Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. METHODS: The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®). RESULTS: Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively. CONCLUSIONS: We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms.

Palladium nanoparticles induce disturbances in cell cycle entry and progression of peripheral blood mononuclear cells: paramount role of ions.

There is concern about the possible toxicity of palladium nanoparticles (Pd-NP), as they are released in the environment through many applications. We previously studied the toxicity of Pd-NP at high concentrations; here we address the possible toxicity of Pd-NP at low, subtoxic doses. In particular, we have exposed normal human PBMC entering into the first in vitro mitotic division to Pd-NP and to Pd(IV) ions to evaluate ROS generation and cell cycle progression. We have measured a statistically significant increase of intracellular ROS in Pd(IV) exposed cells, but not in Pd-NP exposed cells. TEM revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles, which appeared more conspicuous in cells exposed to Pd(IV) ions than to Pd-NP. Pd-NP were visible in the cytoplasm of Pd-NP exposed cells. Pd-NP addition was associated with a significant increase of cells within the G0/G1-phase and a significant reduction in GS- and G2/M-phases. Cells exposed to Pd(IV) ions showed a significant amplification of these cell cycle alterations. These results suggest that ions, per se or released by NPs, are the true inducers of Pd toxicity. It will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations.

JNK and macroautophagy activation by bortezomib has a pro-survival effect in primary effusion lymphoma cells.

Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER) stress and activated autophagy in Primary Effusion Lymphoma (PEL) cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK), resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

Activation of dendritic cells by tumor cell death.

A growing number of studies indicate that cell death can be either immunogenic or not, depending on its modalities, the type and the activation state of the cells, and finally, the environment where it happens. Increased understanding of the immunogenicity of cancer cell death will significantly improve the outcome of chemotherapeutic treatments.

Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes.

The transfer of melanin from melanocytes to keratinocytes is upregulated by UV radiation and modulated by autocrine and paracrine factors. Among them, the keratinocyte growth factor (KGF/FGF7) promotes melanosome transfer acting on the recipient keratinocytes through stimulation of the phagocytic process. To search for possible differences in the melanosome uptake of keratinocytes from different skin color, we analyzed the uptake kinetics and distribution pattern of fluorescent latex beads in primary cultures of light and dark skin-derived keratinocytes stimulated with KGF and we compared the direct effect of KGF on the melanosome transfer in co-cultures of human primary melanocytes with light and dark keratinocytes. KGF-promoted melanosome transfer was more significant in light keratinocytes compared to dark, due to an increased expression of KGF receptor in light skin keratinocytes. Colocalization studies performed by confocal microscopy using FITC-dextran as a phagocytic marker and fluorescent beads as well as inhibition of particle uptake by cytochalasin D, revealed that beads internalization induced by KGF occurs via actin-dependent phagocytosis. 3D image reconstruction by fluorescence microscopy and ultrastructural analysis through transmission electron microscopy showed differences in the distribution pattern of the beads in light and dark keratinocytes, consistent with the different melanosome distribution in human skin.

The maturation potential of NK cell clones toward autologous dendritic cells correlates with HMGB1 secretion.

Interaction of NK cells with autologous immature dendritic cells (iDCs) results in reciprocal activation. We have previously reported that NK cells trigger iDC to polarize and secrete IL-18; in turn, DC-activated NK cells secrete the nuclear protein/proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis. However, activated NK cells can also kill iDC. To investigate whether effector and maturative properties may coexist or segregate in different NK subsets, human NK cell clones were generated and analyzed for their effects on iDC. We found that the ability of different NK cell clones to induce iDC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from nucleus to cytoplasm. "Maturative" NK cell clones secrete HMGB1 spontaneously. It is interesting that secretion is strongly enhanced by engagement of the surface molecule NKp30 but only slightly induced by triggering of the activating NK receptor CD16. However, culturing freshly isolated NK cells for 1 week with low doses of anti-CD16 triggers the relocation of HMGB1 from nucleus to cytoplasm and its spontaneous secretion, resulting in a stronger maturation potential of the NK cells. Together, our data indicate that NK cells comprise functionally different subsets, endowed with different capacities to secrete HMGB1 and to induce maturation of autologous iDC. Nonetheless, maturation properties can be modulated by different stimuli. This suggests that depending on the environmental stimuli, NK/iDC interaction can lead to different outcomes, thus influencing immune response.

Collagen-induced platelet shape change is not affected by positive feedback pathway inhibitors and cAMP-elevating agents.

Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK.

Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.

Blocking the activity of IL-1 beta has entered the clinical arena of treating autoimmune diseases. However, a successful outcome of this approach requires a clear definition of the mechanisms controlling IL-1 beta release. These are still unclear as IL-1 beta, lacking a secretory signal peptide, follows a nonclassical pathway of secretion. Here, we analyze the molecular mechanism(s) undergoing IL-1 beta processing and release in human monocytes and provide a unifying model for the regulated secretion of the cytokine. Our data show that in a first step, pro-caspase-1 and endotoxin-induced pro-IL-1 beta are targeted in part to specialized secretory lysosomes, where they colocalize with other lysosomal proteins. Externalization of mature IL-1 beta and caspase-1 together with lysosomal proteins is then facilitated by extracellular ATP. ATP triggers the efflux of K(+) from the cell, followed by Ca(2+) influx and activation of three phospholipases: phosphatidylcholine-specific phospholipase C and calcium-independent and -dependent phospholipase A(2). Whereas calcium-independent phospholipase A(2) is involved in processing, phosphatidylcholine-specific phospholipase C and calcium-dependent phospholipase A(2) are required for secretion. Dissection of the events that follow ATP triggering allowed to demonstrate that K(+) efflux is responsible for phosphatidylcholine-specific phospholipase C induction, which in turn allows the rise in intracellular free calcium concentration required for activation of phospholipase A(2). This activation is ultimately responsible for lysosome exocytosis and IL-1 beta secretion.

KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum, intermediate compartment, and Golgi complex.

Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, where O-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms.

The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.