In addition, down-regulation of LAMP1/2 have been previously shown to sensitize cells BX-795 to lysosomal mediated death pathways , 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 Selleckchem Dinaciclib of LMP exist, reviewed in , 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 . Cathepsins mediate crosstalk between the lysosome to the mitochondria , where a caspase-dependent pathway is stimulated with cytochrome c release and suPF299 nmr peroxide production . With larger insults, a caspase-independent death pathway may be followed with release of cathepsins, cytosolic acidification, and mafosfamide caspase-2 activation . ROS production due to either pathway can act as both an effector and initiator of cell death. Amongst known inducers of LMP, oxidative stress itself ultimately leads to lipid peroxidation of the membrane with permeabilization . Thus, production of ROS following treatment can amplify LMP. Protection against ROS can be by antioxidants or intracellular enzymes such as superoxide dimutase, catalase, and glutathione peroxidase. NAC is an small diffusible, hydrophobic antioxidant that is a precursor to glutathione, a cellular thiol-reducing agent oxidized by glutathione peroxidase in the reduction of hydrogen peroxide to water. In this study, NAC protected against cell death by SW43 to a greater extent than α-toco, while α-toco protected against PB282 more than NAC.