Incomplete autophagy and increased cholesterol synthesis during neuronal cell death caused by a synthetic cannabinoid, CP-55,940.

There is a propensity for synthetic cannabinoid abuse to spread worldwide. CP-55,940, a synthetic cannabinoid having the ability to activate both CB1 and CB2 receptors, has been shown to induce cell death in neurons as well as other cells. Here we investigate molecular events underling the adverse effects of CP-55,940 on neuronal cells. Exposure of mouse neuroblastoma Neuro2a cells to 10-50 µM CP-55,940 results in concentration-dependent cell death that is not accompanied by an induction of apoptosis. CP-55,940 also stimulates autophagy, but the stimulation is not followed by an increase in autophagic degradation. Transcriptome analysis using DNA microarray revealed the increased expression of genes for the cholesterol biosynthesis pathway that is associated with the activation of SREBP-2, the master transcriptional regulator of cholesterol biosynthesis. However, free cholesterol is localized mainly to cytoplasmic structures, although it is localized to the plasma membrane in healthy cells. Thus, cellular trafficking of cholesterol seems to be somewhat disrupted in CP-55,940 stimulated cells. These results show for the first time that CP-55,940 stimulates autophagy as well as cholesterol biosynthesis, although not all the processes involved in the cellular response to CP-55,940 seem to be complete in these cells.

Increased fatty acid synthesis and disturbed lipid metabolism in Neuro2a cells after repeated cocaine exposure: A preliminary study.

Chronic use of cocaine prompts neurodegeneration and neuroinflammation. Lipids play pivotal roles in neuronal function and pathology. Although evidence correlates cocaine use with the alteration of lipid metabolism in blood and brain, the precise mechanism remains to be elucidated. In this study, we explore the effect of cocaine on neuronal fatty acid profiles in vitro. Neuro2a cells following seven days of repeated exposure to cocaine (0, 600, 800, 1000 µM) showed apoptosis-irrelevant cell death, dysregulated autophagy, activation of atypical endoplasmic reticulum stress response, increased saturated and unsaturated fatty acid synthesis, and disrupted lipid metabolism. These preliminary findings indicated the association between lipid metabolism and cocaine-induced neurotoxicity, which should be beneficial for understanding the neurotoxicity of cocaine.

Thymic involution caused by repeated cocaine administration includes apoptotic cell loss followed by ectopic adipogenesis.

Cocaine abuse has a negative impact on the immune system. To investigate the adverse effects of binge cocaine administration on lymphoid organs such as thymus and spleen, we examined the effects of repeated intravenous (i.v.) administration of cocaine on rats. Sprague Dawley rats (male, 8 weeks old) received 20 mg/kg body weight of cocaine hydrochloride per day for 7 or 14 days. In addition to a significant loss in the weight of the spleen, consistent with our previous intraperitoneal (i.p.) injection model of binge cocaine abuse (50 mg/kg cocaine for 7 days), we also found a significant loss of weight as well as apparent shrinkage of the thymus in the cocaine group. Transcriptome analysis of the thymus revealed increased expressions of genes involved in apoptosis, such as Ifi27 and Traf2, as well as decreased expressions of several genes related to lipid metabolism, such as Cd36, Adipoq, Scd1, and Fabp4, in the thymus of the cocaine group (7 days), suggesting an apoptotic loss of thymic cells as well as alterations in lipid metabolism. Paradoxically, cocaine activates PPARγ, a key transcriptional factor activating lipid metabolism, although ectopic adipogenesis was scarcely observed in the thymus. Further analysis of rats administered 20 mg/kg cocaine for 14 days revealed ectopic adipogenesis, which was accompanied with the activation of PPARγ as well as increased expression of Adipoq and Fabp4, in the thymus. Taken together, these results indicate that repeated cocaine administration induces thymic involution, which is initiated by the loss of thymic cells through apoptosis and subsequent ectopic adipocyte development.

Patulin and LL-Z1640-2 induce apoptosis of cancer cells by decreasing endogenous protein levels of Zic family member 5.

Zic family member 5 (ZIC5) is a transcription factor that promotes the survival of several cancer cell types. As ZIC5 is expressed at minimal levels in normal human adult tissues, it is a potential therapeutic target. In this study, we screened a chemical library containing 3398 compounds that includes pre-existing drugs and compounds with known effects to identify ZIC5 inhibitors. In the first screening, 18 hit compounds decreased GFP intensity in melanoma A375 cells overexpressing GFP-tagged ZIC5. In the second screening, five compounds that attenuated ZIC5 protein levels in A375 cells were identified. Among them, LL-Z1640-2 and patulin selectively induced apoptosis in melanoma cells expressing ZIC5, while only inducing very low levels of apoptosis in normal human melanocytes, which have no detectable ZIC5 expression. LL-Z1640-2 and patulin also induced apoptosis in BRAF inhibitor-resistant melanoma, pancreatic cancer, cholangiocarcinoma and colorectal cancer cells. LL-Z1640-2- and patulin-mediated suppression of melanoma proliferation were rescued by ZIC5 overexpression. These results suggest that LL-Z1640-2 and patulin are promising compounds that decrease ZIC5 expression to induce apoptosis in cancer cells.

Sustained splenic contraction after daily cocaine administration in rats.

The purpose of this study is to examine the effect of repeated cocaine administration on the whole body of rats. Rats (male, 6 weeks old, Sprague Dawley) were injected intraperitoneally with cocaine (50 mg/kg) once a day for 1, 3 or 7 days, and major organs (heart, liver, lung, brain, kidney, spleen) were excised from the sacrificed animals. During autopsy, we found a reduction in spleen size, but not other organs, in cocaine-administered rats as compared to control rats. This reduction became to be noticed at 3 day and easily perceived at 7 day. No marked changes were observed in other organs examined. H&E and EMG staining showed a tendency for a decrease in the number of red blood cells (RBCs) as well as an increase in collagen fibers in the spleens of rats treated repeatedly with cocaine. Transcriptome analysis indicated that repeated cocaine administration depletes RBCs from the spleen. Immunoblot analysis showed that cocaine increases the phosphorylation of myosin light chain (MYL) as well as the levels of transgelin, both of which are involved in the contraction of myofibrils. Collectively, these results show that repeated cocaine administration results in sustained contraction of the spleen, which leads to the release of RBCs from the spleen into circulation.