Unknown,Transcriptomics,Genomics,Proteomics

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C4b-binding protein protects beta-cells from islet amyloid polypeptide induced cytotoxicity.

ABSTRACT: Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-Î²-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-ÎºB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis. The INS-1 cells were grown as 5 separate clones for 10 passages before plating in a 12-well plate (Nunc) at 100.000 cells per well and grown in complete RPMI 1640 medium to 70% confluency for approximately 48 h. The cells were then challenged by adding 77 Î¼M monomeric IAPP alone or together with C4BP (0.6 Î¼M). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 Î¼M) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).

ORGANISM(S): Rattus norvegicus

SUBMITTER: Petter Storm

PROVIDER: E-GEOD-79224 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Sigma-2-ligands (S2L) are characterized by high-affinity interaction with their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes following chemical conjugation (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across pancreatic, ovarian, and synovial cancer cell lines. While C6 and C10 displayed distinct cytotoxicities (extended aliphatic side chains), C0 (no aliphatic side chain) and ISO1 (structurally unrelated) were essentially non-toxic, enabling us to investigate the unique mechanism of S2L cytotoxicity. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced an increase in intralysosomal free cholesterol, a corresponding increase in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking and processing, and cytotoxicity due to cholesterol excess, a phenomenon that contrasts the effects of inhibiting NPC1 function. RNA-sequencing revealed that gene clusters involved in cholesterol homeostasis and ER stress response were activated exclusively by cytotoxic S2L. ER stress markers were confirmed on qPCR, and ER stress modulators inhibited or enhanced cytotoxicity of C10 confirming the involvement of ER stress response in the cytotoxicity of S2L. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) which were also confirmed with qPCR and at the protein level and were found to be a pro-survival mechanism activated by ER stress response. Moreover, inhibiting the processes downstream this adaptive response with simvastatin resulted in synergistic treatment outcomes when combined with C10 in vitro and in vivo. Of note, the conjugates retained the S2L-mediated cholesterol phenotype of their parental ligands, which suggests that disrupting cholesterol homeostasis might contribute to the overall cytotoxicity of the drug conjugates. Furthermore, these phenomena were seen exclusively with cytotoxic S2L. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

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C4b-binding protein protects beta-cells from islet amyloid polypeptide induced cytotoxicity.

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