Μελέτη των μηχανισμών δράσης της Βρωμο-αμίνης Τ στη φλεγμονή και τον καρκίνο

Abstract

Inflammation is associated with the development of acute and chronic diseases including cancer. Towards unveiling novel therapeutic options for patients with such complications, N‑bromotaurine (TauNHBr) is considered a potential anti‑inflammatory and antimicrobial agent; however, its therapeutic efficacy is hindered due to its relatively poor stability. To address this challenge, researchers have designed a stable active bromine compound named Bromamine T (BAT) and demonstrated that BAT exerts similar properties to TauNHBr. Initially, the aim of this study was to evaluate whether BAT and taurine (Tau) can display protective properties against lipopolysaccharide (LPS)‑mediated inflammation both in vitro and in vivo. Towards elucidating the anti-inflammatory effect of either agent, we employed Real-time PCR, immunofluorescence and hematoxylin & eosin experiments. Our results have shown that BAT is more effective than Tau in vitro, by using LPS‑stimulated murine J774.A1 macrophages (Mφs) and in vivo, by using an LPS‑mediated air‑pouch murine model. In particular, BAT and Tau can attenuate acute inflammation, by reducing mRNA levels of pro‑inflammatory mediators secreted by LPS-treated J774A.1 Mφs. To gain a comprehensive picture concerning the anti-inflammatory effect of either agent, our immunofluorescence experiments have shown that the underlying molecular mechanism of BAT and Tau is to prevent the nuclear translocation of activated NF‑κB transcription factor and reduce its high protein expression levels. Indeed, the protective effect of BAT appears to be superior to that of Tau in LPS‑induced J774A.1 Mφs. Furthermore, BAT and Tau seem to inhibit the LPS‑mediated inflammation in vivo, by reducing total cell and polymorphonuclear leukocyte (PMN) infiltration as well as the pouch wall thickness in mice bearing LPS-induced air-pouch. In parallel, BAT and Tau can prevent the secretion of pro‑inflammatory cytokines obtained from exudates of mice with LPS-induced air-pouch. Consistent with in vitro results, mice with LPS-mediated air-pouch treated with 9mg BAT show superior effect on hindering LPS-induced inflammation as opposed to those treated with 9mg Tau. Besides, Tau has been shown to inhibit cancer growth but, the mechanisms that underlie its growth-inhibitory effects remain obscure in both colon and breast cancer. Our further aim was to examine the cytotoxic effect of BAT and Tau on carcinogenesis. To approach that question, we used Crystal Violet, colony formation, flow cytometry and Western blot experiments to evaluate the effect of BAT and Tau on the apoptosis and autophagy of cancer cells. Xenograft experiments were used to determine the in vivo cytotoxicity of either agent. To our knowledge, this is the first study that provides convincing evidence about the greater anti-cancer effect of BAT than that of Tau and sheds light on the underlying molecular mechanisms of either agent. In particular, BAT and Tau inhibit the growth of human colon, breast, cervical and skin cancer cell lines in a dose and time-dependent manner. Our results support that BAT and Tau exert the strongest cytotoxic effect on RKO colon and MDA-MB-468 breast cancer cells, with the most promising results arising in colon cancer cells. In all cases, BAT is shown to be more effective than Tau, up to 100-fold in a statistically significant manner. Of great interest is that neither treatment with BAT nor with Tau exerts any sign of toxicity in Wharton-Jelly mesenchymal stem cells (WJ-MSCs) or HepG2 cells, thereby raising the possibility to use either agent as a putative therapeutic intervention. In colon cancer, further experiments have supported that BAT causes death, through induction of mitochondrial apoptosis and autophagy, due to the oxidative damage and the activation of distinct mitogen-activated protein kinase (MAPK) family members to a greater extent than Tau does. In particular, it has been shown that BAT increases the phosphorylation of c-Jun N-terminal kinases (JNK1/2), p38 MAPK, and extracellular-signal-regulated kinases (ERK1/2), thereby inducing mitochondrial apoptosis and autophagy in RKO cells. In contrast, it has been demonstrated that Tau activates JNK1/2 and ERK1/2, kinases, thus inducing mitochondrial apoptosis. Both substances can reduce the protein expression levels of phosphorylated Akt (Ser473). In response to BAT and Tau, DNA damage response (DDR) occurs because the total protein expression levels of phosphorylated p53 (Ser 15) and the total protein expression levels of phosphorylated H2A.X (Ser 139) are increased in BAT and Tau-treated RKO cells, as opposed to those of negative control (NC) cells. In this way, the p53 transcription factor can be rescued from rapid degradation, ultimately leading to the transcriptional transactivation of p53 target genes. Consistent with in vitro results, it has been proved that BAT and Tau display a strong cytotoxic effect of BAT by attenuating the growth of RKO xenografts in severe combined immunodeficient (SCID) mice. Comparing the excised tumor volume of groups, the mean tumor volume in the BAT-treated mice is smaller compared with that of the Tau-treated mice in a statistically significant manner. Taken together, BAT can be a novel therapeutic modality in both inflammation and cancer.

Η ταυρίνη (taurine, Tau) έχει αποδειχθεί ότι αναστέλλει την ανάπτυξη του καρκίνου. Ωστόσο, οι μηχανισμοί της ογκοκατασταλτικής της δράσης παραμένουν ασαφείς τόσο στον καρκίνο του παχέος εντέρου, όσο και στον καρκίνο του μαστού. Παράλληλα, έχει αποδειχθεί πως η Ν-βρωμοταυρίνη (N-bromotaurine, TauBr) και η Βρωμο-αμίνη Τ (bromamine T, BAT) έχουν ισχυρή αντιφλεγμονώδη δράση. Στην παρούσα διδακτορική διατριβή διερευνώνται για πρώτη φορά συγκριτικά οι μοριακοί μηχανισμοί των αντιφλεγμονωδών και των αντικαρκινικών δράσεων της Βρωμο-αμίνης T και της ταυρίνης. Αρχικά, η παρούσα μελέτη υποστηρίζει την ισχυρότερη προστατευτική δράση της Βρωμο-αμίνης T στην φλεγμονή που επάγεται από λιποπολυσακχαρίτη (lipopolysaccharide, LPS), in vitro στα LPS-επαγόμενα J774.A1 μακροφάγα και in vivo στους LPS-επαγόμενους ραχιαίους αεροθύλακες ποντικών σε σχέση με εκείνη της ταυρίνης. Ο μηχανισμός της προστατευτικής δράσης της Βρωμο-αμίνης T στη φλεγμονή βασίζεται στην απενεργοποίηση του μεταγραφικού παράγοντα NF‑κB. Επιπλέον, τα αποτελέσματα μας τεκμηριώνουν την κατασταλτική δράση της Βρωμο-αμίνης T στην ανάπτυξη των καρκινικών κυττάρων του παχέος εντέρου, του μαστού, του τραχήλου της μήτρας και του δέρματος με δόσο- και χρόνο-εξαρτώμενο τρόπο. Σε σύγκριση με τη ταυρίνη, η Βρωμο-αμίνη T έχει τη μεγαλύτερη κυτταροτοξική δράση στα καρκινικά κύτταρα του παχέος εντέρου RKO και στα καρκινικά κύτταρα του μαστού MDA-MB-468. Περαιτέρω πειράματα στα καρκινικά κύτταρα RKO υποστηρίζουν ότι η Βρωμο-αμίνη Τ προκαλεί το θάνατο μέσω επαγωγής της μιτοχονδριακής απόπτωσης και της αυτοφαγίας, από τις οξειδωτικές βλάβες και την ενεργοποίηση των κινασών από μιτογόνα (mitogen-activated protein kinase, MAPK), σε μεγαλύτερο βαθμό σε σχέση με τη ταυρίνη. Τα παραπάνω in vitro αποτελέσματα της έντονης κυτταροτοξικής δράσης της Βρωμο-αμίνης T επιβεβαιώνονται με in vivo πειράματα ξενομοσχευμάτων σε ανοσοανεπαρκείς ποντικούς. Συμπερασματικά, η Βρωμο-αμίνη Τ μπορεί να χρησιμοποιηθεί ως μία νέα εν δυνάμει φαρμακευτική ουσία στην αντιμετώπιση της φλεγμονής και του καρκίνου.