High dose of extracellular ATP switched autophagy to apoptosis in anchorage-dependent and anchorage-independent hepatoma cells

Extracellular adenosine triphosphate (eATP) transduces purinergic signal and plays an important regulatory role in many biological processes, including tumor cell growth and cell death. A large amount of eATP exists in the fast-growing tumor center and inflammatory tumor microenvironment. Tumor cells could acquire anoikis resistance and anchorage independence in tumor microenvironment and further cause metastatic lesion. Whether such a high amount of eATP has any effect on the anchored and non-anchored tumor cells in tumor microenvironment has not been elucidated and is investigated in this study. Our data showed that autophagy helped hepatoma cells to maintain survival under the treatment of no more than 1 mM of eATP. Only when eATP concentration reached a relatively high level (2.5 mM), cell organelle could not be further maintained by autophagy, and apoptosis and cell death occurred. In hepatoma cells under treatment of 2.5 mM of eATP, an AMP-activated protein kinase (AMPK) pathway was dramatically activated while mTOR signaling pathway was suppressed in coordination with apoptosis. Further investigation showed that the AMPK/mTOR axis played a key role in tipping the balance between autophagy-mediated cell survival and apoptosis-induced cell death under the treatment of eATP. This work provides evidence to explain how hepatoma cells escape from eATP-induced cytotoxicity as well as offers an important clue to consider effective manipulation of cancer.     

Induction of apoptosis of detached oral squamous cell carcinoma cells by safingol. Possible role of Bim, focal adhesion kinase and endonuclease G

The protein kinase C (PKC) inhibitor safingol increased rounding and detachment of human oral squamous cell carcinoma (SCC) cells in monolayer cultures. When dissociated cells were incubated in the presence of safingol, cell adhesion was prevented and cell viability was lost gradually, while most cells survived in the absence of safingol even if their attachment was blocked by coating the culture plates with polyhydroxyethyl methacrylate. Flow cytometric analysis and agarose gel electrophoresis of cellular DNA revealed an increase in the proportion of sub-G₁ cells and DNA fragmentation, indicating that safingol induced apoptosis of dissociated cells. During the induction of apoptosis in cell suspensions by safingol, there was an increase of the pro-apoptotic BH-3 only protein Bim and decrease of pro-survival Bcl-2 family proteins Bcl-xL and mitochondrial pro-apoptogenic factor endonuclease G translocated to the nucleus. The level of phosphorylated focal adhesion kinase (FAK) required for cell survival also rapidly decreased, followed by a decrease in the protein level. The introduction of siRNA against PKCα into SAS cells resulted in an increase of Bim, a decrease of Bcl-xL, the translocation of endonuclease G, and a decrease in the phosphorylation of FAK. These results suggest that Bim, Bcl-xL, FAK and endonuclease G are involved in safingol-induced apoptosis of detached oral SCC cells. Safingol can be used to induce apoptosis with cell detachment, anoikis, of oral SCC cells.     

Foregut separation and tracheo-oesophageal malformations: The role of tracheal outgrowth, dorso-ventral patterning and programmed cell death

Foregut division—the separation of dorsal (oesophageal) from ventral (tracheal) foregut components—is a crucial event in gastro-respiratory development, and frequently disturbed in clinical birth defects. Here, we examined three outstanding questions of foregut morphogenesis. The origin of the trachea is suggested to result either from respiratory outgrowth or progressive septation of the foregut tube. We found normal foregut lengthening despite failure of tracheo-oesophageal separation in Adriamycin-treated embryos, whereas active septation was observed only in normal foregut morphogenesis, indicating a primary role for septation. Dorso-ventral patterning of Nkx2.1 (ventral) and Sox2 (dorsal) expression is proposed to be critical for tracheo-oesophageal separation. However, normal dorso-ventral patterning of Nkx2.1 and Sox2 expression occurred in Adriamycin-treated embryos with defective foregut separation. In contrast, Shh expression shifts dynamically, ventral-to-dorsal, solely during normal morphogenesis, particularly implicating Shh in foregut morphogenesis. Dying cells localise to the fusing foregut epithelial ridges, with disturbance of this apoptotic pattern in Adriamycin, Shh and Nkx2.1 models. Strikingly, however, genetic suppression of apoptosis in the Apaf1 mutant did not prevent foregut separation, indicating that apoptosis is not required for tracheo-oesophageal morphogenesis. Epithelial remodelling during septation may cause loss of cell-cell or cell-matrix interactions, resulting in apoptosis (anoikis) as a secondary consequence.     

Functional cytoplasmic domains of the Mac-1 integrin receptor in phorbol ester-treated U937 cells

The integrin receptor Mac-1 regulates adherence and survival of activated tissue macrophages but the underlying molecular mechanisms are poorly understood. Phorbol ester-induced macrophagic differentiation in U937 cells leads to surface expression of Mac-1 and its activation as well. We have attempted to determine essential amino acids for these activities in the cytoplasmic regions of CD11b and CD18 subunits by deletion mutagenesis. There was complete correlation between adherence and survival. Those deletions that lead to loss of adherence and enhanced apoptosis are truncation of CD11b before the MSEGG sequence; CD18 internal deletion of either the membrane-proximal residues before the NPLF sequence or the NPLF sequence itself; CD18 truncation of the C-terminal residues after the NPLF sequence. Unexpectedly, when the NPLF sequence and the C-terminal residues were removed together by truncation, the adherent, antiapoptotic properties were restored. These results were discussed in terms of protein interaction with Mac-1 cytoplasmic regions.     

Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C(2)C(12) myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5alpha and Hic-5beta, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5alpha is permissive to differentiation while expression of either Hic-5beta or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C(2)C(12) myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.     

An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK

Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V⁺H⁻), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V⁺H⁻ decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V⁺H⁻. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V⁺H⁻. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.     

Cytochemical and ultrastructural study of anoikis and secondary necrosis in enterocytes detached in vivo

Detachment-induced apoptosis of enterocytes (anoikis) has not been investigated in vivo. Here we describe anoikis of fish enterocytes following detachment in a septicemia by Photobacterium damselae subsp. piscicida, or following injection of its exotoxin. The in vivo study was complemented with an ex vivo time-lapse analysis using conditions duplicating the in vivo situation. Linings of enterocytes detached from intestine mucosa dissociate into isolated enterocytes which undergo caspase 3-mediated anoikis with cell rounding, loss of polarization, condensation of chromatin and fragmentation of the nuclear envelope, early swelling of mitochondria with rupture of the outer membrane, and brush border disappearance. One mechanism for brush border loss was shedding of apoptotic bodies incorporating the apical part of the enterocyte. Brush border disappearance was also associated with disassembly of the F-actin microvillar core and involved re-absorption into the cell, or expansion and vesiculation followed by shedding of microvillar fragments. The enterocyte anoikis terminates by secondary necrosis and lysis due to lack of elimination by phagocytosis of apoptosing enterocytes. The conditions prevailing in vivo in the gut lumen accelerate enterocyte secondary necrosis. Our results underscore the importance of analyzing anoikis under conditions similar to those occurring in vivo.     

Enhancing survival,engraftment, and osteogenic potential of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to their plasticity and easiness of sourcing. MSC-based treatments are generally considered a safe procedure, however, the long-term results obtained up to now are far from satisfactory. The main causes of these therapeutic limitations are inefficient homing, engraftment, and osteogenic differentiation. Many studies have proposed modifications to improve MSC engraftment and osteogenic differentiation of the transplanted cells. Several strategies are aimed to improve cell resistance to the hostile microenvironment found in the recipient tissue and increase cell survival after transplantation. These strategies could range from a simple modification of the culture conditions, known as cell-preconditioning, to the genetic modification of the cells to avoid cellular senescence. Many efforts have also been done in order to enhance the osteogenic potential of the transplanted cells and induce bone formation, mainly by the use of bioactive or biomimetic scaffolds, although alternative approaches will also be discussed. This review aims to summarize several of the most recent approaches, providing an up-to-date view of the main developments in MSC-based regenerative techniques.     

Lysosomal Degradation of CD44 Mediates Ceramide Nanoliposome-induced Anoikis and Diminished Extravasation in Metastatic Carcinoma Cells

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.