Green tea catechin extract in intervention of chronic breast cell carcinogenesis induced by environmental carcinogens

Sporadic breast cancers are mainly attributable to long-term exposure to environmental factors, via a multi-year, multi-step, and multi-path process of tumorigenesis involving cumulative genetic and epigenetic alterations in the chronic carcinogenesis of breast cells from a non-cancerous stage to precancerous and cancerous stages. Epidemiologic and experimental studies have suggested that green tea components may be used as preventive agents for breast cancer control. In our research, we have developed a cellular model that mimics breast cell carcinogenesis chronically induced by cumulative exposures to low doses of environmental carcinogens. In this study, we used our chronic carcinogenesis model as a target system to investigate the activity of green tea catechin extract (GTC) at non-cytotoxic levels in intervention of cellular carcinogenesis induced by cumulative exposures to pico-molar 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P). We identified that GTC, at a non-cytotoxic, physiologically achievable concentration of 2.5 μg/mL, was effective in suppressing NNK- and B[a]P-induced cellular carcinogenesis, as measured by reduction of the acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth, increased cell mobility, and acinar-conformational disruption. We also detected that intervention of carcinogen-induced elevation of reactive oxygen species (ROS), increase of cell proliferation, activation of the ERK pathway, DNA damage, and changes in gene expression may account for the mechanisms of GTC's preventive activity. Thus, GTC may be used in dietary and chemoprevention of breast cell carcinogenesis associated with long-term exposure to low doses of environmental carcinogens.     

Grape seed proanthocyanidin suppression of breast cell carcinogenesis induced by chronic exposure to combined 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone and benzo[a]pyrene

Breast cancer is the most common type of cancer among women in northern America and northern Europe; dietary prevention is a cost‐efficient strategy to reduce the risk of this disease. To identify dietary components for the prevention of human breast cancer associated with long‐term exposure to environmental carcinogens, we studied the activity of grape seed proanthocyanidin extract (GSPE) in suppression of cellular carcinogenesis induced by repeated exposures to low doses of environmental carcinogens. We used combined carcinogens 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and benzo[a]pyrene (B[a]P), at picomolar concentrations, to repeatedly treat noncancerous, human breast epithelial MCF10A cells to induce cellular acquisition of cancer‐related properties of reduced dependence on growth factors, anchorage‐independent growth, and acinar‐conformational disruption. Using these properties as biological target endpoints, we verified the ability of GSPE to suppress combined NNK‐ and B[a]P‐induced precancerous cellular carcinogenesis and identified the minimal, noncytotoxic concentration of GSPE required for suppressing precancerous cellular carcinogenesis. We also identified that hydroxysteroid‐11‐beta‐dehydrogenase 2 (HSD11B2) may play a role in NNK‐ and B[a]P‐induced precancerous cellular carcinogenesis, and its expression may act as a molecular target endpoint in GSPE's suppression of precancerous cellular carcinogenesis. And, the ability of GSPE to reduce gene expression of cytochrome‐P450 enzymes CYP1A1 and CYP1B1, which can bioactivate NNK and B[a]P, possibly contributes to the preventive mechanism for GSPE in suppression of precancerous cellular carcinogenesis. Our model system with biological and molecular target endpoints verified the value of GSPE for the prevention of human breast cell carcinogenesis induced by repeated exposures to low doses of multiple environmental carcinogens. © 2010 Wiley‐Liss, Inc.     

Precancerous model of human breast epithelial cells induced by NNK for prevention

Epidemiological investigations have suggested that exposure to tobacco and environmental carcinogens increase the risk of developing human breast cancer. In light of the chronic exposure of human breast tissues to tobacco and environmental carcinogens, we have taken an approach of analyzing cellular changes of immortalized non-cancerous human breast epithelial MCF10A cells during the acquisition of cancerous properties induced by repeated exposure to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at a low concentration of 100 pM. We found that accumulated exposures of MCF10A cells to NNK result in progressive development of cellular carcinogenesis from a stage of immortalization to precancerous sub-stages of acquiring a reduced dependence on growth factors and acquiring anchorage-independent growth. Using Matrigel for MCF10A cells to form size-restricted acini, we detected that exposures to NNK resulted in altered acinar conformation. Analysis of gene expression profiles by cDNA microarrays revealed up- and down-regulated genes associated with NNK-induced carcinogenesis. Using this cellular carcinogenesis model as a target system to identify anticancer agents, we detected that grape seed proanthocyanadin extract significantly suppressed NNK-induced carcinogenesis of MCF10A cells. Our studies provide a carcinogenesis-cellular model mimicking the accumulative exposure to carcinogens in the progression of human breast epithelial cells to increasingly acquire cancerous properties, as likely occurs in the development of precancerous human breast cells. Our cellular model also serves as a cost-efficient, in vitro system to identify preventive agents that inhibit human breast cell carcinogenesis induced by chronic exposures to carcinogens.     

Intervention of human breast cell carcinogenesis chronically induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens, such as those in the diet, through a multistep disease process progressing from non-cancerous to premalignant and malignant stages. The chemical carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines found in high-temperature cooked meats and is recognized as a mammary carcinogen. However, the PhIP's mechanism of action in breast cell carcinogenesis is not clear. Here, we demonstrated, for the first time, that cumulative exposures to PhIP at physiologically achievable, pico to nanomolar concentrations effectively induced progressive carcinogenesis of human breast epithelial MCF10A cells from a non-cancerous stage to premalignant and malignant stages in a dose- and exposure-dependent manner. Progressive carcinogenesis was measured by increasingly- acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth, acinar-conformational disruption, proliferation, migration, invasion, tumorigenicity with metastasis and increased stem-like cell populations. These biological changes were accompanied by biochemical and molecular changes, including upregulated H-Ras gene expression, extracellular signal-regulated kinase (ERK) pathway activation, Nox-1 expression, reactive oxygen species (ROS) elevation, increased HIF-1α, Sp1, tumor necrosis factor-α, matrix metalloproteinase (MMP)-2, MMP-9, aldehyde dehydrogenase activity and reduced E-cadherin. The Ras-ERK-Nox-ROS pathway played an important role in not only initiation but also maintenance of cellular carcinogenesis induced by PhIP. Using biological, biochemical and molecular changes as targeted endpoints, we identified that the green tea catechin components epicatechin-3-gallate and epigallocatechin-3-gallate, at non-cytotoxic doses, were capable of suppressing PhIP-induced cellular carcinogenesis and tumorigenicity.     

Precancerous carcinogenesis of human breast epithelial cells by chronic exposure to benzo[a]pyrene

To understand carcinogenesis of human breast epithelial cells induced by chronic exposure to environmental pollutants, we studied biological and molecular changes in progression of cellular carcinogenesis induced by accumulated exposures to the potent environmental carcinogen benzo[a]pyrene (B[a]P). Increasing exposures of human breast epithelial MCF10A cells to B[a]P at picomolar concentrations resulted in cellular transformation from a noncancerous stage to precancerous substages, in which cells acquired the cancerous abilities of a reduced dependence on growth factors, anchorage-independent growth, and disruption in acini formation on reconstituted basement membranes. Using cDNA microarrays, we detected seven upregulated genes related to human cancers in B[a]P-transformed MCF10A cells. Using this model, we verified that green tea catechin significantly (P < 0.05) suppressed B[a]P-induced carcinogenesis. Our studies indicate that this cellular model may serve as a cost-efficient, in vitro system, mimicking the chronic carcinogenesis of breast cells that likely occurs in early stages of carcinogenesis in vivo, to identify agents that inhibit cellular carcinogenesis.     

Mutagenesis induced by oral carcinogens in lacZ mouse (MutaMouse) tongue and other oral tissues.

Animal models for carcinogenesis of the oral cavity are limited, although this disease is often fatal or disfiguring and its incidence in the USA is approximately 30 000 cases/year. Short-term whole-animal models for this disease should prove valuable in the investigation of factors affecting oral carcinogenesis. In this study we observed that a group of oral carcinogens are clearly mutagenic in the lacZ transgenic mouse oral cavity. The carcinogens 4-nitroquinoline-N-oxide (4-NQO), benzo[a]pyrene (B[a]P), N-nitroso-N-methylurea (NMU), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), nitrosonornicotine (NNN) and 7,12-dimethylbenzanthracene (DMBA) were all mutagenic in a mixture of pooled oral tissues (gingival, buccal, pharyngeal and sublingual) and in the tongue. All agents except DMBA (which was swabbed in the oral cavity) and B[a]P (by gavage) were given in drinking water for 2-4 weeks followed by a 2 week expression period before killing. With one exception, groups of 4-5 female mice were treated. The doses and mutant fractions (MF) in DNA isolated from pooled oral tissues (in mutants/10(5) p.f.u. +/- SD) were: 4-NQO (20-80 microg/ml, over 4 weeks) 78 +/- 16; B[a]P (five doses of 125 mg/ml) 33.2 +/- 10.9; NMU (20-80 microg/ml over 4 weeks) 7.8 +/- 2.6; NNK (0.1 mg/ml, weeks 1-2, 0.2 mg/ml, weeks 3-4) 9.1 +/- 3.0; NNN (same dose as NNK) 9.2 +/- 1.6 and DMBA (0.5 mg/ml in corn oil, 3 weeks) 7.1 +/- 2.7. The corresponding value for untreated controls was 3.2 +/- 1.8. Values for induced mutagenesis in tongue from the same animals were similar except for 4-NQO which was about twice as potent in tongue. Mutagenesis by several compounds was compared in other organs. B[a]P was assayed in lung and kidney and was about twice as mutagenic in oral tissues as in lung, but several times less mutagenic in kidney. Lung, but not kidney is a target organ for B[a]P-induced carcinogenesis in the mouse. NNK was somewhat more mutagenic in lung (MF of 15.0 +/- 5.5) than in oral tissues, corresponding with previous reports on carcinogenesis by NNK. Mutagenesis induced by NNN was also assayed in esophagus, a target organ in rodents, and was similar to that in oral tissue. In all cases the MF in untreated control group was about 3-4. These results suggest that: (i) the oral cavity has a significant capacity for metabolic activation of carcinogens; (ii) DNA damage in the oral cavity can be converted to mutations; and (iii) there is significant target organ specificity. The results also tend to support the concept that the anatomical components of the upper aerodigestive tract, in general, behave similarly with respect to genotoxicity. As carcinogenesis is believed to involve mutagenesis, this study demonstrates the utility of the lacZ mouse for investigations involving initiation of carcinogenesis of the oral cavity.     

Tobacco Smoke Carcinogens Induce DNA Repair Machinery Function Loss: Protection by Carbon Nanotubes

Purpose: DNA damage is a continuous process occurring within the cells caused by intrinsic and extrinsic factors, but it gets repaired regularly. If the DNA repair process is faulty, the incidences of damages/mutations can accumulate in cells resulting in cell transformation. It is hypothesized that the negative variations in DNA repair pathways in even at one point viz. genetic, translational or posttranslational stage may fairly be crucial for the beginning and development of carcinogenesis. Therefore, we investigated the potential of tobacco specific nitrosamines (TSNs) related carcinogens to interact with the enzymes involved in DNA repair mechanisms in the current study. Methods: The derivatives of cigarettes’ smoke like NNK and NNAL are very well known and recognized carcinogens. Therefore, almost 120 enzymes playing crucial role in the DNA repair process have been analysed for their reactivity with NNK and NNAL. Results: The molecular docking study helped to screen out,  07 possible DNA repair enzyme targets for NNK, and 12for NNAL. Present study revealed the loss of activity of DNA repair enzymes in the presence of NNK and NNAL, and this accumulation may induce the tendency of DNA damage which can lead the transformation of exposed normal cells in to cancerous cells. This study also demonstrated the protective potential of nanoparticles like SWCNTs/MWCNTs against TSN’s induced toxicity; here SWCNT against NNK (-17.16 Kcal/Mol) and MWCNT against NNK -17.01 Kcal/Mol were showing maximum binding affinities than the known biomolecular target of NNK 1UGH (Uracil-DNA glycosylase,-7.82Kcal/Mol). Conclusion: CNTs can be applied as chemo-preventive agents against environmental and tobacco induced carcinogens owing to their scavenging potential and warrants for in vivo and in vitro experimental validation of the results obtained from the present study.     

Antimutagenic and anticarcinogenic effects of betel leaf extract against the tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK).

Earlier studies showed that betel leaf inhibits the mutagenic action of standard mutagens like benzo[a]pyrene and dimethylbenz[a]anthracene. Since tobacco-specific nitrosamines are the major carcinogens present in unburnt forms of tobacco, we studied the effect of an extract of betel leaf on the mutagenic and carcinogenic actions of one of the most potent, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK). Betel-leaf extract and hydroxychavicol suppressed the mutagenicity of NNK in both the Ames and the micronucleus test. In studies in mice, betel-leaf extract reduced the tumorigenic effects of NNK by 25%. Concurrent treatment with the extract also inhibited the decreases in levels of vitamin A in liver and plasma induced by NNK. Betel leaf thus has protective effects against the mutagenic, carcinogenic and adverse metabolic effects of NNK in mice.     

BRCA1 and TP53 mutation spectrum of breast carcinoma in an ethnic population of Kashmir, an emerging high-risk area

Breast cancer shows geographical variation in its incidence, even within areas of ethnic homogeneity. Kashmir valley (India), over past few years, witnesses an increase in incidence and occurrence of familial, early onset, and male breast cancer in its unexplored ethnic population. Here, we make a preliminary attempt to estimate the nature and frequency of BRCA1 and TP53 gene mutations of breast cancer patients from Kashmir. PCR-SSCP analysis followed by direct sequencing revealed the presence of only two germline intronic variations (c.199+67T>C and c.5396+187T>C) in BRCA1 gene in only 5.26% (2/38) patients while as 44% (11/25) of sporadic breast cancer patients harboured significant amount of somatic mutations in TP53 (p=0.0074; OR=0.053). The 17 mutations found in TP53 in 11 patients, comprised of 13 substitutions [11 single-base (9 transitions+2 transversions), 1 double-base and 1 complex] and four insertions. The 11 substitutions represent missense mutations, leading to aminoacid substitution while as rest two were silent mutations. The four insertions represented three frame-shifts and one non-sense mutation. The mutation effect data was found to be significant (p=0.0002). Significant amount of mutations were found in exon 6 (p=0.04; OR=0.273) and a combination of exons 6 and 7 (p=0.0145; OR=14.22) of TP53. Comparison of mutation profile with other ethnic populations and regions reflected both differences and similarities indicating co-exposure to a unique set of risk factors. The differences could be due to exposure to particular environmental carcinogens; different lifestyle, reproductive pattern; dietary or cultural practices of Kashmiri women that need further investigations. The infrequent presence of germline BRCA1 mutations in our study agree with the idea that a great proportion of moderate risk breast cancer population could be due to the susceptibility genes distinct from BRCA1. However, high frequency of somatic TP53 gene mutations implicates TP53 as a predominant factor for breast carcinogenesis in moderate risk ethnic Kashmiri population. The study also suggests TP53 as a potential molecular marker and prognostic tool, at least in a subset of sporadic breast tumors.     

Risk Analysis of Environmental Chemicals on Lung Carcinogenesis

Lung cancer is one of the most common cancers in the world, and the incidence of lung cancer is increasing.Risk analysis of environmental chemicals on lung carcinogenesis is particularly important. Detection of chemopreventive agents of lung carcinogenesis is also important to reduce our risk of lung cancer. For that purpose, it is necessary to establish reliable in vivo animal models of lung carcinogenesis. The A/J mouse is a mouse strain sensitive to lung carcinogens, and also develops spontaneous lung tumors without any chemical treatment. We have demonstrated that a treatment of 4-(methylnitrosamino)-1-(3-pyridyle)-1-butanone (NNK), a tobacco specific nitrosamine, or 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (MeIQx), a heterocyclic amine, induced lung tumors in the female A/J mouse in 16 and 32 weeks. The lung tumors developed in the A/J mouse are histopathologically classified as adenocarcinomas, adenomas, and alveolar cell hyperplasias. Some of these types of lung cancer are similar to those of human lung cancer. We also investigated the chemopreventive effects of bovine LF (bLF) on different phases of NNK-induced lung tumorigenesis in A/J mice. The A/J mouse is very useful mouse strain as a reliable in vivo model, which can be used for risk analysis of lung carcinogenesis.     

Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk

The breast cancer resistance protein (BCRP/ABCG2) usually protects the body from a wide variety of environmental and dietary xenotoxins by reducing their net uptake from intestine and by increasing their hepatobiliary, intestinal and renal elimination. BCRP is also highly expressed in lactating mammary glands in mice, and this expression is conserved in cows and humans. As a result, BCRP substrates can be secreted into milk. We investigated whether different classes of dietary carcinogens are substrates of Bcrp1/BCRP and the implications for systemic exposure and breast milk contamination. Using polarized cell lines, we found that Bcrp1 transports the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the potent human hepatocarcinogen aflatoxin B1, and decreases their cellular accumulation up to 10-fold. In vivo pharmacokinetic studies showed that [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin B1 plasma levels were substantially lower in wild-type compared with Bcrp1-/- mice, after both oral and intravenous administration, demonstrating that Bcrp1 restricts systemic exposure to these carcinogens. Moreover, Bcrp1 mediates transfer of [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin into milk, with 3.4+/-0.6, 2.6+/-0.3 and 3.8+/-0.5-fold higher milk to plasma ratios, respectively, in lactating wild-type versus Bcrp1-/- mice. We have thus identified Bcrp1/BCRP as one of the molecular mechanisms by which heterocyclic amines and aflatoxin are transferred into milk, thereby posing a health risk to breast-fed infants and dairy consumers. Paradoxically, Bcrp1/BCRP appears to have both protective and adverse roles with respect to exposure to dietary carcinogens.     

Mesenchymal and stem-like cell properties targeted in suppression of chronically-induced breast cell carcinogenesis

Stem-like cells and the epithelial-to-mesenchymal transition (EMT) program are postulated to play important roles in various stages of cancer development, but their roles in breast cell carcinogenesis and intervention remain to be clarified. We investigated stem-like cell- and EMT-associated properties and markers in breast epithelial cells chronically exposed to low-dose 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene in the presence and absence of the preventive agents green tea catechins and grape seed extract. Our results indicate that stem-like cell- and EMT-associated properties and markers should be seriously considered as new cancer-associated indicators for detecting breast cell carcinogenesis and as endpoints for intervention of carcinogenesis.     

Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates.

B[a]P (benzo[a]pyrene) has been used as a prototype carcinogenic PAH since its isolation from coal tar in the 1930's. One of its diol epoxides, BPDE-2, is considered its ultimate carcinogen on the basis of its binding to DNA, mutagenicity and extreme pulmonary carcinogenicity in newborn mice. However, BPDE-1 has a similar binding to DNA and mutagenicity but it is not carcinogenic. In addition, BPDE-2 is a weak carcinogen relative to B[a]P when repeatedly applied to mouse skin, the conventional assay site. Its carcinogenicity is increased when applied once as an initiator followed repeatedly by a promoter. This indicates a major role for promotion in carcinogenesis by PAHs. Promotion itself is a 2-stage process, the second of which is selective propagation of the initiated cells. Persistent hyperplasia underlies selection by promoters. The non-carcinogenicity of BPDE-1 has yet to be resolved. PAHs have long been considered the main carcinogens of cigarette smoke but their concentration in the condensate is far too low to account by themselves for the production of skin tumors. The phenolic fraction does however have strong promotional activity when repeatedly applied to initiated mouse skin. Several constituents of cigarette smoke are co-carcinogenic when applied simultaneously with repeated applications of PAHs. Catechol is co-carcinogenic at concentrations found in the condensate. Since cigarette smoking involves protracted exposure to all the smoke constituents, co-carcinogenesis simulates its effects. Both procedures, however, indicate a major role for selection in carcinogenesis by cigarette smoke. That selection may operate on endogenous mutations as well as those induced by PAHs. There are indications that the nicotine-derived NNK which is a specific pulmonary carcinogen in animals contributes to smoking-induced lung cancer in man. Lung adenoma development by inhalation has been induced in mice by the gas phase of cigarette smoke. The role of selection has not been evaluated in either of these cases.     

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung

Dietary phenethyl isothiocyanate (PEITC) and a mixture of dietary PEITC and benzyl isothiocyanate (BITC) inhibit lung tumorigenesis in A/J mice induced by a mixture of the tobacco smoke carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we tested the hypothesis that inhibition of tumorigenesis by these isothiocyanates was due to inhibition of DNA adduct formation. We quantified the following pulmonary DNA adducts: N2-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl]deoxyguanosine (BPDE-N2-dG) from B[a]P; and O(6)-methylguanine (O(6)-mG) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts from NNK. Initial experiments demonstrated that there were no effects of B[a]P on NNK-DNA adduct formation, or vice versa, and established by way of a time course study the appropriate sacrifice intervals for the main experiment. Dietary PEITC, or dietary BITC plus PEITC, inhibited the formation of HPB-releasing DNA adducts of NNK at several of the time points examined. There were no effects of dietary isothiocyanates on levels of O(6)-mG or BPDE-N2-dG. These results, which are consistent with previous studies in rats and with tumor inhibition data in mice, support a role for inhibition of HPB-releasing DNA adducts of NNK as a mechanism of inhibition of tumorigenesis by dietary PEITC and BITC plus PEITC. However, the observed inhibition was modest, suggesting that other effects of isothiocyanates are also involved in chemoprevention in this model.     

Biomolecular markers as intermediate end points in chemoprevention trials of upper aerodigestive tract cancer

Head-and-neck squamous-cell cancer (HNSCC) is an important public-health problem, accounting for approximately 40,300 new cancer cases and 12,000 cancer deaths annually in the United States (Greenlee et al., [2000]). Patients with early-stage disease are often cured with surgery or radiotherapy but are at high risk for second primary tumor (SPT) development (Lippman and Hong, [1989]), and the majority of patients present with advanced disease, for which the outcomes have not markedly improved despite advances in combined-modality therapy (Vokes et al., [1993]). HNSCC arises from transformation of the genetic material of normal cells, followed by successive genetic alterations in a multistep fashion, leading to clonal evolution of progeny cells with a proliferative advantage (Vogelstein and Kinzler, [1993]), induced by tobacco carcinogens (Slaughter et al., [1953]). Chemoprevention aims at reversal of this process through re-regulation of growth and differentiation and possibly elimination of genetically and phenotypically aberrant clones. Chemoprevention studies in upper aerodigestive tract (UADT) cancers are based on these fundamental premises and the identification of molecular genetic and biologic cellular changes. These alterations represent biomarkers of the carcinogenesis process and ultimately, if validated, could serve as intermediate end points for these studies.     

Permeation and reservoir formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) across porcine esophageal tissue in the presence of ethanol and menthol

Environmental influences may affect carcinogen absorption and residency in the tissues of the aero-digestive tract. We quantified the effect of ethanol and menthol on the rates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) absorption using a fully validated in vitro diffusion system, capable of accurately and precisely quantifying tobacco carcinogen permeation and reservoir formation in porcine esophageal mucosa. Confocal microscopy was employed to visualize the location of B[a]P in the exposed membranes. Markedly different extents of permeation and reservoir formation for the tobacco carcinogens were recorded in the presence of ethanol and menthol. The water-soluble NNK permeated the membrane rapidly, while the lipophilic B[a]P did not appreciably diffuse through the tissue. Significantly different extents of reservoir formation were observed for the different carcinogens and in the presence of the different penetration-enhancer solvents. Alcohol (at 5% concentration) did not influence the permeation or reservoir formation of NNK. A mentholated donor solution (0.08%) both decreased the flux of NNK and significantly increased the tissue reservoir formation. The magnitude of the reservoir formed by B[a]P was relatively extensive (even though membrane permeation rates were negligible), being greatest in the presence of both ethanol and menthol. This suggests synergy between the two penetration-enhancer species acting on this carcinogen. Confocal microscopy studies confirmed that there was an appreciable intra-cellular, and specifically nuclear, association of the B[a]P species during the reservoir formation process. The aqueous solubility of the diffusing species and the presence of penetration enhancers appeared to be key factors in the absorption and cellular binding processes. The results presented support the hypothesis that the use of mentholated cigarettes, or the concomitant consumption of alcohol while smoking, may have marked effects on the fate of tobacco chemicals. This finding may help to explain elevated rates of esophageal squamous cell carcinoma in African Americans.     

High frequency of HIF-1α overexpression in BRCA1 related breast cancer

Hypoxia is a hallmark of cancer. Hypoxia inducible factor-1α (HIF-1α) is the key regulator of the hypoxia response. HIF-1α is overexpressed during sporadic breast carcinogenesis and correlated with poor prognosis. Little is known on the role of HIF-1α in hereditary breast carcinogenesis. A recent study suggests a role for BRCA1 in HIF-1α regulation. We therefore examined the expression of HIF-1α in BRCA1 related breast cancers. By immunohistochemistry we studied expression of HIF-1α and some of its downstream targets in 30 hereditary invasive breast cancers in comparison with 200 sporadic controls. HIF-1α overexpression was significantly more frequent in BRCA1 related breast cancers (27/30, 90%) than in sporadic controls (88/200, 44%) (P < 0.0001). 19/30 (63%) of BRCA1 tumors showed perinecrotic (hypoxia induced) and 8/30 (27%) a diffuse HIF-1α overexpression pattern, the latter more likely related to genetic alterations in oncogenes and tumor suppressor genes. In contrast, sporadic breast cancer HIF-1 expressing tumors showed an inverse ratio of perinecrotic/diffuse expression (31 vs. 69%, P = 0.0002). Glut-1 and CAIX, downstream HIF1 targets, were expressed in 27/30 (90%) and 15/21 (71%) of hereditary cases versus 54/183 (29%) and 24/183 (13%) in sporadic cases. p300 levels, necessary for HIF-1 downstream activation, were significantly higher in hereditary cases (20/21, 95%) compared to sporadic cases (91/183, 50%, P = 0.0001). In conclusion, in BRCA1 germline mutation related breast cancer, functional HIF-1α overexpression is seen at a much higher frequency than in sporadic breast cancer, mostly hypoxia induced. This points to an important role of hypoxia and its key regulator HIF-1α in hereditary breast carcinogenesis.     

Superoxide-mediated proteasomal degradation of Bcl-2 determines cell susceptibility to Cr(VI)-induced apoptosis

Hexavalent chromium [Cr(VI)] compounds are redox cycling environmental carcinogens that induce apoptosis as the primary mode of cell death. Defects in apoptosis regulatory mechanisms contribute to carcinogenesis induced by Cr(VI). Activation of apoptosis signaling pathways is tightly linked with the generation of reactive oxygen species (ROS). Likewise, ROS have been implicated in the regulation of Cr(VI)-induced apoptosis and carcinogenicity; however, its role in Cr(VI)-induced apoptosis and the underlying mechanism are largely unknown. We report that ROS, specifically superoxide anion (.O(-)(2), mediates Cr(VI)-induced apoptosis of human lung epithelial H460 cells. H460 rho(0) cells that lack mitochondrial DNA demonstrated a significant decrease in ROS production and apoptotic response to Cr(VI), indicating the involvement of mitochondrial ROS in Cr(VI)-induced apoptosis. In agreement with this observation, we found that Cr(VI) induces apoptosis mainly through the mitochondrial death pathway via caspase-9 activation, which is negatively regulated by the antiapoptotic protein Bcl-2. Furthermore, .O(-)(2) induced apoptosis in response to Cr(VI) exposure by downregulating and degrading Bcl-2 protein through the ubiquitin-proteasomal pathway. This study reveals a novel mechanism linking .O(-)(2) with Bcl-2 stability and provides a new dimension to ROS-mediated Bcl-2 downregulation and apoptosis induction.