Shambhunath Choudhary

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.

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 PhIPs 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.

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]

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.

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.

Oncogenic H-Ras, FK228, and exogenous H2O2 cooperatively activated the erk pathway in selective induction of human urinary bladder cancer j82 cell death

More than 35% of human urinary bladder cancers involve oncogenic H‐Ras activation. The goal of this study was to investigate the role of the ERK pathway in mediating apoptotic signals induced by oncogenic H‐Ras, FK228 treatment, and exogenous H2O2 treatment to increase Nox‐1 elevation, leading to production of intracellular reactive oxygen species (ROS) for inducing apoptosis in human bladder cancer J82 cells. Our study revealed that FK228 combined with exogenous H2O2 cooperatively induced activation of Mek1/2 and Erk1/2 to increase Nox‐1 elevation, intracellular ROS production, caspase activation, and cell death. Expression of oncogenic H‐Ras significantly increased these FK228‐ and exogenous H2O2‐induced effects. Oncogenic H‐Ras‐increased cell susceptibility to FK228 could be alternately achieved by additional treatment with exogenous H2O2. Hence, combined use of FK228 with ROS‐generating agents may apply to therapeutic strategies to preferentially kill malignant cells with or without oncogenic H‐Ras activation. ©2010 Wiley‐Liss, Inc.

Pro-apoptotic activity of oncogenic H-Ras for histone deacetylase inhibitor to induce apoptosis of human cancer HT29 cells

PurposeTo verify the pro-apoptotic activity of oncogenic H-Ras in the increased susceptibility of human cancer cells to histone deacetylase inhibitor (HDACI).MethodsThe pro-apoptotic activity of oncogenic H-Ras(V12) was verified by its ability to increase susceptibility of human colorectal adenocarcinoma HT29 cells to HDACI for inducing apoptosis and growth inhibition, assayed by various methods. The mode of action of HDACI FR901228 was studied by its ability to modulate protein phosphorylation, acetylation, and expression levels in various signaling pathways, measured by Western blot analysis.ResultsActivation of caspase-3, -7, and -8, and serine protease by FR901228 was facilitated by oncogenic H-Ras to induce apoptosis. Expression of H-Ras(V12) changed the intrinsic modulation of Raf in cells responding to FR901228 treatment. Both p21Cip1 and p27Kip1 were induced in FR901228-treated cells arrested in either the G0/G1 or G2/M phase of the cell cycle. Deacetylation of FR901228-induced acetylation of core histones was accelerated by H-Ras(V12) in cells undergoing apoptosis.ConclusionExpression of H-Ras(V12) increased susceptibility of HT29 cells to HDACI FR901228 and Trichostatin A for inducing apoptosis. The pro-apoptotic activity of H-Ras(V12) responding to HDACI indicates a potential value of this new class of anticancer agents in treating Ras-related human cancers.

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.

FK228 and oncogenic H-Ras synergistically induce Mek1/2 and Nox-1 to generate reactive oxygen species for differential cell death

To investigate the mechanism behind the pro-apoptotic ability of oncogenic H-Ras to enhance FK228-induced apoptosis, we primarily used the 10T1/2-TR-H-Ras cell line, in which ectopic expression of oncogenic H-Ras(V12) is controlled by the addition of tetracycline into cultures, and secondarily used oncogenic H-Ras-expressing MCF10A cells in our studies. Our results showed the pro-apoptotic roles of Mek1/2 activation, nicotinamide adenine dinucleotide phosphate-oxidase 1 (Nox-1) elevation, and reactive oxygen species (ROS) production in FK228-induced selective cell death of oncogenic H-Ras-expressing cells versus counterpart cells. We found that although Nox-1 elevation and ROS production played essential roles in oncogenic H-Ras-induced cell proliferation and morphological transformation, the expression of oncogenic H-Ras and FK228 treatment synergistically induced activation of Mek1/2. This activation resulted in differentially increased Nox-1 elevation and ROS production leading to selective cell death of oncogenic H-Ras-expressing cells versus counterpart cells. We also found that FK228 treatment induced mitochondrial ROS and Mek1/2 activation, bypassing Raf-1, to downstream Erk1/2, participating in the induction of selective cell death. Thus, the pro-apoptotic abilities of Mek1/2 and Nox-1 should be considered as potential targets in designing therapeutic protocols using FK228 to assure ROS-mediated cell death for treating cancer cells acquiring Ras activation.