Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.
"In our current model, clustering of ligand bound death receptors was modeled in an effective manner and is governed by a free energy parameter (E dd ). Explicit simulation of lipid-mediated interactions would allow us to simulate novel strategies to target the membrane proximal signaling modules in cancer cells (Gajate and Mollinedo 2011; Song et al. 2007; Thome et al. 2012; Xiao et al. 2011; Gulbins and Kolesnick 2003). We considered the effect of cell-type specific variations in the membrane module on death ligand induced apoptotic activation. "
[Show abstract][Hide abstract] ABSTRACT: Apoptotic death pathways are frequently activated by death ligand induction and subsequent activation of the membrane proximal signaling module. Death receptors cluster upon binding to death ligands, leading to formation of a membrane proximal death-inducing-signaling-complex (DISC). In this membrane proximal signalosome, initiator caspases (caspase 8) are processed resulting in activation of both type 1 and type 2 pathways of apoptosis signaling. How the type 1/type 2 choice is made is an important question in the systems biology of apoptosis signaling. In this study, we utilize a Monte Carlo based in silico approach to elucidate the role of membrane proximal signaling module in the type 1/type 2 choice of apoptosis signaling. Our results provide crucial mechanistic insights into the formation of DISC signalosome and caspase 8 activation. Increased concentration of death ligands was shown to correlate with increased type 1 activation. We also study the caspase 6 mediated system level feedback activation of apoptosis signaling and its role in the type 1/type 2 choice. Our results clarify the basis of cell-to-cell stochastic variability in apoptosis activation and ramifications of this issue is further discussed in the context of therapies for cancer and neurodegenerative disorders.
Systems and Synthetic Biology 03/2014; 8(1):83-97. DOI:10.1007/s11693-013-9124-4
"Caspase-3 is rarely mutated in colorectal cancer (Soung et al, 2004) and is therefore an ideal marker for measuring apoptosis in these tumours. Furthermore, studies have validated the use of active caspase-3 staining as a marker for apoptosis (Sabine et al, 2012) The CC3 mAb used in this study has previously been shown to bind specifically to the cleaved fragment of capase-3 and not to the full-length protein by western blot (Cheong et al, 2003; Garnier et al, 2003; Thome et al, 2012), as well as being validated for use with paraffin-embedded immunohistochemistry (Gown and Willingham, 2002; Patel and Krimm, 2010). Caspase 3 can also be cleaved by other proteases, and this does not always lead to activation. "
[Show abstract][Hide abstract] ABSTRACT: Aim:
The primary aim was to determine the prognostic significance of apoptosis in colorectal tumour cells and tumour-associated stroma. A secondary aim was to determine whether apoptosis was related to immune surveillance.
Immunohistochemistry was performed using monoclonal antibodies recognising cleaved caspase-3 (CC3), cleaved poly (ADP-ribose) polymerase (PARP), p53, Bcl2, MHC-II, B cells (CD16), macrophages (CD68) and T cells (CD3), on a tissue microarray of 462 colorectal tumours.
Kaplan–Meier analysis demonstrated that patients with high expression of CC3 in the tumour or CC3 or cleaved PARP in tumour-associated stroma have a good prognosis. This suggests that tumour stroma is promoting tumourigenesis and that high levels of death within the stroma breaks this link. CC3 levels in the tumour correlated with cleaved PARP and MHC-II expression but not with CD16, CD68, CD3, p53 or Bcl2 expression. CC3 levels on tumour-associated stroma also correlated with cleaved PARP and MHC-II expression but not with CD16, CD68, CD3, p53 or Bcl2 expression. Tumour cells express MHC-II in response to IFN-γ, suggesting that this may be one of the initiators of apoptosis within the good prognosis tumours. Although 73% of the MHC-II-positive tumour had high levels of apoptosis, many tumours had high levels of apoptosis in the absence of MHC-II, implying that this is only one of many causes of apoptosis within tumours. On multivariate analysis, using Cox's proportional hazards model, tumour stage, vascular invasion and expression of CC3 in tumour-associated stroma were shown to be independent markers of prognosis.
This study shows that a high level of apoptosis within colorectal tumour-associated stroma is an independent marker of good prognosis.
British Journal of Cancer 04/2013; 108(10). DOI:10.1038/bjc.2013.166 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100 μM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1 hour of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100 μM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo) - liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than one minute of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14 - 18 minutes, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid rafts microdomains must take place in the plasma membrane.
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