For instance, the structure of the N-(9-(6-Aminohexyl)-9-azabicyclo[3.3.1]-nonan-3��-yl)-N-(2-methoxy-5-methylphenyl) selleck chemicals carbamate hydrochloride (SV119) derivatives contain an alkyl extension with terminal amine group that is not present in the 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-propyl]-piperazine dihydrochloride (PB28) derivatives, a moiety that increases lysosomal membrane insertion and permeabilization [28]. The lysosomal associated membrane proteins 1 and 2 (LAMP1/2) are homologous proteins of the lysosomal membrane [21,29,30], where they are highly glycosylated and to contribute to protection of the lysosomal membrane and its proteins from the hostile constituents such as hydrogen ion and proteases [31].

In addition, down-regulation of LAMP1/2 have been previously shown to sensitize cells to lysosomal mediated death pathways [32], and we wished to confirm that sigma-2 receptor ligands act through a component of this pathway by decreasing LAMP1 expression with a lentivirus driven shRNA in Bxpc3 cells. Transformed cells had weaker lysosomes that retained less LysoTracker and the effect was additive with sigma-2 receptor ligand. Overall LysoTracker Green uptake was decreased as assessed by flow cytometry, which could have occurred by either a decreased number of lysosomes, or increased leakage across the membrane. We believe that the enhanced killing of transformed cells was due to compromise of the membrane integrity rather than decreased number of lysosomes based on the above finding that sigma-2 ligand accumulation in lysosomes is a necessary component of cell death.

LMP mediated cell death has been extensively studied recently in the context of apoptosis induction in cancer cells [22,33,34]. The exact mechanism of LMP is still undetermined, and whether it involves pore formation or selective movement of contents, dyes of increasing molecular weight and size can be differentially released indicating some selectivity to LMP. A large number of known inducers of LMP exist, reviewed in [22], and culminate in the release of proteases such as cathepsin B, D, and L, amonst others. Following treatment with sigma-2 receptor ligands, or hydroxychloroquine, we observed a near doubling of Z-RR-AMC cleavage within one hour, which was inhibited completely by CMA and CA-074-Me, supporting the above finding that uptake of the compound into the lysosome is a critical step in LMP mediated cell death.

Cancer cells can undergo both caspase-dependent and independent pathways of cell death following LMP, depending on the degree of insult [22]. Cathepsins mediate crosstalk between the lysosome to Anacetrapib the mitochondria [35], where a caspase-dependent pathway is stimulated with cytochrome c release and superoxide production [36]. With larger insults, a caspase-independent death pathway may be followed with release of cathepsins, cytosolic acidification, and caspase-2 activation [22].