Like many tumors, malignant mesothelioma exhibits significant chemoresistance and resistance to apoptosis in vivo that is not seen in current in vitro models. To study the mechanisms of this multicellular resistance, biologically relevant in vitro models are necessary. Therefore, we characterized and tested human mesothelioma tissue grown in vitro as tumor fragment spheroids. After 5-10 d in culture, fragments from each of 15 human mesothelioma tumors rounded into spheroids. The tumor fragment spheroids maintained multiple characteristics of the original tumors for up to 3 mo including the presence of viable mesothelioma cells, macrophages, and a collagenrich stroma. In 14-d-old spheroids, mesothelioma cells showed the same proliferation rate and expression of a death receptor, DR5, as in the original tumor. To determine responses to treatment, we treated tumor fragment spheroids grown from three separate tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL) plus cycloheximide, that induced near total apoptosis in three human mesothelioma cell lines (M28, REN, MS-1) grown as monolayers (94 Ϯ 6% apoptosis; mean Ϯ SEM). Compared with mesothelioma cells in monolayers, mesothelioma cells in the spheroids were resistant to TRAIL plus cycloheximide (32 Ϯ 4% apoptosis; mean Ϯ SEM). Apoptotic resistance of mesothelioma cells was significantly reduced by inhibiting either the PI3K/Akt pathway with LY294002 (47 Ϯ 6% apoptosis) or the mTOR pathway with rapamycin (50 Ϯ 17% apoptosis). We conclude that human mesothelioma can be maintained in vitro in a biologically relevant model that exhibits apoptotic resistance, thereby permitting study of its tumor biology and of novel approaches to therapy. Keywords: collagen; death receptor DR5; mTOR; multicellular resistance; PI3K/Akt survival pathway; TNF-related apoptosis-inducing ligand (TRAIL); tumor-associated macrophage; tumor fragment spheroid Resistance to apoptosis, or programmed cell death, is now considered to be a critical step in the generation and maintenance of cancer (1). Resistance to apoptosis may underlie the resistance of tumors to chemotherapy and radiotherapy (2). Mechanisms of resistance have been identified on a cellular level, for example via P-glycoprotein efflux pumps, DNA repair mechanisms, or from expression of anti-apoptotic proteins such as Bcl-2 (3). Additional mechanisms of resistance are now recognized to involve stimuli from the cell's external environment, termed multicellular resistance (3). These multicellular resistance mechanisms have been attributed to cell-cell contacts, cell-matrix contacts, and the three-dimensional shape found in tissues but (Received in original form November 14, 2004 and in final form July 30, 2005) *These authors contributed equally to this work. Two types of in vitro models used to study the complex resistance found in tumors are multicellular spheroids and tumor fragment spheroids (5, 6). In the first, cells are allowed to grow into three-dimensional structures called multicellular spheroids (5). In thes...

With the rapid increase in the use of optogenetics to investigate the nervous system, there is a high demand for a neural interface that enables 2D mapping of electrophysiological neural signals with high precision during simultaneous light stimulation. Here, a gold nanonetwork (Au NN)-based transparent neural electrocorticogram (ECoG) monitoring system is proposed as implantable neural electronics. The neural interface enables accurate 2D mapping of ECoG neural signals without any photoelectric artifact during light stimulation. By using the Au NN, not only the transmittance of the microelectrodes is increased by 81% but also a low electrochemical impedance of 33.9 kΩ at 1 kHz with improved mechanical stability is achieved. It is demonstrated that the transparent microelectrode array records multichannel in vivo neural activities with no photoelectric artifact and a high signal-tonoise ratio. Propagation of neural dynamics of optically driven neural activities is also clearly visualized using the 2D Au NN microelectrode array. This transparent, flexible ECoG microelectrode array is a promising candidate for next-generation in vitro and in vivo neural interface for 2D mapping of neural dynamics.

Tumor necrosis factor -related apoptosis inducing ligand (TRAIL) holds promise for the treatment of tumors; however, many tumors are resistant to TRAIL alone. We previously showed that resistant malignant mesothelioma cells are sensitized to TRAIL-induced apoptosis by diverse toxic insults including chemotherapy, irradiation, or protein translation inhibitors such as cycloheximide. In seeking nontoxic sensitizers for TRAIL, we tested the protein translation inhibitor anisomycin at subtoxic concentrations 10-to 100-fold below those reported to inhibit protein translation. At these low concentrations (25 ng/mL), anisomycin potently and rapidly sensitized mesothelioma cells to TRAIL-induced apoptosis. Moreover, such sensitization occurred in malignant but not in nonmalignant mesothelial cells. Sensitization by anisomycin was dependent on Bid, indicating a role for mitochondrial amplification in the apoptotic synergy with TRAIL signaling. Consistent with this, we found that anisomycin induces rapid accumulation of the BH3-only protein Bim; moreover, small interfering RNA knockdown of Bim inhibits anisomycin-induced sensitization. Bim accumulation seems not to be transcriptional; instead, it is associated with Bim phosphorylation and increased stability, both consistent with the activation of c-jun NH 2 -terminal kinase signals by anisomycin. Overall, our data indicate that the rapid and selective sensitization by anisomycin in mesothelioma cells is mediated by posttranslational potentiation of Bim, which primes the cells for apoptosis via the death receptor pathway. Such subtoxic approaches to sensitization may enhance the value of TRAIL in cancer therapy.

Our results suggest that serum leptin and adiponectin may be associated with gender and obesity regardless of development of asthma.

Background/AimsDiesel exhaust particles (DEPs) lead to elevation of reactive oxygen species, which can activate the nucleotide-binding oligomerization domain-like receptor (NLR) family members containing the pyrin domain 3 (NLRP3)-inf lammasome. In this study, we elucidated whether NLRP3 -inf lammasome is activated by DEPs and whether antioxidants (N-acetylcysteine [NAC]) could inhibit such activation.MethodsRAW 264.7 cells and ex vivo lung tissues explants obtained from elastase-induced emphysema animal models were stimulated with cigarette smoking extract (CSE), DEPs, and lipopolysaccharide, and levels of interleukin-1β (IL-1β), caspase-1 and nucleotide-binding oligomerization domain-like receptor (NLR) family members containing the pyrin domain (NLRP3)-inflammasome were assessed by Western blotting and immunohistochemistry.ResultsNAC and caspase-1 inhibitor suppressed CSE- and DEP-induced secretion of IL-1β in RAW 264.7 cells. The expression levels of the NLRP3-inflammasome and caspase-1 were upregulated in RAW 264.7 cells by stimulation with CSE and DEPs and were inhibited by NAC. CSE and DEPs increased the secretion of IL-1β in lung tissues from both the normal and elastase-induced emphysema groups. The secretion of IL-1β by CSE and DEPs was increased in the elastin-induced emphysema group more than that in the normal group (CSE: 309 ± 19 pg/mL vs. 151 ± 13 pg/mL, respectively, p < 0.05; DEP: 350 ± 24 pg/mL vs. 281 ± 15 pg/mL, respectively, p < 0.05). NAC inhibited CSE- and DEP-induced IL-1β secretion in both the normal and elastase-induced emphysema groups. NLRP3-inflammasome expression as determined by immunohistochemistry was increased by CSE and DEPs in both the normal and elastin-induced emphysema groups, and was suppressed by NAC.ConclusionsThe NLRP3-inf lammasome is activated by DEPs in ex vivo tissue explants from elastase-induced emphysema animal model, and this activation is inhibited by NAC.