Carcinogen 4-nitroquinoline N-oxide Research Articles

Abstract 3077: Leveraging a 3D organoid library to identify novel therapeutic targets during ESCC initiation and progression

Abstract Esophageal squamous cell carcinoma (ESCC) is a devastating and common disease worldwide. Despite recent advances annotating the genetic and epigenetic changes underlying the initiation and development of ESCC and its precursor lesion, esophageal squamous dysplasia, more work is needed to translate these findings into novel therapeutic strategies. This gap is due in part to the lack of tractable models of ESCC initiation and development that can then be manipulated ex vivo for preclinical testing. Currently, the large-scale studies characterizing the aforementioned molecular changes have used primary tissue for their analyses and have subsequently been unable to test the specific hypotheses that arise from their data using the original tissue that they profiled. To address this gap, we have generated a large (n = 54) three-dimensional organoid library from the esophagi of mice treated with the carcinogen 4-Nitroquinoline N-oxide (4-NQO) representing each stage of esophageal squamous cell initiation and progression from early intraepithelial dysplasia through lung metastasis. We have determined that these organoids faithfully recapitulate the molecular biology, histology, and oncogenic phenotype of their tissue of origin. Through integrative analysis of both RNA sequencing and whole exome sequencing data, we have identified several novel therapeutic targets that contribute to ESCC initiation and development including the DNA damage response and NOTCH1 signaling. Together, our work capitalizes on the intersection of large-scale multiomics and sophisticated 3D organoid models to identify novel therapeutic targets for the treatment of a devastating disease. Citation Format: Samuel Flashner, Masataka Shimonosono, Satoshi Takada, Norihiro Matsuura, Yasuto Tomita, Xiao Chen, Alison Taylor, Andres Klein-Szanto, Fatameh Momen-Heravi, J. Alan Diehl, Chao Lu, Hiroshi Nakagawa. Leveraging a 3D organoid library to identify novel therapeutic targets during ESCC initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3077.

(E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) induces cytoprotection in CCD18-Co human colon fibroblast cells through Nrf2/ARE pathway activation

Cytoprotection involving the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important preventive strategy for normal cells against carcinogenesis. In our previous study, the chemopreventive potential of (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) has been elucidated through its cytoprotective effects against DNA and mitochondrial damages in the human colon fibroblast CCD-18Co cell model. Therefore this study aimed to investigate the molecular mechanisms underlying BK3C231-induced cytoprotection and the involvement of the Nrf2/ARE pathway. The cells were pretreated with BK3C231 before exposure to carcinogen 4-nitroquinoline N-oxide (4NQO). BK3C231 increased the protein expression and activity of cytoprotective enzymes namely NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST) and heme oxygenase-1 (HO-1), as well as restoring the expression of glutamate-cysteine ligase catalytic subunit (GCLC) back to the basal level. Furthermore, dissociation of Nrf2 from its inhibitory protein, Keap1, and ARE promoter activity were upregulated in cells pretreated with BK3C231. Taken together, our findings suggest that BK3C231 exerts cytoprotection by activating the Nrf2 signaling pathway which leads to ARE-mediated upregulation of cytoprotective proteins. This study provides new mechanistic insights into BK3C231 chemopreventive activities and highlights the importance of stilbene derivatives upon development as a potential chemopreventive agent.

Inflammatory cell infiltrate associated with primary and transplanted tumours in an inbred model of oral carcinogenesis.

This study characterised the nature of the local cellular immune responses associated with an inbred animal model of oral carcinogenesis. Inbred F344 rats developed moderately- to well-differentiated primary oral squamous cell carcinomas (SCC) after treatment with the carcinogen 4-nitroquinoline N-oxide (4-NQO) in vivo for 5-6 months. The inflammatory cell infiltrate associated with the primary tumours was predominantly of the macrophage lineage (CD45+, Ia+) and contained smaller numbers of CD8+ cells (NK cells, cytotoxic/suppressor T cells), CD5+ cells (T cells) and CD25+ cells (activated cells; T and NK cells). Keratinocyte cell lines were established from three lingual and one palatal SCC. By contrast to normal keratinocytes, tumour-derived cell lines were immortal and independent of 3T3 fibroblast support. All of the tumour-derived cell lines were tumorigenic in athymic (nu/nu) mice and showed contrasting latent periods of tumour development and histological differentiation; normal keratinocyte grafts were non-tumorigenic in athymic mice. Three of four malignant cell lines formed well-differentiated tumours in syngeneic F344 rats; the tumours regressed after 10-14 days. Regressing grafts contained significantly larger numbers of NK cells (CD5-, CD8+) in the inflammatory cell infiltrate compared with that associated with primary tumours (p < 0.04). One malignant cell line and normal keratinocytes were non-tumorigenic in syngeneic hosts. The results demonstrate phenotypic variation in the cell-mediated immune responses associated with the actively growing primary SCC and the regressing tumours in syngeneic hosts and suggest that NK cells, possibly activated by local T cell responses, are important for tumour rejection in this model.

Autocrine production of TGF-alpha and TGF-beta during tumour progression of rat oral keratinocytes.

This study describes a new technique to separate transforming growth factor-alpha (TGF-alpha) and transforming growth factor-beta (TGF-beta) from culture supernatants using ion exchange chromatography; assays of competitive inhibition of ligand binding were used to quantify the amount of growth factor. The method was simple, inexpensive and did not require large volumes of culture medium. The autocrine production of TGF-alpha and TGF-beta was examined in oral keratinocyte cell lines derived from the palatal and lingual mucosa of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO). Escape from cellular senescence (immortality) was associated with a marked increase in TGF-alpha production (cell line R2P) but tumour progression, as reflected by the development of anchorage independence in agarose gels and tumorigenicity in athymic mice, did not result in a consistent increase or decrease of TGF-alpha production compared to normals. Four cell lines (R8AP, R1T, R3T, R1P), with different functional cellular phenotypes, produced two to three times more TGF-alpha than normals. TGF-alpha production was inversely correlated to epidermal growth factor cell surface receptor expression. The autocrine production of TGF-beta was variable with the majority of cell lines producing markedly little TGF-beta; three cell lines (R4T, R8BP, R9T) produced more TGF-beta than normals. The production of TGF-beta was unrelated to tumour progression, the expression of TGF-beta cell surface receptors or TGF-alpha production. The results indicate that the autocrine production of TGF-alpha and TGF-beta are not accurate markers of tumour progression in the rat 4NQO model of oral carcinogenesis.

Formation of a tetracyclic structure between cytosine derivatives and a model metabolite of the carcinogen 4-nitroquinoline N-oxide

Formation of a tetracyclic structure between cytosine derivatives and a model metabolite of the carcinogen 4-nitroquinoline N-oxide

Tumour Progression in experimental oral carcinogenesis is associated with changes in EGF and TGF-β receptor expression and altered responses to these growth factors

This study examined the characteristics of premalignant oral epithelial cell lines derived from non-invasive palatal and lingual mucosa of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) in vivo. In contrast to normal keratinocytes, premalignant epithelial cells had an extended life span, were independent of 3T3 fibroblast support, and expressed variable anchorage independence in gel culture and tumorigenicity in athymic mice. The expression of these functional phenotypes did not correlate with the duration of 4NQO treatment. Keratinocytes from 4NQO-treated tissues predominantly had fewer epidermal growth factor (EGF) receptors than normal controls. The expression of high-affinity EGF receptors paralleled the emergence of the anchorage-independent phenotype and was markedly elevated in tumorigenic cell lines. Cell lines with an extended life span expressed fewer transforming growth factor beta 1 (TGF-beta) receptors than their normal counterparts though the loss of these receptors appeared to be unrelated to either anchorage independence or tumorigenicity. Normal keratinocytes were stimulated and inhibited, in a dose-dependent manner, by EGF and TGF-beta respectively. By contrast, a cell line that was immortal, anchorage dependent and non-tumorigenic showed reduced sensitivity to stimulation by EGF and was inhibited only by high concentrations of TGF-beta. Cells that were immortal, anchorage independent and tumorigenic, however, were refractory to EGF and were inhibited only by high concentrations of TGF-beta. There was no correlation between the expression of EGF or TGF-beta cell surface receptors and the response to ligand binding. The results show that tumour progression in rat oral epithelial cells is associated with a progressive independence of growth factor control. The number and distribution of EGF and TGF-beta receptors may be useful markers in more closely defining the stages of epithelial tumour progression.

Epithelial dendritic cells and connective tissue macrophages in oral corcinogenesis and the effects of systemic Corynebacterium parvum

Epithelial dendritic cells (EDC) and connective tissue macrophages were examined during the induction and growth of oral squamous cell carcinomas in Sprague-Dawley rats treated with the carcinogen 4-nitroquinoline N-oxide (4NQO) and the immune potentiator Corynebacterium parvum. Splenomegaly was induced in all animals receiving C. parvum. Acetone-fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and monoclonal antibodies to rat Ia (MRC OX-6) and macrophage subpopulations (ED1, ED2, ED3). EDC were predominantly Ia+, ED1-, ED2- and ED3-. The lamina propria contained Ia+, ED1+ and ED2+ cells; ED3-reactive cells were rare. ED2+ cells predominated in the interstitial connective tissue of deeper muscle. In the non-invasive tissues, the number of positive cells (Ia+EDC and connective tissue Ia+, ED1+ and ED3+ cells) increased significantly throughout the experimental period (0-9 months), were significantly more prevalent in the test tissues (4NQO, 4NQO + C.parvum, C.parvum) compared to untreated controls and, at 9 months, the carcinogen-treated rats (4NQO, 4NQO + C.parvum) had significantly more Ia+ EDC and connective tissue Ia+ cells than C.parvum controls. Irrespective of the marker under study, there were no significant differences between rats treated with 4NQO or 4NQO + C.parvum at any time during the experimental period. Similarly, intra-epithelial Ia+ and ED1+ cells increased significantly throughout the experimental period in all test groups compared to untreated controls, but no significant differences were evident between carcinogen-treated animals (4NQO, 4NQO + C.parvum) and C.parvum controls. Significant positive correlations between connective tissue Ia+ and ED1+ cells and also intra-epithelial Ia+ and ED1+ cells were present in all experimental groups; connective tissue ED2+ and ED3+ cell numbers did not correlate with any of the other phenotypes and intra-epithelial ED2+ and ED3+ cells were rare/absent. Palatal and/or lingual tumours developed in 80% of carcinogen-treated rats by 9 months and the tumour incidence was similar in rats treated with either 4NQO or 4NQO + C.parvum. There were no significant differences in the number of Ia+ EDC between the infiltrating and the non-invasive overlying epithelium of the lingual carcinomas and the non-invasive lingual epithelium treated with either 4NQO or 4NQO + C.parvum.(ABSTRACT TRUNCATED AT 400 WORDS)

The expression of anchorage independence by malignant rat oral keratinocytes after colony formation in vitro and tumour formation in vivo

The expression of anchorage independence in malignant oral epithelial cells retrieved from colonies formed in agarose and tumours formed in athymic mice was examined. The original epithelial cell lines were derived from lingual and palatal squamous cell carcinomas induced in rats by the carcinogen 4-nitroquinoline N-oxide. The capacity to express anchorage independence varied considerably between the original cell lines and essentially increased with passage in culture. In three out of four colony-derived subpopulations, the colony-forming efficiency was significantly greater than that of the original cell lines. Xenograft subpopulations expressed higher colony-forming efficiencies than their original counterparts in only two of five cell lines. Undifferentiated tumour xenografts resulted in more homogeneous tumour-derived subpopulations, in contrast to the more heterogeneous cell lines from well-differentiated tumours. The findings demonstrate functional diversity within and between malignant rat oral epithelial cell lines and their colony- and xenograft-derived subpopulations.

The effect of 3T3 fibroblasts on the expression of anchorage independence and cornification of oral keratinocytes

This study examined the effect of 3T3 fibroblasts on the expression of anchorage independence and the degree of cornification in early cultures of three carcinoma-derived epithelial cell lines (R59, R63a, R63b) and in one cell line derived from non-malignant dysplastic epithelium where there was no evidence of invasion (R66a). The epithelial cell lines originated from the palatal (R63a, R66a) and the lingual (R59, R63b) mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. In the absence of 3T3 fibroblasts, progressive culture resulted in an increase in the colony forming efficiency (CFE) of R63a, R63b and R59 and a decrease in the percentage of cornified cells in all cell lines. 3T3 fibroblasts caused a decrease in the CFE and the degree of cornification in the 3T3-dependent cell line (R63a), particularly at the lower passages, but these parameters remained essentially unchanged by 3T3 fibroblasts in the 3T3-independent cell lines (R59, R63b). 3T3 fibroblasts did not influence the cornification of R66a and this cell line remained anchorage dependent throughout the study. The results suggest that in malignant cell lines characterised by being independent of 3T3 fibroblasts (R63b, R59) the CFE was inversely correlated to the degree of cornification. However, in the malignant cell line showing a greater dependence on support (R63a) the relationship between CFE and cornification was unclear because these parameters may have been modulated by the presence of 3T3 fibroblasts. The cell line from dysplastic non-invasive tissue (R66a) differed from its malignant counterparts in the fact that CFE and cornification were unaffected by 3T3 fibroblasts despite previous studies showing a dependence on mesenchymal support.

La+ epithelial dendritic cells during oral carcinogenesis in the rat

Epithelial dendritic cells (EDC) were examined during the induction and growth of oral squamous cell carcinomas in rats treated with the carcinogen 4-Nitroquinoline N-oxide (4NQO). Acetone-fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and a monoclonal antibody to rat Ia (MRC OX-6). After 6 months there was a significant increase in Ia+ EDC/mm2 in non-invasive palatal and lingual epithelium compared with untreated and solvent painted controls. Furthermore, after 9 months there were significantly more Ia+ EDC/mm2 in non-invasive lingual epithelium compared with invasive epithelium or the epithelium overlying/adjacent to squamous cell carcinomas of the tongue. Although there were no significant differences of Ia+ EDC/mm2 between infiltrating epithelium of lingual carcinomas and non-invasive epithelium overlying/adjacent to the tumour, these tissues did contain significantly more Ia+ EDC than lingual epithelium from either solvent-only or untreated controls. The results indicate that treatment with 4NQO stimulates an increase in Ia+ EDC numbers which, although remaining higher than in controls, is not maintained within, or adjacent to, sites of neoplastic change.

Transformation of oral keratinocytes in vitro by 4-nitroquinoline N-oxide

Normal rat oral keratinocytes have been transformed with the carcinogen 4-nitroquinoline N-oxide (4NQO) in vitro. The morphology, growth characteristics, ability to grow without anchorage and tumorigenicity in athymic mice was examined in 12 selected cell lines. Each of the lines could be assigned to one of two general groups. The first group of cell lines although showing some morphological signs of transformation and the ability to be subcultured beyond passage 15 were not anchorage independent or able to form tumours in athymic mice. The second group of cell lines showed distinct signs of morphological transformation, could be serially subcultured without 3T3 feeder cells, were anchorage independent and tumorigenic in athymic mice. Anchorage independence was more common at higher passages and with increased 4NQO treatment and correlated well with a decreased reliance on 3T3 feeder cell support. The anchorage-independent phenotype was closely associated with the ability to form tumours in athymic mice. This same sequence of phenotypic changes has been demonstrated in rat oral keratinocytes after 4NQO treatment in vivo indicating that during carcinogenesis, cell populations progress through the same stages whether proliferation occurs in vitro or in vivo. There is some evidence to suggest, however, that the time interval between stages may be altered when carcinogenesis takes place in vitro.

The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.

Loss of epithelial cell surface carbohydrates during experimental oral carcinogenesis in the rat.

Cell surface glycoconjugates were investigated in a rat model of oral chemical carcinogenesis. The lectins Griffonia simplicifolia (GS-I-B4; specific for alpha-D-galactosyl end groups) and Ulex europeus (UEA-I; specific for alpha-L-fucosyl groups) were examined microspectrofluorimetrically in the oral epithelium of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) and compared with those treated with solvent alone. After labelling with GS-I-B4, the fluorescent intensity of the basal and parabasal epithelial cells was significantly less after 9 months of 4NQO treatment and in overt squamous cell carcinomas compared to controls. The fluorescent activity of the spinous epithelial cells in the non-invasive tissues treated with 4NQO and in the well differentiated (sites of keratin elaboration) malignant epithelium of squamous cell carcinomas was unchanged after labelling with UEA-I. UEA-I failed to stain undifferentiated (areas lacking keratin) malignant epithelium. The findings indicate that alpha-D-galactosyl residues are diminished on the membranes of premalignant and malignant rat epithelial cells. The expression of alpha-L-fucosyl groups, however, remains unchanged in premalignant rat oral epithelium and is closely correlated to the presence of keratin in the malignant epithelium of squamous cell carcinomas.

Sebaceous metaplasia of rat oral epithelium during chemical carcinogenesis.

Application of the carcinogen 4-nitroquinoline N-oxide (4NQO) thrice weekly to rat oral mucosa led to development of free sebaceous glands in 91% of all carcinogen-treated animals. The palatal epithelium was the area of predilection. The entire process from the transformation of single basal cells to the formation of the multi-alveoli sebaceous glands could be followed in detail. Solvent-treated animals demonstrated only a few sebaceous glands in the gingiva of the maxillary first molars whereas control animals seemed devoid of these structures. It is suggested that de novo formation of free oral sebaceous glands is caused by a metaplastic process due to local irritants among which the carcinogen 4NQO seems particularly powerful. A correlation between sebaceous metaplasia and carcinogenesis could be demonstrated.

Focal acantholytic dyskeratosis in experimental oral carcinogenesis in rats.

Thrice weekly applications of carcinogen 4-nitroquinoline N-oxide to rat oral mucosa, induced focal acantholytic dyskeratosis (FAD) in about 50% of the animals. Mucosa overlying bone (palate and attached buccal gingiva) was the area of predilection. The number of oral foci per affected animal ranged between 1 and 3. Histologically, the experimental FAD foci showed all the characteristic features described for human focal acantholytic dyskeratosis. FAD appeared unrelated to malignancy development. It is suggested that FAD represents an induced epithelial differentiation or maturation defect caused by a variety of stimuli among which are chemical carcinogens. The present experimental model seems suitable for further histo- and cytopathogenetic studies.

1-acetoxy-4-hydroxyimino-1,4-dihydroquinoline, a reactive intermediate derived from the potent carcinogen 4-nitroquinoline N-oxide

The title compound, a new monoester of the carcinogenic metabolite 4-hydroxyamino-quinoline 1-oxide, has been prepared as its crystalline hydrochloride and its decomposition in acidic and basic media examined.

Influence of zinc on onset and progression of oral carcinogenesis in rats

Three-week-old female rats were fed 0.09 (zinc-deficient), 0.77 (zinc-adequate) or 3.98 (zinc-supplemented) mmol zinc/kg diet in three experimental groups and the palatal mucosa was painted with the water-soluble carcinogen 4-nitroquinoline N-oxide (4NQO) three times a week for 20 weeks. The zinc-supplemented diet seemed to retard the induction of carcinogenesis, whereas a low-zinc diet had the opposite effect. Once initial cellular changes had been induced the supplementary zinc seemed to accelerate their further advancement. Zinc-deficiency in animals fed a copper/zinc low-zinc diet was reflected in the plasma and liver zinc levels as well as in the copper/zinc ratio. The development of cancer was accompanied by a decrease in plasma zinc and an increase in the plasma copper/zinc ratio as well as in the liver zinc. These changes were most remarkable in the zinc-supplemented group.

Effects of the water-soluble carcinogen 4-nitroquinoline N-oxide on hamster lingual mucosa

The results of this study illustrate the variability of tissues by species and site in their responses to carcinogens. The results also suggest that it may be the susceptibility of an individual species or site rather than a fundamental difference in the reactions between fat-soluble and water-soluble carcinogens and the salivary layer which accounts for the known efficiency of 4NQO in producing oral tumors in the rat.

Differential sensitivities to a chemical carcinogen (4-nitroquinoline N-oxide) in mammalian cell lines in vitro

By comparing the sensitivity to a potent carcinogen, 4-NQO on colony-forming ability among three established cell lines in vitro, we have found that porcine kidney stable (PS) cells are more resistant to 4-NQO than the mouse L cells and Ehrlich ascites tumor cells, and mouse L cells are the most sensitive to 4-NQO. However, there are no differences in the incorporation rate of 3H-4-NQO into cells among the 3 cell lines. In a previous study, we have shown that Ehrlich ascites tumor cells were more resistant to UV than the other 2 cell lines, and PS cells were the most sensitive to UV. These results seem to indicate that the effect of UV on cells differs from that of 4-NQO, and also the reactivation process of UV-induced damage differs from that of 4-NQO-induced damage if any. It may be suspected that the difference in the reduction activity to the highly carcinogenic but less cytotoxic intermediate, 4-HAQO from 4-NQO in each cell line, causes differences in sensitivity to 4-NQO among the 3 cell lines.

Interaction of the water-soluble carcinogen 4-nitroquinoline N-oxide with DNA.

Thin-layer chromatography revealed that 4-nitroquinoline N -oxide interacts with native DNA, but not with denatured DNA's or the following macromolecules and nucleic acid derivatives: soluble RNA from Escherichia coli and from yeast, polyguanylate-cytidylate, and precursors of DNA and RNA. Polydeoxyadenylate-thymidylate, polyadenylate, and histone interacted to a lesser degree than native DNA's did. That magnesium ion, sodium ion, and proflavine did not interfere with interaction of carcinogen and DNA suggests that this interaction is not an intercalation. Interaction of 4-nitroquinoline N -oxide with DNA may be related to its carcinogenicity.