Sensitivity of renal cell carcinoma to aminoflavone: role of CYP1A1.

PURPOSE: The aminoflavone analogue (AF) exhibits antitumor activity in vitro, particularly against neoplastic cells of renal origin. We identified cellular correlates of responsiveness to AF in continuous human tumor renal cell carcinoma lines and in tumor cell isolates, termed renal carcinoma cell strains, from patients with clear cell and papillary renal neoplasms. MATERIALS AND METHODS: In vitro antiproliferative activity of AF was evaluated using the sulforhodamine B protein dye assay. In vivo antitumor activity of the drug was determined in mice bearing xenografts. Covalent binding of AF/metabolite(s) was assessed following exposure of cells to AF for 16 hours. CYP1A1 and CYP1B1 mRNA and apoptosis were quantitated by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: AF produced total growth inhibition in vitro in 3 of 6 human tumor renal cell lines at concentrations of 90 to 400 nM. In vivo treatment of mice bearing xenografts of the Caki-1 renal cell carcinoma, sensitive to AF in vitro, resulted in significant antitumor activity, including tumor-free survivors. Studies in 13 renal cell strains isolated from patients with clear cell (9) or papillary (4) renal cell carcinoma indicated that 3 of 4 papillary strains were sensitive to AF compared with 2 of 9 clear cell strains. AF sensitive renal cell lines and strains exhibited induction of CYP1A1 and CYP1B1 gene expression, increased covalent binding of AF metabolite(s) and apoptosis. CONCLUSIONS: AF has noteworthy antitumor activity against certain human tumor renal cell lines in vitro and in vivo, which correlates with drug metabolism to covalently binding metabolites after CYP1A1 and CYP1B1 gene expression. We hypothesize that it leads to apoptosis induction. AF sensitive renal cell strains are predominantly of the papillary histological type. These results are limited by the small numbers of cell lines and cell strains but they are suggestive of the need for further testing in larger collections of cell strains.

DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

Hexachlorobenzene, a dioxin-type compound, increases malic enzyme gene transcription through a mechanism involving the thyroid hormone response element.

Hexachlorobenzene (HCB) is a dioxin-type chemical that acts mainly through the aryl hydrocarbon receptor. Chronic exposure of rats to HCB increases the activity of malic enzyme (ME). In this report, we show that this increase is correlated with an induction of ME messenger RNA (mRNA) levels, with the maximal HCB effect achieved after 9 days of intoxication. This effect is specific for ME, as other liver enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenol pyruvate carboxykinase, and mitochondrial alpha-glycerol-3-phosphate dehydrogenase, are not affected by HCB. The induction of ME mRNA levels is accompanied by an increase in ME promoter activity, as demonstrated by transient transfection experiments performed in rat hepatoma H35 cells. In an attempt to identify the cis-regulatory elements responsible for the HCB effect, different promoter deletions and mutations were used. The results obtained localize the responsive region between positions -315 and -177. This region does not contain either consensus xenobiotic response or activating protein-1 elements, the two main mediators of dioxin compounds described to date. In contrast, a thyroid hormone response element (TRE) is located between -281 to -261. Deletions and mutations of the TRE element do not respond to HCB, demonstrating that this element mediates the response of this dioxin-type compound. As ME gene expression is regulated mainly by thyroid hormones, we next investigated the role of T3 receptor (T3R) in the ME gene transcriptional induction mediated by HCB. Using Scatchard analysis, we show that neither T3R binding features for its ligand nor alpha1 or beta1T3R mRNA levels are changed with the toxic. In gel shift assays, however, we observed that protein/DNA complexes formed on TRE from the ME promoter were induced by HCB. Using an oligonucleotide with a mutation that eliminates the TRE function, we demonstrate a loss of the induced protein/DNA complexes. Together, these data suggest that the dioxin-type compound HCB increases ME gene transcription by modulating the levels of still unidentified nuclear proteins that bind to the TRE element of the ME promoter.

[Effect of thyroid hormones on the modulation of genetic expression of liver cytosolic malic enzyme, in rats poisoned with hexachlorobenzene]. / Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of laboratory animals to HCB triggers porphyria, induction of liver microsomal enzymes, low levels of T4 reproductive dysfunction's, liver and thyroid tumors. Previous findings from our laboratory have shown that HCB increased the activity of the liver thyroid-responsive enzymes: malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) without any change in the mytochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD). In this study we have demonstrated that HCB treatment increased ME mRNA. We also have investigated if HCB affected: a) the thyroid hormone receptor (TR) concentration and binding affinity for its ligands, b) specifically the ME gene expression, or other thyroid hormone responsive enzymes were affected as well, c) Protein/DNA complex formed on the thyroid responsive element (TRE). Livers from female Wistar rats intoxicated with HCB (100 mg/100 g b.w.), for 9 and 15 days, were analyzed. Northern blot hybridization analysis, have demonstrated that ME mRNA levels increased 4 times and 2 times after 9 and 15 days intoxication respectively, without any alterations in the mRNA levels of other thyroid hormone responsive enzymes such as glyceraldheyde 3- phosphate dehydrogenase, phosphoenolpyruvatecarboxikinase and alpha-GPD. These results suggest that HCB affects specifically, ME gene expression. Hepatic T3 and T4 levels evaluated by RIA were not affected by HCB. Scatchard analyses showed that TR affinity and number of sites were not altered after 9 and 15 days of HCB treatment (control, Ka: 1.9 nM, Bmax 3.9 f/mol 100 micrograms DNA: HCD 9 days Ka: 2.1 nM, Bmax 4.5 fmol/100 micrograms DNA: HCB 15 days Ka 1.9 nM. Bmax 5.1 fmol/100 micrograms DNA intoxication, neither at 9 nor at 15 days. Electrophoresis mobility shift assay showed that HCB did not modify nuclear protein extract affinity for the TREs sequence. Our results suggest that TR itself was not directly involved in the induction of ME gene expression by HCB. Nevertheless TR could interact with other transcription factors in the overexpression of ME gene.

Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno / Action of the thyroid hormone on modulation of the gene expression of the liver cytosolic malic enzyme, in intoxicated rats with hexachlorobenzene

El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM. (AU)

Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno / Action of the thyroid hormone on modulation of the gene expression of the liver cytosolic malic enzyme, in intoxicated rats with hexachlorobenzene

El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM.