Maria Cekanova

Multiple mechanisms are involved in 6-gingerol-induced cell growth arrest and apoptosis in human colorectal cancer cells

6‐Gingerol, a natural product of ginger, has been known to possess anti‐tumorigenic and pro‐apoptotic activities. However, the mechanisms by which it prevents cancer are not well understood in human colorectal cancer. Cyclin D1 is a proto‐oncogene that is overexpressed in many cancers and plays a role in cell proliferation through activation by β‐catenin signaling. Nonsteroidal anti‐inflammatory drug (NSAID)‐activated gene‐1 (NAG‐1) is a cytokine associated with pro‐apoptotic and anti‐tumorigenic properties. In the present study, we examined whether 6‐gingerol influences cyclin D1 and NAG‐1 expression and determined the mechanisms by which 6‐gingerol affects the growth of human colorectal cancer cells in vitro. 6‐Gingerol treatment suppressed cell proliferation and induced apoptosis and G1 cell cycle arrest. Subsequently, 6‐gingerol suppressed cyclin D1 expression and induced NAG‐1 expression. Cyclin D1 suppression was related to inhibition of β‐catenin translocation and cyclin D1 proteolysis. Furthermore, experiments using inhibitors and siRNA transfection confirm the involvement of the PKCε and glycogen synthase kinase (GSK)‐3β pathways in 6‐gingerol‐induced NAG‐1 expression. The results suggest that 6‐gingerol stimulates apoptosis through upregulation of NAG‐1 and G1 cell cycle arrest through downregulation of cyclin D1. Multiple mechanisms appear to be involved in 6‐gingerol action, including protein degradation as well as β‐catenin, PKCε, and GSK‐3β pathways.

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.

NNK activates ERK1/2 and CREB/ATF‐1 via β‐1‐AR and EGFR signaling in human lung adenocarcinoma and small airway epithelial cells

We have shown that the tobacco nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is an agonist for β‐adrenergic receptors (β‐ARs) and increased DNA synthesis of human lung adenocarinoma cells with features of bronchiolar Clara cells by binding to these receptors. Using a cell line derived from a human pulmonary adenocarcinoma with Clara cell phenotype (PACC) and immortalized human small airway epithelial cells (HPLD1), the putative cells of origin of this cancer type, our current studies have analyzed signaling initiated by binding of NNK to the β1‐AR. NNK upregulated ERK1/2 and CREB/ATF‐1 phosphorylation in a PKA‐dependent manner in both cell lines. This response was further increased by transient overexpression of the β1‐AR. Pre‐exposure of cells to the selective β1‐AR antagonist, atenolol, attenuated the stimulatory effects of NNK, suggesting the latter upregulated ERK1/2 and CREB/ATF‐1 via this receptor. In vivo labeling and immunoprecipitation assays revealed that NNK phosphorylated the epidermal growth factor receptor (EGFR) at tyrosine residues, 991, 1068 and 1173, an effect inhibited by atenolol. The inhibitor of EGFR‐specific tyrosine kinases, AG1478, reduced NNK ability to stimulate ERK1/2 and CREB/ATF‐1. Genomic analysis of the exons 18–21 of the EGFR genes showed that no mutations were present in either gene. Collectively, our data provide evidence, for the first time, that NNK targets ERK1/2 and CREB/ATF‐1 proteins via dual signaling involving β1‐AR and EGFR pathways in PACCs and their putative cells of origin.

Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

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.

Nonsteroidal Anti-inflammatory Drug-Activated Gene-1 Expression Inhibits Urethane-Induced Pulmonary Tumorigenesis in Transgenic Mice

The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) inhibits gastrointestinal tumorigenesis in NAG-1 transgenic mice (C57/BL6 background). In the present study, we investigated whether the NAG-1 protein would alter urethane-induced pulmonary lesions in NAG-1 transgenic mice on an FVB background (NAG-1Tg+/FVB). NAG-1Tg+/FVB mice had both decreased number and size of urethane-induced tumors, compared with control littermates (NAG-1Tg+/FVB = 16 ± 4 per mouse versus control = 20 ± 7 per mouse, P < 0.05). Urethane-induced pulmonary adenomas and adenocarcinomas were observed in control mice; however, only pulmonary adenomas were observed in NAG-1Tg+/FVB mice. Urethane-induced tumors from control littermates and NAG-1Tg+/FVB mice highly expressed proteins in the arachidonic acid pathway (cyclooxygenases 1/2, prostaglandin E synthase, and prostaglandin E2 receptor) and highly activated several kinases (phospho-Raf-1 and phosphorylated extracellular signal-regulated kinase 1/2). However, only urethane-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation was decreased in NAG-1Tg+/FVB mice. Furthermore, significantly increased apoptosis in tumors of NAG-1Tg+/FVB mice compared with control mice was observed as assessed by caspase-3/7 activity. In addition, fewer inflammatory cells were observed in the lung tissue isolated from urethane-treated NAG-1Tg+/FVB mice compared with control mice. These results paralleled in vitro assays using human A549 pulmonary carcinoma cells. Less phosphorylated p38 MAPK was observed in cells overexpressing NAG-1 compared with control cells. Overall, our study revealed for the first time that the NAG-1 protein inhibits urethane-induced tumor formation, probably mediated by the p38 MAPK pathway, and is a possible new target for lung cancer chemoprevention.

Ethanol and the tobacco-specific carcinogen, NNK, contribute to signaling in immortalized human pancreatic duct epithelial cells.

Objectives: Smoking is a well-documented risk factor for pancreatic cancer. The tobacco-specific nitrosamine, NNK (4-[methylnitrosamino]-1-[3-pyridyl]-1-butanone), significantly induces pancreatic ductal adenocarcinomas in laboratory rodents. Recent observations suggest that ethanol enhances the tumorigenic effects of smoking. Ethanol consumption is associated with the development of chronic pancreatitis, also considered a predisposing factor for pancreatic ductal adenocarcinoma. Because the precise role of ethanol in pancreatic carcinogenesis is not known, this study sought to elucidate the cumulative effects of ethanol and NNK on particular signal transduction pathways that might play a role in cell proliferation in immortalized human pancreatic duct epithelial cells. Methods: The HPDE6-c7 cells are developed from pancreatic duct epithelial cells, which are the putative cells of origin of pancreatic ductal adenocarcinoma. Cell proliferation assays, Western blot, and cyclic adenosine monophosphate assays were used to demonstrate the effects of ethanol and NNK treatments on these cells. Results: Ethanol cotreatments enhanced the NNK-induced proliferation of these cells. This response was inhibited by the adenylyl cyclase, protein kinase A, mitogen-activated protein kinase (p42/p44), and epidermal growth factor receptor-specific tyrosine kinase inhibitors. Cotreatments of NNK and ethanol also increased cyclic adenosine monophosphate accumulation, cAMP response element-binding family of proteins and mitogen-activated protein kinase phosphorylation, and protein kinase A activation. Conclusions: These findings suggest a potential role for these pathways contributing to the development of smoking- and alcohol-related pancreatic carcinogenesis.

Breast Cancer Cell Proliferation Is Inhibited by BAD REGULATION OF CYCLIN D1

Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G1 to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G1 transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser75 and Ser99. Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.

Overexpressed Raf‐1 and phosphorylated cyclic adenosine 3′‐5′‐monophosphatate response element‐binding protein are early markers for lung adenocarcinoma

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer and has a high mortality. The tobacco carcinogen nicotine‐derived nitrosamine 4‐(N‐methyl‐N‐nitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) stimulates the proliferation of human PAC cells and small airway epithelial cells through β‐1 adrenorecptor‐mediated transactivation of the epidermal growth factor receptor (EGFR).

Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders – a relationship to the cellular differentiation and degeneration

BackgroundPelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles.MethodsBiopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38–74), and nine asymptomatic women (age 31–49). Cryostat sections were investigated for p27 protein expression and type I (slow twitch) and type II (fast twitch) fibers.ResultsAll fibers exhibited strong plasma membrane (and nuclear) p27 protein expression. cytoplasmic p27 expression was virtually absent in asymptomatic women. In perimenopausal symptomatic patients (ages 38–55), muscle fibers showed hypertrophy and moderate cytoplasmic p27 staining accompanied by diminution of type II fibers. Older symptomatic patients (ages 57–74) showed cytoplasmic p27 overexpression accompanied by shrinking, cytoplasmic vacuolization and fragmentation of muscle cells. The plasma membrane and cytoplasmic p27 expression was not unique to the muscle cells. Under certain circumstances, it was also detected in other cell types (epithelium of ectocervix and luteal cells).ConclusionsThis is the first report on the unusual (plasma membrane and cytoplasmic) expression of p27 protein in normal and abnormal human striated muscle cells in vivo. Our data indicate that pelvic floor disorders are in perimenopausal patients associated with an appearance of moderate cytoplasmic p27 expression, accompanying hypertrophy and transition of type II into type I fibers. The patients in advanced postmenopause show shrinking and fragmentation of muscle fibers associated with strong cytoplasmic p27 expression.