Cathepsin B Acts as a Dominant Execution Protease in Tumor Cell Apoptosis Induced by Tumor Necrosis Factor

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion

Improved immunolabelling of ultrathin cryosections using antibody conjugated with 1nm gold particles

During the last 5 years the diameter of the gold probes used for immuno-cytochemical staining at the electron microscopical (EM) level has been decreased. The advantage of small diameter gold probes is an overall increased labelling density. The disadvantage is a lower detectability due to the low electron density of smaller gold particles consequently an inconvenient high primary magnification needed for EM examination. Since 1 nm gold particles are barely visible by conventional EM examination the need for enlargement by silverenhancement of the gold particles has increased.In the present study of ultrathin cryosectioned material the results of immunostaining using 5 nm gold conjugated antibody and 1 nm gold conjugated antibodies are compared after silverenhancement of the 1 nm gold particles.Slices of freshly isolated mouse pituitary gland were immersion fixed for 20 min in 2 % glutaraldehyde /2 % paraformaldehyde. Blocks cryoprotected with 2.3 M sucrose were frozen in liquid nitrogen and ultra-cryosectioned on a RMC cryoultra-microtome.</jats:p

Selective depletion of heat shock protein 70 (Hsp70) activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2

Heat shock protein 70 is an antiapoptotic chaperone protein highly expressed in human breast tumors and tumor cell lines. Here, we demonstrate that the mere inhibition of its synthesis by adenoviral transfer or classical transfection of antisense Hsp70 cDNA (asHsp70) results in massive death of human breast cancer cells (MDA-MB-468, MCF-7, BT-549, and SK-BR-3), whereas the survival of nontumorigenic breast epithelial cells (HBL-100) or fibroblasts (WI-38) is not affected. Despite the apoptotic morphology as judged by electron microscopy, the asHsp70-induced death was independent of known caspases and the p53 tumor suppressor protein. Furthermore, Bcl-2 and Bcl-X(L), which protect tumor cells from most forms of apoptosis, failed to rescue breast cancer cells from asHsp70-induced death. These results show that tumorigenic breast cancer cells depend on the constitutive high expression of Hsp70 to suppress a transformation-associated death program. Neutralization of Hsp70 may open new possibilities for treatment of cancers that have acquired resistance to therapies activating the classical apoptosis pathway

Data from Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2

&lt;div&gt;Abstract&lt;p&gt;Expression and activity of lysosomal cysteine cathepsins correlate with the metastatic capacity and aggressiveness of tumors. Here, we show that transformation of murine embryonic fibroblasts with &lt;i&gt;v-H-ras&lt;/i&gt; or &lt;i&gt;c-src&lt;sup&gt;Y527F&lt;/sup&gt;&lt;/i&gt; changes the distribution, density, and ultrastructure of the lysosomes, decreases the levels of lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in an extracellular signal-regulated kinase (ERK)- and cathepsin-dependent manner, and sensitizes the cells to lysosomal cell death pathways induced by various anticancer drugs (i.e., cisplatin, etoposide, doxorubicin, and siramesine). Importantly, &lt;i&gt;K-ras&lt;/i&gt; and &lt;i&gt;erbb2&lt;/i&gt; elicit a similar ERK-mediated activation of cysteine cathepsins, cathepsin-dependent down-regulation of LAMPs, and increased drug sensitivity in human colon and breast carcinoma cells, respectively. Notably, reconstitution of LAMP levels by ectopic expression or by cathepsin inhibitors protects transformed cells against the lysosomal cell death pathway. Furthermore, knockdown of either &lt;i&gt;lamp1&lt;/i&gt; or &lt;i&gt;lamp2&lt;/i&gt; is sufficient to sensitize the cells to siramesine-induced cell death and photo-oxidation–induced lysosomal destabilization. Thus, the transformation-associated ERK-mediated up-regulation of cysteine cathepsin expression and activity leads to a decrease in the levels of LAMPs, which in turn contributes to the enhanced sensitivity of transformed cells to drugs that trigger lysosomal membrane permeabilization. These data indicate that aggressive cancers with high cysteine cathepsin levels are especially sensitive to lysosomal cell death pathways and encourage the further development of lysosome-targeting compounds for cancer therapy. [Cancer Res 2008;68(16):6623–33]&lt;/p&gt;&lt;/div&gt;</jats:p

Supplementary Figures 1-9 from Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2

Supplementary Figures 1-9 from Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2</jats:p

Supplementary Figures 1-9 from Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2

Supplementary Figures 1-9 from Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2</jats:p