Nitensidine A,a guanidine alkaloid from Pterogyne nitens,induces osteoclastic cell death

Nitensidine A is a guanidine alkaloid isolated from Pterogyne nitens, a common plant in South America. To gain insight into the biological activity of P. nitens-produced compounds, we examined herein their biological effects on osteoclasts, multinucleated giant cells that regulate bone metabolism by resorbing bone. Among four guanidine alkaloids (i.e., galegine, nitensidine A, pterogynidine, and pterogynine), nitensidine A and pterogynine exhibited anti-osteoclastic effects at 10 μM by reducing the number of osteoclasts on the culture plate whereas galegine and pterogynidine did not. The anti-osteoclastic activities of nitensidine A and pterogynine were exerted in a concentration-dependent manner, whereas nitensidine A exhibited an approximate threefold stronger effect than pterogynine (IC₅₀ values: nitensidine A, 0.93 ± 0.024 μM; pterogynine, 2.7 ± 0.40 μM). In the present study, the anti-osteoclastic effects of two synthetic nitensidine A derivatives (nitensidine AT and AU) were also examined to gain insight into the structural features of nitensidine A that exert an anti-osteoclastic effect. The anti-osteoclastic effect of nitensidine A was greatly reduced by substituting the imino nitrogen atom in nitensidine A with sulfur or oxygen. According to the differences in chemical structures and anti-osteoclastic effects of the four guanidine alkaloids and the two synthetic nitensidine A derivatives, it is suggested that the number, binding site, and polymerization degree of isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their anti-osteoclastic effects.     

Isopetasin and S-isopetasin as novel P-glycoprotein inhibitors against multidrug-resistant cancer cells

BackgroundA major problem of cancer treatment is the development of multidrug resistance (MDR) to chemotherapy. MDR is caused by different mechanisms such as the expression of the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These transporters efflux xenobiotic toxins, including chemotherapeutics, and they were found to be overexpressed in different cancer types.PurposeIdentification of novel molecules that overcome MDR by targeting ABC-transporters.MethodsResazurin reduction assay was used for cytotoxicity test. AutoDock 4.2. was used for molecular docking. The function of P-gp and BCRP was tested using a doxorubicin uptake assay and an ATPase assay. ROS generation was detected using flow cytometry for the measurement of H₂DCFH-DA fluorescence. Annexin/PI staining was applied for the detection of apoptosis. Bioinformatic analyses were performed using LigandScout 3.12. software and DataWarrior software.ResultsIn our search for new molecules that selectively act against resistant phenotypes, we identified isopetasin and S-isopetasin, which are bioactive natural products from Petasites formosanus. They exerted collateral sensitivity towards leukemia cells with high P-gp expression in CEM/ADR5000 cells, compared to sensitive wild-type CCRF-CEM leukemia cells. Also, they revealed considerable activity towards breast cancer cells overexpressing breast cancer resistance protein, MDA-MB-231-BCRP clone 23. This motivated us to investigate whether the function of P-gp was inhibited. In-silico results showed the compounds bound with high affinity and interacted with key amino acid residues in P-gp . Then, we found that the two compounds increased doxorubicin accumulation in P-gp overexpressing CEM/ADR5000 by three-fold compared to cells without inhibitor. P-gp-mediated drug efflux was ATP-dependent. Isopetasin and S-isopetasin increased the ATPase activity of human P-gp in a comparable fashion as verapamil used as control P-gp inhibitor. As isopetasin and S-isopetasin exerted dual roles, first as cytotoxic compounds and then as P-gp inhibitors, we suggested that their P-gp inhibition is part of a larger complex of mechanisms to induce cell death in cancer patients. P-gp dysfunction induces mitochondrial stress to generate ATP. Upon continuing stress by P-gp inhibition, the mitochondria generate reactive oxygen species (ROS). Initially established for verapamil, this theory was validated in the present study for isopetasin and S-isopetasin, as treatment with the two candidates increased ROS levels in CEM/ADR5000 cells followed by apoptosis.ConclusionOur study highlights the importance of isopetasin and S-isopetasin as novel ROS-generating and apoptosis-inducing P-gp inhibitors.     

Influence of combinations of digitonin with selected phenolics,terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of β-carotene (40 μM) and sanguinarine (1 μM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5 μM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p < 0.01) and 46% (p < 0.01) in Caco-2, and to 61% (p < 0.05) and 1% (p < 0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.     

Cytotoxicity of a naturally occurring furoquinoline alkaloid and four acridone alkaloids towards multi-factorial drug-resistant cancer cells

IntroductionChemotherapy is one of the preferred mode of treatment of malignancies, but is complicated by the expression of diverse resistance mechanisms of cancer cells.MethodsIn the present study, we investigated the cytotoxicity of five alkaloids including a furoquinoline montrofoline (1) and four acridones namely 1-hydroxy-4-methoxy-10-methylacridone (2), norevoxanthine (3), evoxanthine (4), 1,3-dimethoxy-10-methylacridone (5) against 9 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry.ResultsFuroquinoline 1 as well as the acridone alkaloids 2–5 displayed cytotoxic effects with IC₅₀ values below 138 µM on all the 9 tested cancer cell lines. The IC₅₀ values ranged from 41.56 µM (towards hepatocarinoma HepG2 cells) to 90.66 µM [towards colon carcinoma HCT116 (p53^−/−) cells] for 1, from 6.78 µM [towards HCT116 (p53^−/−) cells) to 106.47 µM [towards breast adenocarcinoma MDA-MB-231-pcDNA cells] for 2, from 5.72 µM (towards gliobastoma U87MG.ΔEGFR cells) to 137.62 µM (towards leukemia CCRF-CEM cells] for 3, from 6.11 µM [towards HCT116 (p53^+/+) cells] to 80.99 µM (towards HepG2 cells] for 4, from 3.38 µM (towards MDA-MB-231-BCRP cells) to 58.10 µM (towards leukemia CEM/ADR5000 cells] for 5 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Acridone alkaloid 5 induced apoptosis in CCRF-CEM leukemia cells, mediated by increased ROS production.ConclusionsThe five tested alkaloids and mostly acridone 5 are potential cytotoxic natural products that deserve more investigations to develop novel cytotoxic compounds against multifactorial drug-resistant cancers.     

Redox active copper chelate overcomes multidrug resistance in T-lymphoblastic leukemia cell by triggering apoptosis

Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new molecules that are less toxic to normal cell and preferentially effective against drug resistant malignant cells. In this preclinical study we report the apoptotic potential of copper N-(2-hydroxyacetophenone) glycinate (CuNG) on doxorubicin resistant T lymphoblastic leukaemia cells (CEM/ADR5000). To evaluate the cytotoxic effect of CuNG, we used different normal cell lines (NIH 3T3, Chang liver and human PBMC) and cancerous cell lines (CEM/ADR5000, parental sensitive CCRF-CEM, SiHa and 3LL) and conclude that CuNG preferentially kills cancerous cells, especially both leukemic cell types irrespective of their MDR status, while leaving normal cell totally unaffected. Moreover, CuNG involves reactive oxygen species (ROS) for induction of apoptosis in CEM/ADR5000 cells through the intrinsic apoptotic pathway. This is substantiated by our observation that antioxidant N-acetyle-cysteine (NAC) and PEG catalase could completely block ROS generation and, subsequently, abrogates CuNG induced apoptosis. On the other hand, uncomplexed ligand N-(2-hydroxyacetophenone) glycinate (NG) fails to generate a significant amount of ROS and concomitant induction of apoptosis in CEM/ADR5000 cells. Therefore, CuNG induces drug resistant leukemia cells to undergo apoptosis and proves to be a molecule having therapeutic potential to overcome MDR in cancer.     

Cytotoxicity, anti-angiogenic, apoptotic effects and transcript profiling of a naturally occurring naphthyl butenone, guieranone A

ABSTRACT: BACKGROUND: Malignant diseases are responsible of approximately 13% of all deaths each year in the world. Natural products represent a valuable source for the development of novel anticancer drugs. The present study was aimed at evaluating the cytotoxicity of a naphtyl butanone isolated from the leaves of Guiera senegalensis, guieranone A (GA). RESULTS: The results indicated that GA was active on 91.67% of the 12 tested cancer cell lines, the IC50 values below 4 g/ml being recorded on 83.33% of them. In addition, the IC50 values obtained on human lymphoblastic leukemia CCRF-CEM (0.73g/ml) and its resistant subline CEM/ADR5000 (1.01g/ml) and on lung adenocarcinoma A549 (0.72g/ml) cell lines were closer or lower than that of doxorubicin. Interestingly, low cytotoxicity to normal hepatocyte, AML12 cell line was observed. GA showed anti-angiogenic activity with up to 51.9% inhibition of the growth of blood capillaries on the chorioallantoic membrane of quail embryo. Its also induced apotosis and cell cycle arrest. Ingenuity Pathway Analysis identified several pathways in CCRF-CEM cells and functional group of genes regulated upon GA treatment (P < 0.05), the Cell Cycle: G2/M DNA Damage Checkpoint Regulation and ATM Signaling pathways being amongst the four most involved functional groups. CONCLUSION: The overall results of this work provide evidence of the cytotoxic potential of GA and supportive data for its possible use in cancer chemotherapy.     

8,8-bis-(Dihydroconiferyl)-diferulate displayed impressive cytotoxicity towards a panel of human and animal cancer cells

BackgroundRecalcitrant cancers appear as a major obstacle to chemotherapy, prompting scientists to intensify the search for novel drugs to tackle the cell lines expressing multi-drug resistant (MDR) phenotypes.PurposeThe purpose of this study was to evaluate the antiproliferative potential of a ferrulic acid derivative, 8,8-bis-(dihydroconiferyl)-diferulate (DHCF2) on a panel of 18 cancer cell lines, including various sensitive and drug-resistant phenotypes, belonging to human and animals. The mode of induction of cell death by this compound was further studied.MethodsThe antiproliferative activity, autophagy, ferroptotic and necroptotic cell death were evaluated by the resazurin reduction assay (RRA). CCRF-CEM leukemia cells were used for all mechanistic studies. A caspase-Glo assay was applied to evaluate the activity of caspases. Cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H₂DCFH-DA) were assessed by flow cytometry.ResultsDHCF2 demonstrated impressive cytotoxic effects towards the 18 cancer cell lines tested, with IC₅₀ values all below 6.5 µM. The obtained IC₅₀ values were in the range of 1.17 µM (towards CCRF-CEM leukemia cells) to 6.34 µM (towards drug-resistant HCT116 p53^−/− human colon adenocarcinoma cells) for DHCF2 and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against multidrug-resistant CEM/ADR5000 leukemia cells) for the reference drug, doxorubicin. DHCF2 had IC₅₀ values lower than those of doxorubicin, against CEM/ADR5000 cells and on some melanoma cell lines, such as MaMel-80a cells, Mel-2a cells, MV3 cells and SKMel-505 cells. DHCF2 induced autophagy as well as apoptosis in CCRF-CEM cells though caspases activation, MMP alteration and increase of ROS production.ConclusionThe studied diferulic acid, DHCF2, is a promising antiproliferative compound. It deserves further indepth investigations with the ultimate aim to develop a novel drug to fight cancer drug resistance.     

Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells

Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites which have a wide range of bioactivities. Yet, their ability to reverse multidrug resistance (MDR) in cancer cells has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The MDR reversal potential of the isolated lignans and the underlying mechanism of action were studied using two MDR cancer cell lines, CaCo2 and CEM/ADR 5000 which overexpress P-gp and other ABC transporters. In two-drug combinations of lignans with the cytotoxic doxorubicin, all lignans exhibited synergistic effects in CaCo2 cells and matairesinol, arctiin, lappaol C and lappaol F display synergistic activity in CEM/ADR 5000 cells. Additionally, in three-drug combinations of lignans with the saponin digitonin and doxorubicin MDR reversal activity was even stronger enhanced. The lignans can increase the retention of the P-gp substrate rhodamine 123 in CEM/ADR 5000 cells, indicating that lignans can inhibit the activity of P-gp. Our study provides a first insight into the potential chemosensitizing activity of a series of natural lignans, which might be candidates for developing novel adjuvant anticancer agents.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

New artesunic acid homodimers: Potent reversal agents of multidrug resistance in leukemia cells

To evade the problem of multidrug resistance, hybridization of natural products in dimers is considered as an effective method. After the successful synthesis of three artesunic acid homodimers connected by different types of chemical linkers, we analyzed their activity against human CCRF-CEM and multidrug-resistant p-glycoprotein-overexpressing CEM/ADR 5000 leukemia cells and observed, that multidrug resistant cells were not cross-resistant to the new compounds. Collateral sensitivity was observed for artesunic acid homodimer 2. The obtained results deliver valuable information about the linker's structure which is required for homodimers to be highly cytotoxic.     

Synergism of three-drug combinations of sanguinarine and other plant secondary metabolites with digitonin and doxorubicin in multi-drug resistant cancer cells

We determined the ability of some phytochemicals, including alkaloids (glaucine, harmine, and sanguinarine), phenolics (EGCG and thymol), and terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), alone or in combination with the saponin digitonin to reverse the relative multi-drug resistance of Caco-2 and CEM/ADR5000 cells to the chemotherapeutical agent doxorubicin. The IC₅₀ of doxorubicin in Caco-2 and CEM/ADR5000 was 4.22 and 44.08 μM, respectively. Combination of non-toxic concentrations of individual secondary metabolite with doxorubicin synergistically sensitized Caco-2 and CEM/ADR5000 cells, and significantly enhanced the cytotoxicity of doxorubicin. Furthermore, three-drug combinations (secondary metabolite + digitonin + doxorubicin) were even more powerful. The best synergist was the benzophenanthridine alkaloid sanguinarine. It reduced the IC₅₀ value of doxorubicin 17.58-fold in two-drug combinations (sanguinarine + doxorubicin) and even 35.17-fold in three-drug combinations (sanguinarine + digitonin + doxorubicin) in Caco-2 cells. Thus synergistic drug combinations offer the possibility to enhance doxorubicin efficacy in chemotherapy.     

Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket

P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.     

New glycosides from Tetracentron sinense and their cytotoxic activity

One novel neolignan (tetracentronsine; 1), one new indole alkaloid (=3-(2-hydroxyethyl)-1H-indole-5-O-beta-D-glucopyranoside; 2), and two new phenol derivatives, 3-{2-[(beta-glucopyranosyl)oxy]-4,5-(methylenedioxy)phenyl}propanoic acid (3) and methyl 3-{2-[(beta-glucopyranosyl)oxy]-4,5-(methylenedioxy)phenyl}propanoate (4), together with six known compounds were isolated from the stem bark of Tetracentron sinense. Their structures were determined by spectral analysis, including 1D- and 2D-NMR, and MS analyses. These compounds were tested for their cytotoxic activity against human leukaemia cells in vitro. Among them, compound 2, (E)-3-(4-hydroxyphenyl)-N-[2-(4-hydroxyphenyl)ethyl]prop-2-enamide (5), and maslinic acid (6) showed significant inhibitory activities against human leukaemia cells CCRF-CEM and its multidrug-resistant sub-line, CEM/ADR5000, with IC50 values in a range of 7.1 to 29.7 microM.     

Cytotoxic and new tetralone derivatives from Berchemia floribunda (Wall.) Brongn

Two new alpha-tetralone (=3,4-dihydronaphthalen-1(2H)-one) derivatives, berchemiaside A and B (1 and 2, resp.), and one new flavonoid, quercetin-3-O-(2-acetyl-alpha-L-arabinofuranoside (3), together with ten known flavonoids compounds, eriodictyol (4), aromadendrin (5), trans-dihydroquercetin (6), cis-dihydroquercetin (7), kaempferol (8), kaempferol-3-O-alpha-L-arabinofuranoside (9), quercetin (10), quercetin-3-O-alpha-L-arabinofuranoside or avicularin (11), quercetin 3'-methyl ether, 3-O-alpha-L-arabinofuranoside (12), and maesopsin (13), were isolated from the bark of Berchemia floribunda. Their structures were determined by various NMR techniques and chemical studies. Compounds 3-13 were tested for their cytotoxic activity against human leukemia cells. Among them, kaempferol (8) and maesopsin (13) showed significant inhibitory activities against human leukemia cells CCRF-CEM and its multidrug-resistant sub-line, CEM/ADR5000, with IC(50) values of 14.0, 5.3, 10.2, and 12.3 microM, respectively.     

N-acetylglycoside of oleanolic acid (aridanin) displays promising cytotoxicity towards human and animal cancer cells,inducing apoptotic,ferroptotic and necroptotic cell death

BackgroundThe discovery of novel phytochemicals represents a reasonable approach to fight malignancies, especially those which are resistant to standard chemotherapy.PurposeWe evaluated the cytotoxic potential of a naturally occurring N-acetylglycoside of oleanolic acid, aridanin, on 18 cancer cell lines, including sensitive and drug-resistant phenotypes mediated by P-glycoprotein, BCRP, p53 knockout, deletion-mutated EGFR, or BRAF mutations. Furthermore, metastasizing B16/F10 cells, HepG2 hepatocarcinoma and normal AML12 hepatocytes were investigated. The mechanisms of aridanin-induced cell death was further investigated.MethodsThe resazurin reduction assay (RRA) was applied to evaluate the cytotoxicity, autophagy, ferroptotic and necroptotic cell death. CCRF-CEM leukemia cells were used for all mechanistic studies. A caspase-Glo assay was applied to evaluate the caspase activities. Flow cytometry was applied for the analyses of cell cycle (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1) and reactive oxygen species (ROS; H₂DCFH-DA).ResultsAridanin and doxorubicin (positive control) inhibited the proliferation of all cancer cell lines tested. The IC₅₀ values for aridanin varied from 3.18 µM (CCRF-CEM cells) to 9.56 µM (HepG2 cells). Aridanin had considerably lower IC₅₀ values than that of doxorubicin against multidrug-resistant CEM/ADR5000 cells and melanoma cell lines (MaMel-80a, Mel-2a, MV3, and SKMel-505). Aridanin induced apoptosis in CCRF-CEM cells through increase of ROS levels and MMP breakdown, and to a lesser extent via caspases activation. Aridanin also induced ferroptotic and necroptotic cell death.ConclusionThe present study opens good perpectives for the use of this phytochemical as an anticancer drug to combat multi-facorial resistance to established chemotherapeutics.     

Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site

The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.     

Cytotoxicity and modes of action of 4′-hydroxy-2′,6′-dimethoxychalcone and other flavonoids toward drug-sensitive and multidrug-resistant cancer cell lines

IntroductionResistance of cancer to chemotherapy is a main cause in treatment failure. Naturally occurring chalcones possess a wide range of biological activities including anti-cancer effects. In this work, we evaluated the antiproliferative activity of three chalcones [4′-hydroxy-2′,6′-dimethoxychalcone (1), cardamomin (2), 2′,4′-dihydroxy-3′,6′-dimethoxychalcone (3)], and four flavanones [(S)-(–)-pinostrobin (4), (S)-(–)-onysilin (5) and alpinetin (6)] toward nine cancer cell lines amongst which were multidrug resistant (MDR) types.MethodsThe resazurin reduction assay was used to detect the antiproliferative activity of the studied samples whilst flow cytometry for the mechanistic studies of the most active molecule (1).ResultsIC₅₀ values in a range of 2.54 μM against CEM/ADR5000 leukemia cells to 58.63 μM toward hepatocarcinoma HepG2 cells were obtained with 1. The lowest IC₅₀ values of 8.59 μM for 2 and 10.67 μM for 3 were found against CCRF-CEM cells leukemia cells, whilst the corresponding values were above 80 μM for 4 and 6. P-glycoprotein-expressing and multidrug-resistant CEM/ADR5000 cells were much more sensitive toward compound 1 than toward doxorubicin and low cross-resistance or even collateral sensitivity was observed in other drug-resistent cell lines to this compound. Normal liver AML12 cells were more resistant to the studied compounds than HepG2 liver cancer cells, indicating tumor specificity at least to some extent. Compound 1 arrested the cell cycle between Go/G1 phase, strongly induced apoptosis via disrupted mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in the studied leukemia cell line.ConclusionsChalcone 1 was the best tested cytotoxic molecule and further studies will be performed in order to envisage its possible use in the fight against multifactorial resistant cancer cells.     

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (K_d = 0.18 μm) and an ATPase activity with a K_m of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.     

The carotenoid fucoxanthin can sensitize multidrug resistant cancer cells to doxorubicin via induction of apoptosis,inhibition of multidrug resistance proteins and metabolic enzymes

BackgroundMultidrug resistance (MDR) causes failure of doxorubicin therapy of cancer cells, which develops after or during doxorubicin treatment resulting in cross-resistance to structurally and functionally-unrelated other anticancer drugs. MDR is multifactorial phenomenon associated with overexpression of ATP-binding cassette (ABC) transporters, metabolic enzymes, impairment of apoptosis, and alteration of cell cycle checkpoints. The cancer-prevention of the dietary carotenoid; fucoxanthin (FUC) has been extensively explored. Nevertheless, the underlying mechanism of its action is not full elucidated.Hypothesis/PurposeInvestigation of the underlying mechanism of MDR reversal by the dietary carotenoid fucoxanthin (FUC) and its ability to enhance the doxorubicin (DOX) cytotoxicity in resistant breast (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) cell lines.MethodsThe synergistic interaction of FUC and DOX was evaluated using several techniques, viz.; MTT assay, ABC transporter function assays using FACS and fluorimetry, enzyme activity via spectroscopy and luminescence assays, and apoptosis assay using FACS, and gene expression using RTPCR.ResultsFUC (20 µM) synergistically enhanced the cytotoxicity of DOX and significantly reduced the dose of DOX (FR) in DOX resistant cells (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) to 8.42-(CI= 0.25), 6.28-(CI= 0.32), and 4.56-fold (CI=0.37) (P<0.001). FUC significantly increased the accumulation of DOX more than verapamil in resistant cells by 2.70, 2.67, and 3.95-fold of untreated cells (p<0.001), respectively. A FUC and DOX combination significantly increased the Rho123 accumulation higher than individual drugs by 2.36-, 2.38-, 1.89-fold verapamil effects in tested cells (p<0.001), respectively. The combination of the FUC and DOX decreased ABCC1, ABCG2, and ABCB1 expression. The FUC and DOX combination increased the levels and activity of caspases (CASP3, CASP8) and p53, while decreased the levels and activity of CYP3A4, GST, and PXR in resistant cancer cells. The combination induced early/late apoptosis to 91.9/5.4% compared with 0.0/0.7% of untreated control.ConclusionOur data suggests a new dietary and therapeutic approach of combining the FUC with DOX to overcome multidrug resistance in cancer cells. However, animal experiments should be conducted to confirm the findings before applying the results into clinical trials.