DHP23002 as a next generation oral paclitaxel formulation for pancreatic cancer therapy.

OBJECTIVE: This study aimed to develop a new oral paclitaxel formulation (DHP23002) and to evaluate its absorption and antitumor effects in a pancreatic tumor mouse model. METHODS: To investigate the oral absorption of DHP23002, a newly developed lipid-based orally active paclitaxel formulation, a pharmacokinetic study of DHP23002, was conducted in mice (62.5 and 125 mg/kg). Moreover, to evaluate the antitumor effect of DHP23002 in pancreatic cancer treatment, the drug was administered to female athymic nude mice at 0 (vehicle), 25, 62.5, and 125 mg/kg on alternate days; the efficacy of the agent was compared with the efficacy of intravenous Taxol® injections at 10 mg/kg once per week. After 3 weeks of administration, tumor growth in mice belonging to each group was further monitored for 4 weeks after discontinuing medication. Moreover, to examine paclitaxel (DHP23002) accumulation in the tumor tissue, the amount of paclitaxel in tumor/blood was quantified using liquid chromatography with quadruple-TOF mass spectrometry. RESULTS: In the mouse pharmacokinetic study, oral Taxol® showed a negligible absorption, whereas DHP23002 showed a high absorption rate dependent on dosage, with a bioavailability of approximately 40% at a dose of 62.5 mg/kg. In efficacy-related studies, DHP23002 administration at a dose of 25, 62.5, or 125 mg/kg on alternate days for 3 weeks showed a superior tumor inhibitory effect of 80%, 92%, and 97% in a xenograft mouse model, respectively, after 7 weeks. Paclitaxel accumulation in tumors persisted for >24 h in mice, when orally administered once at doses of 25, 62.5, and 125 mg/kg DHP23002. CONCLUSION: Oral chemotherapy with DHP23002 showed excellent absorption in animals owing to a strong antitumor activity in a pancreatic cancer mouse model. This demonstrates that paclitaxel is largely distributed and persists for a prolonged period at the tumor site owing to oral DHP23002 administration.

Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl Indeno[1,2-b]pyridinol Derivatives as Potent Topoisomerase IIα-Targeted Chemotherapeutic Agents for Breast Cancer.

With the aim of developing new effective topoisomerase IIα-targeted anticancer agents, we synthesized a series of hydroxy- and halogenated 2,4-diphenyl indeno[1,2-b]pyridinols using a microwave-assisted single step synthetic method and investigated structure-activity relationships. The majority of compounds with chlorophenyl group at 2-position and phenol group at the 4-position of indeno[1,2-b]pyridinols exhibited potent antiproliferative activity and topoisomerase IIα-selective inhibition. Of the 172 compounds tested, 89 showed highly potent and selective topoisomerase IIα inhibition and antiproliferative activity in the nanomolar range against human T47D breast (2.6 nM) cancer cell lines. In addition, mechanistic studies revealed compound 89 is a nonintercalative topoisomerase II poison, and in vitro studies showed it had promising cytotoxic effects in diverse breast cancer cell lines and was particularly effective at inducing apoptosis in T47D cells. Furthermore, in vivo administration of compound 89 had significant antitumor effects in orthotopic mouse model of breast cancer.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys.

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.

Preclinical safety evaluation of intravenously administered SAL200 containing the recombinant phage endolysin SAL-1 as a pharmaceutical ingredient.

Phage endolysins have received increasing attention as potent antibacterial agents. However, although safety evaluation is a prerequisite for the drug development process, a good laboratory practice (GLP)-compliant safety evaluation has not been reported for phage endolysins. A safety evaluation of intravenously administered SAL200 (containing phage endolysin SAL-1) was conducted according to GLP standards. No animals died in any of the safety evaluation studies. In general toxicity studies, intravenously administered SAL200 showed no sign of toxicity in rodent single- and repeated-dose toxicity studies. In the dog repeated-dose toxicity test, there were no abnormal findings, with the exception of transient abnormal clinical signs that were observed in some dogs when daily injection of SAL200 was continued for more than 1 week. In safety pharmacology studies, there were also no signs of toxicity in the central nervous and respiratory system function tests. In the cardiovascular function test, there were no abnormal findings in all tested dogs after the first and second administrations, but transient abnormalities were observed after the third and fourth administrations (2 or 3 weeks after the initial administration). All abnormal findings observed in these safety evaluation studies were slight to mild, were apparent only transiently after injection, and resolved quickly. The safety evaluation results for SAL200 support the implementation of an exploratory phase I clinical trial and underscore the potential of SAL200 as a new drug. We have designed an appropriate phase I clinical trial based on the results of this study.

Anti-inflammatory effects of the Zingiber officinale roscoe constituent 12-dehydrogingerdione in lipopolysaccharide-stimulated Raw 264.7 cells.

Ginger has long been used worldwide as a spice, seasoning, and wine and is also used as a traditional medicine. There have been no previous studies of the potential beneficial effects of the ginger constituent 12-dehydrogingerdione (12-DHGD). We investigated the anti-inflammatory effect of 12-DHGD on lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. The cytotoxicity of 12-DHGD was measured using the MTT assay, and production of prostaglandin E2 (PGE2 ) and the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α was measured by ELISA. Production of nitric oxide (NO) was measured using Griess reagent and expression of cyclooxygenase-2 (COX-2) and inducible NO (iNOS) enzymes was assessed by reverse transcriptase-polymerase chain reaction. Treatment of Raw 264.7 cells with 12-DHGD significantly inhibited LPS-stimulated production of NO (at 12-DHGD concentrations of 150 and 200 ng/ml), IL-6 (at 50, 100, 150, and 200 ng/ml), and PGE2 (at 200 ng/ml). Consistent with the effects on NO and PGE2 production, 12-DHGD treatment also inhibited the LPS-stimulated increase in iNOS and COX-2 mRNA levels. However, 12-DHGD did not affect production of IL-1ß or TNF-α in response to LPS. 12-DHGD, a constituent of ginger, is a potent inhibitor of proinflammatory mediator production in Raw 264.7 macrophage cells.

Enhancement of B cell and monocyte populations in rats exposed to chlorpheniramine.

Chlorpheniramine is an anti-histamine agent on IgE-mediated inflammation. In order to investigate the immunomodulatory effects of chlorpheniramine, we assessed the changes of peripheral mononuclear cell populations and other general clinical parameters, including hematology and clinical chemistry, following chlorpheniramine administration in rats. Since prednisolone is commonly co-prescribed with anti-histamine in many hypersensitive reactions, we also examined the changes to compare the results after the prednisolone administration. Chlorpheniramine (50, 100 and 200 µg/kg) and prednisolone (1, 2 and 4 mg/kg) were intramuscularly administered to female Sprague-Dawley (SD) rats 3 times, at intervals of 1 week. Except the clinical signs, such as stiffness and abnormal gait due to the local toxicity at injection sites, no other significant changes in body weights, urinalysis, and macroscopic examination were noted in the animals given chlorpheniramine. On the other hand, white blood cells, especially B cells and monocytes, showed a dose-dependent increase in the chlorpheniramine-treated animals; whereas, the numbers of both B and T cells (helper T and cytotoxic T, NKT cells) were decreased in the prednisolone-treated animals. Taken together, these results suggest that chloropheniramine administration enhances white blood cells in the peripheral blood, mostly due to increases of the B cells and monocytes, but no T cells and NK cells.

Safety Evaluation of Yukmijihwang-tang: Assessment of Acute and Subchronic Toxicity in Rats.

Yukmijihwang-tang (YMJ; Liu wei di huang tang (China), Rokumigan (Japan)) has been used in the treatment of diseases including renal disorder, cognitive vitality, and diabetes mellitus. However, there is very little information regarding the toxicity of YMJ to give an assurance of safety for clinical treatment. To provide safety information for YMJ, we evaluated its acute and sub-chronic toxicity in rats. The single-dose toxicity of YMJ was examined using Sprague-Dawley rats. Rats were treated with YMJ extract orally at 0, 500, 1000, or 2000 mg/kg body weight. After a single administration, clinical signs were observed every day for two weeks, and body weights were measured five times, including an initial measurement on day 1 (the day of administration). In the sub-chronic oral toxicity study, YMJ was administered to rats at 0, 500, 1000, or 2000 mg/kg/day for 13 weeks. Mortalities, clinical signs, body weight changes, food and water consumption, ophthalmologic findings, urinalysis, hematological and biochemical parameters, gross findings, organ weights, and histological examination were monitored during the study period. We found no mortality and no abnormalities in clinical signs, body weights, and necropsy findings for any of the animals in the acute and sub-chronic studies following oral administration in the rat at up to 2000 mg/kg/day YMJ. YMJ may not have any single-dose toxicity; the LD(50) of YMJ was over 2000 mg/kg, and it is safe for rats. The no-observed-adverse-effect-level (NOAEL) was considered to be 2000 mg/kg/day.

Evaluation of safety of the herbal formula Ojeok-san: acute and sub-chronic toxicity studies in rats.

AIM OF THE STUDY: Ojeok-san (OJS; wuji powder in China and goshaku-san in Japan), a widely used herbal formula in traditional Korean medicine and Japanese herbal medicine (Kampo medicine), has been used to treat common cold and illnesses including fatigue and gastrointestinal disorders, but there is very little information on its safety. To provide information on the safety of OJS, we evaluated its acute and sub-chronic toxicity in rats. MATERIALS AND METHODS: The single and sub-chronic toxicity of OJS was examined using male and female Sprague-Dawley rats. The rats were treated with the OJS extract orally at the highest dose level of 2000 mg/kg/day body weight. After single administration, signs of toxicity were observed every hour for the first 6h and every day for two weeks. In the sub-chronic toxicity study, OJS was administered for 13 weeks. Mortality, clinical signs, body weight changes, food and water consumption, ophthalmologic findings, urinalysis, hematological and biochemical parameters, gross findings, organ weights and histological markers were monitored during the study period. RESULTS: We found no mortality and no abnormality in clinical signs, body weight, and necropsy findings for any of the animals in the acute and sub-chronic toxicity study following oral administration of OJS. CONCLUSION: OJS may not have any single dose toxicity. The lethal dose with a 50% mortality rate (LD(50)) was over 2000 mg/kg. The no-observed adverse effects level (NOAEL) was considered to be 2000 and 1000 mg/kg/day for male and female rats, respectively.

Genotoxicity studies on HM10760A, recombinant human erythropoietin conjugated to globin fragment.

HM10760A is a recombinant human erythropoietin chemically conjugated to the N-terminus of human immunoglobulin Fc fragment through a polyethylene glycol linker. HM10760A was shown to have a relatively long half-life, compared with unconjugated recombinant erythropoietin. In this study, the genotoxicity of HM10760A was investigated by using a test battery of three different methods. In the Ames assay, five strains (TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA) were tested at six concentrations of 3.13, 6.25, 12.5, 25, 50, and 100microg/plate. HM10760A did not increase the number of revertant colonies in any tester strains with and without metabolic activation by rat-liver S9 mix. Subsequently, in vitro chromosomal aberration test, using Chinese hamster lung cells, were conducted at the concentrations of 25, 50, and 100microg/mL. HM10760A did not induce chromosomal aberrations either in the short-period (6 hours) test with or without rat-liver S9 mix or in the continuous-treatment (24 hours) test. In the in vivo bone marrow micronucleus assay using the male ICR (imprinting control region) mouse, HM10760A was subcutaneously administered twice at 24-hour intervals at doses of 0, 150, 300, and 600microg/kg. HM10760A produced a slight, but statistically significant, increase in the frequency of micronucleated polychromatic erythrocytes at 600microg/kg. However, no biological significance was assumed, because this value was within the historical control range. From these findings obtained from the genotoxicity assays performed in this study, it appears unlikely that HM10760A acts as a genotoxic agent in vitro and in vivo.

Modulation of macrophage functions by compounds isolated from Zingiber officinale.

Bioactivity-guided fractionation of Zingiber Officinale (zingiberaceae) led us to isolate 14 compounds, -gingerol ( 1), -gingerol ( 2), -gingerol ( 3), -gingerol ( 4), -paradol ( 5), -shogaol ( 6), -shogaol ( 7), 1-dehydro- -gingerdione ( 8), -gingerdione ( 9), hexahydrocurcumin ( 10), tetrahydrocurcumin ( 11), gingerenone A ( 12), 1,7-bis-(4' hydroxyl-3' methoxyphenyl)-5-methoxyhepthan-3-one ( 13), and methoxy- -gingerol ( 14). Using the RAW 264.7 cell line, the inhibitory effects on nitric oxide production induced by lipopolysaccharide and the stimulatory effects on phagocytosis of these compounds were evaluated. Compounds 7, 8, and 9 significantly decreased lipopolysaccharide-induced nitric oxide production, and compounds 7 and 8 significantly reduced inducible nitric oxide synthase expression. Among them, compound 8 also showed significant stimulatory effects on phagocytosis.

In vitro assessment of clastogenicity of mobile-phone radiation (835 MHz) using the alkaline comet assay and chromosomal aberration test.

Recently we demonstrated that 835-MHz radiofrequency radiation electromagnetic fields (RF-EMF) neither affected the reverse mutation frequency nor accelerated DNA degradation in vitro. Here, two kinds of cytogenetic endpoints were further investigated on mammalian cells exposed to 835-MHz RF-EMF (the most widely used communication frequency band in Korean CDMA mobile phone networks) alone and in combination with model clastogens: in vitro alkaline comet assay and in vitro chromosome aberration (CA) test. No direct cytogenetic effect of 835-MHz RF-EMF was found in the in vitro CA test. The combined exposure of the cells to RF-EMF in the presence of ethylmethanesulfonate (EMS) revealed a weak and insignificant cytogenetic effect when compared to cells exposed to EMS alone in CA test. Also, the comet assay results to evaluate the ability of RF-EMF alone to damage DNA were nearly negative, although showing a small increase in tail moment. However, the applied RF-EMF had potentiation effect in comet assay when administered in combination with model clastogens (cyclophosphamide or 4-nitroquinoline 1-oxide). Thus, our results imply that we cannot confidently exclude any possibility of an increased risk of genetic damage, with important implications for the possible health effects of exposure to 835-MHz electromagnetic fields.

Immunosuppressive lignans isolated from Saururus chinensis.

Bioactivity-guided fractionation of Saururus chinensis (Saururaceae) using a lymphoproliferation assay led us to isolate 5 lignans (compounds 1 - 5). Compounds 1 - 5 were identified as sauchinone, (-)-saucerneol, saucerneol C, manassantin A, and manassantin B, respectively, by spectroscopic analyses. The immunosuppressive activities of the active compounds were evaluated using lymphoproliferation, mixed leukocyte response, and Th1/Th2 cytokine assays. The relative potency was in the order: manassantin A, B > (-)-saucerneol > saucerneol C > sauchinone.

Genotoxicity assessment of HM10620 containing recombinant human interferon-alpha.

HM10620 is a recombinant human interferon-alpha (rhIFN-alpha) linked to immunoglobulin via N-terminal-specific non-peptidyl polyethylene glycol linker to improve the in vivo stability of interferon. Potential genotoxic effects of HM10620 in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro chromosomal aberration assay, and the in vivo micronucleus assay. HM10620 did not cause any mutation in the Ames assay tested using five tester strains at six concentrations of 6.25, 12.5, 25, 50, 100, and 200 microg/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay and the in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Chromosomal aberration was not induced at the concentrations of 10, 20, and 40 microg/mL. Also, there was no difference in the incidence of micronucleated polychromatic erythrocytes at doses of 10, 20, and 40 mg/kg in male mice compared with the vehicle control group. Therefore, based on the results obtained from the three studies, it is concluded that HM10620 is not a mutagenic agent in bacterial cells and causes no chromosomal damage in mammalian cells both in vitro and in vivo.

Potential side effects of dendritic cells pulsed with allogenic melanoma cell lysate in mice.

An attempt has been made to investigate the toxicity of cancer immunotherapy based on the dendritic cells pulsed with lysate of allogenic melanoma cell, DM401. Dendritic cells pulsed with lysate of clone M3 were subcutaneously administered once a week eight times to C57BL/6 mice at 0, 2.5, 5, and 10 x 10(7) cells/kg. No changes attributable to the administration were observed in clinical signs and food and water consumption. The administration induced slight increases in body weights, white blood cells, total protein, total cholesterol, triglyceride, phospholipids, and absolute spleen weights, but a slight decrease in albumin/globulin ratio. Microscopic examinations revealed the infiltration of inflammatory cells in the lung, mainly in the pulmonary arteriole, in which the tunica media thickened, and in the pulmonary alveoli and alveolar space. Thickened tunica media of pulmonary arteriole was observed in both males and females at all selected doses. In addition, the subcutis at the test substance-application site showed inflammation and fibrosis. In conclusion, lung is a target organ of DM401, and most of the changes including the findings in lung are considered as the immunomodulatory functions of dendritic cells.

Enhancement of plasmacytoma cell growth by ascorbic acid is mediated via glucose 6-phosphate dehydrogenase.

PURPOSE: We investigated the mechanism by which some types of cancer cells grow faster in the presence of ascorbic acid supplementation. MATERIALS AND METHODS: Adj.PC-5, a mouse plasmacytoma cell, is known to show ascorbic acid-dependent growth and was chosen as a test system. The growth of cancer cells was measured by the colony number on soft agar or the cellular proliferation in suspension culture. The ascorbate level was measured by a high performance liquid chromatography system with an electrochemical detector. Glucose 6-phosphate dehydrogenase was analyzed both on the specific enzyme activity level and on the transcription level by performing Northern blot analysis. RESULTS: Ascorbyl 2-phosphate among the ascorbate derivatives was the most efficient in stimulating cell growth. The intracellular and extracellular ascorbate concentrations following treatment with either ascorbate or ascorbyl 2-phosphate suggest that the superiority of ascorbyl 2-phosphate for stimulating cell growth may be due to its slow conversion to ascorbate in the culture medium. The steady transformation to ascorbate ensures sustained levels of ascorbate in the culture medium and thereby maximizes the growth stimulatory effect of ascorbate. Ascorbyl 2-phosphate markedly enhanced, in a concentration-and time-dependent manner, mRNA synthesis as well as the enzymatic activity of glucose 6-phosphate dehydrogenase, which is known to be a rate-limiting enzyme in cell growth. On the other hand, simultaneous addition of dehydroisoandrosterone, a well- known inhibitor of glucose 6-phosphate dehydrogenase, to the culture medium abrogated the growth stimulation by ascorbyl 2-phosphate, and it also reduced the glucose 6-phosphate dehydrogenase activity proportionately. CONCLUSIONS: The results from this study suggest that enhanced glucose 6-phosphate dehydrogenase activity may at least in part explain the stimulation of cell growth by ascorbate or ascorbyl 2-phosphate.

Identification of potential biomarkers of genotoxicity and carcinogenicity in L5178Y mouse lymphoma cells by cDNA microarray analysis.

In the present study, cDNA microarray analyses were performed with mouse cDNA chips in order to evaluate similarities and differences in the gene expression profiles for compounds differing in their genotoxic and carcinogenic potential. Eight test substances were evaluated, two each from four classes of compounds: genotoxic carcinogens (1,2-dibromoethane and glycidol), genotoxic noncarcinogens (8-hydroxyquinoline and emodin), nongenotoxic carcinogens (methyl carbamate and o-nitrotoluene), and nongenotoxic noncarcinogens (D-mannitol and 1,2-dichlorobenzene). Quadruplicate hybridization experiments were performed in order to identify a set of genes with significant expression changes for these four classes of substances. Twelve genes were consistently altered more than twofold by the genotoxic noncarcinogens while four genes were consistently regulated by the nongenotoxic carcinogens. One gene (Trp63) was identified whose expression was upregulated by all four genotoxic substances regardless of the presence or absence of carcinogenicity; this finding, however, was not confirmed by quantitative real-time RT-PCR. RT-PCR did confirm the change in expression of 9 of 15 genes (60%) identified by microarray analysis. Interestingly, the downregulated genes were least likely to be validated by real-time RT-PCR. Those genes showing more than a twofold change in expression level in response to at least one substance were further analyzed with hierarchical clustering after category assignment of each gene according to its main cellular function. Clustering revealed differences in the gene expression profiles between the genotoxic and nongenotoxic substances for genes involved in cell cycle control, the stress response, and the immune response. However, no clustering specific to all four carcinogenic substances was observed in any of the functional categories. Taken together, these results suggest that gene expression profiling in mouse lymphoma cells can provide valuable information for the evaluation of potential genotoxicity but may have limitations in predicting carcinogenicity.

Apoptotic effect of PP2 a Src tyrosine kinase inhibitor, in murine B cell leukemia.

Src is a non-receptor protein tyrosine kinase that transduces signals regulating cell growth and differentiation. We report here that activation of signaling pathway after blockade of tyrosine phosphorylation by PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, can lead to cell death in murine B cell leukemia, 70Z/3. Death from PP2 occurred by apoptosis as indicated by the induction of caspase activation and annexin V/propidium iodide staining. Interestingly, PP2 was found to be able to enhance the DNA binding activity of nuclear factor kappaB (NF-kappaB) before induction of apoptosis without accompanying by increased phosphorylation of inhibitor of NF-kappaB-alpha (IkappaB-alpha). Additionally, immunoblotting analysis with PP2-treated cell extract demonstrated that, compared to other protein kinase C (PKC) isotypes, the translocation of novel PKC isotypes from the cytosol to membrane fraction was sustained for a longer time. These data suggest that the inhibition of Src-mediated tyrosine phosphorylation by PP2 may tilt the balance between each PKC isotypes, which in turn, activate NF-kappaB transcription factor, leading to apoptosis.

Non-specific inhibition of human cytochrome P450-catalyzed reactions by hemin.

Hemin, a stable form of heme, is known to have an antimutagenic effect. Inhibitory effects of hemin on the cytochrome P450 (CYP)-catalyzed reactions of human liver microsomes and reconstituted systems containing purified CYP and NADPH-cytochrome P450 reductase (NPR) were seen. Hemin non-specifically inhibited all of the microsomal CYP activities examined. Hemin also inhibited 7-ethoxyresorufin O-deethylation, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin O-demethylation, and testosterone 6beta-hydroxylation catalyzed by purified CYPs 1A2, 2D6, and 3A4, with IC50 values of 27, 19, and 2.4 microM, respectively. Hemin also inhibited reduction of cytochrome c and ferricyanide by NPR, as much as 47%. Spectrally detectable CYP was destroyed in human liver microsomes and in a reconstituted system in the presence of hemin and an NADPH-generating system. We propose that the antimutagenic effect of hemin might be due to inhibition of CYP and NPR enzymes involved in the bioactivation of mutagens.

cDNA microarray gene expression profiling of hydroxyurea, paclitaxel, and p-anisidine, genotoxic compounds with differing tumorigenicity results.

The potential application of toxicogenomics to predictive toxicology has been discussed widely, but the utility of the approach remains largely unproven. Using cDNA microarrays, we compared the gene expression profiles produced in mouse lymphoma cells by three genotoxic compounds, hydroxyurea (a carcinogen), p-anisidine (a noncarcinogen), and paclitaxel (carcinogenicity unknown). To minimize the effect of biological variability and technological limitations, quadruplicate observations were made for each compound and a subset of genes yielding reproducible induction/repression was selected for comparison. A method was applied to attach normalized expression data to genes with a low false-discovery rate (<0.1) to yield more confidence regarding differential expression. This analysis identified genotoxicity-specific gene expression. Seven genes were consistently upregulated and 12 downregulated more than 2-fold by the three genotoxic compounds. Using additional genes, the expression pattern induced by the genotoxic noncarcinogen, p-anisidine, was readily distinguished from that associated with the genotoxic carcinogen, hydroxyurea. Comparison of paclitaxel-induced expression data to data for p-anisidine and hydroxyurea suggested that paclitaxel's profile is more similar to the genotoxic noncarcinogen. With further supporting evidence it may be possible to perform large-scale monitoring of gene expression during drug and chemical development that can provide an early warning of potential toxicological responses.

Down-regulation of Raf-1 kinase is associated with paclitaxel resistance in human breast cancer MCF-7/Adr cells.

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance and apoptosis induced by paclitaxel. In the present study, paclitaxel sensitivity, Raf-1 activity and mitogen-activated protein kinases activation were compared in two cell lines: parental human breast cancer cells and its drug resistant variant (MCF-7/Adr) cells. Paclitaxel treatment of parental MCF-7 cells caused a marked inhibition of Raf-1 kinase activity, concomitant with its mobility shift after 18 h exposure. In addition, paclitaxel greatly increased c-Jun N-terminal protein kinase (JNK) activity whereas showing a small enhancing effect on extracellular-regulated kinases (ERK) activity. Interestingly, MCF-7/Adr cells have lower basal Raf-1 activity, yet have much higher basal ERK activity than parental cells. However, it appeared that PD 98059, which turns off ERK through mitogen-activated protein kinase kinase (MEK) inhibition, enhanced basal Raf-1 kinase activity in MCF-7/Adr cells. Thus, the findings suggest that paclitaxel-induced apoptosis is mediated by JNK and occurs in parallel with suppression of the Raf-1 kinase activity in parental MCF-7 cells. In addition, down-regulation of Raf-1 kinase, which can be induced through the sustained ERK activation, may contribute to the development of acquired resistance in MCF-7/Adr cells.