resistant Jurkat cells treated with VP-16. Thus, no changes in permeability transition, loss in inner membrane cardiolipin, generation of reactive oxygen species, or release of cytochrome c were observed in resistant cells treated with VP-16. Further, unlike purified mitochondria from wild type cells, those obtained from resistant cells did not release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. These results identify a defect in mitochondria ability to release intermembrane proteins in response to Bid or Bax as a mechanism of resistance to chemotherapeuetic drugs. Further, the selection of VP-16-resistant mitochondria via elimination of Fas-susceptible cells may suggest the existence of a shared regulatory component between the extrinsic and intrinsic pathways of apoptosis.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.     

Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions

A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position -2 and G at position +6 were observed for the top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2. The VP-16-induced top2(S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-1, whereas the most reversible sites showed no base preference at positions -1 or -2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser740 --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.     

Tyrosinase-Induced Free Radical Formation From VP-16,213: Relationship To Cytotoxicity

Tyrosinase-dependent activation of hydroxybenzenes forms reactive compounds, including catechols and o-quinones, and some of which show antitumor activity against pigmented melanomas. Since VP-16 is a phenoxy-containing antitumor drug, forms free radicals and reactive o-quinones during peroxidative activation, wc evaluated the cytotoxicity of VP-16 to both tyrosinase-containing and non-tyrosinasecontaining tumor cells. Our results show that VP-16 is significantly more cytotoxic to B-16/F-10 melanoma cells than human MCF-7 breast tumor cells. Phenylthiocarbamide, an inhibitor of tyrosinase activity, selectively decreased VP-16 toxicity only in melanoma cells. Furthermore, VP-16 was readily activated to its phenoxy free radical intermediate by purified tyrosinase, indicating tyrosinase may play a role in VP-16 toxicity in pigminted melanomas.     

Novel semisynthetic spin-labeled derivatives of podophyllotoxin with cytotoxic and antioxidative activity

A series of novel spin-labeled podophyllotoxin derivatives were synthesized by reacting the corresponding N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxy carbonyl)-amino acids with 4β-amino-4′-demethylepipodophyllotoxin. The synthesized derivatives 12a–g were evaluated for the partition coefficients, cytotoxicities in vitro against three tumor cell lines (A-549, HL-60, and RPMI-8226) and antioxidative activities in tissues of SD rats by the TBA method. The vast majority of target compounds have shown superior or comparable activities against A-549, HL-60, and RPMI-8226 compared to VP-16, and they have shown more significant antioxidative activities and superior water solubility than VP-16.     

Synthesis and antitumor activity of N-substituted iminodiacetato(1,1-bis[aminomethyl]cyclohexane)platinum(II) complexes

A number of water-soluble N-substituted iminodiacetato(1,1-bis[aminomethyl]cyclohexane)platinum(II) complexes have been synthesized, and their mode of coordination characterized by elemental analysis and infrared data. Preliminary in vitro screening test for antitumor activity of these complexes against L1210 murine leukemia cells were performed. The results indicate that these complexes have an acceptable in vitro cytotoxicity against L1210 leukemia.     

Interactions of the antitumor drug, etoposide, with reduced thiols in vitro and in vivo

The interaction of activated etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), with thiols has been studied both in vitro and in vivo in mice. We have found that both glutathione (GSH) and cysteine rapidly reduce the VP-16 free radical, which results in the regeneration of the parent drug and the oxidation of the thiol. Using spin-trapping and electron spin resonance (ESR) techniques, we have shown that this one-electron/hydrogen donation by thiols forms thiyl radicals (RS.) which are intermediates for the formation of the oxidized thiols. The administration of VP-16 in vivo to mice decreased the total thiol levels in liver and concomitantly increased the formation of oxidized thiols. Furthermore, VP-16 stimulated glutathione reductase in liver. While administration of VP-16 also increased the total thiol pools in kidney, in contrast, no significant effects were observed on lung and heart thiol pools.     

Antitumor effects of human recombinant interleukin-1α and etoposide against human tumor cells: mechanism for synergismin vitro and activityin vivo

Recombinant human interleukin 1α (rh IL-1α) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell linesin vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1α. The purposes of this study were to test our hypothess that these events were critical to the synergy between rhIL-1α and VP-16, to determine whether rhIL-1α and VP-16 synergize to increase superoxide (SO) anion radical productionin vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combinaton against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1ra) before exposure to rhIL-1α, VP-16 and rhIL-1α plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1α, VP-16, and rhIL-1α+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1α and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1α and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1α and VP-16. However, when A375/S0D15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfecton were exposed to rhIL-1α and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1α and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1α and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combnation of IL-1α and VP-16 might prove useful for the treatment of malignant diseasein vivo, if the increased toxicity can be reduced or managed. The US Government’s right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged.     

Combination vincristine and VP-16-213: evaluation of drug sequence

The combination of vincristine and VP-16-213 has been found to have synergistic antitumor activity in a murine system in vivo when the sequence of drug administration was vincristine followed by VP-16-213. To investigate the potential influence of drug scheduling on this synergistic combination, the reverse sequence of drug administration was evaluated. DBA/2 mice were inoculated with 10(6) P-388 murine leukemia cells, after which saline only, VP-16-213 only, vincristine only, or VP-16-213 followed at various time intervals by vincristine, were administered. Probable cure (survival greater than 60 days) was observed in 0/20, 0/20, 0/120, and 46/115 (40%), respectively (p less than 0.001). The proportion of animals attaining probable cure was greatest in the group receiving vincristine 4-72 hours after VP-16-213 (40-50%). Similar results had been obtained previously with the reverse drug sequence. In this animal model, the synergistic antitumor activity of vincristine and VP-16-213 does not appear to be schedule-dependent with respect to the sequence of drug administration.     

Polymerizable Fab' antibody fragments for targeting of anticancer drugs.

We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab' fragments (MA-Fab'). The targeted systems can be directly prepared by copolymerization of the MA-Fab', N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab' and the Fab'-targeted copolymers can effectively bind to target cells. An MA-Fab' (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab', HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer.     

Synthesis,anticancer activity and cytotoxicity of galactosylated epothilone B

Epothilones are the 16-membered macrolide compounds, exhibit microtubule-promoting activity, have the same anti-tumor mechanism as paclitaxel, and are expected to be the ideal substitutes for paclitaxel. However, natural epothilone compounds have been found to have disadvantages such as high toxicity in vivo, poor selectivity to tumor cells, and susceptibility to drug resistance. Herein, epothilone B was synthesized by fermentation, and it was galactosylated by chemical method. The toxicity in vitro of epothilone B and its galactosylated derivative was investigated by the MTT method. The anticancer activity evaluation in vitro was performed using a method similar to the antibody-directed enzyme-prodrug therapy (ADEPT) method. It indicated that the ratio of cytotoxicity between the free epothilone B and the galactosylated epothilone B was about 150. This would lay the foundation for the targeted treatment of cancer with epothilone glycosides.     

Structure-antitumour activity relationships for new platinum complexes

Fourteen platinum (Pt) coordination complexes with different ligands, which include both Pt(II) and Pt(IV) complexes, were prepared, characterized and tested for their in vitro cytotoxic effects on KB cells and for their antitumour activity against some tumour systems (L1210 and P388 leukaemia, ADJ/PC6A plasma cell tumour and Yoshida sarcoma).The majority of the ligands were derivatives of aniline or pyridine, but complexes with tranylcypromine, guanethidine and octodrine were also synthetized.Depending on cytotoxicity the Pt-compounds could be divided into 3 groups. The compounds with a high cytotoxicity (ED₅₀ = 0.1–1 μg/ml) were also active against L1210 and P-388 leukaemia; a correlation between cytotoxicity and antitumour activity was not always observed.In these complexes the oxidation state of the Pt appears to be critical for their activity.