miR-21 and miR-31 Converge on TIAM1 to Regulate Migration and Invasion of Colon Carcinoma Cells

TGF-β promotes cell migration and invasion, an attribute that is linked to the pro-metastasis function of this cytokine in late stage cancers. The LIM 1863 colon carcinoma organoid undergoes epithelial-mesenchymal transition (EMT) in response to TGF-β. This process is markedly accelerated by TNF-α, and we found that the levels of miR-21 and miR-31 were prominently elevated under the synergistic actions of TGF-β/TNF-α. Consistent with this, overexpression of either miR-21 or miR-31 significantly enhanced the effect of TGF-β alone on LIM 1863 morphological changes. More importantly, transwell assays demonstrated the positive effects of both miR-21 and miR-31 in TGF-β regulation of LIM 1863 motility and invasiveness. Elevated levels of miR-21 and miR-31 also enhanced motility and invasiveness of other colon carcinoma cell lines. We present compelling evidence that TIAM1, a guanidine exchange factor of the Rac GTPase, is a direct target of both miR-21 and miR-31. Indeed in LIM 1863 cells, suppression of TIAM1 is required for miR-21/miR-31 to enhance cell migration and invasion. Therefore, we have uncovered miR-21 and miR-31 as downstream effectors of TGF-β in facilitating invasion and metastasis of colon carcinoma cells.     

Cot/tpl2 (MAP3K8) Mediates Myeloperoxidase Activity and Hypernociception following Peripheral Inflammation

Cot/tpl2 (also known as MAP3K8) has emerged as a new and potentially interesting therapeutic anti-inflammatory target. Here, we report the first study of Cot/tpl2 involvement in acute peripheral inflammation in vivo. Six hours after an intraplantar injection of zymosan, Cot/tpl2^−/− mice showed a 47% reduction in myeloperoxidase activity, concomitant with a 46% lower neutrophil recruitment and a 40% decreased luminol-mediated bioluminescence imaging in vivo. Accordingly, Cot/tpl2 deficiency provoked a 25–30% reduction in luminol-mediated bioluminescence and neutrophil recruitment together with a 65% lower macrophage recruitment 4 h following zymosan-induced peritonitis. Significantly impaired levels of G-CSF and GM-CSF and of other cytokines such as TNFα, IL-1β, and IL-6, as well as some chemokines such as MCP-1, MIP-1β, and keratinocyte-derived chemokine, were detected during the acute zymosan-induced intraplantar inflammatory response in Cot/tpl2^−/− mice. Moreover, Cot/tpl2 deficiency dramatically decreased the production of the hypernociceptive ligand NGF at the inflammatory site during the course of inflammation. Most importantly, Cot/tpl2 deficiency significantly reduced zymosan-induced inflammatory hypernociception in mice, with a most pronounced effect of a 50% decrease compared with wild type (WT) at 24 h following intraplantar injection of zymosan. At this time, Cot/tpl2^−/− mice showed significantly reduced NGF, TNFα, and prostaglandin E₂ levels compared with WT littermates. In conclusion, our study demonstrates an important role of Cot/tpl2 in the NGF, G-CSF, and GM-CSF production and myeloperoxidase activity in the acute inflammatory response process and its implication in inflammatory hypernociception.     

Akt-phosphorylated Mitogen-activated Kinase-activating Death Domain Protein (MADD) Inhibits TRAIL-induced Apoptosis by Blocking Fas-associated Death Domain (FADD) Association with Death Receptor 4

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.     

Altered expression of key cellular gene products accompanies development of resistance to nitric oxide

NALM-6 is a pre-B leukemia cell line sensitive to exogenous nitric oxide (NO), which enters into apoptosis during 24 h of exposure to low doses of the NO donors SNAP (100 microM) or DETA-NO (250 microM). By culturing NALM-6 with repeated and increasing concentrations of SNAP, we obtained a variant (NALM-6R) that retains >95% viability and does not enter into apoptosis during 24 h culture in the presence of up to 500 microM SNAP or 750 microM DETA-NO. A power blot screen performed with 277 antibodies on cell lysates from NALM-6 and NALM-6R cultured without NO donors served to determine the altered constitutive expression of 19 proteins in NALM-6R. Proteins affected in the less sensitive cell line NALM6-R are involved in the regulation of apoptosis, the cell cycle, cell interactions, signal transduction, cell morphology, and cell motility. This model shows that repeated exposure of tumor cells to NO may either select NO-resistant cells or contribute to NO-sensitive conversion into NO-resistant cells. The identification of the proteins that are affected during this transition may help us to define the mechanisms that are involved in cell resistance to NO-cytotoxicity which often accompany clinical progression.     

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.     

Addition of anticancer agents enhances freezing-induced prostate cancer cell death: implications of mitochondrial involvement

Recent evidence suggests that the successful treatment of prostate cancer may require adjuvant therapies. Accordingly, a better understanding of the molecular mechanisms involved in current treatments may lead to enhanced efficacy by providing a basis for adjuvant therapies. In this study, we demonstrate that the combination of sub-lethal concentrations of chemotherapeutic agents prior to freezing (-15 degrees C) in a prostate cancer cell (PC-3) model results in enhanced efficacy over either treatment alone. Morphological analysis revealed that necrosis appeared to be the prevalent mode of cell death following adjuvant (in vitro) modeling, yet molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family. Freezing results in a time-dependent increase of the antiapoptotic Bcl-2 protein, while chemotherapy results in an increase of the pro-apoptotic Bax protein. Anti-apoptotic Bcl-2 protein levels increase over 3-fold following exposure to freezing. 5-Fluorouracil (5-FU) causes pro-apoptotic Bax levels to increase 2-fold during the drug exposure. The increase in Bax was also apparent following the combination of 5-FU/freezing, while Bcl-2 levels were maintained at or below control levels. This led to a shift in the Bcl-2 to Bax ratio to a pro-death tendency. Other effective cryo/chemo combinations were also found to provide similar effects. The combination of cisplatin/freezing resulted in a 4-fold increase in the ratio of Bax to Bcl-2 when compared to controls, which represented a 2-fold increase over the 5-FU/freezing-combination model. This increase may contribute to the continued reduction in cell number observed during the 13-day recovery period. Additionally, the addition of an apoptotic caspase inhibitor was not able to protect cultures from cell death following combination treatment. In conclusion, the data suggest that both Bcl-2 and Bax may, not only, play an important role in the efficacy of the cryo/chemo combination, but also the balance between the two may determine the role and extent of system destruction.     

Disruption of a single copy of the p38alpha MAP kinase gene leads to cardioprotection against ischemia-reperfusion

The p38 mitogen-activated protein kinase (p38) is activated in the heart during ischemia-reperfusion. However, it is not clear whether the activation of p38 is the protective response or the kinase mediates the cellular damage by ischemia-reperfusion. We examined the role of p38alpha in ischemia-reperfusion injury by studying p38alpha(+/-) mice. The p38alpha protein level in the p38alpha(+/-) heart was 50+/-8.7% compared with that in the p38alpha(+/+) heart. Upon reperfusion following ischemia for 25min, p38alpha activity was transiently increased. The maximum level of p38 activity in p38alpha(+/-) was 60+/-10.5% compared with that in p38alpha(+/+). In the p38alpha(+/+) heart, 25min ischemia and 2h reperfusion resulted in necrotic injury (37.1+/-2.7% of the area at risk), whereas infarct size was drastically reduced to 7.2+/-0.7% in the p38alpha(+/-) heart. These suggested that p38alpha plays a pivotal role in the signal transduction pathway mediating myocardial cell death caused by ischemia-reperfusion.     

7-Ketocholesterol and staurosporine induce opposite changes in intracellular pH,associated with distinct types of cell death in ECV304 cells

Incubation of ECV304 cells with 7-ketocholesterol, a lipid component of oxidized low-density lipoproteins, caused a concentration- and time-dependent decrease in the number of viable cells. Other cholesterol oxides, 7 beta-hydroxycholesterol and 25-hydroxycholesterol, but not cholesterol, were only weakly cytotoxic. No evidence for activation of caspase-3 and -8, DNA laddering, or release of cytochrome c from mitochondria into the cytoplasm was obtained in 7-ketocholesterol-treated cells, indicating that cell death was not due to apoptosis. As a positive control for apoptosis, ECV304 cells were treated with staurosporine, which indeed caused significant activation of caspase-3 activity, DNA laddering, and cytochrome c release. Cellular morphology and actin cytoskeletal organization were distinctly different after exposure to the two drugs. Furthermore, staurosporine caused intracellular acidification, whereas 7-ketocholesterol induced a significant alkalinization, which was abolished by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid. In conclusion, in ECV304 cells 7-ketocholesterol induces some typical hallmarks of necrotic cell death but not of apoptosis.     

Induction of Reactive Oxygen Species in Neurons by Haloperidol

Abstract: Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT-22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT-22 cells are equally sensitive to HP with an IC₅₀ of 35 µ M in the primary neurons and 45 µ M in HT-22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as β-estradiol and vitamin E, lowers the level of ROS and protects diol and vitamin E, lowers the level of ROS and protects HT-22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca²⁺, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.     

Cellular responses of the skin and changes in plasma cortisol levels of trout (Oncorhynchus mykiss) exposed to acidified water

The skin structure and the plasma cortisol levels of trout, Oncorhynchus mykiss, were examined during 7 days of exposure to water of pH 5. By day-4 and-7, the thickness of the epidermis was significantly (P<0.05) less in acid exposed fish than in controls, and degenerative cells were common in the upper epidermal layers. Many epidermal cells exhibited signs of necrosis, and by day-7 many apoptotic cells were also present. Secretory vesicles of high electron density were abundant in the filament cells of the 3–4 outermost layers of epidermis, and intercellular spaces had increased. Mitotic figures occureed throughout the epidermis, with the exception of the outermost cell layer. Mucous cells became elongated after day-1, and later, newly differentiating mucous cells could be seen close to the skin surface, and many mucocytes contained mucosomes of high electron density. Rodlet cells were occasionally seen. Chloride cells appeared similar to those of control fish. Many leucocytes, mainly macrophages and lymphocytes, had penetrated the epidermis via the highly undulating basal lamina, and at day-7, numerous apoptotic lymphocytes were found. In the dermis, melanosomes became dispersed in the cytoplasmic extensions of melanocytes which were present in the epidermis of all acid-exposed fish. Iridocytes were rate after day-4, while fibroblasts were abundant and secreted large amounts of collagen. After 1 day of exposure to acidified water, a significant (P<0.05) elevation of the plasma cortisol level had occurred, but this subsequently declined, and had returned to control values by day-7. The changes in skin structure, however, remained throughout the whole exposure period.