Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones

1,2-Dithiol-3-thiones, reported constituents of cruciferous vegetables, are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, and chemoprotective activities. The effects of dietary administration of a substituted 1,2-dithiol-3-thione, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione], a potent antischistosomal agent, on aflatoxin B1 (AFB1) metabolism, DNA adduct formation, and hepatic tumorigenesis were examined in male F344 rats. Rats were fed graded doses of oltipraz (0.01-0.1%) for 4 wk. During the second and third wk of oltipraz feeding rats were gavaged with 250 micrograms of AFB1/kg five times a wk. Rats were finally restored to control diet 1 wk after cessation of AFB1 dosing. At 4 months focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transpeptidase activity. Treatment with oltipraz at all doses reduced by greater than 90% the volume of liver occupied by gamma-glutamyl transpeptidase-positive foci. Levels of AFB1 bound to hepatic DNA were reduced between 40 and 80% in animals fed increasing doses of dietary oltipraz (0.01-0.1%) for 1 wk prior to a single exposure to AFB1. Feeding of the higher levels of oltipraz led to marked increases in the specific activity of glutathione S-transferases, presumably serving to facilitate the detoxication of the ultimate electrophilic form of AFB1, the 8,9-oxide. At low dietary concentrations of oltipraz (0.01%), the only inductive effects seen were on the activities of selected cytochrome P-450 monooxygenases. Therefore, the protection afforded by oltipraz may be due to both the enhancement of electrophile detoxication pathways as well as modified oxidative metabolism of AFB1. In in vitro metabolism studies with hepatic post-mitochondrial supernatant, low-dose oltipraz pretreatment facilitated the oxidative production of aflatoxins P1 and Q1, but not M1, from AFB1. High-dose (0.1%) oltipraz pretreatment enhanced the primary metabolism of AFB1 to aflatoxins P1, M1, and Q1 as well as the formation of chloroform-insoluble metabolites. Feeding studies with a series of 1,2-dithiol-3-thione and 1,2-dithiol-3-one derivatives of oltipraz demonstrated that the inductive activity for cytochrome P-450-dependent monooxygenases and electrophile detoxication enzymes, such as glutathione S-transferases, could be readily separated by minor modifications of the 1,2-dithiol-3-thione structure. The unsubstituted 1,2-dithiol-3-thione nucleus strongly induced electrophile detoxication enzymes, but not the monooxygenases, and was the most effective inhibitor of the binding of AFB1 to hepatic DNA in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of phase II enzymes by 3H-1,2-dithiole-3-thione: dose-response study in rats

Derivatives of 3H-1,2-dithiole-3-thione (D3T) are known to protect against a variety of chemical carcinogens. There is evidence that this chemoprotective effect depends, at least in part, on the ability of these compounds to increase tissue activities of phase II detoxification enzymes. In the present study, D3T was dosed to rats at daily doses of between 0.98 and 125 micromol/kg/day for 5 days. The activity of two phase II enzymes, quinone reductase and glutathione S-transferase, were then assayed in the liver, spleen, kidneys, lungs, heart, urinary bladder, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum and colon plus rectum of the animals. D3T was particularly effective in increasing enzyme activities in the stomach and duodenum, with significant effects being recorded at a dose-level of only 0.98 micromol/kg/day. At slightly higher dose-levels, increases were recorded in other segments of the small and large intestine and in the urinary bladder. D3T caused enlargement of the liver, kidneys, stomach and intestinal tract of the animals at the higher dose-levels, but no other toxic effects were recorded. D3T is a very effective inducer of phase II enzymes, showing significant effects at lower dose-levels than any other compound for which dose-response data are available. The inductive potency of D3T makes it a most promising candidate for use as a chemoprotective agent.     

1,2-dithiole-3-thione and its structural analogue oltipraz are potent inhibitors of dibenz

Dithiolethiones are currently one of the most promising classes of cancer chemopreventive agents that exhibit antitumorigenic properties at numerous organ sites against several classes of carcinogens. In the current study, we examined the effects of 2 dithiolethiones, 1,2-dithiole-3-thione (D3T) and its structural analogue oltipraz, on DNA adduction induced by the potent mammary carcinogen dibenzo-[a,l]pyrene (DBP) in vivo. Female Sprague-Dawley rats were provided dietary D3T and oltipraz (500 ppm each) for I week followed by a single intragastric dose of DBP (8 micromol/kg body weight) and killed 5 days later. D3T inhibited DBP-DNA adduction from 78% to 82% in all tissues examined, while oltipraz was equally effective in the lung and liver but less effective in the mammary glands, inhibiting DBP-DNA adduction by nearly 60%. These data coupled with their broad anti-tumor specificity support the use of D3T and oltipraz as cancer-preventive agents in clinical trials.     

Potent inhibition of aflatoxin-induced hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione.

1,2-Dithiole-3-thiones are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, radioprotective and chemoprotective properties. Several substituted 1,2-dithiole-3-thiones are used medicinally and one of these, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione], has been recently shown to be an inhibitor of aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Structure-activity studies have been undertaken to probe the mechanisms by which dithiolethiones inhibit carcinogenesis. Such studies revealed that unsubstituted 1,2-dithiole-3-thione was more effective than oltipraz at inhibiting aflatoxin-DNA adduct formation in vivo and at inducing electrophile detoxication enzymes in cell culture. In the present studies the effects of dietary administration of 1,2-dithiole-3-thione on the induction of xenobiotic metabolizing enzymes and inhibition of aflatoxin-induced hepatic tumorigenesis were examined. Male F344 rats were fed graded doses of 1,2-dithiole-3-thione (0.001-0.03%) for 4 weeks. During the second and third weeks of 1,2-dithiole-3-thione feeding, rats were dosed by gavage with 250 micrograms of AFB1/kg five times a week. Rats were then restored to control AIN-76A diet 1 week after cessation of AFB1 dosing. At 4 months, focal areas of hepatocellular alteration were identified and quantified by staining sections of liver for gamma-glutamyltranspeptidase (GGT) activity and glutathione S-transferase P (GST-P) expression. Treatment with 1,2-dithiole-3-thione at the lowest dose (0.001%) reduced by greater than 80% the volume of liver occupied by GGT or GST-P foci; higher dietary concentrations provided greater than 98% reductions in the volume per cent of these markers for presumptive preneoplastic lesions. All dietary concentrations of 1,2-dithiole-3-thione resulted in significant elevations in hepatic GST activities. In accord with the protective effects against tumorigenesis, 4- to 6-fold increases in the specific activities of aflatoxin-glutathione conjugation were observed in cytosols prepared from livers of animals fed 1,2-dithiole-3-thione. By contrast, 1,2-dithiole-3-thione did not have any detectable inductive effects on hepatic microsomal cytochrome P450 levels or activities. Dietary administration of 1,2-dithiole-3-thione also elevated activities of GSTs and other phase II enzymes in several extrahepatic organs. This broad pattern of induction of detoxication enzymes by 1,2-dithiole-3-thione supports the potential widespread use of this compound as a protective agent against chemical carcinogenesis and other forms of electrophile toxicity.     

1,2-Dithiol-3-thione analogs: effects on NAD(P)H:quinone reductase and glutathione levels in murine hepatoma cells

The 1,2-dithiol-3-thiones are a class of five-membered cyclic sulfur compounds which have chemotherapeutic and chemoprotective properties. The parent 1,2-dithiol-3-thione nucleus and a series of six substituted analogs all induced NAD(P)H: quinone reductase (EC 1.6.99.2) activity and elevated glutathione levels in Hepa 1c1c7 murine hepatoma cells in culture thereby enhancing detoxification potential. These analogs included monosubstituted derivatives with phenyl, p-methoxyphenyl or 2-pyrazinyl groups at C-4 or C-5, and disubstituted compounds bearing phenyl or 2-pyrazinyl moieties at C-5 and an additional methyl group at C-4. This system can be used as an in vitro model for the study of the specificity and mechanism of action of the 1,2-dithiol-3-thiones as already demonstrated for several other classes of chemoprotective agents. The 1,2-dithiol-3-thiones also elevated quinone reductase and glutathione levels in the Hepa 1c1c7 cell mutants (BPrc1 and TAOBPrc1) that are defective in aryl hydrocarbon receptor functions. We conclude that the 1,2-dithiol-3-thiones are largely concerned with the stimulation of metabolic inactivation of electrophiles.     

Molecular dosimetry of urinary aflatoxin-N7-guanine and serum aflatoxin-albumin adducts predicts chemoprotection by 1,2-dithiole-3-thione in rats.

Hepatocellular carcinoma has one of the poorest 5 year survival rates of any human cancer. Preventive measures offer the best possibility of ameliorating this disease and chemoprotective agents are being developed for this purpose. The dithiolethiones, including oltipraz and the unsubstituted molecule 1,2-dithiole-3-thione, have been shown to be potent inhibitors of aflatoxin-induced hepatic tumorigenesis in rats. However, subsequent evaluation of dithiolethiones or other chemoprotective agents in human clinical trials will require the development of intermediate, non-invasive biomarkers to evaluate the efficacy of these interventions. In this study, levels of molecular dosimetry biomarkers for determining genotoxic damage caused by aflatoxin B1 have been measured in a chronic exposure model with male F344 rats wherein half the animals were fed a diet supplemented with 0.03% 1,2-dithiole-3-thione to lower their risk for tumors and the other half were fed unsupplemented AIN-76A diet and were at high risk for tumor development. Levels of hepatic aflatoxin-DNA adducts, serum aflatoxin-albumin adducts and excreted aflatoxin-N7-guanine adducts in urine were determined following multiple administrations of 250 micrograms aflatoxin B1/kg body wt on days 0-4 and 7-11 to assess the use of the serum and urinary biomarkers as indices of chemoprotective efficacy. In the rats fed 1,2-dithiole-3-thione, the overall diminutions in the levels of hepatic DNA adducts, urinary aflatoxin-N7-guanine and serum aflatoxin-albumin adducts over the 2 week exposure period were 76, 62 and 66% respectively. This parallelism in reductions of levels of biomarkers relative to target organ DNA adduct burden suggests that these biomarkers are predictive short-term, non-invasive measures for assessing the efficacy of chemoprotective interventions in experimental studies and can be applied to human clinical trials directed at populations at high risk for aflatoxin exposure and primary hepatocellular carcinoma.     

Resistance ot cisplatin and analogues: mechanisms and potential clinical implications

Summary In view of the important role of cisplatin (CDDP) in cancer chemotherapy, the frequent occurrence of resistance to the drug is a major clinical problem. The main cause for unresponsiveness of a tumor to CDDP is thought to be cellular drug resistance, which may be caused by (1) a decreased uptake of CDDP, (2) an increase in metallothioneins, (3) an increase in glutathione and/or glutathione-S-transferase, (4) increased DNA repair, or (5) increased tolerance to unrepaired lesions in DNA. Several mechanisms may be concomitantly operative. However, almost all data on CDDP resistance are derived from cell lines or experimental animal systems, and it is uncertain whether they are relevant for human tumors. Possible methods for overcoming CDDP resistance in cancer patients include the use of high-dose CDDP or carboplatin or of different formulations of platinum derivatives, the regional administration of CDDP, the inducement of hyperthermia, the depletion of glutathione by buthionine-S-R-sulfoximine (BSO), or the use of platinum analogues. The development of methods to detect and classify CDDP resistance in human tumor samples is urgently required for the development of modalities to overcome resistance.     

Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts

The mutagenic potential of the epoxide metabolites of butadiene(BD) was measured at the tk and hprt loci in TK6 human lymphoblastoidcells. TK6 cells were exposed for 24 h to 0–400 µM1,2-epoxybutene (EB), 0–800 µM 3,4-epoxy-1,2-butanediol(EBD), or 0–6 µM 1,2,3,4-diepoxybutane (DEB). Treatedcells were allowed to grow for several days and then seededin medium containing either 6-thioguanine or trifluorothymidineto select for hprt^– or tk^–/– mutants, respectively.All three metaboiltes were mutagenic at both loci, with DEBexhibiting activity at concentrations approximately 100-foldlower than EB or EBD. At the hprt locus, an induced mutationfrequency of 5 x 10^–6 (approximately twice backgroundhprt^– frequency) was produced by treatment with 3.5 µMDEB, 150 µM EB and 450 µM EBD. At the tk locus,a similar increase in mutation frequency (total tk^–/–frequency) was produced by treatment with 1.0 µM DEB,100 µM EB and 350 µM EBD. Each epoxide tested wascapable of inducing slow growth tk^–/– mutants. Thismutant phenotype, as shown previously by others, results fromlarge alterations in the tk region which completely remove theactive tk allele. In addition, Southern blot analysis revealedthat approximately half of DEB-induced hprt^– mutants displayedloss of wild-type hprt restriction fragments. No statisticallysignificant increase in the fraction of hprt deletions amongEB mutants was observed. The ability of DEB to induce deletionsmay be related to its ability to form DNA-DNA and DNA-proteincross-links.     

Inhibitory effects of 5-(2-pyrazinyl)-4-methyl-1,2-dithioI-3-thione (Oltipraz) on carcinogenesis induced by benzo[a]pyrene, diethylnitrosamine and uracil mustard

5-(2-Pyrazinyl)-4-methyl-1, 2-dithiol-3-thione (Oltipraz) wasstudied for its capacity to inhibit carcinogen-induced neoplasiain female ICR/Ha mice. When administered by oral intubation48 h prior to benzo[a]pyrene (BP), also given by oral intubation,Oltipraz inhibited the occurrence of pulmonary adenomas andtumors of the forestomach. The ratio of the number of tumorsoccurring in the mice receiving Oltipraz to that of the correspondingcontrols was: lung, 0.36 and forestomach0.38. Inhibition alsooccurred when Oltipraz was given p.o. 24 h prior to BP. In otherexperiments, oral administration of Oltipraz 48 h prior to p.o.administration of diethylnitrosamine or uracil mustard inhibitedpulmonary adenoma formation but to a lesser extent than withBP as the carcinogen. The low toxicity of Oltipraz found previously,coupled with evidence of protective effects against chemicallydiverse carcinogens, suggests that this compound should be studiedfurther for its possible use as an agent for the chemopreventionof neoplasia.     

Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry.

Previous studies have demonstrated that dietary administration of the schistosomicidal drug 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) ameliorates the hepatotoxicity of aflatoxin B1 (AFB1). Notably, mortality, altered hepatic function, hepatic AFB1-DNA adduct levels, and expression of hepatic enzyme-altered foci were markedly reduced in the rat by concurrent feeding of oltipraz during exposures to AFB1. Collectively, these studies prompted us to evaluate the chemoprotective properties of oltipraz against AFB1-induced liver cancer. In addition, preliminary molecular dosimetry studies were undertaken to determine the utility of measurements of urinary aflatoxin-N7-guanine excretion as a marker of relative risk for hepatocarcinogenesis in AFB1-exposed rats. For the carcinogenesis studies, 5-wk-old male F344 rats were randomly divided into two groups. One group (55 rats) received the AIN-76A diet, and the other group (56 rats) received the AIN-76A diet supplemented with 0.075% oltipraz. The oltipraz-supplemented diet was fed for 4 wk. Beginning 1 wk after starting the experimental diets, all rats in both groups received 25 micrograms of AFB1/rat/day by gavage for 5 days per wk over the next 2 wk. One wk following cessation of dosing with AFB1, oltipraz was removed from the diet, and all rats were fed the AIN-76A diet for the remainder of the experiment. At 3 mo after dosing, livers of ten sentinel rats from each group were analyzed for the burden of gamma-glutamyltranspeptidase-positive foci. In accord with previous findings, rats fed the oltipraz-supplemented diet exhibited substantial reductions in the focal burden (97% reduction; P less than 0.05) of these AFB1-induced lesions. The remaining rats were maintained for the cancer study until they became moribund or the termination of the experiment at 23 mo. Gross liver lesions were identified at autopsy and confirmed by microscopic evaluation. An 11% incidence of hepatocellular carcinoma was observed in the AFB1-treated, control diet-fed rats. An additional 9% of this group had hepatocellular adenomas. Oltipraz afforded complete protection against both AFB1-induced hepatocellular neoplasms. Using Kaplan-Meier survival analyses, rats in the oltipraz group had a significantly (P less than 0.02) longer life span and an increased survival free of liver tumors (P less than 0.0002). Molecular dosimetry studies used rats fed either the oltipraz-supplemented or control diet for 1 wk and then challenged with a single dose of AFB1 to examine the initial rates of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 excreted in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physiologically based pharmacokinetic modeling of 1,3-butadiene, 1,2- epoxy-3-butene, and 1,2:3,4-diepoxybutane toxicokinetics in mice and rats

1,3-Butadiene (BD) is a more potent tumor inducer in mice than in rats. BD also shows striking differences in metabolic activation, with substantially higher blood concentrations of 1,2:3,4-diepoxybutane (butadiene diepoxide; BDE) in BD-exposed mice than in similarly exposed rats. The objective of this study was to develop a single mechanistic model structure capable of describing BD disposition in both species. To achieve this objective, known pathways of 1,2-epoxy-3-butene (butadiene monoepoxide; BMO) and BDE metabolism were incorporated into a physiologically based pharmacokinetic model by scaling rates determined in vitro. With this model structure, epoxide clearance was underestimated for both rats and mice. Improved simulation of blood epoxide concentrations was achieved by addition of first-order metabolism in the slowly perfused tissues, verified by simulation of data on the time course for BMO elimination after i.v. injection of BMO. Blood concentrations of BD were accurately predicted for mice and rats exposed by inhalation to constant concentrations of BD. However, if all BD was assumed to be metabolized to BMO, blood concentrations of BMO were overpredicted. By assuming that only a fraction of BD metabolism produces BMO, blood concentrations of BMO could be predicted over a range of BD exposure concentrations for both species. In vitro and in vivo studies suggest an alternative cytochrome P-450-mediated pathway for BD metabolism that does not yield BMO. Including an alternative pathway for BD metabolism in the model also gave accurate predictions of blood BDE concentrations after inhalation of BD. Blood concentrations of BMO and BDE observed in both mice and rats are best explained by the existence of an alternative pathway for BD metabolism which does not produce BMO.      

Quantification of DNA and hemoglobin adducts of 3,4-epoxy-1,2-butanediol in rodents exposed to 3-butene-1,2-diol

1,3-Butadiene (BD) is a confirmed rodent carcinogen and a suspect human carcinogen that forms mutagenic epoxide metabolites during biotransformation. Species differences in the roles of individual DNA reactive intermediates in BD mutagenicity and carcinogenicity are not completely understood. Evidence suggests that 1,2:3,4-diepoxybutane (DEB) is responsible for the mutagenic effect induced by exposures to low concentrations of BD in mice and that metabolites of 3-butene-1,2-diol (BD-diol) are involved in the mutagenicity at high exposures in both mice and rats. Two reactive metabolites, 3,4-epoxy-1,2-butanediol (EB-diol) and hydroxymethylvinyl ketone (HMVK), are formed during the biotransformation of BD-diol and could potentially be involved in BD-diol associated mutagenicity. To examine the role of EB-diol in BD-diol mutagenicity we have evaluated the dosimetry of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) and N-(2,3,4-trihydroxybutyl)valine (THB-Val) in female B6C3F1 mice and female F344 rats exposed by inhalation to 0, 6, 18 and 36 p.p.m. BD-diol for 4 weeks (6 h/day x 5 days/week). Results showed higher levels of both THB-Gua and THB-Val in mice than in rats. An evaluation of THB-Gua adducts showed virtually no differences between liver and lung for either species, suggesting that EB-diol is stable and is freely circulated. The data also indicated that THB adduct formation began to plateau around 18 p.p.m. in both species. Most importantly, the shape of the dose-response curve for THB adduct formation mimicked the one observed for hypoxanthine-guanine phosphoribosyltransferase (Hprt) mutation frequency. This showed that THB adducts, which are not thought to be responsible for causing the mutations, are good quantitative indicators of mutagenicity in rodents exposed to BD-diol. Although the potential contribution of HMVK still needs to be evaluated, the data suggest that EB-diol is responsible, at least in part, for BD-diol associated mutagenicity in rodents.     

Control of proliferating potential of myeloid leukemia cells during long-term treatment with vitamin D3 analogues and other differentiation inducers in combination with antileukemic drugs: in vitro and in vivo studies

Growth inhibition of murine and human myeloid leukemia cells by differentiation inducers during long-term culture was examined to improve the strategy for therapy of myeloid leukemia by differentiation inducers. When the effect of 1 alpha,25-dihydroxyvitamin D3, a typical differentiation inducer, on proliferation of mouse myeloid leukemia M1 cells was examined at a constant product of time and concentration (480 nM in 20 days), the continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3 was the most effective for inhibition of cell proliferation. After 20 days, the cumulative cell number was reduced about 3 X 10(5) times by continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. Similar results were obtained when M1 cells were treated continuously with dexamethasone. M1 cells resistant to 1 alpha,25-dihydroxyvitamin D3 appeared about 25 days after the start of continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. On the other hand, when M1 cells were treated continuously with 1 alpha,25-dihydroxyvitamin D3 and noncytotoxic doses of antileukemic drugs such as 1-beta-D-arabinofuranosylcytosine and daunomycin, resistant cells did not appear for at least 35 days. A similar effect of 1 alpha,25-dihydroxyvitamin D3 and antileukemic drugs on cell proliferation was observed with the human monoblast-like cell line U937. The survival of syngeneic SL mice inoculated with M1 cells was prolonged more by treatment with both 1 alpha-hydroxyvitamin D3 and daunomycin than by treatment with either drug alone. These results suggest that continuous treatment with both differentiation inducers and certain antileukemic drugs may be more effective therapeutically than treatment with a differentiation inducer alone.     

Cisplatin and platinum analogues: recent advances.

Cisplatin is a critically important antineoplastic agent employed in treating patients who have a broad spectrum of neoplasms. Resistance to this agent poses a serious clinical dilemma. The objective of this review is to summarize the principal mechanisms that contribute to cytotoxicity by this agent and underlie resistance. Knowledge of these processes provides a rational basis for developing therapeutic strategies to circumvent resistance.     

eIF3c: A potential therapeutic target for cancer

Cancer cells are rapidly evolving due to their unstable genome, which contributes to the development of new cancer clones with different gene expression profile (GEP). Manipulating the expression of the genes vital for the progression of the disease is essential to overcome its heterogeneity. However, targeting overexpressed genes, retrieved from GEP analysis, would be efficient for a specific kind of a malignancy. Alternatively, manipulating the expression of genes that are part of a fundamental mechanism in the cell would be effective against a wide range of malignancies. To test this hypothesis we characterized, using RNAi approaches, the therapeutic potential of the housekeeping eIF3c gene in five different cancer cell lines NCI-ADR/RES (NAR), HeLa, MCF7, HCT116 and B16F10. eIF3c is one of the core subunit of the eukaryote translation initiation factor (eIF) 3 complex, which has a crucial role in the translation initiation process. In this study, we demonstrated that eIF3c is vital to translation initiation in vivo, as its downregulation decreases the global protein synthesis and causes a polysome run-off. In addition, reducing the expression of eIF3c mediates G₀/G₁ or G₂/M arrest in a tissue dependent manner, which leads to a reduction in cell proliferation and eventually to cell death. Moreover, we demonstrated the efficiency of the hyaluronan (HA)-coated lipid-based nanoparticles (LNPs) platform to deliver eIF3c-siRNAs to mouse melanoma cells. Taking together, our results emphasize the importance of seeking ubiquitously expressed housekeeping genes such as eIF3c rather than tumor associated overexpressed genes as therapeutic targets for the heterogeneous malignancies.     

Interaction of p53 and DNA-PK in response to nucleoside analogues: potential role as a sensor complex for DNA damage.

Therapeutic nucleoside analogues such as ara-C, gemcitabine, and fludarabine exert their cytotoxic activity against cancer cells mainly by incorporation into DNA and disruption of further DNA synthesis, resulting in the triggering of apoptosis. However, the molecules that recognize the incorporated analogues in DNA and subsequently initiate the downstream cellular responses remain to be identified. Here, we report that the DNA-dependent protein kinase (DNA-PK) and p53 are able to form a protein complex that interacts with the gemcitabine-containing DNA and plays a role in signaling to apoptotic pathways. DNA-PK/Ku and p53 were copurified in a protein fraction that binds to gemcitabine-containing DNA in preference to normal DNA. Immunoprecipitation experiments revealed that the two proteins physically associate in a complex. Treatment with gemcitabine resulted in an increase of DNA-PK and p53 protein and an increase in the phosphorylation of p53 at Ser15. Furthermore, confocal microscopy demonstrated a colocalization of DNA-PK and p53 to the nucleus in cells treated with gemcitabine. The nuclear localization of the DNA-PK/p53 complex was coincident with the induction of apoptosis in these cells. Although the wild-type p53 present in the protein complex exhibited 3'-5' exonuclease activity, it was incapable of excising the incorporated gemcitabine from DNA. The binding of the p53/DNA-PK complex to DNA substantially blocked further DNA synthesis by DNA polymerases alpha and epsilon in vitro, indicating a stalling of this complex at the site of drug incorporation. These data suggest that DNA-PK and p53 may form a sensor complex that detects the disruption of DNA replication caused by nucleoside analogue incorporation and may subsequently signal for apoptosis.     

Chronic daily low dose of 4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (Oltipraz) in patients with previously resected colon polyps and first degree female relatives of breast cancer patients.

The chemoprevention agent oltipraz, one of the most active chemopreventive compounds in preclinical studies, has been shown to induce glutathione-S-transferase (GST) activity in animals. Oltipraz was evaluated in a Phase I trial at daily oral doses of 20 mg (L1), 50 mg (L2), and 100 mg (L3) and twice weekly doses of 125 mg (L4) taken for 6 months with 6 patients entered at L1 and L2 and 7 patients entered at L3 and L4 (26 subjects: 19 females and 7 males). The subject population included patients with previously resected colon polyps and first-degree female relatives of breast cancer patients. Patients with resected colon polyps underwent rectal biopsy for GST and glutathione (GSH) analyses. Of the 26 subjects, the following completed 6 months of therapy: 4 of 6 patients (L1), 4 of 6 patients (L2), 5 of 7 patients (L3), and 4 of 7 patients (L4). Toxicities were mild to severe and included: gastrointestinal symptoms, photosensitivity/heat intolerance, and neurological symptoms. Monthly plasma samples were obtained 2-3 h after oltipraz ingestion with minimally detectable plasma concentrations at L1. There was a significant difference in mean oltipraz concentration across the four doses, with no significant differences in mean oltipraz concentration over time. Rectal tissue and lymphocyte GSH and GST were variable, with no significant difference in mean levels across doses. At the 100-mg/day dose (L3), 1 patient experienced significant increase in rectal tissue GSH and GST activity, whereas 3 additional patients (L1 and L4) had >50% increase in tissue GSH. Lymphocyte GSH level was significantly related to plasma oltipraz concentration. There were no significant correlations between plasma oltipraz concentration and lymphocyte GST level nor any significant correlation between plasma concentration and percentage of change in tissue GSH or GST. Further investigation of dose/schedule and biological end points is ongoing.     

Evidence for a defective mitochondrial membrane in 1,2-dimethylhydrazine-induced colon adenocarcinoma in rat: Enhanced lipid peroxidation potential in vitro

Enhanced lipid peroxidation potential was measured in Holtzman rat colon tumors induced by chronic subcutaneous injection of 1,2-dimethylhydrazine as compared with normal colonic tissue. The peroxidation potentials were determined in the mitochondrial cellular components by measuring the ferrous-ascorbate induced formation of malondialdehyde. The tumor mitochondria were found to peroxidize at a rate 8–10-fold higher than the comparable normal tissue components. In addition, we found that the mitochondria from the cancer cells exhibited reduced NADH-cytochrome c reductase activity. These observations suggest an involvement of non-enzymatic free radical flux in DMH-induced carcinogenesis, which may be the result of structurally altered mitochondrial membranes.