Doxorubicin is a useful antineoplastic drug with multiple mechanisms of cytotoxicity. One such mechanism involves the reductive bioactivation of the quinone ring to a semiquinone radical, which can exert direct toxic effects and/or undergo redox cycling. We hypothesized that human NADPH-cytochrome p450 reductase (CYPRED) catalyzes doxorubicin reduction and that overexpression of this enzyme sensitizes human breast cancer cell lines to the aerobic cytotoxicity of doxorubicin. cDNA-expressed human CYPRED catalyzed doxorubicin reduction, measured as the rate of doxorubicin-stimulated NADPH consumption. Using a bank of 17 human liver microsomal samples, the rate of doxorubicin reduction correlated with CYPRED catalytic activity and CYPRED protein immunoreactivity. Diphenyliodonium chloride, a mechanism-based inactivator of CYPRED, inhibited CYPRED activity and doxorubicin reduction in human liver microsomes with similar concentration dependence. Stably transfected clones of MDA231 human breast cancer cells overexpressing human CYPRED immunoreactive protein and catalytic activity showed enhanced sensitivity to the aerobic cytotoxicity of tirapazamine, a bioreductive drug known to be activated by CYPRED; however, no sensitization to the cytotoxic effects of doxorubicin was observed. Although human CYPRED is an important catalyst of doxorubicin reduction, overexpression of this enzyme does not confer enhanced sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin.
The anthracycline doxorubicin has little activity against colorectal cancers. It is hypothesized that this is attributable to a multifactorial resistance mechanism in which the glutathione S-transferases (GST) may play a role. We studied the relationship between GST expression and doxorubicin resistance in four human colon adenocarcinoma cell lines (HT-29, LoVo, SW620, and Caco-2), with the goal of modulating GST activity to overcome resistance. Caco-2 cells were the most resistant to doxorubicin, showing an IC50 value approximately 80- to 90-fold higher than HT-29 or LoVo and 600-fold higher than SW620. Total GST catalytic activity was significantly higher in Caco-2 cells compared with the other lines. All four cell lines expressed GST-pi at the catalytic activity, protein, and mRNA levels; however, no significant differences were observed among the cell lines. GST-mu expression was not detectable at the protein and mRNA levels, and the four cell lines displayed very low catalytic activity toward a GST-mu-selective substrate. Caco-2 cells showed a unique, highly expressed GST-alpha-immunoreactive band that was not detected in the other lines; however, the glutathione peroxidase activity of Caco-2 cells was the lowest among the four cell lines. Neither ethacrynic acid nor glutathione analogues that function as GST class-selective inhibitors were able to potentiate the cytotoxic effects of doxorubicin in these colon cancer cell lines, as demonstrated in both microplate colorimetric and clonogenic assays. The multidrug resistance-associated protein and P-glycoprotein were either not detectable or expressed at such low levels that they are not likely to contribute to the differences in doxorubicin sensitivity observed among these cell lines.
Heme biosynthesis in hepatocytes is controlled by a free heme pool, which regulates δ-aminolevulinic acid synthase. Porphyrinogenic chemicals deplete the regulatory free heme pool by interacting with cytochrome P-450 thereby inhibiting heme biosynthesis and/or causing heme breakdown. Recent developments allow us to predict which groups of chemicals are likely to be porphyrinogenic. One group is exemplified by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine. Heterocyclic compounds of this type cause mechanism-based inactivation of cytochrome P-450, leading to the formation of N-alkylporphyrins, with ferrochelatase-inhibitory activity resulting in lowering the free heme pool. Allylisopropylacetamide exemplifies a second group. Such compounds containing a terminal olefinic or acetylenic group, cause mechanism-based inactivation of cytochrome P-450. In the process, the heme moiety of cytochrome P-450 is destroyed and the free heme pool is lowered. A third group is exemplified by planar polyhalogenated or polycyclic aromatic hydrocarbons. These compounds induce specific cytochrome P-450 isozymes but are poor substrates. Active oxygen is formed, which interacts with a hepatic substrate to form a uroporphyrinogen decarboxylase inhibitor. Inhibition of this enzyme leads to depletion of the free heme pool.—Marks, G. S.; McCluskey, S. A.; Mackie, J. E.; Riddick, D. S.; James, C. A. Disruption of hepatic heme biosynthesis after interaction of xenobiotics with cytochrome P-450. FASEB J. 2: 2774-2783; 1988.
The aryl hydrocarbon receptor (AHR) has physiological roles in the absence of exposure to exogenous ligands and mediates adaptive and toxic responses to the environmental pollutant, 2,3,7,8‐tetracholorodibenzo ‐p‐ dioxin (TCDD). A readily metabolized AHR agonist, 3‐methylcholanthrene, disrupts expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 ( Cyp2d9 ), a male‐specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). Using TCDD as an essentially non‐metabolized AHR agonist and Ahr −/− mice as a model to determine the AHR‐dependence of biological responses, we show that two mouse hepatic STAT5b target genes, Cyp2d9 and major urinary protein 2 ( Mup2 ), are suppressed by TCDD in an AHR‐dependent manner. TCDD also decreased hepatic mRNA levels for GH receptor, Janus kinase 2, and STAT5a/b with AHR‐dependence. Without inducing selected hepatic inflammatory markers, TCDD caused AHR‐dependent induction of Cyp1a1 and NADPH‐cytochrome P450 oxidoreductase ( Por ) and suppression of Cyp3a11 . In vehicle‐treated mice, basal mRNA levels for CYP2D9, CYP3A11, POR, serum amyloid protein P, and MUP2 were influenced by Ahr genetic status. We conclude that AHR activation per se leads to dysregulation of hepatic GH signaling components and suppression of some, but not all, STAT5b target genes. [Support: CIHR]
Aromatic hydrocarbons (AHs) elicit toxic and adaptive responses via the aryl hydrocarbon receptor (AHR). AHs suppress the transcription of the growth hormone-regulated, male-specific rat hepatic cytochrome P450 2C11 gene (CYP2C11) via an unknown mechanism. We hypothesize that the in vivo suppression of CYP2C11 by AHs is mediated by the gene’s promoter and 5′-flank. Following bioinformatic analysis of putative transcription factor (TF) binding sites, we cloned up to 10kb of the CYP2C11 5′-flank into a promoterless luciferase plasmid. Deletion analysis of CYP2C11-luciferase reporter constructs showed that a region between −2.4kb and −1.3kb mediated a paradoxical induction by AHs, particularly in human HepG2 cells. In the context of a heterologous promoter, this region did not appear to possess enhancer activity. Since suppression may only occur in an environment containing an intact endocrine system and the full complement of TFs necessary for CYP2C11 regulation, our study is now focused on live rats. Preliminary in vivo studies using high-volume tail vein injection to deliver plasmid to the liver of live rats demonstrated measurable basal luciferase activity in rat liver homogenates; AH-induced luciferase activity was also observed using a positive control plasmid under AHR control. The effects of anesthesia and high-volume tail vein injection on endogenous hepatic CYP2C11 and CYP1A1 mRNA levels in vehicle and AH-treated rats were examined. Our novel CYP2C11-luciferase constructs will be studied in live rats challenged with AHs to identify key regulatory sequences mediating CYP2C11 suppression. [Support: CIHR]
Abstract We are investigating the mechanisms by which aromatic hydrocarbons, such as 3-methylcholanthrene (MC), suppress hepatic cytochrome P450 2C11 (CYP2C11) gene expression. CYP2C11 is an enzyme expressed in the liver of male rats and is regulated by a pulsatile pattern of GH secretion. We have previously shown that MC attenuates the stimulatory effect of GH on CYP2C11 expression in hypophysectomized male rats. In follow-up studies we evaluated the effect of MC on GH-stimulated signal transducer and activator of transcription 5b (STAT5b) phosphorylation, nuclear translocation, and DNA-binding activity. GH-stimulated increases in hepatic nuclear STAT5b and phospho-STAT5b levels were not different between groups of hypophysectomized rats receiving MC or vehicle. This observation was corroborated at the DNA-binding level by EMSA. We also measured GH-induced STAT5b activation in the H4IIE rat hepatoma cell line. STAT5b DNA-binding activity detected in GH-treated cells was not affected by MC. Immunocytochemistry experiments revealed no effect of MC on GH-stimulated STAT5b nuclear translocation in H4IIE cells. These in vivo and in vitro data suggest that interference with GH-stimulated STAT5b activation does not constitute a mechanism by which MC attenuates the stimulatory effect of GH on CYP2C11 gene expression.
3-Methylcholanthrene (MC) is a readily metabolized aryl hydrocarbon receptor (AHR) agonist. MC disrupts expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 (Cyp2d9), a male-specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). To determine if these effects of MC depend on hepatic microsomal P450–mediated activity, we examined biologic responses to MC treatment in liver Cpr–null (LCN) mice with hepatocyte-specific conditional deletion of NADPH-cytochrome P450 oxidoreductase (POR). MC caused mild induction of Por and a hepatic inflammatory marker in wild-type mice, whereas MC caused strong induction of AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 in wild-type and LCN mice. Two mouse hepatic STAT5b target genes, Cyp2d9 and major urinary protein 2 (Mup2), were suppressed by MC in wild-type mice, and the CYP2D9 mRNA response was maintained in LCN mice. In wild-type mice only, MC decreased hepatic GH receptor (GHR) mRNA but increased GHR protein levels. There was an apparent impairment of STAT5 phosphorylation by MC in wild-type and LCN mice, but large interanimal variation prevented achievement of statistical significance. In vehicle-treated mice, basal levels of MUP2 mRNA, GHR mRNA, GHR protein, and the activation status of extracellular signal-regulated kinase 2 and Akt were influenced by hepatic Por genetic status. These results indicate that the effects of MC on hepatic GH signaling components and target genes are complex, involving aspects that are both dependent and independent of hepatic microsomal P450–mediated activity.
The aryl hydrocarbon receptor (AHR) is activated by 3-methylcholanthrene (MC), a polycyclic aromatic hydrocarbon, and environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Adrenalectomized (ADX) rats have decreased hepatic AHR protein and lower levels of MC-induced CYP1B1 mRNA. To further characterize the effects of decreased AHR protein and the response to MC in ADX rats, we measured AHR-mediated responses in the liver of sham-operated (SHAM) and ADX rats, 6 and 54 h after MC treatment. CYP1A2 mRNA was suppressed by 46 to 60% 4 days after ADX in vehicle-treated animals. AHR mRNA was induced 4-fold 6 h after MC in SHAM rats, but no induction was observed in ADX rats. The MC-induced 7-ethoxyresorufin O-deethylation (EROD) activity in ADX rats was 35% of the activity in the MC-treated SHAM group at 6 h. At 54 h after treatment, the induction of EROD activity by MC was more pronounced in ADX rats than at 6 h. To assess the overall capacity for hepatic P450-mediated metabolism, we measured NADPH-cytochrome P450 oxidoreductase (POR) activity. POR activity was decreased by 50% after ADX. We have shown that the response to MC in ADX rats is suppressed for some, but not all, AHR-mediated responses and that reduced POR activity after ADX could contribute to a decreased capacity for P450-dependent metabolism. The current study contributes to our understanding of how adrenal-dependent factors modulate the AHR pathway and the response to MC in vivo.
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