Lycorine and homolycorine derivatives for chemo-sensitizing resistant human ovarian adenocarcinoma cells.

BACKGROUND: Multidrug resistance is the major obstacle to cancer chemotherapy. Modulation of P-glycoprotein and drug combination approaches have been considered important strategies to overcome drug resistance. PURPOSE: Aiming at generating a small library of Amaryllidaceae-type alkaloids to overcome drug resistance, two major alkaloids, isolated from Pancratium maritimum, lycorine (1), and 2α-10bα-dihydroxy-9-O-demethylhomolycorine (2), were derivatized, giving rise to nineteen derivatives (3 - 21). METHODS: The main chemical transformation of lycorine resulted from the cleavage of ring E of the diacetylated lycorine derivative (3) to obtain compounds that have carbamate and amine functions (5 - 16), while acylation of compound 2 provided derivatives 17 - 21. Compounds 1 - 21 were evaluated for their effects on cytotoxicity, and drug resistance reversal, using resistant human ovarian carcinoma cells (HOC/ADR), overexpressing P-glycoprotein (P-gp/ABCB1), as model. RESULTS: Excluding lycorine (1) (IC50 values of 1.2- 2.5 µM), the compounds were not cytotoxic or showed moderate/weak cytotoxicity. Chemo-sensitization assays were performed by studying the in vitro interaction between the compounds and the anticancer drug doxorubicin. Most of the compounds have shown synergistic interactions with doxorubicin. Compounds 5, 6, 9 - 14, bearing both carbamate and aromatic amine moieties, were found to have the highest sensitization rate, reducing the dose of doxorubicin 5-35 times, highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (4 - 6, 9 - 14, and 21), able of re-sensitizing resistant cancer cells, were further evaluated as P-gp inhibitors. Compound 11, which has a para­methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 9-11 and 13 behaved as verapamil, suggesting competitive inhibition of P-gp. At the same time, none of these compounds affected P-gp expression at the mRNA or protein level. CONCLUSIONS: This study provided evidence of the potential of Amaryllidaceae alkaloids as lead candidates for the development of MDR reversal agents.

Lycorine Carbamate Derivatives for Reversing P-glycoprotein-Mediated Multidrug Resistance in Human Colon Adenocarcinoma Cells.

Multidrug resistance (MDR) is a major challenge in cancer chemotherapy. Aiming at generating a small library of anticancer compounds for overcoming MDR, lycorine (1), a major Amaryllidaceae alkaloid isolated from Pancratium maritimum, was derivatized. Thirty-one new compounds (2-32) were obtained by chemical transformation of the hydroxyl groups of lycorine into mono- and di-carbamates. Compounds 1-32 were evaluated as MDR reversers, through the rhodamine-123 accumulation assay by flow cytometry and chemosensitivity assays, in resistant human colon adenocarcinoma cancer cells (Colo 320), overexpressing P-glycoprotein (P-gp, ABCB1). Significant inhibition of P-gp efflux activity was observed for the di-carbamate derivatives, mainly those containing aromatic substituents, at non-cytotoxic concentrations. Compound 5, bearing a benzyl substituent, and compounds 9 and 25, with phenethyl moieties, were among the most active, exhibiting strong inhibition at 2 µM, being more active than verapamil at 10-fold higher concentration. In drug combination assays, most compounds were able to synergize doxorubicin. Moreover, some derivatives showed a selective antiproliferative effect toward resistant cells, having a collateral sensitivity effect. In the ATPase assay, selected compounds (2, 5, 9, 19, 25, and 26) were shown to behave as inhibitors.

Amaryllidaceae-Type Alkaloids from Pancratium maritimum: Apoptosis-Inducing Effect and Cell Cycle Arrest on Triple-Negative Breast Cancer Cells.

Aiming to find Amaryllidaceae alkaloids against breast cancer, including the highly aggressive triple-negative breast cancer, the phytochemical study of Pancratium maritimum was carried out. Several Amaryllidaceae-type alkaloids, bearing scaffolds of the haemanthamine-, homolycorine-, lycorine-, galanthamine-, and tazettine-type were isolated (3-11), along with one alkamide (2) and a phenolic compound (1). The antiproliferative effect of compounds (1-11) was evaluated by the sulforhodamine B assay against triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, breast cancer cells MCF-7, and the non-malignant fibroblast (HFF-1) and breast (MCF12A) cell lines. The alkaloids 3, 5, 7, and 11 showed significant growth inhibitory effects against all breast cancer cell lines, with IC50 (half-maximal inhibitory concentration) values ranging from 0.73 to 16.3 µM. The homolycorine-type alkaloid 7 was selected for further investigation in MDA-MB-231 cells. In the annexin-V assay, compound 7 increased cell death by apoptosis, which was substantiated, in western blot analyses, by the increased expression of the pro-apoptotic protein Bax, and the decreased expression of the anti-apoptotic protein Bcl-xL. Consistently, it further stimulated mitochondrial reactive oxygen species (ROS) generation. The antiproliferative effect of compound 7 was also associated with G2/M cell cycle arrest, which was supported by an increase in the p21 protein expression levels. In MDA-MB-231 cells, compound 7 also exhibited synergistic effects with conventional chemotherapeutic drugs such as etoposide.

Bioactive compounds from the African medicinal plant Cleistochlamys kirkii as resistance modifiers in bacteria.

Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. The aim of this study was to find resistance modifiers in C. kirkii for Gram-positive and Gram-negative model bacterial strains. One of the most important resistance mechanisms in bacteria is the efflux pump-related multidrug resistance. Therefore, polycarpol (1), three C-benzylated flavanones (2-4), and acetylmelodorinol (5) were evaluated for their multidrug resistance-reverting activity on methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Escherichia coli AG100 and AG100 A strains overexpressing and lacking the AcrAB-TolC efflux pump system. The combined effects of antibiotics and compounds (2 and 4) were also assessed by using the checkerboard microdilution method in both S. aureus strains. The relative gene expression of the efflux pump genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. The inhibition of quorum sensing was also investigated. The combined effect of the antibiotics and compound 2 or 4 on the methicillin-sensitive S. aureus resulted in synergism. The most active compounds 2 and 4 increased the expression of the efflux pump genes. These results suggested that C. kirkii constituents could be effective adjuvants in the antibiotic treatment of infections.

Cleistochlamys kirkii chemical constituents: Antibacterial activity and synergistic effects against resistant Staphylococcus aureus strains.

ETHNOPHARMACOLOGICAL RELEVANCE: Cleistochlamys kirkii (Benth) Oliv., (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. AIMS OF THE STUDY: To find antibacterial lead compounds from C. kirkii and provide scientific validation for its use in traditional medicine. MATERIALS AND METHODS: Through bioassay-guided fractionation, nine compounds (1-9), with different scaffolds, were isolated from the methanol extract of C. kirkii whose structures were identified by spectroscopic methods. Compounds 1-9 were evaluated for their in vitro antibacterial activity against a panel of eight Gram-positive, including five drug-resistant strains of Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and two Gram-negative bacteria strains. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. A chemosensitization assay, using the checkerboard method, was also performed in order to evaluate the type of interaction of compounds with antibiotics/compounds against two S. aureus resistant strains (ATCC 9144 and CIP 106760) and a susceptible strain (ATCC 6538). RESULTS: Dichamanetin (3), a rare C-benzylated flavanone, was very active against all the Gram-positive strains tested, displaying MIC values in the range of 1-7.5 µg/mL. The C-benzylated flavanones chamanetin (1), isochamanetin (2), and the α,ß-unsaturated lactone (-)-cleistenolide (6) also showed relevant antibacterial activity against some of the Gram-positive strains assayed. Compounds 4, 5, and 7-9 have shown no significant activity at the concentration ranges tested. In the combination with antibiotics, polycarpol (8) (MIC 125 µg/mL) showed a strong synergistic effect against the methicillin-resistant S. aureus ATCC 9144. When combined with oxacillin (MIC 125 µg/mL), compound 8 reduced the MIC to 1.5 µg/mL (FICI=0.11). Similarly, it reduced the MIC of amoxicillin (MIC 250 µg/mL) to 7.5 µg/mL (FICI=0.18). Synergy was also obtained when this compound was combined with both ß-lactam antibiotics (FICI=0.30) and with vancomycin (FICI=0.24) against vancomycin-intermediate S. aureus (VISA) CIP 106760. Remarkable, compound 8 was also able to reduce synergistically the MIC value of dichamanetin (3) (FICI=0.18) against this strain. CONCLUSIONS: These results suggested that C. kirkii constituents may be valuable as a leads for restoring antibiotic activity against resistant S. aureus strains.