In vitro and in vivo anticancer activity of flavonoids and congeners

Flavonoids are plant derived products that have been reported to demonstrate cytotoxic activity in vitro while some have entered clinical trials. Recently, kaempferol glycoside was shown to interfere with MAPK signal transduction pathway. MAPK pathway regulates proliferation, survival, growth and motility of cells. p90 ribosomal S6 kinase (RSK) constitutes a family of protein kinases downstream of the MAP kinase cascade and has been shown to regulate cancer progression. We have investigated 16 different flavonols, including flavonol glycosides and their peracetylated derivatives for their ability to induce cytotoxicity and cell cycle perturbations towards solid tumor cells. Our aim was to approach the most cytotoxic flavonol. Further on we proceeded with the investigation of its mechanism of action and whether this compound acts downstream to MAPK pathway via RSK inhibition. Finally, we have evaluated the antitumor activity of peracetylated tiliroside (Tac) in the human tumor xenograft / immunodeficient mouse model and its relation to the regulation of the MAPK pathway. Antiproliferative activity was investigated using the SRB assay according to the NCI. Further, cell cycle perturbations were monitored by flow cytometry using propidium iodide staining. COMPARE algorithm was further employed for an initial evaluation of the mechanism of action. Tac induced perturbations on cell cycle were examined on the HCT116 cancer cell line using FACS analysis. Western blot was utilized for the detection of caspase activation and changes in the levels of kinases involved in MAPK. The in vivo antitumor activity of Tac was also evaluated against HCT116 xenografts. The examined flavonols showed weak activity while acetylation did not enhance their activity. Of all compounds tested Tac demonstrated significant antiproliferative and cytotoxic activity. Cell cycle perturbations were found similar between most compounds while induction of apoptosis was significant only on cells treated with Tac. COMPARE analysis of Tac activity revealed great similarities with DNA damaging agents. FACS analysis indicated that Tac treatment resulted in a delay swift of cells from G2- to M-phase, subsequent blockage in G1 phase, and apoptosis through caspase activation. Under these experimental conditions Tac did not affect MARKCS or ERK1/2 phosphorylation levels but did alter RSK’s activity as this was depicted by the elevated eEF2 phosphorylation. Intraperitoneal (ip) administration of Tac at the maximum tolerated dose (MTD), following a [(Q1D5) x 2] schedule significantly suppressed growth of HCT116 tumors in xenografts. Findings suggest that there is virtually limited linkage of the – OH groups of the aromatic ring B and of their acetylation, to the in vitro anticancer activity of the tested flavonols. In contrast, acetylation of tiliroside greatly improved the activity of this congener. Tac induced apoptotic cell death to HCT116 cell seems the result of a mechanism involving MAPK inhibition. COMPARE analysis revealed similarities of Tac mechanism of action to that of DNA damaging agents, thus linking RSK activity to DNA damage. Taken together the in vitro and in vivo results evaluate acetylated kaempferol glucosides as lead compounds for the development of substrate specific anticancer agents.Τα φλαβονοειδή είναι φυτικά προϊόντα που έχουν δείξει αξιόλογη in vitro κυτταροτοξική δράση ενώ κάποια έχουν εισέλθει σε κλινικές μελέτες. Πρόσφατες μελέτες υπέδειξαν ένα γλυκοσίδη της καιμπφερόλης να εμπλέκεται στο μονοπάτι μεταγωγής σημάτων MAPK. Το MAPK ρυθμίζει τον πολλαπλασιασμό, την επιβίωση, την ανάπτυξη και την κινητικότητα των κυττάρων. Η p90 ριβοσωμική S6 κινάση (RSK) βρίσκεται κατάρρους της κινάσης ERK στο μονοπάτι MAPK και ρυθμίζει την ανάπτυξη των καρκινικών όγκων. Μελετήσαμε την ικανότητα 16 ουσιών; φλαβονολών, γλυκοσιδών και περακετυλιωμένα παράγωγα τους, να προάγουν κυτταροτοξική δράση και να επάγουν διαταραχές στον κυτταρικό κύκλο ανθρώπινων στέρεων όγκων. Απώτερος σκοπός ήταν η ανεύρεση της πιο κυτταροτοξικής φλαβονόλης. Στην συνέχεια, εξετάσαμε το μηχανισμό δράσης της ουσίας σε in vitro αλλά και in vivo πρότυπα ανθρώπινου καρκίνου, καθώς και την εμπλοκή της δράσης αυτής στο μονοπάτι σηματοδότησης MAPK μέσω αναστολής της δράσης της κινάσης RSK. Η αντιπολλαπλασιαστική δράση των ουσιών εξετάσθηκε με τη μεθοδολογίας της SRB. Οι διαταραχές στον κυτταρικό κύκλο εξετάσθηκαν με κυτταρομετρητή ροής ύστερα από χρώση με προπίδιο του ιωδίου. Ο αλγόριθμος COMPARE χρησιμοποιήθηκε για μια εκτίμηση του μηχανισμού δράσης της πιο δραστικής ουσίας, περακετυλιωμένος τιλιροσίδης (Tac), ενώ ο κυτταρικός θάνατος και οι διαταραχές στον κυτταρικό κύκλο μελετήθηκαν έναντι της καρκινικής σειράς HCT116, με κυτταρομετρία ροής. Η ενεργοποίηση των κασπασών και η μελέτη μηχανισμού δράσης εξετάσθηκε με ανοσοστύπωση. Η in vivo δράση του Tac εξετάσθηκε ξανά έναντι της καρκινικής σειράς HCT116. Οι φλαβονόλες και τα παράγωγα τους έδειξαν μέτρια αντιπολλαπλασιαστική δράση, ενώ η ακετυλίωση δεν βελτίωσε την δραστικότητα των παράγωγων τους. Από όλες τις ουσίες το Tac έδειξε την πιο αξιοσημείωτη αντιπολλαπλασιαστική δράση. Οι διαταραχές στον κυτταρικό κύκλο ήταν περίπου ίδιες για όλες τις ουσίες παρόλα αυτά κυτταρικός θάνατος επήλθε μονάχα στα κύτταρα που είχαν επωαστεί με Tac. O αλγόριθμος COMPARE υπέδειξε όμοιο μηχανισμό δράσης του Tac με γενοτοξικές ουσίες. Η μελέτη του κυτταρικού κύκλου έδειξε ότι το Tac επιφέρει καθυστερημένη μετάβαση των κυττάρων από την φάση G2/M με παράλληλη φραγή στην G1 ενώ ο προγραμματισμένος κυτταρικός θάνατος φάνηκε εξαρτώμενος από τη δράσης των κασπασών. Κάτω από τις συγκεκριμένες πειραματικές συνθήκες το Tac δεν επηρέασε τη δράση κινασών ανάρρους της RSK παρόλα αυτά έδειξε ότι πιθανά αναστέλλει τη δράση της RSK όπως φάνηκε από την ενεργοποίηση του παράγοντα eEF2. Ενδοπεριτονιακή χορήγηση του Tac στα ποντίκια μείωσε αξιοσημείωτα το ρυθμό ανάπτυξης των καρκινικών κυττάρων του παχέως εντέρου, HCT116. Τα διαφορετικά δομικά χαρακτηριστικά των φλαβονολών αλλά και η ακετυλίωσης τους υπέδειξαν ελάχιστη επιρροή στην δράση τους. Σε αντίθεση, η ακετυλίωση του τιλιροσίδη ευνόησε αξιοσημείωτα τη δράση του προϊόντος επιφέροντας απόπτωση στα καρκινικά κύτταρα η οποία δύναται να είναι αποτέλεσμα της εμπλοκής της ουσίας στο μονοπάτι MAPK. Τα ευρήματα από το COMPARE υπέδειξαν ομοιότητες στη δράση του Tac με ουσίες που παρεμποδίζουν την φυσιολογική εξέλιξη του γενετικού υλικού προσδίδοντας μια σχέση ανάλογη μεταξύ της γενοτοξικής δράσης της ουσίας και της επαγόμενης αναστολής της δράσης της RSK. Τα in vitro και in vivo αποτελέσματα αξιολογούν του γλυκοζίτες της καιμπφερόλης ως πρόδρομα μόρια για την ανάπτυξη στοχευμένων αντικαρκινικών φαρμάκων

Study of the relationship between sigma receptor expression levels and some common sigma ligand activity in cancer using human cancer cell lines of the nci-60 cell line panel

Sigma (σ) receptors have attracted great interest since they are implicated in various cellular functions and biological processes and diseases, including various types of cancer. The receptor family consists of two subtypes: sigma-1 (σ1) and sigma-2 (σ2). Both σ receptor subtypes have been proposed as therapeutic targets for various types of cancers, and many studies have provided evidence that their selective ligands (agonists and antagonists) exhibit antiproliferative and cytotoxic activity. Still, the precise mechanism of action of both σ receptors and their ligands remains unclear and needs to be elucidated. In this study, we aimed to simultaneously determine the expression levels of both σ receptor subtypes in several human cancer cell lines. Additionally, we investigated the in vitro antiproliferative activity of some widely used σ1 and σ2 ligands against those cell lines to study the relationship between σ receptor expression levels and σ ligand activity. Finally, we ran the NCI60 COMPARE algorithm to further elucidate the cytotoxic mechanism of action of the selected σ ligands studied herein

P90 ribosomal S6 kinases : A bona fide target for novel targeted anticancer therapies?

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases. They are downstream effectors of the Ras/ERK/MAPK signaling cascade. ERK1/2 activation directly results in the phosphorylation of RSKs, which further, through interaction with a variety of different downstream substrates, activate various signaling events. In this context, they have been shown to mediate diverse cellular processes like cell survival, growth, proliferation, EMT, invasion, and metastasis. Interestingly, increased expression of RSKs has also been demonstrated in various cancers, such as breast, prostate, and lung cancer. This review aims to present the most recent advances in the field of RSK signaling that have occurred, such as biological insights, function, and mechanisms associated with carcinogenesis. We additionally present and discuss the recent advances but also the limitations in the development of pharmacological inhibitors of RSKs, in the context of the use of these kinases as putative, more efficient targets for novel anticancer therapeutic approaches

Potential of the dual mTOR kinase inhibitor AZD2014 to overcome paclitaxel resistance in anaplastic thyroid carcinoma.

PURPOSE Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. METHODS Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. RESULTS Rho- cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho- cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. CONCLUSIONS Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies

A promising natural product, pristimerin, results in cytotoxicity against breast cancer stem cells in vitro and xenografts in vivo through apoptosis and an incomplete autopaghy in breast cancer

Several natural products have been suggested as effective agents for the treatment of cancer. Given the important role of CSCs (Cancer Stem Cells) in cancer, which is a trendy hypothesis, it is worth investigating the effects of pristimerin on CSCs as well as on the other malignant cells (MCF-7 and MDA-MB-231) of breast cancer. The anti-growth activity of pristimerin against MCF-7 and MCF-7s (cancer stem cell enriched population) cells was investigated by real time viability monitorization (xCELLigence System (R)) and ATP assay, respectively. Mode of cell death was evaluated using electron and fluorescence microscopies, western blotting (autophagy, apoptosis and ER-stress related markers) and flow cytometry (annexin-V staining, caspase 3/7 activity, BCL-2 and PI3K expressions). Pristimerin showed an anti-growth effect on cancer cells and cancer stem cells with IC50 values ranging at 0.38-1.75 mu M. It inhibited sphere formation at relatively lower doses (<1.56 mu M). Apoptosis was induced in MCF-7 and MCF-7s cells. In addition, extensive cytoplasmic vacuolation was observed, implying an incompleted autophagy as evidenced by the increase of autophagy-related proteins (p62 and LC3-II) with an unfolded protein response (UPR). Pristimerin inhibited the growth of MCF-7 and MDA-MB-231-originated xenografts in NOD.CB17-Prkdc(scid)/J mice. In mice, apoptosis was further confirmed by cleavage of PARP, activation of caspase 3 and/or 7 and TUNEL staining. Taken together, pristimerin shows cytotoxic activity on breast cancer both in vitro and in vivo. It seems to represent a robust promising agent for the treatment of breast cancer. Pristimerin's itself or synthetic novel derivatives should be taken into consideration for novel potent anticancer agent(s). (C) 2017 Elsevier Ltd. All rights reserved