Diphtheria toxin mutant CRM197-mediated transcytosis across blood-brain barrier in vitro.
Blood-brain barrier (BBB) is specialized to limit brain drug delivery. Cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin, could act as a diphtheria toxin receptor-specific carrier protein and deliver drugs across the BBB. CRM197 has previously been shown to inhibit phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells. We therefore investigated the effects and mechanisms underlying the potential of CRM197, not only as a carrier protein for targeted drug delivery to the brain, but also for inducing signaling to affect endocytosis in endothelial cells. The hCMEC/D3 cell line had been used to establish a BBB in vitro model; the transport efficiency of CRM197 was analyzed both by association and transcytosis experiments. CRM197 was shown to prefer apical-to-basal transcytosis, which involved the caveolae-mediated pathway. The uptake of CRM197 conjugates by endothelial cells reached equilibrium after 60 min of treatment. The caveolin-1 mRNA and protein expression levels were significantly increased by CRM197. The up-regulation of caveolin-1 may be mediated by CRM197 via a PI3K/Akt dependent pathway and reduction of the phospho-FOXO1A (forkhead box O) transcription factor. Our results indicate that carrier protein CRM197-mediated delivery across the BBB is involved in the induction of FOXO1A transcriptional activity and upregulation of caveolin-1 expression.
Wang
Ping
P
Department of Neurobiology, China Medical University, Shenyang 110001, China.
Xue
Yixue
Y
Shang
Xiuli
X
Liu
Yunhui
Y
D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000378:metabolism*; D015153:Blotting, Western; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D051242:Caveolin 1; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D050505:Mutant Proteins; D012333:RNA, Messenger; C028581:CRM197 (non-toxic variant of diphtheria toxin); D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt