Potential interference of aluminum chlorohydrate with estrogen receptor signaling in breast cancer cells

Aluminum salts are widely used as the active antiperspirant in underarm cosmetic. Experimental observations indicate that its long term application may correlate with breast cancer development and progression. This action is proposed to be attributed, among others, to aluminum possible estrogen-like activities. In this study we showed that aluminum, in the form of aluminum chlorohydrate (ACH), caused increase in estrogen receptor alpha (ERα) protein levels, in ERα-positive MCF-7 cells. This effect was accompanied by moderate activation of Estrogen Response Elements (ERE)-driven reporter gene expression and 20%-50% increase in certain estrogen responsive, ERE-independent genes expression. Genes affected were ERα, p53, cyclin D1, and c-fos, crucial regulators of breast cancer development and progression. ACH-induced genes expression was eliminated in the presence of the estrogen antagonist: ICI 182780, in MCF-7 cells, whereas it was not observed in ERα-negative MDA-MB-231 breast cancer cells, indicating aluminum interference with estrogen signaling. Moreover, ACH caused increase in the perinuclear localization of estrogen receptor alpha in MCF-7 breast cancer cells and increase in the mitochondrial Bcl-2 protein, possibly affecting receptors-mediated mitochondrial actions and mitochondrial-dependent apoptosis. ACH-induced perinuclear localization of estrogen receptor beta was also observed in MDA-MB-231. Our findings indicate that aluminum actions on estrogen receptors protein level and subcellular localization possibly affect receptors-mediated actions and thus, aluminum interference with estrogen signaling

Potential Dissociative Glucocorticoid Receptor Activity for Protopanaxadiol and Protopanaxatriol

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist

The druggability of the ATP binding site of glycogen phosphorylase kinase probed by coumarin analogues

Glycogen phosphorylase kinase (PhK) converts by phosphorylation, the inactive glycogen phosphorylase (GPb) into active GPa in the glycogenolytic pathway. It is a complex enzyme comprising of the catalytic (γ) and three regulatory subunits (α, β, δ) forming a hexadecamer with stoichiometry (αβγδ)4. Several studies have indicated PhK as a promising target for the development of antihyperglycemics as its inhibition blocks glycogenolysis in liver and a potential therapeutic target for cancer against pathological angiogenesis and tumor progression. The identification of compounds that inhibit the kinase through their direct binding to its catalytic site is an effective approach to identify bioactive molecules of therapeutic significance. Towards this, the structure of the N-terminal kinase domain (residues 1–298) of the catalytic γ subunit of PhK (PhKγtrnc) has been determined by X-ray crystallography while staurosporine and indirubin analogues have been characterized as potent inhibitors targeting the ATP binding site. In this study, a series of 38 synthetic analogues of naturally occurring coumarins were screened for inhibition of PhKγtrnc, in vitro, using a photometric assay. The IC50 values of the two most potent compounds were determined for PhKγtrnc and the pharmacologically relevant target, human liver isoform (PHKG2A). Their cellular efficacy and toxicity in HepG2 cells were further assessed ex vivo. Docking experiments and the structural comparison with previously described inhibitors reveal the binding mode of the coumarin scaffold at a no hinge region of the ATP site of PhK and the role of a conserved β3-Lys in binding. The experimental findings provide structural insights with implications to the kinase targeting and drug design

Estrogen receptor beta as direct regulator of mitochondrial function

Estrogens are crucial regulators of the nervous system physiology, exerting anti-oxidant, anti-apoptotic and anti-inflammatory actions. They act via binding to their cognate nuclear, mitochondrial and membrane estrogen receptors (ERs), but also in an ER-independent manner. Recently, the neuroprotective effect of mitochondrial ERs, especially mitochondrial ERβ (mtERβ), has been documented. The exact mechanism of mtERβ action in this organelle has not been fully elucidated and is the subject of study by various research groups. Considering the significant role of mitochondria in the maintenance of neurons survival and function, the characterization of the role and the biochemical mechanisms of the actions of mtERβ in neural cells is of great importance. In this context, the aim of this PhD thesis was the characterization of the direct action of mtERβ on several mitochondrial processes, such as mitochondrial transcription, energy production, anti-oxidant defense, mitochondria-associated apoptosis, as well as the elucidation of mtERβ effects on carcinogenesis, cell differentiation and defense against neurotoxic agents. To this aim, a stable mouse neuroblastoma cell line Neuro-2a (N2A) overexpressing a mitochondrial-targeted ERβ (mtERβ) fused with the green fluorescent protein (N2AmtGFPERβ), and a control Ν2Α cell line stably overexpressing a mitochondrial-targeted GFP (N2ΑmtGFP), was generated. The produced stable cell lines along with the human neuroblastoma cell line SH-SY5Y, that exhibits considerable endogenous ERβ protein levels with mitochondrial ERβ localization as well, were cultured in the presence or absence of a specific inhibitor of nuclear transcription or differentiation- or neurotoxicity-inducing factors, in order to study the role of mtERβ in mitochondrial function, by applying real-time PCR, fluorescence microscopy, immunocytochemistry and Western blotting, measurement of ATP levels or / and MTT assays. Moreover, stable cell lines were used for the development of a xenograft mouse model, for in vivo tumorigenesis studies. The results of this PhD thesis revealed the direct involvement of mtERβ in the activation of mitochondrial transcription and ATP production, even in the presence of a specific inhibitor of nuclear polymerase and in the presence or absence of estradiol (Estradiol, E2), in both the SH-SY5Y and N2AmtGFPERβ cells. Furthermore, investigation of the role of mtERβ in the regulation of neural cell differentiation uncovered the involvement of mtERβ in neuronal differentiation as indicated by the statistically significant increase in the neuronal length and Tuj-1 protein levels in mtERβ-overexpressing cells, compared to control cells. Moreover, the increased anti-apoptotic and anti-oxidant defense of mtERβ-overexpressing N2A cells, in the presence of apoptosis- and oxidative stress- inducing factors compared to the control cells, demonstrate the direct involvement of mtERβ in the regulation of apoptosis and oxidative stress, verifying the neuroprotective effect of mtERβ. The actions and effects of the mtERβ actions were confirmed and further investigated by applying a xenograft mouse model of mtERβ-overexpressing N2A cells. In tumorigenesis studies, the direct action of mtERβ in the regulation of mitochondrial transcription was also verified. Thus, in mtERβ expressing tumors the increased synthesis of OXPHOS subunits was observed that possible lead to the enhancement of energy production through the respiratory chain - OXPHOS. Our findings indicate that these mtERβ-induced actions contribute to the inhibition of carcinogenesis, through direct or indirect regulation of other biochemical mechanisms such as, cellular energy metabolism, mitophagy and cell cycle, thereby reversing the trends for metabolic reprogramming observed in Warburg effect. Finally, the study of both the potential estrogenic action of aluminum (Aluminium, Al) and the involvement of this action in Al-induced neurotoxicity, highlighted the Al-induced reduction in ERβ protein levels. This action may be linked to the observed involvement of Al in the phosphorylation at S118 residue of ERα, which cause the inhibition of ERα proteolytic degradation and the increase of ERα protein levels. Increased ERα levels, as we know from the literature, induce the reduction in ERβ protein levels and consequently ERβ-induced neuroprotective action. In addition, the neurotoxic effect of Al could be attributed to the observed Al-induced increase in mitochondrial localization of ERβ, possibly triggering mitochondrial metabolism and increasing production of ROS and induction of apoptosis. In summary, the results of the present PhD thesis support the direct action of mtERβ in the regulation of mitochondrial function in both normal and pathological conditions, rendering mtERβ a potential pharmaceutical target for mitochondrial related diseases, such as cancer and neurodegenerative diseases.Τα οιστρογόνα αποτελούν βασικούς ρυθμιστές της λειτουργίας του νευρικού συστήματος, ασκώντας αντιοξειδωτικές, αντιαποπτωτικές και αντιφλεγμονώδεις δράσεις. Οι δράσεις αυτές των οιστρογόνων επιτελούνται μέσω πυρηνικών και μεμβρανικών ERs (Estrogen Receptors, ERs), αλλά και ανεξαρτήτως ERs. Τελευταία αναδεικνύεται και η νευροπροστατευτική δράση των μιτοχονδριακών ERs, και κυρίως του μιτοχονδριακού ERβ (mitochondrial ERβ, mtERβ). Ο ακριβής μηχανισμός δράσης του mtERβ στο συγκεκριμένο οργανίδιο δεν έχει διευκρινιστεί πλήρως και αποτελεί αντικείμενο μελέτης αρκετών ερευνητικών ομάδων. Συνυπολογίζοντας το σημαντικό ρόλο των μιτοχονδρίων στη διατήρηση της βιωσιμότητας και της εύρυθμης λειτουργίας των νευρώνων αναδεικνύεται η σημαντικότητα του χαρακτηρισμού του ρόλου και των μηχανισμών άμεσης δράσης του mtERβ σε νευρικά κύτταρα. Με βάση τα προαναφερθέντα στην παρούσα διδακτορική διατριβή μελετήθηκε η άμεση δράση του mtERβ σε μια πληθώρα μιτοχονδριακών διεργασιών, όπως η μιτοχονδριακή μεταγραφή, η παραγωγή ενέργειας, η αντιοξειδωτική άμυνα και η σχετιζόμενη με το μιτοχόνδριο απόπτωση, καθώς και οι επιπτώσεις των επαγόμενων από τον mtERβ δράσεων στην καρκινογένεση, στην κυτταρική διαφοροποίηση, και στην άμυνα έναντι νευροτοξικών παραγόντων. Για την επίτευξη των στόχων της παρούσας διδακτορικής διατριβής κατασκευάστηκε κυτταρική σειρά νευροβλαστώματος ποντικού Neuro-2a (Ν2Α) που υπερεκφράζει τον ERβ υπό μορφή χιμαιρικής πρωτεΐνης με την πράσινη φθορίζουσα πρωτεΐνη (Green fluorescent protein, GFP) με μιτοχονδριακή στόχευση (Ν2AmtGFPERβ), καθώς και κυτταρική σειρά αναφοράς, που υπερεκφράζει σταθερά την πρωτεΐνη mtGFP (Ν2ΑmtGFP). Οι κυτταρικές αυτές σειρές, μεταξύ άλλων, χρησιμοποιήθηκαν για τη διεξαγωγή in vivo πειραμάτων καρκινογένεσης σε μύες (xenograft mouse model). Επιπρόσθετα, σε μια πληθώρα πειραματικών διαδικασιών, χρησιμοποιήθηκε η κυτταρική σειρά νευροβλαστώματος ανθρώπου SH-SY5Y καθώς εμφανίζει, σε σύγκριση με αντίστοιχες κυτταρικές σειρές νευροβλαστώματος, αυξημένα ενδογενή πρωτεϊνικά επίπεδα και μιτοχονδριακή εντόπιση του ERβ. Στις προαναφερθείσες κυτταρικές σειρές (Ν2ΑmtGFP, Ν2ΑmtGFPERβ και SH-SY5Y) πραγματοποιήθηκαν μελέτες που περιλάμβαναν αρχικά καλλιέργεια κυττάρων παρουσία και απουσία ειδικού αναστολέα πυρηνικής μεταγραφής, παρουσία και απουσία παραγόντων διαφοροποίησης και επαγωγής νευροτοξικότητας, και στη συνέχεια εφαρμογή διαφόρων μεθόδων όπως PCR πραγματικού χρόνου, μικροσκοπία φθορισμού, ανοσοφθορισμό και ανοσοαποτύπωση κατά Western με χρήση ειδικών αντισωμάτων, μέτρηση των επιπέδων ATP και δοκιμασίες ελέγχου κυτταροτοξικότητας ΜΤΤ. Τα αποτελέσματα της παρούσας διδακτορικής διατριβής αναδεικνύουν την άμεση εμπλοκή του mtERβ στην ενεργοποίηση της μιτοχονδριακής μεταγραφής και της παραγωγής ATP μέσω της αναπνευστικής αλυσίδας – οξειδωτικής φωσφορυλίωσης (OXPHOS), δεδομένου ότι παρατηρήθηκε ενεργοποίηση της μιτοχονδριακής μεταγραφής, παρουσία αναστολέα πυρηνικής μεταγραφής και παρουσία ή απουσία οιστραδιόλης (17β-estradiol, Ε2), και αύξηση των επιπέδων της μιτοχονδριακής ATP τόσο σε κύτταρα SHSY-5Y, όσο και σε κύτταρα N2AmtGFPERβ. Επιπλέον μελετήθηκε ο ρόλος του υποδοχέα στη ρύθμιση μηχανισμών διαφοροποίησης νευρικών κυττάρων, χρησιμοποιώντας μοντέλο ανάπτυξης κυττάρων σε συνθήκες διαφοροποίησης, όπου παρατηρήθηκε η διαφοροποίηση των κυττάρων που υπερεκφράζουν τον mtERβ, όπως υποδεικνύει η παρατηρούμενη αύξηση του μήκους των νευρώνων και των πρωτεϊνικών επιπέδων του δείκτη διαφοροποίησης βIII τουμπουλίνη (βIII-tubulin, Tuj-1) στη συνθήκη αυτή, σε σύγκριση με τα κύτταρα αναφοράς. Επίσης, η αυξημένη αντιαποπτωτική και αντιοξειδωτική άμυνα των κυττάρων Ν2Α, που υπερεκφράζουν τον mtERβ, σε συνθήκες επαγωγής απόπτωσης και οξειδωτικού στρες, αποδεικνύουν την άμεση εμπλοκή του mtERβ στη ρύθμιση της απόπτωσης και του οξειδωτικού στρες, επαληθεύοντας έτσι την νευροπροστατευτική δράση του υποδοχέα. Οι δράσεις και οι επιπτώσεις των δράσεων αυτών του mtERβ επαληθεύτηκαν και διερευνήθηκαν περαιτέρω πραγματοποιώντας in vivo πειράματα καρκινογένεσης σε μύες. Κατά τη διεξαγωγή των μελετών αυτών επαληθεύτηκε η άμεση δράση του υποδοχέα στη ρύθμιση της μιτοχονδριακής μεταγραφής, η οποία οδηγεί στην αυξημένη σύνθεση υπομονάδων OXPHOS και κατ’ επέκταση στην ενίσχυση της παραγωγής ενέργειας μέσω της αναπνευστικής αλυσίδας – OXPHOS. Επίσης διαπιστώθηκε ότι οι προαναφερθείσες δράσεις του mtERβ συντελούν στην αναστολή της καρκινογένεσης, μέσω άμεσης ή έμμεσης ρύθμισης και άλλων βιοχημικών μηχανισμών όπως του κυτταρικού ενεργειακού μεταβολισμού, της μιτοφαγίας και του κυτταρικού κύκλου, αναστρέφοντας έτσι τάσεις για μεταβολικό επαναπρογραμματισμό που έχουν παρατηρηθεί στο φαινόμενο Warburg. Τέλος, η μελέτη τόσο της πιθανής οιστρογονικής δράσης του αργιλίου (Aluminum, Al) όσο και της εμπλοκής αυτής στην εκδήλωση της νευροτοξικότητας του αργιλίου, ανέδειξε την επαγόμενη από το Al μείωση των επιπέδων του ERβ. Η δράση αυτή πιθανώς να οφείλεται στην παρατηρούμενη εμπλοκή των ιόντων Al στην κατάσταση φωσφορυλίωσης του ERα στο αμινοξύ S118, που οδηγεί σε αναστολή της πρωτεολυτικής του αποικοδόμησης και συνεπώς σε αύξηση των πρωτεϊνικών του επιπέδων, τα οποία επάγουν με τη σειρά τους, όπως γνωρίζουμε από τη βιβλιογραφία, τη μείωση των επιπέδων του ERβ και κατά συνέπεια τη μείωση της νευροπροστατευτικής του δράσης. Επίσης η νευροτοξική δράση του Al θα μπορούσε να αποδοθεί στην παρατηρούμενη από το Al αύξηση της μιτοχονδριακής εντόπισης του ERβ, η οποία πιθανόν να οδηγεί σε υπερλειτουργία της αναπνευστικής αλυσίδας – OXPHOS, αυξημένη παραγωγή ROS και επαγωγή απόπτωσης. Συνοψίζοντας τα αποτελέσματα της παρούσας διδακτορικής διατριβής υποστηρίζουν την άμεση δράση του mtERβ στη ρύθμιση της μιτοχονδριακής λειτουργίας τόσο σε φυσιολογικές όσο και παθολογικές συνθήκες αναδεικνύοντας τον ως έναν εν δυνάμει φαρμακευτικό στόχο για τη ρύθμιση της μιτοχονδριακής λειτουργίας σε σχετιζόμενες με μιτοχόνδρια παθήσεις όπως καρκινογένεση και νευροεκφυλιστικές νόσοι

Regulation of Energy Metabolism and Anti-Inflammatory Activities of Mastiha Fractions from Pistacia lentiscus L. var. chia

Pistacia lentiscus L. var. chia resin (Chios Mastiha), the first natural chewing gum, is widely used in Mediterranean cuisine and has been used in traditional medicine from ancient times. Regarding its chemical composition, Chios Mastiha is known to be rich in triterpenes. Triterpenes have a similar structure to glucocorticoids (GCs), the steroid hormones that exert strong anti-inflammatory activities and play crucial roles in the regulation of cellular metabolism. To simplify the characterization of the bioactive compounds of Mastiha resin, three different polarity fractions were isolated and were further analyzed regarding their main chemical composition and an assessment of their biological activities. The biological assessment focused on the evaluation of the potential anti-proliferative, anti-inflammatory, and apoptotic activities as well as the possible interference of the three different polarity Mastiha fractions with the glucocorticoid receptor signaling, with the aim of characterizing the biochemical mechanisms of the actions of the Mastiha fraction. Applying MTT cell viability assay, luciferase/beta-galactosidase assay, and Western blot analysis showed that Chios Mastiha apolar, medium-polar, and polar fractions reduced the HEK293 cell viability in a dose-dependent manner, possibly by mitochondrial-mediated induction of apoptosis. Medium-polar and polar Mastiha fractions also suppressed the GR and NF-kappa B transcriptional activation and the p65 protein levels. These activities were accompanied by the modulation of protein levels of regulatory molecules playing a crucial role in cellular energy homeostasis, such as GR, phosphoenolpyruvate carboxykinase (PEPCK), and/or peroxisome proliferator-activated receptor alpha (PPAR alpha), and by the induction of phosphorylation and the activation of the AMP-activated protein kinase (AMPK). The medium-polar fraction was found to be enriched in triterpenes, such as lupeol, 24Z-masticadienonic acid methyl ester, and 24Z-isomasticadienonic acid methyl ester, and it was the most active one, so we propose that triterpenes in medium-polar fraction are possibly the bioactive compounds responsible for Mastiha's regulatory actions on energy metabolism and anti-inflammatory activities via interference with GR, NF-kappa B, and AMPK signaling. This highlights its potential applications in many fields of pharmaceutical, cosmetic, and nutraceutical interest

Anti-Apoptotic and Antioxidant Activities of the Mitochondrial Estrogen Receptor Beta in N2A Neuroblastoma Cells

Estrogens are steroid hormones that play a crucial role in the regulation of the reproductive and non-reproductive system physiology. Among non-reproductive systems, the nervous system is mainly affected by estrogens due to their antioxidant, anti-apoptotic, and anti-inflammatory activities, which are mediated by membranous and nuclear estrogen receptors, and also by non-estrogen receptor-associated estrogen actions. Neuronal viability and functionality are also associated with the maintenance of mitochondrial functions. Recently, the localization of estrogen receptors, especially estrogen receptor beta, in the mitochondria of many types of neuronal cells is documented, indicating the direct involvement of the mitochondrial estrogen receptor beta (mtERβ) in the maintenance of neuronal physiology. In this study, cell lines of N2A cells stably overexpressing a mitochondrial-targeted estrogen receptor beta were generated and further analyzed to study the direct involvement of mtERβ in estrogen neuroprotective antioxidant and anti-apoptotic actions. Results from this study revealed that the presence of estrogen receptor beta in mitochondria render N2A cells more resistant to staurosporine- and H2O2-induced apoptotic stimuli, as indicated by the reduced activation of caspase-9 and -3, the increased cell viability, the increased ATP production, and the increased resistance to mitochondrial impairment in the presence or absence of 17-β estradiol (E2). Thus, the direct involvement of mtERβ in antioxidant and anti-apoptotic activities is documented, rendering mtERβ a promising therapeutic target for mitochondrial dysfunction-associated degenerative diseases

Apoptotic, Anti-Inflammatory Activities and Interference with the Glucocorticoid Receptor Signaling of Fractions from Pistacia lentiscus L. var. chia Leaves

In this study acetonic extracts of leaves of Pistacia lentiscus L. var. chia (mastiha tree) grown in the south as well as in the north Chios Greek island were isolated and further fractionated to give three different polarity fractions: apolar, medium-polar, and polar. The isolated fractions were assessed as regards their main composition, cytotoxic, anti-inflammatory activities, and interference with the glucocorticoid receptor (GR) signaling, applying cytotoxic assay, luciferase assays, and Western blot analysis of apoptosis-, energy-, and inflammation-associated molecules. Differences in cell viability have been detected among different polarity leaf fractions as well as among fractions of different plant origin with polar fractions showing the highest cytotoxicity. Fractions-induced anti-inflammatory activities and suppressive effects on the dexamethasone (DEX)-induced GR transcriptional activation were unveiled. The partition protocol of leaves fractions applied uncovers the enhanced glucocorticoid-associated biological activities of the medium-polar fractions, which may be associated with their enrichment in the triterpenoids that showed structural similarity with the glucocorticoids. A reduction in GR protein levels is observed by the fraction which is shown to be associated with the medium polar-induced proteolytic degradation of the receptor. In addition, the enhanced cytotoxic, anti-inflammatory, and potential anti-glycemic activities of the fractions from the Southern P. lentiscus L. that exclusively produce the mastiha resin, is revealed, indicating that leaves fractions from mastiha tree, similarly to mastiha tree resin, may have the potential to be further analyzed for their potent applications in the pharmaceutical cosmetic and nutraceutical fields

Multidisciplinary docking, kinetics and X-ray crystallography studies of baicalein acting as a glycogen phosphorylase inhibitor and determination of its’ potential against glioblastoma in cellular models

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and glioblastoma at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (K i  = 32.54 μM), human liver GPa (K i  = 8.77 μM) and rabbit muscle GPb (K i  = 5.66 μM) isoforms. It is also an effective inhibitor of glycogenolysis (IC 50  = 119.6 μM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC 50 values of ∼20–55 μM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O6-methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP–baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against glioblastoma is suggested