Breast Cell Carcinogenesis Research Articles

Gadd45g initiates embryonic stem cell differentiation and inhibits breast cell carcinogenesis

Many self-renewal-promoting factors of embryonic stem cells (ESCs) have been implicated in carcinogenesis, while little known about the genes that direct ESCs exit from pluripotency and regulate tumor development. Here, we show that the transcripts of Gadd45 family genes, including Gadd45a, Gadd45b, and Gadd45g, are gradually increased upon mouse ESC differentiation. Upregulation of Gadd45 members decreases cell proliferation and induces endodermal and trophectodermal lineages. In contrast, knockdown of Gadd45 genes can delay mouse ESC differentiation. Mechanistic studies reveal that Gadd45g activates MAPK signaling by increasing expression levels of the positive modulators of this pathway, such as Csf1r, Igf2, and Fgfr3. Therefore, inhibition of MAPK signaling with a MEK specific inhibitor is capable of eliminating the differentiation phenotype caused by Gadd45g upregulation. Meanwhile, GADD45G functions as a suppressor in human breast cancers. Enforced expression of GADD45G significantly inhibits tumor formation and breast cancer metastasis in mice through limitation of the propagation and invasion of breast cancer cells. These results not only expand our understanding of the regulatory network of ESCs, but also help people better treatment of cancers by manipulating the prodifferentiation candidates.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) induce epigenetic alterations and promote human breast cell carcinogenesis in vitro

Gene–environment interactions are involved in the development of breast cancer, the tumor type that accounts for the majority of the cancer-related deaths among women. Here, we demonstrate that exposure to PFOS (10 µM) and PFOA (100 µM)—two contaminants ubiquitously found in human blood—for 72 h induced breast epithelial cell (MCF-10A cell line) proliferation and alteration of regulatory cell-cycle proteins (cyclin D1, CDK6, p21, p53, p27, ERK 1/2 and p38) that persisted after a multitude of cell divisions. The contaminants also promoted cell migration and invasion by reducing the levels of E-cadherin, occludin and β-integrin in the unexposed daughter cells. The compounds further induced an increase in global DNA methylation and differentially altered histone modifications, epigenetic mechanisms implicated in tumorigenesis. This mechanistic evidence for PFOS- and PFOA-induced malignant transformation of human breast cells supports a role of these abundant contaminants in the development and progression of breast cancer. Increased knowledge of contaminant-induced effects and their contribution to breast tumorigenesis is important for a better understanding of gene–environment interactions in the etiology of breast cancer.

Abstract P6-07-05: Estrogen-induced carcinogenesis of normal breast cells via regulating NRF2-mediated pathway

Abstract Background: Breast cancer is the most common malignancy in women and is known to be associated with steroid estrogen (E2). The importance of E2 in the etiology of breast cancer has been widely recognized. Most postmenopausal breast cancer is E2-dependent, mainly due to the continuous growth and proliferation of potent biological E2. E2-induced oxidative stress is involved in this carcinogenic process. The objective of the present study was to characterize the mechanism of E2-induced carcinogenesis of normal breast cells.Methods: MCF-10A and MCF-12A cells were treated with different doses of E2 for 48h, the cells were harvested and extracted for MTT assay to detect cell viability. MCF-10A and MCF-12A cells were treated with E2 for up to 48h. The 8-Hydroxydeoxyguanosine (8-OhdG) and lactate dehydrogenase (LDH) leakage were measured using the Oxiselect Oxidative DNA damage ELISA kit and LDH assay kits, respectively. The Annexin V Apoptosis Detection Kit FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were used to detect cell apoptosis. Colony formation and migration assays were performed to examine the effect on colony formation and migration of cells by E2. Intracellular reactive oxygen species (ROS) status was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method. Levels of oxidative stress markers were determined by measuring total superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (TAC) using commercial kits. Western blot analysis and RT-qPCR were conducted to respectively evaluate protein and mRNA expressions of the nuclear factor erythroid 2 like 2 (NRF2), HO-1 and NQO1.Results: In this study, we explored the E2-induced carcinogenesis of normal breast cells and uncovered the signaling pathways involved in these processes. Our results demonstrate that 5nM E2 treatment induces cell viability, colony formation, and migration, reduces cell apoptosis. The results of LDH and 8- OhdG tests reveal that E2 could reduce LDH and 8-ohdg levels. The results of ROS level and related oxidative stress indicators identity that E2 could reduce the expression of ROS, SOD, GPX, CAT, and TAC. Furthermore, the Western blot analysis and RT-qPCR results show that E2 could induce the expression of NRF2, HO-1, and NQO1.Conclusions: Taken together, these results suggest that E2-induced breast carcinogenesis via induction of NRF2-mediated pathways.Keywords: Estrogen, MCF-10A, MCF-12A, ROS, NRF2. Funding: Guangdong Natural Science Foundation No. 2017A030313719;Foundation Project of Guangzhou University of Traditional Chinese Medicine, No. XKP2019002. Citation Format: Dandan Chen, Yuzhu Zhang, Ling Zhu, Renxiong Wei, Qianjun Chen. Estrogen-induced carcinogenesis of normal breast cells via regulating NRF2-mediated pathway [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-05.

Effects of dienogest on breast : MCF cell line data

Dienogest (DNG) is a widely used progestin which is safe and effective for long-term management of endometriosis. However, its association to breast cells remains to be elucidated. We perform this study to investigate whether in vitro treatment of DNG can cause any biologic changes on MCF cell line (human estrogen receptor (ER)-positive breast cancer cell line) experiments. A laboratory study. Following in vitro culture of MCF cells, we treated those cells and compared cell viability and the expression of several markers have shown to be increased in breast cancer cells between with estradiol alone and estradiol with DNG. Cell viability was measured utilizing MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and the expression of PCNA (proliferating cell nuclear antigen) and PAK4 (p21 activated kinase 4) was measured by western blot analyses. VEGF (vascular endothelial growth factor) and IL (interleukin)-32 were analyzed by ELISA, and MMP2 (matrix metalloproteinase 2) activity was assayed by zymography. In vitro treatment of MCF7 cells led to an increased cell viability by estradiol alone and decreased by both estradiol and DNG after 24 and 48-hour culture. The expression of PCNA after 48 hours showed the same result. VEGF and IL-32 were also significantly increased with estradiol and decreased following DNG treatment. However, there was no significant changes in MMP2 activity and PAK4 expression. These findings suggest that DNG may have inhibitory effects on carcinogenesis of breast cells by suppressing specific biologic changes treated by estradiol. However, further study is necessary using normal human breast cells.

Long-term exposure to extremely low-dose of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induce non-malignant breast epithelial cell transformation through activation of the a9-nicotinic acetylcholine receptor-mediated signaling pathway.

Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in cases of second-hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non-tumorigenic breast epithelial cell line, HBL-100 used for a time-course assay, was exposed to very low levels of either Nic or NNK, or both. The time-course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL-100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48 hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL-100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9-nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells, increase Nanog expression and mammosphere-forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.

TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro.

Lifeguard (LFG) is a transmembrane protein which is highly expressed in tissues of the hippocampus and the cerebellum, especially during postnatal development. This protein is responsible for the protection of neurons against Fas-induced apoptosis, and the same effect can be seen in tumor cells derived from mastocarcinoma. However, the molecular function of LFG and its regulation in the carcinogenesis of human breast cells remains to be elucidated. In the present study, we investigated the connection of the interaction of LFG within an array analysis of over 9,000 different proteins. Results showed an interaction between the proteins tripartite motif-containing 21 (TRIM21) and LFG and a negative regulatory effect of TRIM21 towards LFG on the protein level. Furthermore, Fas-induced apoptosis decreased upon the addition of TRIM21 to the cultured cells. These results revealed TRIM21 to be a negative modulator of LFG in cells of mastocarcinoma in vitro. For all analyses, MDA-MB-231 cells were used. The interaction of TRIM21 and LFG was analyzed by co-immunoprecipitation. To examine changes in regulatory processes, western blot analyses, real-time PCR, activity of apoptotic process and flow cytometric analyses were carried out.

Reactive oxygen species-mediated breast cell carcinogenesis enhanced by multiple carcinogens and intervened by dietary ergosterol and mimosine

Most breast cancers occur sporadically due to long-term exposure to low-dose carcinogens in the diet and the environment. Specifically, smoke, polluted air, and high-temperature cooked meats comprise multiple carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[α]pyrene (B[α]P), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). We sought to determine if these carcinogens act together to induce breast cell carcinogenesis, and if so, whether noncytotoxic dietary agents could intervene. We demonstrated that coexposure to physiologically achievable doses of NNK, B[α]P, and PhIP (NBP) holistically enhanced initiation and progression of breast cell carcinogenesis. Reactive oxygen species (ROS) and activation of the ERK pathway were transiently induced by NBP in each exposure, and cross talk between reinforced ROS elevation and ERK activation played an essential role in increased DNA oxidation and damage. After cumulative exposures to NBP, this cross talk contributed to enhanced initiation of cellular carcinogenesis and led to enhanced acquisition of cancer-associated properties. Using NBP-induced transient changes, such as ROS elevation and ERK pathway activation, and cancer-associated properties as targeted endpoints, we revealed, for the first time, that two less-studied dietary compounds, ergosterol and mimosine, at physiologically achievable noncytotoxic levels, were highly effective in intervention of NBP-induced cellular carcinogenesis. Combined ergosterol and mimosine were more effective than individual agents in blocking NBP-induced transient endpoints, including ROS-mediated DNA oxidation, which accounted for their preventive ability to suppress progression of NBP-induced cellular carcinogenesis. Thus, dietary components, such as mushrooms containing ergosterol and legumes containing mimosine, should be considered for affordable prevention of sporadic breast cancer associated with long-term exposure to environmental and dietary carcinogens.

Chronic exposure to combined carcinogens enhances breast cell carcinogenesis with mesenchymal and stem-like cell properties.

Breast cancer is the most common type of cancer affecting women in North America and Europe. More than 85% of breast cancers are sporadic and attributable to long-term exposure to small quantities of multiple carcinogens. To understand how multiple carcinogens act together to induce cellular carcinogenesis, we studied the activity of environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), and dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) using our breast cell carcinogenesis model. Our study revealed, for the first time, that combined NNK and B[a]P enhanced breast cell carcinogenesis chronically induced by PhIP in both non-cancerous and cancerous breast cells. Co-exposure was more potent than sequential exposure to combined NNK and B[a]P followed by PhIP in inducing carcinogenesis. Initiation of carcinogenesis was measured by transient endpoints induced in a single exposure, while progression of carcinogenesis was measured by acquisition of constitutive endpoints in cumulative exposures. Transient endpoints included DNA damage, Ras-Erk-Nox pathway activation, reactive oxygen species elevation, and increased cellular proliferation. Constitutive endpoints included various cancer-associated properties and signaling modulators, as well as enrichment of cancer stem-like cell population and activation of the epithelial-to-mesenchymal transition program. Using transient and constitutive endpoints as targets, we detected that a combination of the green tea catechins ECG and EGCG, at non-cytotoxic levels, was more effective than individual agents in intervention of cellular carcinogenesis induced by combined NNK, B[a]P, and PhIP. Thus, use of combined ECG and EGCG should be seriously considered for early intervention of breast cell carcinogenesis associated with long-term exposure to environmental and dietary carcinogens.

Abstract 1585: Chronic induction of breast cell carcinogenesis by multiple environmental and dietary carcinogens

Abstract Breast cancer, the most common type of cancer among North American and European women, is mostly sporadic and attributable to long-term exposure to small quantities of multiple carcinogens. To understand how multiple carcinogens act together to induce breast cell carcinogenesis, we investigated the activity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the environmental carcinogen benzo[a]pyrene (B[a]P), and the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Non-cancerous human breast epithelial MCF10A cells were exposed to NNK, B[a]P, and PhIP sequentially or in combination and then analyzed for the acquisition of cancerous properties/endpoints. Transient endpoints included DNA damage, Ras-Erk-Nox pathway activation, reactive oxygen species (ROS) elevation, and increased cell proliferation. Constitutive endpoints included reduced dependence on growth factors, anchorage-independent growth, Ras-Erk-Nox pathway activation, and increased ROS and cell proliferation. To detect agents that can intervene with cellular carcinogenesis induced by NNK, B[a]P, and PhIP, we investigated the activity of green tea catechins effective in blocking these cancerous endpoints. Our study revealed, for the first time, that combined NNK and B[a]P enhanced breast cell carcinogenesis chronically induced by PhIP. Co-exposure was more potent than sequential exposure to induce initiation of cellular carcinogenesis measured by induction of transient endpoints in a single exposure and progression of carcinogenesis measured by cellular acquisition of constitutive endpoints in cumulative exposures. Our study also revealed that combined ECG and EGCG, at non-cytotoxic levels, was more effective than individual agents in intervention of cellular carcinogenesis induced by combined NNK, B[a]P, and PhIP. Thus, co-exposure to NNK, B[a]P, and PhIP should be critically examined in epidemiological studies to reveal the impact of these carcinogens in the development of sporadic breast cancer. Use of combined ECG and EGCG should be seriously considered for chemoprevention of breast cell carcinogenesis associated with long-term exposure to environmental and dietary carcinogens. Citation Format: Lenora A. Pluchino, Hwa-Chain R. Wang. Chronic induction of breast cell carcinogenesis by multiple environmental and dietary carcinogens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1585. doi:10.1158/1538-7445.AM2014-1585

Effect of green tea catechins on breast carcinogenesis

Catechins (flavan-3-oils) are the main flavonoids present in green tea. The potential cancer chemopreventive and therapeutic properties of green tea catechins have been the focus of research efforts in the last two decades. This systematic review aims to generate in vitro and in vivo data on the effect of green tea catechins on breast carcinogenesis. Electronic databases were searched with the appropriate search terms. Existing evidence suggests that green tea catechins modulate breast cell carcinogenesis. The effect of green tea catechins on breast cell carcinogenesis has been investigated in different experimental models and under different experimental conditions, that is, carcinogen investigated, green tea catechin dosage regimen, treatment with green tea extract versus pure synthetic EGCG, and time point of treatment with green tea catechins in relation to the exposure to the carcinogen. Although the effect of green tea catechins was not always statistically significant, the protective effect of green tea catechins was demonstrated in all the trials, suggesting that treatment with green tea catechins should be further investigated in the clinical setting of chemoprevention of high-risk women. However, it should be emphasized that the reported actions of green tea catechins are observed in high concentrations that are difficult to achieve in the clinical setting. This drawback could be overcome by designing green tea catechins with better bioavailability and/or by cotreatment combining breast cancer endocrine treatment with green tea catechins.

Sensitising Breast Cancer Cells to Chemotherapy by Down Regulation of Lifeguard

Introduction: Lifeguard (LFG) is an anti-apoptotic protein that inhibits programmed cell death mediated by Fas in tumour cells. The exact mechanism of action and the molecular function from LFG in the carcinogenesis of human breast cells is not clear. But the expression of LFG mRNA correlates with LEF-1 transcription factor activity. Methods: In the present study, chemotherapeutic-induced apoptotic effects were studied using MCF-7 cells as an in vitro test model. Molecular (Western blot and RT-PCR) techniques were used to investigate LFG expression. To investigate the breast cancer cell proliferation in presence of siRNA-LFG we performed fluorescent cell viability assays. Results: The results indicated that, a decrease of LFG expression using siRNA correlates with an increased sensitivity to Trastzumab and Erlotinib. Moreover, cell cycle analysis of LEF-1 siRNA-transfected human breast cancer cells revealed a significant arrest in G2 phase. Conclusion: Taken together, our results indicate a pivotal role of LFG in the regulation of apoptosis in MCF-7 breast cancer cells.

Breast Cell Carcinogenesis Model for Intervention

Carcinogenesis is a multiyear, multistep, multipath disease process with progressive genetic and epigenetic alterations and associated tissue damage. More than 85% of breast cancers are sporadic and attributable to long-term exposure to small quantities of environmental carcinogenic substances. We have developed a chronically-induced cellular carcinogenesis model to mimic sporadic breast cancer development. This model allows us to identify carcinogenic substances, at physiologically-achievable low levels, capable of inducing breast cell carcinogenesis. With this model, we are able to identify cellular, biochemical, and molecular endpoints transiently and constitutively induced by single and cumulative exposures, respectively, thereby identifying carcinogenic substances involved in induction and progression of carcinogenesis. Using our model, we are also able to identify preventive agents effective in blocking these transient and constitutive carcinogenic endpoints. Thus, use of our model system will help accelerate development of novel strategies for targeted intervention of sporadic breast cancer in association with chronic exposure to low-dose carcinogenic substances, leading to a comprehensive reduction of breast cancer incidence and mortality.

Induction of human breast cell carcinogenesis by triclocarban and intervention by curcumin

More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens and co-carcinogens. To identify co-carcinogens with abilities to induce cellular pre-malignancy, we studied the activity of triclocarban (TCC), an antimicrobial agent commonly used in household and personal care products. Here, we demonstrated, for the first time, that chronic exposure to TCC at physiologically-achievable nanomolar concentrations resulted in progressive carcinogenesis of human breast cells from non-cancerous to pre-malignant. Pre-malignant carcinogenesis was measured by increasingly-acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth and increased cell proliferation, without acquisition of cellular tumorigenicity. Long-term TCC exposure also induced constitutive activation of the Erk–Nox pathway and increases of reactive oxygen species (ROS) in cells. A single TCC exposure induced transient induction of the Erk–Nox pathway, ROS elevation, increased cell proliferation, and DNA damage in not only non-cancerous breast cells but also breast cancer cells. Using these constitutively- and transiently-induced changes as endpoints, we revealed that non-cytotoxic curcumin was effective in intervention of TCC-induced cellular pre-malignancy. Our results lead us to suggest that the co-carcinogenic potential of TCC should be seriously considered in epidemiological studies to reveal the significance of TCC in the development of sporadic breast cancer. Using TCC-induced transient and constitutive endpoints as targets will likely help identify non-cytotoxic preventive agents, such as curcumin, effective in suppressing TCC-induced cellular pre-malignancy.

Abstract 2581: Intervention of chronically-induced human breast cell carcinogenesis.

Abstract The purpose of our study was to investigate the ability of dipyridamole (DPM) [2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-(5,4-d)-pyrimidine] to intervene in breast cell carcinogenesis induced by chronic exposure to environmental carcinogens. More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens through a multistep and multiyear disease process. Therefore, it is urgent to identify preventive agents effective in intervention of cellular carcinogenesis. DPM, an antioxidant and a synthetic inhibitor of phosphodiesterase, is approved by the Food and Drug Administration to inhibit platelet aggregation and vasoconstriction. DPM is also able to enhance the cytotoxicity of anticancer agents, such as cisplatin, adriamycin, and 5-fluorouracil, in various cell types. However, its ability to block breast cell carcinogenesis has not been determined. Using our chronically-induced breast cell carcinogenesis model as a target system, we found that DPM, at a non-cytotoxic physiologically-achievable dose of 10 nmol/L, effectively blocked breast cell carcinogenesis induced by chronic exposure to three unrelated carcinogens: 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), benzo[a]pyrene (B[a]P), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The ability of DPM to block H-Ras upregulation, thus blocking ERK pathway activation, reactive oxygen species (ROS) elevation, and DNA damage in each exposure, may account for its mechanisms in intervention of carcinogenesis induced by chronic exposures to PhIP. Likewise, DPM's ability to block ROS elevation and DNA damage may account for its mechanisms in intervention of carcinogenesis chronically induced by NNK and B[a]P, as well. Our study revealed, for the first time, the novel ability of DPM to block breast cell carcinogenesis induced by three unrelated carcinogens. DPM should be seriously considered as a chemopreventive agent in development of strategies for reducing the risk of sporadic breast cancer associated with long-term exposure to environmental carcinogens. Citation Format: Shambhunath Choudhary, Shilpa Sood, Hwa-Chain Robert Wang. Intervention of chronically-induced human breast cell carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2581. doi:10.1158/1538-7445.AM2013-2581

Abstract 3706: Intervention of human breast cell carcinogenesis chronically induced by 3, 4, 4’-trichlorocarbanilide.

Abstract The purpose of this study was to investigate the ability of antimicrobial agent 3,4,4’-trichlorocarbanilide (TCC) to induce breast cell carcinogenesis, and then identify preventive agents to intervene in TCC-induced breast cell carcinogenesis. TCC is widely used in personal care products such as bath soaps, detergents, and cleansing lotions. Recent studies suggest that TCC acts as an endocrine disruptor to enhance the ability of steroid hormones (estrogen or testosterone) to induce estrogen and androgen receptor-mediated gene expression. Due to its environmental persistence and wide use, there are concerns about TCC's possible impact on human health. To determine whether TCC exposure may contribute to the development of breast cancer, we used our chronically-induced breast cell carcinogenesis model to determine the ability of TCC to induce progressive carcinogenesis of human breast epithelial MCF10A cells to acquire cancer-associated properties. We found that chronic exposure of MCF10A cells to TCC at physiologically-achievable nanomolar levels resulted in cellular acquisition of the cancer-associated properties of reduced dependence on growth factors, anchorage independent growth, and increased cell proliferation. These cellular changes were accompanied by upregulation of the ERK pathway, Nox1-induced ROS production, and DNA damage. We also found that curcumin, a component of turmeric extracted from the rhizome of the Indian herb Curcuma longa, was able to suppress TCC-induced cellular carcinogenesis. Our study revealed, for the first time, that chronic exposure to TCC was able to induce breast cell carcinogenesis, and TCC-induced cellular carcinogenesis was preventable by curcumin. Citation Format: Shilpa Sood, Shambhunath Choudhary, Hwa-Chain R. Wang. Intervention of human breast cell carcinogenesis chronically induced by 3, 4, 4’-trichlorocarbanilide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3706. doi:10.1158/1538-7445.AM2013-3706

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.

Dipyridamole intervention of breast cell carcinogenesis

Dietary prevention is a cost-efficient strategy to reduce the risk of human cancers. More than 85% breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens through a multistep and multiyear disease process. We used our chronically induced cellular carcinogenesis model as a target to search for preventive agents capable of blocking breast cell carcinogenesis. Dipyridamole (DPM), at a non-cytotoxic physiologically achievable dose of 10 nmol/L, effectively blocked breast cell carcinogenesis induced by cumulative exposures to three unrelated carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The ability of DPM to block H-Ras upregulation, thus blocking ERK pathway activation, reactive oxygen species (ROS) elevation, and DNA damage in each exposure, may account for its mechanisms in intervention of carcinogenesis induced by cumulative exposures to PhIP. Likewise, DPM's ability to block ROS elevation and DNA damage may account for its mechanisms in intervention of carcinogenesis chronically induced by NNK and B[a]P, as well. DPM is approved by the Food and Drug Administration to control platelet aggregation and vasoconstriction in patients. Our study revealed, for the first time, the novel ability of DPM to block breast cell carcinogenesis induced by three unrelated carcinogens. DPM should be seriously considered as a chemopreventive agent in development of strategies for reducing the risk of sporadic breast cancer associated with long-term exposure to environmental carcinogens.

Abstract 5467: Green tea catechin intervention of human breast cell carcinogenesis chronically induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Abstract The purpose of our study was to investigate the mechanism by which dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces breast cell carcinogenesis. More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental factors, such as carcinogens in the diet, through a multi-step disease process progressing from non-cancerous to premalignant and malignant stages. The dietary carcinogen PhIP is one of the most abundant heterocyclic amines found in high-temperature cooked meats and is recognized as a mammary carcinogen. Here, we demonstrated, for the first time, that cumulative exposures to PhIP at physiologically-achievable, pico- to nano-molar 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, ERK pathway activation, Nox-1 expression and reactive oxygen species (ROS) elevation. 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, at non-cytotoxic doses, were capable of suppressing PhIP-induced cellular carcinogenesis and tumorigenicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5467. doi:1538-7445.AM2012-5467

Abstract 5446: Green tea catechin intervention of reactive oxygen species-mediated ERK pathway activation, DNA damage, and chronically-induced breast cell carcinogenesis

Abstract Long-term exposure to low doses of environmental carcinogens contributes to sporadic human breast cancers. Epidemiologic and experimental studies indicate that green tea catechins (GTCs) may interfere with breast cancer development. We have been developing a chronically-induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous, human breast epithelial MCF10A cells to physiologically-achievable pico-molar concentrations of environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), to progressively induce cellular acquisition of cancer-associated properties, as measurable endpoints. The model is then used as a target to identify non-cytotoxic preventive agents effective in suppressing cellular carcinogenesis. Here, we report a two-step strategy that initially used short-term endpoints that were transiently-induced by a single exposure to NNK and B[a]P. These short-term endpoints were used as targets to detect the ability of individual GTC components to block carcinogenesis. We subsequently used long-term endpoints that were constantly-induced by cumulative exposures to carcinogens as targets to verify and compare the activity of individual GTC components in suppressing carcinogenesis. We detected that a single exposure to NNK and B[a]P resulted in elevation of reactive oxygen species (ROS), leading to ERK pathway activation and induction of cell proliferation and chromosomal DNA damage. GTC components, at non-cytotoxic levels, were able to suppress chronically-induced cellular carcinogenesis by blocking carcinogen-induced ROS elevation, ERK activation, cell proliferation, and DNA damage in each exposure cycle. Our model may help accelerate the identification of agents to prevent carcinogenesis induced by long-term exposure to environmental carcinogens, thereby safely and effectively reducing the health risk of sporadic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5446. doi:1538-7445.AM2012-5446

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.