Anti-inflammatory effects of pioglitazone on iron-induced oxidative injury in the nigrostriatal dopaminergic system.

AIMS: Transition metals, oxidative stress and neuroinflammation have been proposed as part of a vicious cycle in central nervous system neurodegeneration. Our aim was to study the anti-inflammatory effect of pioglitazone, a peroxisome proliferative activated receptor-γ agonist, on iron-induced oxidative injury in rat brain. METHODS: Intranigral infusion of ferrous citrate (iron) was performed on anaesthetized rats. Pioglitazone (20 mg/kg) was orally administered. Oxidative injury was investigated by measuring lipid peroxidation in the substantia nigra (SN) and dopamine content in the striatum. Western blot assay and DNA fragmentation were employed to study the involvement of α-synuclein aggregation, neuroinflammation as well as activation of endoplasmic reticulum (ER) and mitochondrial pathways in iron-induced apoptosis. RESULTS: Intranigral infusion of iron time-dependently increased α-synuclein aggregation and haem oxygenase-1 levels. Furthermore, apoptosis was demonstrated by TUNEL-positive cells and DNA fragmentation in the iron-infused SN. Systemic pioglitazone was found to potentiate iron-induced elevation in nuclear peroxisome proliferative activated receptor-γ levels. However, pioglitazone inhibited iron-induced α-synuclein aggregation, elevations in interleukin-1ß and interleukin-6 mRNA levels as well as increases in oxygenase-1, cyclo-oxygenase II, nitric oxide synthase and ED-1 protein levels, an indicator of activated microglia. Moreover, iron-induced DNA laddering as well as activation of ER and mitochondrial pathways were attenuated by pioglitazone. In addition, pioglitazone decreased iron-induced elevation in lipid peroxidation in the infused SN and depletion in striatal dopamine level. CONCLUSIONS: Our results suggest that pioglitazone prevents iron-induced apoptosis via both ER and mitochondrial pathways. Furthermore, inhibition of α-synuclein aggregation and neuroinflammation may contribute to the pioglitazone-induced neuroprotection in central nervous system.

Role of autophagy in protection afforded by hypoxic preconditioning against MPP+-induced neurotoxicity in SH-SY5Y cells.

A sublethal preconditioning has been proposed as a neuroprotective strategy against several CNS neurodegenerative diseases. In this study, the involvement of autophagy in the protection provided by hypoxic preconditioning against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity was studied in SH-SY5Y neuroblastoma cells. In contrast to the cytotoxicity of 0.1% oxygen, 1% oxygen hypoxia for 24h did not cause significant cell death. A transient increase in LC3-II level, a biomarker of autophagy, was demonstrated during hypoxic treatment. At the same time, 8-h hypoxia increased fluorescence due to monodansylcadaverine, a specific dye for autophagosomes, in the treated cells. Co-incubation with bafilomycin A1 (10 nM) further increased hypoxia-induced LC3-II levels but 3-methyladenine (3-MA; 10 mM) reduced the elevation in LC3-II levels induced by 8-h hypoxia. Moreover, 8-h hypoxia increased free radical formation and nuclear HIF-1alpha level. Glutathione was found to diminish hypoxia-induced LC3-II elevation. In contrast to the elevated LC3-II level, 8-h hypoxia significantly decreased mitochondrial mass. Furthermore, a rebound elevation in mitochondrial mass was observed under 8-h hypoxia and subsequent 12-h normoxia. Prior hypoxia attenuated the MPP(+)-induced elevation in LC3-II levels and cell death. Moreover, hypoxic pretreatment inhibited MPP(+)-induced activation of caspase-3 and DNA fragmentation. Co-incubation with 3-MA during hypoxia prevented the protection afforded by hypoxic preconditioning against MPP(+)-induced increases in LC3-II levels and neurotoxicity. Taken together, our results suggest that sublethal hypoxia induces autophagy that is mediated by oxidative stress. Furthermore, autophagy may be involved in the protection provided by hypoxic preconditioning against MPP(+)-induced neurotoxicity, indicating a neuroprotective role of autophagy in hypoxic preconditioning.

Melatonin inhibits arsenite-induced peripheral neurotoxicity.

In this study, the effect of melatonin on sodium arsenite (arsenite)-induced peripheral neurotoxicity was investigated using dorsal root ganglion (DRG) explants. After 24-hr incubation, arsenite (30 microm) consistently elevated the expression of heat shock protein 70 and haeme oxygenase-1, two well-known stress proteins, in the treated DRG explants. Co-incubation with melatonin (4 and 20 mm) concentration-dependently attenuated arsenite-induced elevation in stress proteins. Furthermore, melatonin inhibited arsenite-induced phosphorylation of p38 and DNA fragmentation. Inhibition by melatonin of arsenite-induced apoptosis was mediated via inactivating both endoplasmic reticulum (ER) and mitochondrial pathways. In the ER pathway, melatonin suppressed arsenite-induced elevation in activating transcription factor-6 and CCAAT/enhancer-binding protein homologous protein in the nuclear fraction of the treated DRG explants. Moreover, melatonin attenuated arsenite-induced activation of caspase 12, an ER-specific enzyme. In the mitochondrial pathway, arsenite-induced increases in Bcl-2 levels and cytosolic cytochrome c were reduced by melatonin. At the same time, melatonin inhibited arsenite-induced activation of caspase 3 in the treated DRG explants. Compared with glutathione and N-acetyl cysteine, melatonin was more potent than either in inhibiting arsenite-induced elevation in stress proteins. Taken together, our study demonstrates that melatonin is protective against arsenite-induced neurotoxicity in DRG explants. In addition, melatonin prevented arsenite-induced apoptosis via suppression of ER and mitochondrial activation. Our data suggest that melatonin is potentially a therapy for arsenite-induced peripheral neuropathy.

Arsenite-induced cytotoxicity in dorsal root ganglion explants.

Peripheral neuropathy is common in people chronically overexposed to arsenic. We studied sodium arsenite (arsenite)-induced cytotoxicity in dorsal root ganglion (DRG) explants. Incubation with arsenite concentration- and time-dependently increased the expression of stress proteins, heat shock protein 70, and heme oxygenase-1 in DRG explants. Furthermore, apoptosis was involved in the arsenite-induced cytotoxicity in the treated DRG. Elevation in cytosolic cytochrome c levels and reduction in procaspase 3 levels suggested an involvement of the mitochondrial pathway in arsenite-induced apoptosis in this preparation. At the same time, increases in the activating transcription factor-4 and C/EBP homologous protein and reduction in procaspase 12 levels indicated activation of the endoplasmic reticulum (ER) pathway in the arsenite-induced cytotoxicity in DRG explants. Salubrinal (30 microM), an ER inhibitor, was found to attenuate arsenite-induced DNA fragmentation and reduction in procaspase 12 in DRG explants. Cytotoxic effects by arsenite, sodium arsenate (arsenate), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were compared, and the potency was as follows: arsenite >>> arsenate>MMA and DMA. Recombinant adenovirus vectors encoding glial-cell-derived neurotrophic factor (AdGDNF) genes allowed a stable delivery of GDNF genes to the infected cells in DRG explants. Applied in this manner, AdGDNF was found to inhibit arsenite-induced DNA fragmentation in DRG explants. Moreover, AdGDNF attenuated the arsenite-induced reduction in procaspases 3 and 12 levels. Taken together, our study demonstrates that arsenite is capable of inducing cytotoxicity in DRG explants. Both ER and mitochondria pathways are involved in the arsenite-induced apoptosis in DRG explants. Glial-cell-derived neurotrophic factor appears to be protective against arsenite-induced peripheral neuropathy.

N-acetylcysteine attenuates arsenite-induced oxidative injury in dorsal root ganglion explants.

Chronic exposure to arsenic causes health problems, including peripheral neuropathy. Oxidative stress is one of the mechanisms underlying arsenic-induced neurotoxicity. For this report, we studied the protective effect of N-acetylcysteine (NAC) on arsenic-induced oxidative injury in dorsal root ganglion (DRG) explants. After 24-h incubation, NAC concentration-dependently attenuated arsenite-induced depletion in glutathione (GSH) content and increases in the ratio of oxidized GSH/reduced GSH (GSSG/GSH ratio) in DRG explants. Furthermore, NAC inhibited arsenite-induced elevation in the expression of stress proteins, such as heat shock protein 70 and heme oxygenase 1, as well as arsenite-induced phosphorylation of p38 mitogen-activated protein kinase. Incubation with NAC ameliorated arsenite-induced apoptosis by abolishing both mitochondrial and endoplasmic reticulum (ER) pathways. In the mitochondrial pathway, NAC attenuated arsenite-induced elevation in Bcl-2 level and cytosolic cytochrome c, as well as arsenite-induced reduction in procaspase-3 levels. In the ER pathway, NAC suppressed arsenite-induced increases in activating transcription factor 6 and C/EBP homologous protein in the nuclear fraction. Furthermore, arsenite-induced reductions in procaspase-12 and elevation in BIP and caspase-12, an ER-specific enzyme, were prevented after NAC incubation. Taken together, our results demonstrate that NAC is neuroprotective against arsenite-induced oxidative injury in DRG explants. Furthermore, NAC inhibits arsenite-induced toxicity by inhibiting ER and mitochondrion activation. Our data indicate that NAC is potentially therapeutic for arsenite-induced peripheral neuropathy.

Endoplasmic reticulum stress is involved in arsenite-induced oxidative injury in rat brain.

The mechanism underlying sodium arsenite (arsenite)-induced neurotoxicity was investigated in rat brain. Arsenite was locally infused in the substantia nigra (SN) of anesthetized rat. Seven days after infusion, lipid peroxidation in the infused SN was elevated and dopamine level in the ipsilateral striatum was reduced in a concentration-dependent manner (0.3-5 nmol). Furthermore, local infusion of arsenite (5 nmol) decreased GSH content and increased expression of heat shock protein 70 and heme oxygenase-1 in the infused SN. Aggregation of alpha-synuclein, a putative pathological protein involved in several CNS neurodegenerative diseases, was elevated in the arsenite-infused SN. From the breakdown pattern of alpha-spectrin, both necrosis and apoptosis were involved in the arsenite-induced neurotoxicity. Pyknotic nuclei, cellular shrinkage and cytoplasmic disintegration, indicating necrosis, and TUNEL-positive cells and DNA ladder, indicating apoptosis was observed in the arsenite-infused SN. Arsenite-induced apoptosis was mediated via two different organelle pathways, mitochondria and endoplasmic reticulum (ER). For mitochondrial activation, cytosolic cytochrome c and caspase-3 levels were elevated in the arsenite-infused SN. In ER pathway, arsenite increased activating transcription factor-4, X-box binding protein 1, C/EBP homologues protein (CHOP) and cytosolic immunoglobulin binding protein levels. Moreover, arsenite reduced procaspase 12 levels, an ER-specific enzyme in the infused SN. Taken together, our study suggests that arsenite is capable of inducing oxidative injury in CNS. In addition to mitochondria, ER stress was involved in the arsenite-induced apoptosis. Arsenite-induced neurotoxicity clinically implies a pathophysiological role of arsenite in CNS neurodegeneration.

Melatonin attenuates arsenite-induced apoptosis in rat brain: involvement of mitochondrial and endoplasmic reticulum pathways and aggregation of alpha-synuclein.

In the present study, the protective effect of melatonin on sodium arsenite (arsenite)-induced apoptosis was investigated. Local infusion of arsenite elevated lipid peroxidation and depleted glutathione content in the infused substantia nigra (SN), as well as reduced striatal dopamine content. Systemic administration of melatonin diminished arsenite-induced oxidative injury. Furthermore, melatonin attenuated arsenite-induced increases in heat shock protein 70 and heme oxygenase-1 as well as phosphorylation of p38 mitogen-activated protein kinase and elevations in cyclooxygenase II and inducible nitric oxide synthase expression. Inhibition by melatonin of arsenite-induced apoptosis was determined by its attenuation of DNA fragmentation and terminal deoxytransferase-mediated dUTP-nick end labeling's positive cells in the infused SN of melatonin-treated rats. Melatonin reduced arsenite-induced apoptosis through mitochondrial and endoplasmic reticulum (ER) pathways. In the mitochondrial pathway, systemic melatonin inhibited arsenite-induced elevations in Bcl-2 and cytosolic cytochrome c as well as arsenite-induced reductions in procaspase-3 levels and elevations in active caspase-3 levels in the infused SN. Regarding the ER pathway, melatonin attenuated arsenite-induced elevations in activating transcription factor-4, CCAAT/enhancer binding protein (C/EBP) homologues protein, X-bon binding protein (XBP-1) and cytosolic immunoglobulin binding protein (BIP) as well as reductions in procaspase 12 levels. Moreover, aggregation of alpha-synuclein was reduced in the arsenite-infused SN of melatonin-treated rats. Our in vitro data showed that melatonin ameliorated arsenite-induced lipid peroxidation. Taken together, our data suggest that melatonin is neuroprotective against arsenite-induced oxidative injury in the nigrostriatal dopaminergic system of rat brain. Furthermore, the neuroprotective effects by melatonin on arsenite-induced apoptosis were mediated via inhibiting both mitochondrial and ER pathways. Accordingly, melatonin may be therapeutically useful for the treatment of arsenite-induced apoptosis in central nervous system.

Antioxidative effect of vitamin D3 on zinc-induced oxidative stress in CNS.

Antioxidative mechanisms of vitamin D3 were evaluated both in vitro and in vivo. A 4-h incubation of brain homogenates at 37 degrees C increased the formation of Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Incubation with vitamin D3 dose-dependently suppressed auto-oxidation. The antioxidative potency for inhibiting zinc-induced lipid peroxidation was as follows: vitamin D3 > Trolox (a water-soluble analogue of vitamin E) > or = beta-estradiol > melatonin. In the presence of high dose of desferrioxamine, a metal chelator, vitamin D3 attenuated auto-oxidation. These in vitro data indicate that vitamin D3 may act as a terminator of the lipid peroxidation chain reaction. The antioxidative effect of vitamin D3 on zinc-induced oxidative injury was verified using local infusion of vitamin D3 in vivo. Intranigral infusion of zinc elevated lipid peroxidation in the infused substantia nigra and depleted striatal dopamine content at 7 days after infusion. Furthermore, elevated cytosolic cytochrome c and DNA ladder, indicatives of apoptosis, were demonstrated in the infused substantia nigra. Simultaneous infusion of vitamin D3 and zinc prevented oxidative injury and apoptosis induced by zinc alone. The involvement of glia-derived neurotrophic factor (GDNF) expression was excluded since vitamin D3 did not alter GDNF level in the infused substantia nigra at 24 h or 4 days after intranigral infusion of vitamin D3. Our results suggest that vitamin D3, independent of upregulation of GDNF expression, may acutely prevent zinc-induced oxidative injuries via antioxidative mechanisms.

Molecular cloning and characterization of full-length cDNAs encoding a novel high-molecular-weight Dermatophagoides pteronyssinus mite allergen, Der p 11.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. METHODS: The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. RESULTS: Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. CONCLUSIONS: This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients.

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.

Serum specific IgE reactivity to recombinant Der f 11 in asthmatic children.

This study was designed to examine the prevalence of positive serum IgE reactivity to the recombinant group 11 Dermatophagoides farinae allergen (rDer f 11) in asthmatic children in Taiwan. Using immunoblot analysis in a preliminary study of 18 asthmatic children, 13 (72.2%) reacted positively to rDer f 11 and 16 (88.9%) showed positive reactivity to D. farinae extracts. The allergenicity of rDer f 11 was further evaluated with in vivo skin tests and in vitro IgE immunodot assays in 24 mite skin-test-positive asthmatic children. Whereas 17 (70.8%) had positive skin tests to rDer f 11, 18 (75.0%) had positive serum IgE reactivity to rDer f 11. A good coincidence (87.5%) between the immunodot assay and the skin test was confirmed in these asthmatic children. Moreover, the prevalence of serum IgE reactivity to rDer f 11 was further investigated in a large panel of 49 mite skin-test-positive asthmatic children. Again, 38 (77.6%) had positive serum IgE reactivity to rDer f 11 in immunodot assays. Taken together the positive IgE reactivity to rDer f 11 in immunodot analysis ranged from 75 to 77.6% in two groups of 73 mite skin-test-positive asthmatic children. High incidence of serum IgE antibodies specific for rDer f 11 in the present study suggests that Der f 11 is a novel major allergen of house dust mites.

Significance of specific IgG subclass antibodies to house dust mites in asthmatic children.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), the major components of house dust, are considered to be etiologic factors of extrinsic asthma. The relationships between immunoglobulin (Ig) G subclass antibodies specific for Dp (or Df) were compared in specific IgE-positive and -negative asthmatic children. METHODS: Serum levels of IgG and IgE subclass antibodies specific for Dp and Df were studied in 52 children (age, 3-13 years; mean age, 8.4 years) with asthma using enzyme-linked immunosorbent assays. The skin prick test was also used in diagnosis of the reactivity of allergic disease. RESULTS: The levels of serum-specific IgG1 and IgG2 to Dp and Df in mite-specific IgE-(or skin-test) positive asthmatic children were significantly higher than those in mite-specific IgE- (or skin test) negative children (p < 0.01). Significant correlations between the level of the specific IgE and IgG1 (r = 0.6067; p = 0.0001) or IgG2 (r = 0.5851; p = 0.0002) to Dp, and IgG1 (r = 0.3823; p = 0.0214) or IgG2 (r = 0.5057; p = 0.0017) to Df were found. The specific IgE level and skin test reactivity showed a high correlation of greater than 96% (50/52). CONCLUSIONS: The levels of mite-specific IgG subclasses were partially compatible to specific IgE levels and skin test reactivity. We conclude that house dust mite allergy is significantly associated with specific IgG1, IgG2 and IgE responses.

Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11.

BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.

Isolation and characterization of a novel 98-kd Dermatophagoides farinae mite allergen.

BACKGROUND: Exposure to allergens from house dust mites is a significant cause of immediate hypersensitivity. Thus far, the active mite allergens defined are low molecular weight (MW) proteins or glycoproteins. However, other important mite allergens remain to be investigated. In this study a high MW mite antigen with a high IgE-binding activity was characterized. METHODS: An anti-Dermatophagoides farinae (Df) monoclonal antibody, mAb642, which recognized a 98-kd allergenic mite protein, was used for affinity chromatography. The purified Df642 was characterized biochemically and immunologically. RESULTS: Competitive ELISA demonstrated that mAb642 was inhibited by the interaction between serum IgE from allergic patients and Df642 antigen in a dose-dependent fashion. The IgE reactivity to both 98-kd and 92-kd components was removed or diminished by preincubation of asthmatic sera with Df642-coated CNBr-activated cellulose-4B gel. Two-dimensional immunoblot analysis revealed that there are at least 4 isoforms of Df642 that represent a minor component in the crude mite extract. The allergenicity of Df642 was assayed by IgE immunoassay with a large panel of 67 sera from asthmatic patients with positive skin reactions, and Df 642 showed positive IgE reactivity with more than 80% of the sera tested. Thus it should be classified as an important allergen. In addition, amino acid sequence analysis revealed that Df642 shares more than 50% homology with paramyosin from invertebrates. CONCLUSION: We have identified and characterized a 98-kd house dust mite allergen that showed greater than 80% IgE reactivity with sera from patients allergic to mites. This is the first high MW allergen characterized to date, and it shares high sequence homology with paramyosins in invertebrates.

Effects of tamoxifen and retinoic acid on cell growth and c-myc gene expression in human breast and cervical cancer cells.

The effects of estradiol, tamoxifen, and retinoic acid on the proliferation of breast and cervical cancer cells were investigated. Estrogen stimulated only MCF-7 cell growth, whereas tamoxifen and retinoic acid inhibited the proliferation of all cells studied. Northern blot analysis indicated that estradiol up-regulates c-myc mRNA level in all cell lines studied regardless of the estrogen receptor status in the cells. On the contrary, tamoxifen inhibits c-myc gene expression in all cell lines studied except in MCF-7 cells where the c-myc transcript was not affected. The inhibitory effect of tamoxifen on c-myc gene expression and cell proliferation in estrogen receptor-negative cells suggest an estrogen receptor-independent mechanism. The results also suggest that different mechanisms are involved in the regulation of cell growth and c-myc gene expression in different cancer cells by estrogen and tamoxifen.

Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate.

PURPOSE: The aim of this study was to assess an immunotoxin, monoclonal antibody C27-abrin A chain conjugate (MAAAC), that might be effective in the treatment of colorectal carcinoma. METHODS: The immunotoxin was prepared by a specific monoclonal antibody against carcinoembryonic antigen (CEA), monoclonal antibody C27, linked to N-succinimidyl-3-(2-pyridyldithio)propionate and then coupled covalently to the toxic abrin-A chain to synthesize MAAC. The therapeutic role of this immunotoxin in suppressing the in vitro and in vivo growth of CEA-secreting human colorectal cancer cells (LS174T) was assayed by methods of protein biosynthesis inhibition, cell colony proliferation, and treatment of tumor cells before and after inoculation in nude mice. RESULTS: We found that MAAC effectively suppressed the growth of LS174T in culture medium and completely eradicated cells in inoculated nude mice. In contrast, irrelevant immunotoxin antiferritin-abrin A chain conjugate and isotype-matched monoclonal immunoglobin (MOPC21IgG1)-abrin A chain conjugate did not cause such effects. The in vitro toxicity was highly specific because the conjugate (MAAC) inhibited de novo protein biosynthesis, impeded growth, and caused death of cells possessing surface CEA determinants. The 50 percent inhibition dose values of the conjugate for colonogenic survival and for protein biosynthesis in LS174T cells were 0.09 microgram/ml and 0.06 microgram/ml, respectively. Colon survival was inhibited 96.3 percent after prolonged MAAC treatment. MAAC showed selective cytotoxicity; the inhibitory effect of MAAC to the CEA-secreting LS174T cells over the CEA-nonsecreting human embryonic kidney cells was 16-fold. CONCLUSION: These results indicate that MAAC may be of benefit in therapy during or soon after resection of colorectal carcinoma or in patients who have micrometastasis.