Global oncology research and training at US National Cancer Institute-designated cancer centres: results of the 2021 Global Oncology Survey.

Global oncology research and training are crucial to address the growing global burden of cancer, which largely and increasingly occurs in low-income and middle-income countries. To better understand global oncology activities at the 71 National Cancer Institute (NCI)-designated cancer centres, the US NCI Centre for Global Health regularly surveys cancer centre directors, global oncology leads, and principal investigators in 36 US states and the District of Columbia. The survey results complement internal and publicly available data about global oncology research funded directly by the US National Institutes of Health to provide a comprehensive catalogue of global oncology research, training, and activities led by NCI-designated cancer centres. 91% (61 of 67) of responding cancer centres reported global oncology activities not directly funded by the National Institutes of Health. The survey results indicate that global oncology is an important priority at cancer centres and provide a valuable resource for these centres, researchers, collaborators, trainees, and the NCI and other funders.

Patterns of Enrollment in Cancer Treatment Trials During the COVID-19 Pandemic at National Cancer Institute-Designated Cancer Centers.

ABSTRACT: The COVID-19 pandemic posed unprecedented strain on enrollment to cancer clinical trials and their conduct. Here, we highlight an analysis using information from the National Cancer Institute (NCI) Clinical Trials Reporting Program database to describe enrollment patterns to interventional cancer treatment trials at NCI-Designated Cancer Centers during the pandemic. Enrollment to cancer treatment trials at NCI-Designated Cancer Centers decreased precipitously early in the pandemic and has not yet fully returned to the 2019 baseline as of mid-2021. We discuss possible reasons for this and how some of the changes in clinical trial conduct implemented during the pandemic may become part of the standard conduct of NCI-supported clinical trials and broaden access to trials.

A National Map of NCI-Designated Cancer Center Catchment Areas on the 50th Anniversary of the Cancer Centers Program.

BACKGROUND: In 1971, the National Cancer Act created a process to recognize the leadership, facilities, and research efforts at cancer centers throughout the United States. Toward this goal, each NCI-designated cancer center defines and describes a catchment area to which they tailor specific scientific and community engagement activities. METHODS: The geographically defined catchment areas of 63 NCI-designated comprehensive and clinical cancer centers are collated and presented visually. In addition, the NCI-designated cancer center catchment areas are geographically linked with publicly available data sources to aggregate sociodemographic and epidemiologic characteristics across the NCI Cancer Centers Program. RESULTS: The national map portrays the size, shape, and locations for 63 catchment areas of the 71 NCI-designated cancer centers. The findings illustrate the geographic extent of the NCI Cancer Centers Program during the 50th anniversary of the National Cancer Act. CONCLUSIONS: NCI-designated cancer centers occupy a prominent role in the cancer control ecosystem and continue to perform research to address the burden of cancer among their local communities. The strength of the NCI Cancer Centers Program is partly defined by the scope, quality, and impact of community outreach and engagement activities in the catchment areas. IMPACT: The collation and geographic presentation of the distinct, but complementary, NCI-designated cancer center catchment areas are intended to support future research and community outreach activities among NCI-designated cancer centers. See related commentary by Vadaparampil and Tiro, p. 952.

Delivery of Financial Navigation Services Within National Cancer Institute-Designated Cancer Centers.

Background: Cancer centers have a responsibility to help patients manage the costs of their cancer treatment. This article describes the availability of financial navigation services within the National Cancer Institute (NCI)-designated cancer centers. Methods: Data were obtained from the NCI Survey of Financial Navigation Services and Research, an online survey administered to NCI-designated cancer centers from July to September 2019. Of the 62 eligible centers, 57 completed all or most of the survey, for a response rate of 90.5%. Results: Nearly all cancer centers reported providing help with applications for pharmaceutical assistance programs and medical discounts (96.5%), health insurance coverage (91.2%), assistance with nonmedical costs (96.5%), and help understanding medical bills and out-of-pocket costs (85.9%). Although other services were common, in some cases they were only available to certain patients. These services included direct financial assistance with medical and nonmedical costs and referrals to outside organizations for financial assistance. The least common services included medical debt management (63.2%), detailed discussions about the cost of treatment (54.4%), and guidance about legal protections (50.1%). Providing treatment cost transparency to patients was reported as a common challenge: 71.9% of centers agreed or strongly agreed that it is difficult to determine how much a cancer patient's treatment will cost, and 70.2% of oncologists are reluctant to discuss financial issues with patients. Conclusions: Cancer centers provide many financial services and resources. However, there remains a need to build additional capacity to deliver comprehensive financial navigation services and to understand the extent to which patients are referred and helped by these services.

Population Health Assessment in NCI-Designated Cancer Center Catchment Areas.

In May 2016, the NCI announced supplemental funding for NCI-Designated Cancer Centers to conduct research to better characterize populations within cancer center catchment areas. The initiative was intended to support primary data collection efforts to better define and describe cancer center catchment areas using a multilevel population health framework. The long-term goal was to facilitate collaborations in which researchers, providers, public health practitioners, and nonprofit organizations could utilize the data to develop or expand applied cancer control research, planning, and implementation, with an emphasis on local health disparities and communication inequalities. This CEBP Focus issue on "Population Health Assessment in Cancer Center Catchment Areas" highlights the results from those catchment area data collection efforts. Articles highlight research from surveys conducted to define and describe cancer center catchment areas using both probability and nonprobability designs and a variety of sampling techniques, survey modes, and data linkages. Some articles report pooled analyses of data collected by multiple cancer centers to highlight local versus national comparisons based on standardized behavioral and demographic measures. Others examine rural-urban differences in measures relevant to cancer prevention and control. The studies published in this Focus will provide the field with a myriad of methodologic approaches for defining and describing cancer center catchment areas.

Resveratrol inhibits the deleterious effects of diet-induced obesity on thymic function.

Obesity is associated with an increased risk of infectious diseases. It has been shown to have deleterious effects on cell-mediated immunity, including reducing thymocyte numbers and altering responses of thymocytes to pathogens. In the current study, we examined the efficacy of the antiobesity phytochemical resveratrol in preventing the deleterious effects of a high-fat diet on thymic anatomy and function. Compared to C57Bl/6 male mice fed a low-fat diet, mice on a high-fat diet had a significant increase in thymic weight and lipid content, and a disrupted anatomy, including a reduction of the medullary compartment and absence of a corticomedullary junction. There were a decrease in thymic cellularity and mature T-cell output, and a disrupted T-cell maturation, as evidenced by increased double-negative and decreased single- and double-positive thymocytes. Mice that had been fed resveratrol along with a high-fat diet had a dose-dependent reversal in all these parameters. Western blots from thymi showed that obese mice had lower levels of the key stimulators of lipid metabolism, phospho-5' adenosine monophosphate-activated protein kinase and its downstream target, carnitine palmitoyl transferase-1; this was restored to normal levels in resveratrol-fed mice. Resveratrol also reversed an increase in glycerol-3-phosphate acyltransferase-1, the enzyme that catalyzes the first step in triglycerol synthesis. Taken together, these results indicate that resveratrol is a potent inhibitor of the deleterious effects of diet-induced obesity on thymic anatomy and function, and this may hold promise in preventing obesity-related deficits in cell-mediated immunity.

Glycerol-3-phosphate acyltransferase-1 gene ablation results in altered thymocyte lipid content and reduces thymic T cell production in mice.

Glycerol 3-phosphate acyltransferase-1 (GPAT-1) catalyzes the initial and rate-limiting step in de novo glycerophospholipid and triacylglycerol (TAG) biosynthesis. We have previously shown that peripheral T cell proliferation and cytokine production is altered in GPAT-1 gene-ablated (KO) mice. This finding is important in light of the reduction in GPAT-1 activity associated with aged T cells. To determine if the mechanism for altered peripheral T cell function is linked to altered T cell development, we assessed thymic function in 3, 6 and 16-week old GPAT-1 KO compared to wild type (WT) mice. At 16 weeks of age, there was a significant reduction in thymic T cell production in KO compared to WT mice but not at 6 weeks of age. The reduced thymic T cell production was associated with altered thymic development as confirmed by increased numbers of double-negative (DN) thymocytes and a significant reduction in the double positive (DP) thymocytes suggesting a developmental block at the DN stage. This change was accompanied by an increase in the single positive CD4 subset. These changes were associated with reduced glycerophospholipid mass while thymic cortex and medulla architecture was not altered by GPAT-1 KO. Taken together, these data suggest that GPAT-1 deletion is capable of reducing the number of new T cells produced via alterations in membrane receptor function rather than by causing deleterious changes within the thymic microenvironment explaining in part the observed alterations in peripheral T cell function.

Pomegranate extract induces cell cycle arrest and alters cellular phenotype of human pancreatic cancer cells.

BACKGROUND: Pomegranate extract (PE) is a standardized whole-fruit extract of pomegranate, a fruit with known anticancer properties. MATERIALS AND METHODS: PANC-1 and AsPC-1 human pancreatic cancer cells were used as in vitro models to test the effects of PE. RESULTS: PE treatment induced cell cycle arrest and inhibited cell proliferation in PANC-1 cells. PE treatment increased the proportion of cells lacking CD44 and CD24 expression, which are associated with increased tumor-initiating ability, demonstrating that PE altered cell phenotype. PE was more effective in inhibiting the proliferation of PANC-1 cells than the clinically used dose of paclitaxel. Similar results were obtained in the AsPC-1 cell line. Individual pomegranate phytochemicals were only modestly effective in inhibiting cell proliferation, suggesting that unidentified phytochemicals are responsible for the inhibitory effect of PE. CONCLUSION: These data suggest that PE is a promising candidate for further preclinical testing for treatment of human pancreatic cancer.

Retinol inhibits aromatase activity and expression in vitro.

Aromatase converts androgens into estrogens and is thought to supply a local source of estrogen that facilitates the growth of hormone-responsive tumor cells. Inhibition of aromatase is therefore an important chemopreventive strategy. We investigated the effect of retinol and selected retinoids on the activity and expression of aromatase in two human carcinoma cell lines in vitro. Retinol (ROH) and all-trans retinoic acid (ATRA) significantly inhibited aromatase activity in a concentration-dependent manner in microsomes isolated from JEG-3 human placental carcinoma cells, whereas 9-cis and 13-cis retinoic acid had significant inhibitory activity only at the highest concentrations tested. Similar results were observed in an assay of cellular aromatase activity in MCF-7 human breast cancer cells. Enzyme kinetic studies by double-reciprocal plot demonstrated that ROH inhibited microsomal aromatase activity in a mixed manner. In addition, ROH suppressed both the basal and cAMP-induced expression of aromatase mRNA in MCF-7 cells and inhibited transcription controlled by a cAMP-responsive element. These results suggest that aromatase activity and expression are a molecular target of ROH and chemopreventive retinoids, an activity that may underlie, in part, their inhibitory effects on hormone-dependent cancer.

Pomegranate extract inhibits the proliferation and viability of MMTV-Wnt-1 mouse mammary cancer stem cells in vitro.

Pomegranate (Punica granatum L.) is known to possess anticancer activities. The effects of a standardized extract of pomegranate (PE) on a mouse mammary cancer cell line (designated WA4) derived from mouse MMTV-Wnt-1 mammary tumors were examined in this study. The WA4 cell line has been previously characterized as containing a majority of cells possessing stem cell characteristics. PE inhibited the proliferation of WA4 cells in a time- and concentration-dependent manner. This was due to an arrest of cell cycle progression in the G0/G1 phase. PE was also cytotoxic to quiescent WA4 cells in a concentration-dependent manner at concentrations >10 microg/ml. PE treatment of WA4 cells resulted in an increase in caspase-3 enzyme activity in a time- and concentration-dependent manner, indicating that the cytotoxic effect of PE was due to the induction of apoptosis. We tested the effect of several individual phytochemicals derived from PE on WA4 cells. Ellagic acid, ursolic acid and luteolin caused a time- and concentration-dependent reduction of cell proliferation and viability, suggesting that they contribute to the inhibitory effect of PE, while caffeic acid had no effect. Cancer stem cells, which are highly resistant to conventional chemotherapeutic agents, are thought to be the origin of both primary and secondary breast tumors, and thus are a critical target in both breast cancer therapy and prevention. These data suggest that PE, which is a proven and safe dietary supplement, has promise as an treatment against breast cancer by preventing proliferation of cancer stem cells.

Sulindac and its metabolites induce carcinogen metabolizing enzymes in human colon cancer cells.

Sulindac is a nonsteroidal antiinflammatory drug that has been demonstrated to be a potent chemopreventive agent against colorectal cancer in both human and animal models. In vivo, sulindac may be reversibly reduced to the active antiinflammatory compound, sulindac sulfide, or irreversibly oxidized to sulindac sulfone. Sulindac has also been shown to inhibit polycyclic aromatic hydrocarbon (PAH)-induced cancer, but the molecular mechanisms of its antitumor effect remain unclear. In this study, we investigated the effects of sulindac and its metabolites on the expression of enzymes that metabolize and detoxify PAHs in 2 human colon cancer cell lines, LS180 and Caco-2. Sulindac and sulindac sulfide induced a sustained, concentration-dependent increase in CYP enzyme activity as well as an increase in the mRNA levels of CYP1A1, CYP1A2 and CYP1B1. Sulindac and sulindac sulfide induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear CYP1A1 RNA and verified by the use of actinomycin D as a transcription inhibitor. Chromatin immunoprecipitation assays demonstrated that sulindac and sulindac sulfide also increased the nuclear level of activated aryl hydrocarbon receptor, the transcription factor which mediates CYP expression. Additionally, sulindac and both metabolites increased the activity and mRNA expression of the carcinogen detoxification enzyme NAD(P)H:quinone oxidoreductase, as well as the expression of UDP-glucuronosyltransferase mRNA. These results show an overall upregulation of carcinogen metabolizing enzymes in colon cancer cells treated with sulindac, sulindac sulfide and sulindac sulfone that may contribute to the established chemoprotective effects of these compounds.

A novel synthetic analogue of a constituent of Isodon excisus inhibits transcription of CYP1A1, -1A2 and -1B1 by preventing activation of the aryl hydrocarbon receptor.

We investigated the effect of a novel synthetic analogue of a constituent from the Chinese medicinal herb Isodon excisus, 3-(3-methoxy-phenyl)-N-(3, 4, 5-trimethoxy-phenyl)-acrylamide (compound 343), on the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in human hepatoma HepG2 cells. We found that compound 343 inhibited the upregulation of cytochrome P-450 (CYP) enzyme activity in cells treated with the AhR ligands and potent carcinogens, dimethylbenz[a]anthracene (DMBA) or 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Compound 343 also inhibited the DMBA- or TCDD-induced increase in CYP1A1, -1A2 and -1B1 mRNA levels. Carcinogen-induced transcription of CYP genes was also suppressed by compound 343, as measured by a reporter gene controlled by the xenobiotic-responsive element (XRE). This was confirmed by measuring the amount of carcinogen-induced CYP1A1 heterogeneous nuclear RNA. Compound 343 blocked the DMBA- or TCDD-induced activation of the AhR DNA-binding capacity for the XRE, as measured by a chromatin immunoprecipitation assay. Compound 343 also inhibited CYP enzyme activity in microsomes isolated from DMBA- or TCDD-treated cells, as well as the activity of recombinant CYP1A1, -1A2 and -1B1, indicating that compound 343 directly inhibits CYP enzymes. These results indicate that compound 343 is both a potent inhibitor of carcinogen-induced CYP enzyme expression, as well as a direct inhibitor of CYP enzymes.

Sulindac regulates the aryl hydrocarbon receptor-mediated expression of Phase 1 metabolic enzymes in vivo and in vitro.

Sulindac, a widely used non-steroidal anti-inflammatory drug (NSAID), has been shown to inhibit chemically induced carcinogenesis in animal models. In the present study, we have investigated the molecular mechanism by which sulindac affects the activity and expression of the enzymes that mediate the initial detoxification steps of many environmental carcinogens, the cytochromes P450 1A1, 1A2 and 1B1. Sulindac treatment of Sprague-Dawley rats resulted in a dose-dependent increase in hepatic cytochrome P450 (CYP) enzyme activity and in the expression of hepatic CYPs 1A1 and 1B1 mRNA. In the HepG2 human liver cancer cell line, sulindac caused a sustained, dose-dependent increase in CYP enzyme activity. Sulindac treatment resulted in a profound, dose-dependent increase in CYP 1A1 mRNA and a modest increase in 1A2 mRNA. The increase in CYP 1A1 mRNA induced by sulindac was, like enzyme activity, sustained for several days after the initial treatment. Sulindac induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear 1A1 RNA and by actinomycin D chase experiment. Since the transcription of CYP1A1 is under the control of the aryl hydrocarbon receptor (AhR), we examined the ability of sulindac to activate the receptor. Sulindac bound to the AhR, as measured by ligand-binding assay, and induced the binding of the AhR with the xenobiotic-responsive element present in the promoter region of the CYP1A1 gene. These results are the first demonstration that NSAIDs modulate carcinogen metabolic enzymes and provide a novel mechanism to explain the established chemopreventive activity of sulindac.

The antitumor drug candidate 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole induces NF-kappaB activity in drug-sensitive MCF-7 cells.

2-(4-Amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203) potently inhibits MCF-7 breast cancer cell growth in part by activating the aryl hydrocarbon receptor (AhR) signaling pathway. Ligands for the AhR (i.e. dioxin) have also been shown to modulate the NF-kappaB signaling cascade, affecting physiological processes such as cellular immunity, inflammation, proliferation and survival. The objective of this study was to investigate the effect of 5F 203 treatment on the NF-kappaB signaling pathway in breast cancer cells. Exposure of MCF-7 cells to 5F 203 increased protein-DNA complex formation on the NF-kappaB-responsive element as determined by electrophoretic mobility shift assay, but this effect was eliminated in MDA-MB-435 cells, which are resistant to the antiproliferative effects of 5F 203. An increase in NF-kappaB-dependent transcriptional activity was confirmed by a significant increase in NF-kappaB-dependent reporter activity in sensitive MCF-7 cells, which was absent in resistant MDA-MB-435 cells and AhR-deficient subclones of MCF-7 cells. Inhibition of NF-kappaB activation enhanced the increase in xenobiotic response element-dependent reporter activity in MCF-7 cells when treated with 5F 203. The drug candidate 5F 203 also induced mRNA levels of IL-6, an NF-kappaB-responsive gene, in MCF-7 cells, but not in MDA-MB-435 cells, as determined by quantitative RT-PCR. These findings suggest that 5F 203 activation of the NF-kappaB signaling cascade may contribute to 5F 203-mediated anticancer activity in human breast cancer MCF-7 cells.

Aryl hydrocarbon receptor activation of an antitumor aminoflavone: basis of selective toxicity for MCF-7 breast tumor cells.

Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; NSC 686288) demonstrates differential antiproliferative activity in the National Cancer Institute's anticancer drug screen. We demonstrate here that MCF-7 human breast cancer cells are sensitive to aminoflavone both in vitro and when grown in vivo as xenografts in athymic mice. As previous work has indicated that aminoflavone requires metabolic activation by cytochrome P450 1A1 (CYP1A1), we investigated the effect of aminoflavone on CYP1A1 expression and on the aryl hydrocarbon receptor (AhR), a transcriptional regulator of CYP1A1. In aminoflavone-sensitive but not aminoflavone-resistant cells, the drug caused a 100-fold induction of CYP1A1 mRNA and a corresponding increase in ethoxyresorufin-O-deethylase activity. An AhR-deficient variant of the MCF-7 breast carcinoma, AH(R100), with diminished CYP1A1 inducibility, exhibits cellular resistance to aminoflavone and is refractory to CYP1A1 mRNA induction by the drug. The increase in CYP1A1 mRNA in the aminoflavone-sensitive MCF-7 breast tumor cell results from transcriptional activation of xenobiotic-responsive element (XRE)-controlled transcription. Aminoflavone treatment causes a translocation of the AhR from the cytoplasm to the nucleus with subsequent formation of AhR-XRE protein DNA complexes. In contrast to the aminoflavone-sensitive MCF-7 cells, the resistant cell lines (MDA-MB-435, PC-3, and AH(R100)) demonstrated constitutive nuclear localization of AhR. Additionally, aminoflavone failed to induce ethoxyresorufin-O-deethylase activity, CYP1A1 transcription, AhR-XRE complex formation, and apoptosis in aminoflavone-resistant cells. These results suggest that the cytotoxicity of aminoflavone in a sensitive breast tumor cell line is the result of the engagement of AhR-mediated signal transduction.

The drug salicylamide is an antagonist of the aryl hydrocarbon receptor that inhibits signal transduction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant, that has been linked with a variety of deleterious effects on human health, including increased cancer rates and reproductive anomalies. The detrimental effects of TCDD are mediated via the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of the carcinogen-activating enzymes cytochromes P-450 (CYP) 1A1, 1A2, and 1B1. In the present study, we examined the ability of synthetic derivatives of salicylic acid to affect TCDD-stimulated AhR-mediated signal transduction in human hepatoma HepG2 cells. Salicylamide (SAL), an analgesic drug, caused a potent and long-lasting inhibition of TCDD-induced CYP enzyme activity. Acetylsalicylic acid (aspirin) and the naturally occurring phytochemical salicylic acid had no effect on CYP activity. SAL inhibited the increase in CYP1A1, -1A2, and -1B1 mRNA levels that occurs on exposure to TCDD. TCDD-induced transcription of these genes was also inhibited by SAL, but not by aspirin or salicylic acid, as demonstrated by luciferase reporter assays. The transcription of the CYP1 family of genes is regulated by the interaction of TCDD-activated AhR with the xenobiotic-responsive element present in the promoter regions of these genes. As shown by electrophoretic mobility shift assay, SAL completely blocked the binding of TCDD-activated AhR to the xenobiotic responsive element. Also, SAL substantially blocked the binding of TCDD to the cytosolic AhR. These results demonstrate that SAL, a commonly used analgesic, is a potent inhibitor of AhR-mediated signal transduction, and may be an effective agent in the prevention of TCDD-associated disease.

Dehydroepiandrosterone inhibits the expression of carcinogen-activating enzymes in vivo.

We investigated the effect of the steroid hormone dehydroepiandrosterone (DHEA) on the hepatic expression and activity of carcinogen-activating enzymes, the cytochromes P450 (CYP) 1A1, 1A2 and 1B1, in Sprague-Dawley rats. In animals fed DHEA at 200 or 400 mg/kg body weight every other day for 2 weeks prior to exposure to the aryl hydrocarbon dimethylbenz[a]anthracene (DMBA, 5 mg/kg), there was a dose-dependent decrease in hepatic CYP activity, as measured by ethoxyresorufin-O (EROD) assay, from 37.1 to 22.9 and 14.7 pmoles/min/10 microg microsomes, respectively. DHEA did not directly inhibit microsomal EROD activity, however, leading us to investigate its effects on enzyme expression. To test this, we examined protein and mRNA levels of the enzymes. Western blot for CYP1A1 and CYP1A2 showed that DHEA inhibited the increase in hepatic CYP1A1 and CYP1A2 enzyme levels that are normally induced by DMBA. DMBA-induced increase in expression of CYP1A1, CYP1A2 and CYP1B1 mRNA was similarly blunted in DHEA-treated animals. DHEA was also able to significantly reduce the basal expression of CYP1A1 and CYP1A2 but not of CYP1B1. These results indicate that DHEA regulates the expression and, hence, the activity of hepatic carcinogen-activating enzymes in vivo, and this may be an important mechanism of its chemopreventive activity.

Resistance of MCF-7 cells to dimethylbenz(a)anthracene-induced apoptosis is due to reduced CYP1A1 expression.

We have developed a series of aryl hydrocarbon (AH)-resistant cell lines derived from MCF-7 human breast epithelial cancer cells by continuous exposure to the AH benzo[a]pyrene. These cell lines display cross-resistance to the mammary carcinogen dimethylbenz[a]anthracene (DMBA). Apoptosis induced by exposure to DMBA is greatly decreased in the resistant cell lines compared to the wild-type, in proportion to the level of resistance. Apoptosis induced by DMBA could be blocked by inhibitors of DMBA metabolism such as alpha-naphthoflavone and diosmetin. We therefore examined the resistant cell lines for their ability to metabolize DMBA and for the formation of DMBA-DNA adducts, and found that both parameters were decreased compared to wild-type cells in proportion to the level of resistance. When exposed to DMBA or 2,3,7,8-tetrachlorodibenzo-p-dioxin, the resistant cell lines have a diminished capacity to carry out ethoxyresorufin-O-deethylation, indicating that the induction of cytochrome P450 1A1 (CYP1A1) enzyme is impaired. We therefore examined the expression of the CYP1A1 gene, and found reduced levels of both CYP1A1 mRNA and CYP1A1-promoter controlled transcription in resistant cells compared to the wild-type. The deleterious effects of AHs are believed to be mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which regulates CYP1A1 expression. Resistant cell lines had a reduced expression of the AhR, as measured at the mRNA and protein levels. These data demonstrate that AH resistance in these cells is mediated by changes in the signal transduction pathway which regulates CYP1A1 expression.

Inhibition of carcinogen-activating enzymes by 16alpha-fluoro-5-androsten-17-one.

In the present study, we examined the effect of a synthetic analogue of the chemopreventive hormone dehydroepiandrosterone, 16alpha-fluoro-5-androsten-17-one, also known as fluasterone, on the activity and expression of carcinogen-activating enzymes in MCF-7 cells. The increase in cytochrome P450 (CYP) 1A1 and 1B1 activity, as measured by ethoxyresorufin-O-deethylase activity, in cells treated with the carcinogens dimethylbenzanthracene (DMBA) or 2,3,5,7-tetrachlorodibenzo-p-dioxin (TCDD), was inhibited by cotreatment with fluasterone. However, treatment of the cells with fluasterone after induction with DMBA or TCDD failed to decrease enzyme activity, indicating that inhibition was not the result of direct enzyme inhibition. Therefore, we examined the effect of fluasterone on gene expression at the mRNA level. Both DMBA and TCDD caused a dramatic increase in the amount of CYP1A1 and CYP1B1 mRNA, the two major isoforms involved in carcinogen activation in these cells. In cells cotreated with fluasterone, however, there was a dose-dependent decrease in CYP1A1 and CYP1B1 mRNA. Fluasterone also inhibited the basal level of CYP1A1 mRNA but not CYP1B1. Fluasterone inhibited the rate of CYP1A1 promoter-controlled transcription, indicating that it affects the transcriptional regulation of the gene. Actinomycin D chase experiments showed that fluasterone also caused an increase in the degradation of CYP1A1 mRNA, while leaving CYP1B1 mRNA unaffected. These results indicate that fluasterone inhibits the increase in the expression of CYP1A1 normally caused by exposure to carcinogens by both transcriptional and post-transcriptional mechanisms and that CYP1B1 expression is not susceptible to the same post-transcriptional mechanism.