Detection of carcinogen-induced bladder cancer by fluorocoxib A.

BACKGROUND: Conventional cystoscopy can detect advanced stages of bladder cancer; however, it has limitations to detect bladder cancer at the early stages. Fluorocoxib A, a rhodamine-conjugated analog of indomethacin, is a novel fluorescent imaging agent that selectively targets cyclooxygenase-2 (COX-2)-expressing cancers. METHODS: In this study, we have used a carcinogen N-butyl-N-4-hydroxybutyl nitrosamine (BBN)-induced bladder cancer immunocompetent mouse B6D2F1 model that resembles human high-grade invasive urothelial carcinoma. We evaluated the ability of fluorocoxib A to detect the progression of carcinogen-induced bladder cancer in mice. Fluorocoxib A uptake by bladder tumors was detected ex vivo using IVIS optical imaging system and Cox-2 expression was confirmed by immunohistochemistry and western blotting analysis. After ex vivo imaging, the progression of bladder carcinogenesis from normal urothelium to hyperplasia, carcinoma-in-situ and carcinoma with increased Ki67 and decreased uroplakin-1A expression was confirmed by histology and immunohistochemistry analysis. RESULTS: The specific uptake of fluorocoxib A correlated with increased Cox-2 expression in progressing bladder cancer. In conclusion, fluorocoxib A detected the progression of bladder carcinogenesis in a mouse model with selective uptake in Cox-2-expressing bladder hyperplasia, CIS and carcinoma by 4- and 8-fold, respectively, as compared to normal bladder urothelium, where no fluorocoxib A was detected. CONCLUSIONS: Fluorocoxib A is a targeted optical imaging agent that could be applied for the detection of Cox-2 expressing human bladder cancer.

Aligned nanofiber material supports cell growth and increases osteogenesis in canine adipose-derived mesenchymal stem cells in vitro.

Tissue engineering shows great promise for the treatment of degenerative diseases, including bone repair. Polymer nanofibers provide a three-dimensional (3-D) scaffold for attachment and growth of mesenchymal stem cells. Increasing evidence supports that fiber alignment on scaffolds plays a major role in the viability and differentiation of stem cells. We compared the cell viability of canine adipose tissue-derived mesenchymal stem cells (cADMSCs) cultured in the aligned- (NanoAligned™) and random- (NanoECM™) oriented polycaprolactone (PCL) nanofiber-coated plates to control polystyrene tissue culture plates using a proliferation assay. Ability of the plates to induce differentiation of cADMSCs into osteocytes, adipocytes, and neurons was evaluated based on expression of the osteocyte markers, COL1A1 and osterix; adipocyte markers PPARγ2 and LPL; and neuronal marker nestin using RT-PCR. Proliferation results demonstrated that aligned-oriented PCL nanofiber-coated plates were more suitable substrate for cADMSCs after 7 days in culture compared to random-oriented PCL nanofiber-coated or control plates. Additionally, we demonstrated that both 3-D PCL nanofiber-coated plates were a better scaffold for cADMSCs differentiation into osteocytes compared to control plates. In conclusion, our results confirm that PCL nanofiber is a suitable tissue engineering material for use in regenerative medicine for canine patients in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1780-1788, 2018.

Inhibition of the PI3K/AKT Pathway Sensitizes Oral Squamous Cell Carcinoma Cells to Anthracycline-Based Chemotherapy In Vitro.

Anthracycline-based chemotherapy, such as doxorubicin (Dox), while effective against many solid tumors, is not widely used for head and neck cancers. In this study, we evaluated the efficacy of Dox, and its derivative AD198 in human, canine, and feline oral squamous cell carcinomas cells (OSCC) in vitro. Dox and AD198 had significant an anti-proliferative effect on human, canine, and feline OSCC cells in dose-dependent manner. AD198 inhibited cell proliferation more effectively than Dox in tested OSCC cells. In the human oral squamous cell carcinoma SCC25 cells, Dox and AD198 increased the production of reactive oxygen species and subsequently increased apoptosis through activation of caspase signaling pathway. Dox and AD198 increased activation of AKT, ERK1/2, and p38 MAPK signaling pathways in tested OSCC cells by dose-dependent manner. The efficacy of Dox and AD198 treatments in inhibition of cell proliferation was increased in tested OSCC when combined with PI3K/AKT inhibitor, LY294002 treatment. Inhibition of PI3K/AKT reduced Dox- and AD198-induced activation of ERK1/2 and further increased Dox- and AD198-induced phosphorylation of p38 MAPK in OSCC. Our results suggest that the anthracycline therapies, such as Dox or AD198, can be more effective for treatment of OSCC when combined with inhibitors of the PI3K/AKT pathway. J. Cell. Biochem. 118: 2615-2624, 2017. © 2016 Wiley Periodicals, Inc.

Molecular targets in urothelial cancer: detection, treatment, and animal models of bladder cancer.

Bladder cancer remains one of the most expensive cancers to treat in the United States due to the length of required treatment and degree of recurrence. In order to treat bladder cancer more effectively, targeted therapies are being investigated. In order to use targeted therapy in a patient, it is important to provide a genetic background of the patient. Recent advances in genome sequencing, as well as transcriptome analysis, have identified major pathway components altered in bladder cancer. The purpose of this review is to provide a broad background on bladder cancer, including its causes, diagnosis, stages, treatments, animal models, as well as signaling pathways in bladder cancer. The major focus is given to the PI3K/AKT pathway, p53/pRb signaling pathways, and the histone modification machinery. Because several promising immunological therapies are also emerging in the treatment of bladder cancer, focus is also given on general activation of the immune system for the treatment of bladder cancer.

Evaluation of the role of the cyclooxygenase signaling pathway during inflammation in skin and muscle tissues of ball pythons (Python regius).

OBJECTIVE To determine degrees of production of cyclooxygenase (COX)-1 and -2 and other mediators of inflammation in noninflamed and inflamed skin and muscle tissues in ball pythons (Python regius). ANIMALS 6 healthy adult male ball pythons. PROCEDURES Biopsy specimens of noninflamed skin and muscle tissue were collected from anesthetized snakes on day 0. A 2-cm skin and muscle incision was then made 5 cm distal to the biopsy sites with a CO2 laser to induce inflammation. On day 7, biopsy specimens of skin and muscle tissues were collected from the incision sites. Inflamed and noninflamed tissue specimens were evaluated for production of COX-1, COX-2, phosphorylated protein kinase B (AKT), total AKT, nuclear factor κ-light-chain-enhancer of activated B cells, phosphorylated extracellular receptor kinases (ERKs) 1 and 2, and total ERK proteins by western blot analysis. Histologic evaluation was performed on H&E-stained tissue sections. RESULTS All biopsy specimens of inflamed skin and muscle tissues had higher histologic inflammation scores than did specimens of noninflamed tissue. Inflamed skin specimens had significantly greater production of COX-1 and phosphorylated ERK than did noninflamed skin specimens. Inflamed muscle specimens had significantly greater production of phosphorylated ERK and phosphorylated AKT, significantly lower production of COX-1, and no difference in production of COX-2, compared with production in noninflamed muscle specimens. CONCLUSIONS AND CLINICAL RELEVANCE Production of COX-1, but not COX-2, was significantly greater in inflamed versus noninflamed skin specimens from ball pythons. Additional research into the reptilian COX signaling pathway is warranted.

Effects of environmental carcinogen benzo(a)pyrene on canine adipose-derived mesenchymal stem cells.

Dogs and their owners share the same environment and are subjected to similar environmental risk factors for developing breast cancer. Adipose tissue-derived mesenchymal stem cells (ADMSCs) may affect development and progression of breast cancer. In this study, we evaluated the effects of environmental carcinogen benzo(a)pyrene (BaP) on proliferation and differentiation of ADMSCs isolated from dogs. We characterized eight canine ADMSC lines and studied the effects of BaP on cell proliferation and differentiation. BaP did not inhibit cell proliferation of ADMSCs; however, BaP significantly inhibited differentiation potential of ADMSCs into adipocytes. BaP down-regulated AhR protein levels; however, increased its translocation from the cytoplasm to nucleus and suppressed PPARγ expression during adipogenesis. BaP increased the expression of AhR signaling pathway protein, cytochrome P450 (CYP1A1) in ADMSCs. Our data suggest that canine ADMSCs are susceptible to the environmental carcinogen BaP through AhR and PPARγ signaling pathways and may contribute to canine mammary carcinogenesis.

Phosphatidylinositol- 3-kinase inhibitor induces chemosensitivity to a novel derivative of doxorubicin, AD198 chemotherapy in human bladder cancer cells in vitro.

BACKGROUND: Doxorubicin (Dox) is widely used to treat progressed bladder cancer after transurethral resection. The use of Dox-chemotherapy has been limited due to induced drug resistance and cumulative cardiotoxic effects. N-benzyladriamycin-14-valerate (AD198), a novel derivative of Dox, has a potential to become a more effective treatment than Dox by overcoming drug resistance and cardio-toxicity as shown in the rodent model of lymphoma in vivo. The purpose of this study was to compare the efficacy of Dox and AD198 and explore their mechanisms in inhibition on human bladder cancer cells in vitro. METHODS: We evaluated the effects of Dox and AD198 on cell viability of human transitional cell carcinoma (TCC) cell lines T24 and UMUC3 by MTS assay in vitro. The effects of Dox and AD198 on cell apoptosis were determined by caspase 3/7 assay, generation of reactive oxygen species (ROS), and Western Blotting (WB) analysis. RESULTS: AD198 was more effective than Dox in inhibition of cell viability of T24 and UMUC3 cells in vitro. Both Dox and AD198 significantly increased the generation of ROS and induced apoptosis in caspase-dependent and -independent manner in T24 and UMUC3 cells. AD 198 induced significantly higher production of ROS as compared to Dox in human TCC cells. Dox and AD198 activated the pro-apoptotic p38 MAPK pathway; however, on the other hand also increased phosphorylation of AKT, an anti-apoptotic signaling pathway, in T24 and UMUC3 cells. Combined treatment of PI3K inhibitor (LY294002) with Dox or AD198 inhibited cell viability of T24 and UMUC3 cells more effectively than any of drug treatments alone. CONCLUSIONS: These data suggest that AD198 as novel derivative of Dox, could be a used as effective treatment for bladder cancer. Dox and AD198 induced PI3K/AKT signaling pathway that is a one of the indicators of pro-survival and possible drug-resistance mechanisms of chemotherapies in bladder cancer. Combined therapies of Dox or AD198 with inhibitors of PI3K/AKT signaling pathway might lead to more effective treatment outcome for patients diagnosed with bladder cancer based on our in vitro experiments.

A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro.

Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo.

Animal models and therapeutic molecular targets of cancer: utility and limitations.

Cancer is the term used to describe over 100 diseases that share several common hallmarks. Despite prevention, early detection, and novel therapies, cancer is still the second leading cause of death in the USA. Successful bench-to-bedside translation of basic scientific findings about cancer into therapeutic interventions for patients depends on the selection of appropriate animal experimental models. Cancer research uses animal and human cancer cell lines in vitro to study biochemical pathways in these cancer cells. In this review, we summarize the important animal models of cancer with focus on their advantages and limitations. Mouse cancer models are well known, and are frequently used for cancer research. Rodent models have revolutionized our ability to study gene and protein functions in vivo and to better understand their molecular pathways and mechanisms. Xenograft and chemically or genetically induced mouse cancers are the most commonly used rodent cancer models. Companion animals with spontaneous neoplasms are still an underexploited tool for making rapid advances in human and veterinary cancer therapies by testing new drugs and delivery systems that have shown promise in vitro and in vivo in mouse models. Companion animals have a relatively high incidence of cancers, with biological behavior, response to therapy, and response to cytotoxic agents similar to those in humans. Shorter overall lifespan and more rapid disease progression are factors contributing to the advantages of a companion animal model. In addition, the current focus is on discovering molecular targets for new therapeutic drugs to improve survival and quality of life in cancer patients.

Animal model of naturally occurring bladder cancer: characterization of four new canine transitional cell carcinoma cell lines.

BACKGROUND: Development and further characterization of animal models for human cancers is important for the improvement of cancer detection and therapy. Canine bladder cancer closely resembles human bladder cancer in many aspects. In this study, we isolated and characterized four primary transitional cell carcinoma (K9TCC) cell lines to be used for future in vitro validation of novel therapeutic agents for bladder cancer. METHODS: Four K9TCC cell lines were established from naturally-occurring canine bladder cancers obtained from four dogs. Cell proliferation rates of K9TCC cells in vitro were characterized by doubling time. The expression profile of cell-cycle proteins, cytokeratin, E-cadherin, COX-2, PDGFR, VEGFR, and EGFR were evaluated by immunocytochemistry (ICC) and Western blotting (WB) analysis and compared with established human bladder TCC cell lines, T24 and UMUC-3. All tested K9TCC cell lines were assessed for tumorigenic behavior using athymic mice in vivo. RESULTS: Four established K9TCC cell lines: K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly were confirmed to have an epithelial-cell origin by morphology analysis, cytokeratin, and E-cadherin expressions. The tested K9TCC cells expressed UPIa (a specific marker of the urothelial cells), COX-2, PDGFR, and EGFR; however they lacked the expression of VEGFR. All tested K9TCC cell lines confirmed a tumorigenic behavior in athymic mice with 100% tumor incidence. CONCLUSIONS: The established K9TCC cell lines (K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly) can be further utilized to assist in development of new target-specific imaging and therapeutic agents for canine and human bladder cancer.

Piroxicam inhibits Masitinib-induced cyclooxygenase 2 expression in oral squamous cell carcinoma cells in vitro.

Development and characterization of animal models for human cancers is important for the improvement of diagnosis and therapy. The oral squamous cell carcinoma (OSCC) of domestic animals resembles human OSCC in many aspects; thus, cell lines derived from OSCC of cats and dogs are a valuable model for human OSCC. We characterized 1 feline OSCC (FeOSCC-Sidney) and 1 canine OSCC (K9OSCC-Abby) cell line and compared their characteristics with human OSCC cell line hSCC-25. We calculated the doubling time of the new OSCC cell lines and evaluated the expression profiles of cancer-related markers and cell-cycle proteins such as c-kit, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, epidermal growth factor receptor, cyclooxygenase (COX)-1, COX-2, and p27 by immunocytochemistry and Western blot analysis. We evaluated the effects of novel receptor tyrosine kinase inhibitor (Masitinib, AB1010) and the nonsteroidal anti-inflammatory drug piroxicam on the previously mentioned OSCC cells. Interestingly, AB1010 increased expression levels of COX-2 in all tested OSCCs. Cotreatment of piroxicam with Masitinib significantly inhibited cell proliferation of OSCC as compared to either drug alone through the c-kit and AKT signaling pathways. Piroxicam inhibited Masitinib-induced COX-2 expression in all tested OSCCs. Therefore, targeting these two signaling pathways simultaneously was more efficient for inhibition of OSCCs across these species.

Molecular imaging of cyclooxygenase-2 in canine transitional cell carcinomas in vitro and in vivo.

The enzyme COX-2 is induced at high levels in tumors but not in surrounding normal tissues, which makes it an attractive target for molecular imaging of cancer. We evaluated the ability of novel optical imaging agent, fluorocoxib A to detect urinary bladder canine transitional cell carcinomas (K9TCC). Here, we show that fluorocoxib A uptake overlapped with COX-2 expression in primary K9TCC cells in vitro. Using subcutaneously implanted primary K9TCC in athymic mice, we show specific uptake of fluorocoxib A by COX-2-expressing K9TCC xenograft tumors in vivo. Fluorocoxib A uptake by COX-2-expressing xenograft tumors was blocked by 70% (P < 0.005) when pretreated with the COX-2 selective inhibitor, celecoxib (10 mg/kg), 4 hours before intravenous administration of fluorocoxib A (1 mg/kg). Fluorocoxib A was taken up by COX-2-expressing tumors but not by COX-2-negative human UMUC-3 xenograft tumors. UMUC-3 xenograft tumors with no expression of COX-2 showed no uptake of fluorocoxib A. In addition, fluorocoxib A uptake was evaluated in five dogs diagnosed with TCC. Fluorocoxib A specifically detected COX-2-expressing K9TCC during cystoscopy in vivo but was not detected in normal urothelium. Taken together, our findings show that fluorocoxib A selectively bound to COX-2-expressing primary K9TCC cells in vitro, COX-2-expressing K9TCC xenografts tumors in nude mice, and heterogeneous canine TCC during cystoscopy in vivo. Spontaneous cancers in companion animals offer a unique translational model for evaluation of novel imaging and therapeutic agents using primary cancer cells in vitro and in heterogeneous cancers in vivo.

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.

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.

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 2.5 µg/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 GTC's 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.

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.

PURPOSE: This 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. METHODS: Human 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. RESULTS: Erk1/2, 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. CONCLUSION: Oncogenic 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.

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.

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 GSPE's 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.

Spo0B of Bacillus anthracis - a protein with pleiotropic functions.

Spo0B is an important component of the phosphorelay signal transduction pathway, the pathway involved in the initiation of sporulation in Bacillus subtilis. Bioinformatic, phylogenetic and biochemical studies showed that Spo0B of Bacillus anthracis has evolved from citrate/malate kinases. During the course of evolution, Spo0B has retained the characteristic histidine kinase boxes H, N, F, G(1) and G(2), and has acquired nucleotide-binding domains, Walker A and Walker B, of ATPases. Owing to the presence of these domains, autophosphorylation and ATPase activity was observed in Spo0B of B. anthracis. Mutational studies showed that among the six histidine residues, His13 of the H-box is involved in the autophosphorylation activity of Spo0B, whereas Lys33 of the Walker A domain is associated with the ATPase activity of the protein. Thermodynamic and binding studies of the binding of Mg-ATP to Spo0B using isothermal titration calorimetry (ITC) suggested that the binding is driven by favorable entropy changes and that the reaction is exothermic, with an apparent dissociation constant (K(d)) equal to 0.02 mm. The value of the dissociation constant (K(d) = 0.05 mm) determined by the intrinsic fluorescence of trytophan of Spo0B was similar to that obtained by ITC studies. The purified Spo0B of B. anthracis also showed nucleoside diphosphate kinase-like activity of phosphate transfer from nucleoside triphosphate to nucleoside diphosphate. This is the first evidence for Spo0B of B. anthracis as an enzyme with histidine kinase and ATPase activities, which may have important roles to play in sporulation and pathogenesis.