A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 μM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 μM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5-40 μM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 μM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell.
COBISS.SI-ID: 27111975
The endocytic pathway is a system specialized for the uptake of compounds from the cell microenvironment for their degradation. It contains an arsenal of hydrolases, including proteases, which are normally enclosed in membrane-bound organelles, but if released to the cytosol can initiate apoptosis signaling pathways. Endogenous and exogenous compounds have been identified that can mediate destabilization of lysosomal membranes, and it was shown that lysosomal proteases are not only able to initiate apoptotic signaling but can also amplify the apoptotic pathways initiated in other cellular compartments. The endocytic pathway also receives cargo destined for degradation via the autophagic pathway. By recycling energy and biosynthetic substrates, and by degrading damaged organelles and molecules, the endocytic system assists the autophagic system in resisting apoptotic stimuli. Steps leading to lysosomal membrane permeabilization and subsequent triggering of cell death as well as the therapeutic potential of intervention in lysosomal membrane permeabilization are discussed.
COBISS.SI-ID: 26621479
Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (StfB) in mammary cancer, StfB null mice were crossed with transgenic PyMT mammary cancer model mice. We show that ablation of StfB resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of StfB-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed that upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking StfB exhibited a higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for StfB in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.
COBISS.SI-ID: 26964263
The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker, in HEK293 cells. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides, results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms.
COBISS.SI-ID: 26842407
Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-2) are extracellular calcium-binding proteins that belong to the BM-40 family of proteins. In this work we have identified a highly basic glycosaminoglycan-binding region in the extracellular calcium-binding (EC) domain of the SMOCs. Size-exclusion chromatography showed that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate with equilibrium dissociation constants in the lower micromolar range. Furthermore, we showed that the SMOC-1 EC domain supports the adhesion of epithelial HaCaT cells. Since heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.
COBISS.SI-ID: 36550661