Early prevention of human breast cancer associated with chronic exposure to environmental carcinogens by dietary bioactive components is an underinvestigated area. The current challenge in identification of environmental carcinogens in breast cancer development is the low doses of carcinogens involved in transformation of breast cells from noncancerous to precancerous and cancerous stages through a multiyear disease process. Our precancerous carcinogenesis-cellular model mimics chronic carcinogenesis of human breast cells induced by accumulated exposures to environmental carcinogens, and our model is able to reveal the potency of environmental carcinogens at low doses of picomolar ranges to induce cells to acquire cancer-related properties. Although accumulated exposures of noncancerous breast epithelial MCF10A cells to individual [15
] or combined NNK and B[a]P did not result in transforming cells to acquire the cancerous ability of tumorigenicity, NNK- and B[a]P-exposed cells acquired the cancer-related abilities of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption. Aberrantly increased cell survivability acquired to reduce a dependence on growth factors and to promote anchorage-independent growth can render cells into tumorigenic transformation [30
]. Cellular acquisition of acinar-conformational disruption is highly related to breast cells at precancerous stages, such as DCIS, in breast cancer development [26
]. Long-term exposure to low doses of NNK and B[a]P is conceivable to induce cells entering into a precancerous, but not cancerous, stage in chronic development of human breast cancer. Using the acquired cancer-related properties of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption as biological target endpoints, we were able to verify the ability of GSPE to suppress precancerous carcinogenesis induced by repeated exposures of cells to NNK and B[a]P in an exposure-and dose-dependent manner. GSPE alone was able to suppress approximately 50% of NNK- and B[a]P-induced precancerous carcinogenic properties as measured by cellular acquisition of cancer-related properties of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption. Such findings demonstrate that multiple, complementary preventive agents are required to enhance the blockage of NNK- and B[a]P-induced precancerous carcinogenesis. Our working system will continue to provide access to the finding of bioactive components for suppression of precancerous carcinogenesis, leading to identification of agents providing benefits for prevention of breast cancer.
Investigating molecular targets for GSPE in suppression of cellular carcinogenesis, we identified that increased HSD11B2 gene expression served as a molecular target endpoint for GSPE in suppression of NNK- and B[a]P-induced precancerous carcino-genesis. In our previous reports, upregulated gene expression of HSD11B2 was detected in MCF10A cells exposed to either B[a]P [15
] or NNK [16
]. The upregulated HSD11B2 gene expression in NNK-exposed cells was significantly reduced in cells protected by GSPE [16
]. The HSD11B2 gene expression appears to be a common molecular target endpoint of NNK and B[a]P and may also serve as a molecular target endpoint for GSPE in suppression of carcinogen-exposed cells. In this study, we showed that HSD11B2 gene expression and protein level were highly elevated in carcinogen-exposed MCF10A cells acquiring cancer-related properties induced by repeated exposures to both NNK and B[a]P, but the upregulated HSD11B2 levels were significantly reduced in GSPE-protected, carcinogen-exposed cells, as were the acquired cancer-related abilities of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption. In addition, we furthered our investigation to show that knocking down the upregulated expression in NB-P20 cells with validated HSD11B2-specific siRNAs not only reduced the elevated HSD11B2 protein level but also suppressed the acquired cancer-related ability of reduced dependence on growth factors. Apparently, upregulated HSD11B2 expression played a contributing role in NNK- and B[a]P-induced precancerous cellular carcinogenesis; thus, protection of HSD11B2 gene expression from induction by NNK and B[a]P was important in GSPE suppression of NNK- and B[a]P-induced precancerous carcinogenesis. The human HSD11B2
gene encodes a 45 kDa protein exhibiting dehydrogenase activity, which is able to deactivate glucocorticoids; expression of HSD11B2 results in inhibition of antiproliferative activity of glucocorticoids [40
]. Upregulation of HSD11B2 is detected in breast cancer cell lines and breast tumors [40
], and inhibition of HSD11B2 by glycyrrhetinic acid reduces cell proliferation of breast cancer PMC42 cells [42
]. Thus, upregulation of HSD11B2 is postulated to play a pro-proliferative role in breast cancer cell growth through suppression of the antiproliferative activity of glucocorticoids [40
]. However, although HSD11B2 was increased in carcinogen-exposed cells, we did not detect any increased cell proliferation acquired by carcinogen-exposed cells maintained in medium containing hydrocortisol (). Accordingly, upregulated HSD11B2 expression may play a novel role in cellular acquisition of cancer-related properties induced by chronic exposure to low doses of environmental carcinogens. Protection of HSD11B2 expression from upregulation may contribute to GSPE's ability to suppress cellular carcinogenesis. However, the mechanisms for HSD11B2 involvement in NNK- and B[a]P-induced precancerous cellular carcinogenesis and in GSPE-induced suppression of cellular carcino-genesis remain to be elucidated.
Studies showed that GSPE inhibits the growth of human breast cancer cells in culture
]. In contrast, we did not detect any activity of GSPE at 40 μg/mL to inhibit cell growth or to induce cell death of either parental MCF10A [16
] or NNK- and B[a]P-exposed cells; instead, GSPE at noncytotoxic concentrations of 2.5–40 μg/mL was able to reduce NNK- and B[a]P-induced cellular acquisition of cancer-related properties. Studying the preventive, noncytotoxic mechanisms for GSPE in counteracting the carcinogenic activity of NNK and B[a]P, we detected the ability of GSPE to reduce the levels of CYP1A1 and CYP1B1 in MCF10A cells. CYP1A1 and CYP1B1 have been shown to act as bioactivating enzymes for activation of NNK and B[a]P [43
]. Our finding revealed, for the first time, that GSPE was able to reduce CYP1A1 and CYP1B1 contents in breast cells exposed to NNK and B[a]P. It is possible that reduction of CYP1A1 and CYP1B1 contents may contribute to the ability of GSPE to suppress cellular acquisition of cancer-related properties induced by NNK and B[a]P. However, whether reduction of CYP1A1 and CYP1B1 contents is the noncytotoxic, preventive mechanism for GSPE in suppression of NNK- and B[a]P-induced precancerous cellular carcinogenesis remains to be determined.
Several cell systems have been developed to study cellular carcinogenesis induced by high concentrations of environmental carcinogens. For example, Russo's group used DMBA, B[a]P, methyl-N
-nitro-nitrosoguanidine, and N
-nitrosourea at micromolar concentrations to induce cellular carcinogenesis of MCF10 cells for studying gene mutations [55
]. Caruso's group used B[a]P at 1 μM to induce acquisition of anchorage-independent growth and chromosomal alterations, but not tumorigenicity, in MCF10A [23
]. Narayan et al. [57
] reported that a single-dose treatment of MCF10A cells with cigarette smoke condensate at 1 μg/mL concentration results in acquired anchorage-independent growth. Liu and Lin [58
] showed that repeated treatment of MCF10A cells with estrogenic zeranol induces cellular acquisition of anchorage-independent growth and estrogen receptor β gene expression. All these model systems can be used in a preclinical assessment of a potential preventive agent of carcinogenesis associated with occupational exposure to high doses of carcinogens. Our working system presents unique features of low-dose, exposure-dependent chronic carcinogenesis and distinct biological and molecular target endpoints for identifying dietary bioactive components, such as GSPE, and understanding their mechanisms for early prevention of precancerous carcinogenesis in breast cancer induced by repeated exposures to carcinogens at low doses as in environmental exposure. Using this model, we detected that GSPE at concentrations of 2.5–40 μg/mL was able to reduce NNK- and B[a]P-induced cellular acquisition of cancer-related abilities. Applying this cellular model will accelerate the identification of dietary bioactive components for the formulation of combined supplements that can effectively reduce the health risk of human cancers from long-term exposure to carcinogens present in environmental pollution. On the other hand, our model revealed no synergistic nor additive effects of combined use of NNK and B[a]P together on the chronic induction of cellular acquisition of cancer-related properties. Whether long-term exposure of cells to these carcinogens together increases the qualitative complexity, but not the quantitative enhancement, in alterations of genetic and epigenetic machineries in cellular carcinogenesis remains to be studied.