Corticosteroid-induced chemotherapy resistance in urological cancers.

PURPOSE: Glucocorticoids such as dexamethasone are widely used for medication of urological diseases, e.g., as cotreatment of advanced prostate cancer, to improve appetite, weight loss, fatigue, relieve bone pain, diminish ureteric obstruction, to reduce the production of adrenal androgens, as an antiemetic in patients undergoing chemo- and/or radiotherapy together with serving as "standard" therapy arm in randomized studies. While the potent pro-apoptotic properties and the supportive effects of glucocorticoids to tumor therapy in lymphoid cells are well studied, the impact to growth of prostate and other urological carcinomas is unknown. METHODS: We isolated cells from surgical resections of 21 prostate tumors and measured apoptosis and viability in these primary cells and 17 established cell lines from human prostate, bladder, renal cell and testicular carcinomas. RESULTS: We found that dexamethasone induces resistance regarding exposure to several cytotoxic agents such as taxol, gemcitabine, cisplatin, 5-FU and gamma-irradiation in 86% of the freshly isolated prostate tumors and in 100% of the established urological cell lines. No difference in dexamethasone-mediated protection was found in normal, benign and malignant prostate tumors. CONCLUSIONS: These data show for the first time that dexamethasone induced therapy resistance in urological carcinomas is not the exception but a more common phenomenon and implicate that glucocorticoids may have two faces in cancer therapy, a beneficial and a dangerous one.

Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells.

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, alpha-tocopherol (TOCO) and N-acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells.

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor.

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 androgen response element. The application of this cell line in an endogenous Androgen Receptor-mediated LUciferase eXpression assay (AR-LUX) was validated. An EC50 value of 86 pM was determined for the standard androgen R1881 with a detection limit of 46 pM. Other androgens like dihydrotestosterone, 17beta-trenbolone, and bolasterone also induced luciferase expression, while anti-androgens suppressed these responses. As expected, AR-mediated responses were also elicited by high concentrations of the steroids progesterone, 17beta-estradiol, d-aldosterone, and dexamethasone, with observed EC50 values 10 to 350,000 times higher than that for R1881. A unique feature of the AR-LUX assay is that effects on modulation of active endogenous AR-levels are reliably reflected in the luciferase induction response, as exemplified by vitamin D, all-trans-retinoic acid, epigallocatechin gallate, and forskolin. This feature is especially useful when assessing complex mixtures, e.g., environmental samples or natural compound libraries. From these data it is concluded that the AR-LUX assay is a reliable in vitro test system for the detection and quantification of AR-mediated biological effects. The 96-well plate format makes the assay particularly suitable for high-throughput screening.

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells.

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.

Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons.

Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix. Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested. In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways.

Isolation of a bovine full length cytochrome P450 (CYP3A) cDNA sequence and its functional expression in V79 cells.

From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6ß-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6ß-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2ß and 12ß position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.

Inhibition of tolbutamide 4-methylhydroxylation by a series of non-steroidal anti-inflammatory drugs in V79-NH cells expressing human cytochrome P4502C10.

1. To study the role of cytochrome P4502C10 in the metabolism of the non-steroidal antiinflammatory drugs (NSAIDs) diclofenac, phenylbutazone, fenoprofen, ibuprofen, flurbiprofen, ketoprofen and naproxen, a cell line was developed stably expressing CYP2C10 cDNA. A retroviral vector construct, containing a human CYP2C10 cDNA, was transfected in V79-NH Chinese hamster lung cells by calcium phosphate co-precipitation. Sublines stably expressing human cytochrome P450 cDNA were established by selection with the neomycin analogue G418. 2. Enzymatic activity of CYP2C10 was detected by 4-methylhydroxylation of tolbutamide. This activity was inhibited to background levels by preincubation with the CYP2C9/10 inhibitor sulphaphenazole. 3. Preincubations with the NSAIDs ketoprofen, phenylbutazone, flurbiprofen and diclofenac (all 250 microM) caused a decrease in 4-methylhydroxylation of tolbutamide (500 microM), significantly different from control values (p < 0.05). Inhibition of this activity was not seen in preincubations with the NSAIDs fenoprofen, ibuprofen and naproxen (250 microM). 4. The V79-NH CYP2C10 cell line we have developed has been shown to be a useful tool to predict drug-drug interactions.

Stable expression of human cytochrome P450 3A4 in conjunction with human NADPH-cytochrome P450 oxidoreductase in V79 Chinese hamster cells.

V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs was shown by Northern and Western analyses. Activity was tested by 6 beta-hydroxylation of testosterone for cytochrome P450 3A4 and by cytochrome c reduction for NADPH-cytochrome P450 reductase. Five V79 cell lines were obtained expressing cytochrome P450 3A4, human NADPH-cytochrome P450 oxidoreductase, and both. Cytochrome P450 3A4 activity depended highly on cytochrome P450 reductase activity, with lowest activity when only the parental Chinese hamster cytochrome P450 reductase was present, 5- and 10-fold higher when coexpressed with the human NADPH-cytochrome P450 reductase. Correspondingly, cytotoxic and genotoxic potency of aflatoxin B1 was increased by orders of magnitudes when human cytochrome P450 3A4 was coexpressed with the human NADPH-cytochrome P450 reductase. The effect of NADPH-cytochrome P450 reductase coexpression on cytochrome P450 3A4 activity was also tested by nifedipine oxidation and midazolam hydroxylation. Nifedipine oxidation was increased about 10-fold, 1-hydroxylation of midazolam and 4-hydroxylation of midazolam were increased 15-fold.

Development of human cytochrome P450-expressing cell lines: application in mutagenicity testing of ochratoxin A.

With the aim to assess the involvement of distinct forms of cytochrome P450 enzymes in the activation of procarcinogens, we have developed by means of retroviral infection a series of NIH/3T3 cell lines stably expressing human CYP1A1, CYP1A2, CYP2C10, CYP2D6, and CYP2E1 cDNA. The levels of cytochrome P450 enzyme activities were determined using specific substrates. An increase in specific catalytic activity could be observed in all cell lines compared to background activity in vector-infected cells. Furthermore, we developed a test system in which we are able to combine P450-expressing cells with a shuttle vector containing the lacZ' gene, which serves as a reporter gene for mutations. Using this system, we investigated the cytotoxicity and mutagenicity of the mycotoxin ochratoxin A. Natural occurrence of ochratoxin A in food commodities has been linked to an increased incidence of urinary tract tumors in certain geographic regions. Although biotransformation seems to play a crucial role in ochratoxin A toxicity, the possible contribution of metabolites to genotoxicity and carcinogenicity remained unelucidated. We have demonstrated that the mutation frequency of ochratoxin A was increased dependent upon concentration in NIH/3T3 cell lines, stably expressing human CYP1A1, CYP1A2, CYP2C10, and CYP3A4. In contrast, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and CYP2E1-expressing cells was an increase of mutation frequency observed.

Mutagenicity and genotoxicity of the mycotoxin ochratoxin A.

Natural occurrence of the mycotoxin ochratoxin A in food commodities has been linked to endemic diseases in certain human populations, where a high incidence of nephropathy is observed (Balkan endemic nephropathy). The increase of renal disease is accompanied with a high risk for urinary tract tumours. Despite epidemiological and experimental evidence for the carcinogenicity of ochratoxin A the underlying mechanism needs to be established. The pivotal role of cytochrome P450 in the mutagenicity of ochratoxin A could be demonstrated in experiments with cell lines stably expressing the human cytochrome P450 enzymes. CYP1A1, 1A2, 2C10 and 3A4, which were able to activate the non-mutagenic ochratoxin A into mutagenic metabolites. In the cell lines the bacterial lacZ' gene was used as reporter gene for mutagenicity. Sequencing of the lacZ' gene resulted in the detection of large deletions. In addition, in metabolically competent rat hepatocytes an increase of single strand breaks could be observed by means of the DNA alkaline elution assay. These DNA alterations could be related to biotransformation processes, indicating extensive metabolism of ochratoxin A. The discrepancies found between microsomal and cellular metabolism leads to the conclusion that ochratoxin A mediated mutagenicity requires additional processing of cytochrome P450 derived metabolism.

Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

A NIH/3T3 cell line stably expressing human cytochrome P450-3A4 used in combination with a lacZ' shuttle vector to study mutagenicity.

An NIH/3T3 cell line, stably expressing human cytochrome P450-3A4 (CYP3A4) cDNA has been developed. This cell line was used in combination with a shuttle vector, containing the bacterial lacZ' gene as reporter gene, to study mutagenicity. Ethylmethanesulphonate and aflatoxin B1 were used as model agents to test this system. The mutation frequency of ethylmethanesulphonate increased concentration dependently and was the same in CYP3A4-expressing cells as in parental NIH/3T3 cells, demonstrating that CYP3A4 activity has no influence on the mutagenicity of ethylmethanesulphonate. The mutation frequency of aflatoxin B1 increased concentration dependently only in the CYP3A4-expressing cells and not in parental nor in vector-transfected cells. This increase in mutation frequency could be completely inhibited by ketoconazole, an inhibitor of cytochrome P450 activity, demonstrating the role of CYP3A4 in the activation of aflatoxin B1. The system described in this paper opens the possibility to study the capacity of single human cytochrome P450s to activate xenobiotics into mutagenic metabolites. Since activation, phase II metabolism, DNA repair and an endpoint for mutations are all present in one cell, this system will be useful in screening as well as in mechanistic studies.

Transgenic cell lines and changes in gene function and expression: Useful tools in toxicity testing?

Molecular biology provides the toxicologist with powerful tools that can be used in the development and use of in vitro techniques in toxicity testing. As an example, a number of cDNAs coding for human cytochrome P-450 enzymes have been cloned, incorporated in eukaryotic expression vectors and introduced in cell lines. These cell lines can be used to study cytochrome P-450-mediated promutagen activation. To this end, a shuttle vector carrying a bacterial gene, for example beta-galactosidase (lacZ), as a reporter gene for mutations, is introduced in the cytochrome P-450 expressing cell lines. After exposure to putative mutagenic compounds, the shuttle vector is rescued and screened for mutations in bacteria. This system proves to be sensitive and versatile and can be applied to a vast array of cell lines. Another application of molecular biology is the construction of detector cells. Responsive elements are cloned into chloramphenicol acetyltransferase expression vectors or into firefly luciferase expression vectors and introduced in cell lines. These cells can be used in mechanistic studies towards the gene-regulating properties of, for example, dioxins, but also as reporter cells for determination of dioxin contamination.