Hwa-Chain Robert Wang

Green tea catechin intervention of reactive oxygen species-mediated ERK pathway activation and chronically induced breast cell carcinogenesis

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 intervene 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 bioachievable picomolar concentrations of environmental carcinogens, such as 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 end points. The model is then used as a target to identify non-cytotoxic preventive agents effective in suppression of cellular carcinogenesis. Here, we demonstrate, for the first time, a two-step strategy that initially used end points that were transiently induced by short-term exposure to NNK and B[a]P as targets to detect GTCs capable of blocking the acquisition of cancer-associated properties and subsequently used end points constantly induced by long-term exposure to carcinogens as targets to verify GTCs capable of suppressing carcinogenesis. We detected that short-term exposure to NNK and B[a]P resulted in elevation of reactive oxygen species (ROS), leading to Raf-independent extracellular signal-regulated kinase (ERK) pathway activation and subsequent induction of cell proliferation and DNA damage. These GTCs, 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 preventive agents to intervene in carcinogenesis induced by long-term exposure to environmental carcinogens, thereby safely and effectively reducing the health risk of sporadic breast cancer.

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 GSPEs 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.

Proapoptotic ability of oncogenic H-Ras to facilitate apoptosis induced by histone deacetylase inhibitors in human cancer cells

More than 35% of human urinary bladder cancers involve oncogenic H-Ras activation. In addition to tumorigenic ability, oncogenic H-Ras possesses a novel proapoptotic ability to facilitate the induction of apoptosis by histone deacetylase inhibitors (HDACI). HDACIs are a new class of anticancer agents and are highly cytotoxic to transformed cells. To understand the connection between the selectivity of HDACIs on transformed cells and the proapoptotic ability of oncogenic H-Ras to facilitate HDACI-induced apoptosis, we introduced oncogenic H-Ras into urinary bladder J82 cancer cells to mimic an acquisition of the H-ras gene activation in tumor development. Expression of oncogenic H-Ras promoted J82 cells to acquire tumorigenic ability. Meanwhile, oncogenic H-Ras increased susceptibility of J82 cells to HDACIs, including FR901228 and trichostatin A, for inducing apoptosis. The caspase pathways, the B-Raf and extracellular signal-regulated kinase pathway, p21Cip1 and p27Kip1, and core histone contents are regulated differently by FR901228 in oncogenic H-Ras–expressed J82 cells than their counterparts in parental J82 cells, contributing to the increased susceptibility to the induction of selective apoptosis. Our results lead us to a suggestion that HDACIs activate the proapoptotic ability of oncogenic H-Ras, indicating a potential therapeutic value of this new class of anticancer agents in the control of human urinary bladder cancer that has progressed to acquire oncogenic H-Ras. [Mol Cancer Ther 2007;6(3):1099–111]

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 100xa0pM. 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, inxa0vitro system to identify preventive agents that inhibit human breast cell carcinogenesis induced by chronic exposures to carcinogens.

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 2.5u2009µg/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 GTCs 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.

Role of reactive oxygen species in proapoptotic ability of oncogenic H-Ras to increase human bladder cancer cell susceptibility to histone deacetylase inhibitor for caspase induction

PurposeReveal mechanisms for the novel proapoptotic ability of oncogenic H-Ras to increase cell susceptibility to a histone deacetylase inhibitor (HDACI) FR901228 for inducing caspase activation and selective apoptosis.MethodsHuman urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal differential induction of intracellular reactive oxygen species (ROS), caspase activation, and apoptosis by HDACI FR901228. ROS levels and caspase-8, -9, and -3/7 activities were measured by flow cytometry and luminescence assays, respectively. Specific inhibitors were used to suppress caspases and ROS. Western blot analysis determined modulators of caspase pathways.ResultsROS, caspase activity, and cell death was differentially increased by FR901228 in oncogenic H-Ras-expressing J82 versus parental cells. Blocking ROS resulted in reduced FR901228-induced cell death and caspase activation. Suppression of caspase-8 resulted in reduced FR901228-activated caspase-9 and -3/7. Suppression of caspase-9 resulted in reduced FR901228-activated caspase-3/7. Although FR901228 induced an ROS-dependent increase of FasL, FasL failed to induce caspase activation and cell death.ConclusionIncreased ROS played an important role in the activation of the extrinsic and intrinsic caspase pathways to cooperatively induce executioner caspase-3/7 through a novel FasL-independent pathway in FR901228-induced selective apoptosis of oncogenic H-Ras-expressing J82 versus parental cells.

Reactive oxygen species-mediated therapeutic control of bladder cancer

Urinary bladder cancer is the fifth most common cancer in the US and the most costly cancer to manage because it requires life-long surveillance to monitor for recurrence and advanced progression. Urothelial carcinomas account for more than 90% of urinary bladder cancer cases. Transurethral resection and intravesical chemotherapy or immunotherapy are effective short-term treatments of urothelial carcinoma, but long-term management has not yet been optimized. Recent therapeutic strategies emphasize the targeted interference with aberrantly-regulated signaling modulators that result from genomic alterations. However, targeted therapeutic agents might not distinguish cancer cells from their normal counterparts, resulting in undesirable adverse effects. Thus, a new approach for the treatment of urothelial carcinoma has been suggested that differentially augments cancer-associated events, leading to selective death of cancer cells but not normal cells. Many aberrantly-regulated signaling modulators are associated with the elevation of reactive oxygen species (ROS), and an increasing number of studies report agents with the ability to induce ROS in cancer cells. Accordingly, therapeutic augmentation of ROS to a lethal level in cancer cells only would induce selective death of tumor cells but not normal cells, leading to a highly effective chemotherapy strategy for urothelial carcinoma.

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 (Pu2009<u20090.05) 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.

Distinct roles of the ERK pathway in modulating apoptosis of Ras-transformed and non-transformed cells induced by anticancer agent FR901228

Ectopic expression of oncogenic H‐Ras in cells results in increases of cell susceptibility to the anticancer agent FR901228. Investigating the roles of Ras‐induced pathways in FR901228‐induced apoptosis, we have found that the phosphatidylinositol 3‐kinase pathway plays an anti‐apoptotic role, whereas the stress‐activated protein kinase p38 pathway plays a pro‐apoptotic role in FR901228‐induced apoptosis. Interestingly, the extracellular signal‐regulated kinase (ERK) pathway plays an anti‐apoptotic role in non‐transformed cells; however, it plays a pro‐apoptotic role in Ras‐transformed cells in response to FR901228 treatment. An essential role of the ERK pathway in regulating caspase‐3 contents may contribute to its pro‐apoptotic role in Ras‐transformed cells.

Differential induction of reactive oxygen species through Erk1/2 and Nox-1 by FK228 for selective apoptosis of oncogenic H-Ras-expressing human urinary bladder cancer J82 cells

PurposeThis study sought to reveal mechanisms for differential regulation of reactive oxygen species (ROS) in histone deacetylase inhibitor FK228-induced selective apoptosis of oncogenic H-Ras-expressing human cancer cells.MethodsHuman urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal FK228-induced differential Erk1/2 activation, Nox-1 elevation, ROS production, glutathione (GSH) depletion, caspase activation, and apoptosis. Specific inhibitors were used to suppress Nox-1 activity and ROS production. Mek1/2 inhibitor was used to suppress Erk1/2 activation. Validated-specific siRNAs were used to knock down Nox-1. ROS levels, GSH levels, and caspase-3/7 activities were measured by GSH assay, flow cytometry and luminescence assays, respectively. Western blot analysis determined levels of Erk1/2 and Nox-1.ResultsErk1/2, Nox-1, ROS, caspase-3/7, and cell death were differentially induced, whereas GSH was differentially depleted by FK228 in oncogenic H-Ras-expressing J82 versus parental cells. Blockage of the ERK pathway resulted in suppressing oncogenic H-Ras- and FK228-induced Nox-1 elevation, ROS production, caspase activation, and cell death. Knockdown of Nox-1 by specific siRNAs reduced FK228-induced ROS production, caspase activation, and cell death.ConclusionOncogenic H-Ras expression and FK228 treatment synergistically induced the ERK pathway, resulting in differentially increased Nox-1 elevation, ROS production, and GSH depletion, leading to differential caspase activation and cell death in oncogenic H-Ras-expressing J82 versus parental cells.