Multidrug-resistant Subline Research Articles

Synthesis, structural and computational study, DNA binding and cytotoxic activity of Cu(II) complexes of 6- and 7-chloro-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-2-furoyl-hydrazones

Three new coordination complexes of Cu(II) ions made from two hydrazone ligands, 7-chloro-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-2-furoyl-hydrazone (HL1) and 6-chloro-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-2-furoyl-hydrazone (HL2) have been synthesized and fully characterized by spectroscopic techniques. Their crystal and molecular structures revealed distorted square pyramidal mononuclear complexes: [(L1)Cu(H2O)2](NO3)·3H2O, 1(NO3), [(L2)Cu(H2O)2](NO3)·2H2O·CH3OH, 2(NO3), and [(L2)Cu(NO3)(CH3OH)]·2CH3OH, 3, comprising the ligand (L1 and L2) in tridentate fashion (ONO) with two water molecules in 1+ and 2+, and a single methanol molecule and a nitrate ion in 3 in their respective copper coordination spheres. EPR spectra in frozen methanol revealed the occurrence of several species arising from different coordination environments. A detailed DFT investigation on the energetics of solvents exchange (H2O, MeOH, and DMSO) and simulation of the EPR parameters showed that the exchange processes occur easily in solution. The value gz indicated the occurrence of a dimeric aggregate for 2+. The new copper complexes exhibited a noticeable antiproliferative activity with IC50 values in the micromolar range against HCT-15, H157, BxPC3, PNS-1, and A431 cell lines and they were found to be 3-fold more effective than cisplatin against pancreatic PSN-1 cell lines. Cross-resistance tests on A2780 and LoVo cancer cell lines and the corresponding multidrug or oxaliplatin resistant sublines showed that complexes 1(NO3) and 2(NO3) were equally cytotoxic to sensitive and resistant cells, thus overcoming multidrug and oxaliplatin resistance.

Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline.

To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids. The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was generated by the stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of doxorubicin (Dox) for 5 months. Half-inhibitory concentrations (IC50) for Dox, vinblastine (Vin), and paclitaxel (PTX) were calculated by a colorimetric MTS-based assay. Crystal violet staining was used to assess cellular viability, whereas the proliferation capacities of cancer cells were monitored in real-time by the i-Celligence system. Expression of apoptotic markers (e.g., cleaved PARP and caspase-3), DNA repair proteins (e.g., ATM, DNA-PK, Nbs1, Rad51, MSH2, etc.), and certain ABC transporters (P-glycoprotein, MRP1, ABCG2, etc.) was assessed by western blotting and real-time PCR. Flow cytometry was used to examine the fluorescence intensity of Dox and ABC-transporter substrates (e.g., Calcein AM and CMFDA) and to assess their excretion to define the activity of specific ABC-transporters. To confirm OS resistance to Dox in vivo, xenograft experiments were performed. An OS subline generated by a stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of Dox resulted in an increase in the IC50 for Dox, Vin, and PTX (~6-, 4-, and 30-fold, respectively). The acquisition of chemoresistance in vitro was also evidenced by the lack of apoptotic markers (e.g., cleaved PARP and caspase-3) in resistant OS cells treated with the chemotherapeutic agents indicated above. The development of the multidrug resistance (MDR) phenotype in this OS subline was due to the overexpression of ABCB1 (i.e., P-glycoprotein) and ABCC1 (i.e., multidrug resistance protein-1, MRP-1), which was evidenced on both mRNA and protein levels. Due to increased expression of MDR-related proteins, resistant OS exhibited an excessive efflux of Dox. Moreover, decreased accumulation of calcein AM, a well-known fluorescent substrate for both ABCB1 and ABCC1, was observed for resistant OS cells compared to their parental SaOS-2 cell line. Importantly, tariquidar and cyclosporin, well-known ABC inhibitors, retained the intensity of Dox-induced fluorescence in resistant SAOS-2 cells. Furthermore, in addition to the increased efflux of the chemotherapeutic agents from Dox-resistant OS cells, we found higher expression of several DNA repair proteins (e.g., Rad51 recombinase, Mre11, and Nbs1, activated forms of ATM, DNA-PK, Chk1, and Chk2, etc.), contributing to the chemoresistance due to the excessive DNA repair. Lastly, the in vivo study indicated that Dox has no impact on the SaOS-2 Dox-R xenograft tumor growth in a nude mouse model. An acquired resistance of OS to the chemotherapeutic agents might be due to the several mechanisms undergoing simultaneously on the single-cell level. This reveals the complexity of the mechanisms involved in the secondary resistance of OS to chemotherapies.

Modes of Action of a Novel c-MYC Inhibiting 1,2,4-Oxadiazole Derivative in Leukemia and Breast Cancer Cells.

The c-MYC oncogene regulates multiple cellular activities and is a potent driver of many highly aggressive human cancers, such as leukemia and triple-negative breast cancer. The oxadiazole class of compounds has gained increasing interest for its anticancer activities. The aim of this study was to investigate the molecular modes of action of a 1,2,4-oxadiazole derivative (ZINC15675948) as a c-MYC inhibitor. ZINC15675948 displayed profound cytotoxicity at the nanomolar range in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells. Multidrug-resistant sublines thereof (i.e., CEM/ADR5000 and MDA-MB-231-BCRP) were moderately cross-resistant to this compound (<10-fold). Molecular docking and microscale thermophoresis revealed a strong binding of ZINC15675948 to c-MYC by interacting close to the c-MYC/MAX interface. A c-MYC reporter assay demonstrated that ZINC15675948 inhibited c-MYC activity. Western blotting and qRT-PCR showed that c-MYC expression was downregulated by ZINC15675948. Applying microarray hybridization and signaling pathway analyses, ZINC15675948 affected signaling routes downstream of c-MYC in both leukemia and breast cancer cells as demonstrated by the induction of DNA damage using single cell gel electrophoresis (alkaline comet assay) and induction of apoptosis using flow cytometry. ZINC15675948 also caused G2/M phase and S phase arrest in CCRF-CEM cells and MDA-MB-231-pcDNA3 cells, respectively, accompanied by the downregulation of CDK1 and p-CDK2 expression using western blotting. Autophagy induction was observed in CCRF-CEM cells but not MDA-MB-231-pcDNA3 cells. Furthermore, microarray-based mRNA expression profiling indicated that ZINC15675948 may target c-MYC-regulated ubiquitination, since the novel ubiquitin ligase (ELL2) was upregulated in the absence of c-MYC expression. We propose that ZINC15675948 is a promising natural product-derived compound targeting c-MYC in c-MYC-driven cancers through DNA damage, cell cycle arrest, and apoptosis.

Anticancer and chemosensitizing activities of stilbenoids from three orchid species.

Recently, we have isolated and identified several bioactive flavonoids and stilbenoids with potential anticancer activity from Thai orchids. In this study, we further investigated the cytotoxic and chemosensitizing activities of these phytochemicals (namely, pinocembrin, cardamonin, isalpinin, galangin, pinosylvin monomethyl ether, 2,3'-dihydroxy-5'-methoxystilbene, (E)-2,5'-dihydroxy-2'-(4-hydroxybenzyl)-3'-methoxystilbene, 2,3-dihydroxy-3',5'-dimethoxystilbene, 2,3'-dihydroxy-5,5'-dimethoxystilbene, 3,4'-dihydroxy-5-methoxystilbene and batatasin III) against breast cancer MCF7 cells and its two multidrug resistant (MDR) sublines (MCF7/DOX and MCF7/MX). Cytotoxicity was determined with MTT assay for the estimation of the half maximal cytotoxic concentrations (IC50). Effects of the test compounds on activities of efflux transporters (BCRP, P-gp, MRP1, and MRP2) were evaluated with substrate accumulation assays using fluorometry and flow cytometry analysis. Out of these 11 test compounds, the stilbene pinosylvin monomethyl ether displayed its cytotoxicity specifically toward MCF7 cells (IC50 = 6.2 ± 1.2μM, 72-h incubation) with 4.96 folds higher than normal fibroblast. Its potency decreased in MCF7/DOX and MCF7/MX cells by 3.94 and 7.38 folds, respectively. Our transporter assay indicated that this stilbene significantly reduced the activities of P-gp, MRP1, and MRP2, but not BCRP. After 48-h co-incubation, this stilbene (at 2μM) synergistically increased doxorubicin- and mitoxantrone-mediated cytotoxicity in MCF7, MCF7/DOX, and MCF7/MX cells potentially by increasing the intracellular level of cytotoxic drug. Pinosylvin monomethyl ether could sensitize breast cancer cells to chemotherapy and overcome MDR, in part, via the inhibition of drug efflux transporters.

Palladium (II), platinum (II) and silver (I) complexes with oxazolines: Their synthesis, characterization, DFT calculation, molecular docking and antitumour effects

Six new Pd(II), Pt(II) and Ag(I) complexes, (1);{Pd (L1)]2C6H4}2Cl4} (2); Pt(L2)(DMSO)Cl; 3; {PtL5]2C6H4}2·PhCOO-⋅11NO3-; 4; {[Pt(L4)]2C6H4}; the binuclear cyclometalated complex the polymer chain (5); {[PtL5]C6H4}·NO3-}; and the polymeric silver species (6); Zn(L6)2·AgNO3·CHCl3 were synthesized and thoroughly characterized using X-ray diffraction and spectroscopic techniques (L1=(S,S)-1,4-i-PrOx]2C6H4}2Cl4, L2=Di(2,2-bis(4R-isopropyl-4,5-dihydro-oxazol-2-yl)acetonitrile) zinc (II) (BR1);L3= 1,4-bis(4R-benzyl-4,5-dihydro-oxazol-2-yl)benzene (AR2); L4= 1,4-bis(4R-benzyl-4,5-dihydro-oxazol-2-yl)benzene，L5=1,4-bis(4R-benzyl-4,5-dihydro-oxazol-2-yl)-benzene，L6=Di(2,2-bis(4S-isopropyl-4,5-dihydrooxazol-2-yl)acetonitrile) zinc (II).Complexes 1-6 showed cytotoxic effects against human tumour cell lines, including a multidrug-resistant subline. Oxazoline and Pd complex 1 induced apoptosis in A549 cells. DFT calculations were also performed to exhibit the excellent bioactivity of complex 1 against A549, MDA-MB-231, and KB cells. Complex 1, with the best docking score and a stable interaction network within the binding site of the G-quadruplex, could stably interact with the G-quadruplex. Additionally, complex 1 was further used in the animal experiment of human lung adenocarcinoma cells in nude mice. By comparing with the model control group, the tumour volume, relative tumour volume and relative tumour proliferation rate T/C decreased significantly in the cisplatin group and compound 1 (complex 1) group.

Discovery of C20-Diterpenoid Alkaloid Kobusine Derivatives Exhibiting Sub-G1 Inducing Activity.

Although many diterpenoid alkaloids have been evaluated recently for antiproliferative activity against human cancer cell lines, little data have been offered relating to the antiproliferative effects of hetisine-type C20-diterpenoid alkaloids, such as kobusine (1), likewise as their derivatives. A total of 43 novel diterpenoid alkaloid derivatives (2–10, 2b, 3a, 3b, 6a–16a, 7b, 9b, 10b, 13, 15–26, 15b, 18a, 23a, 27a) were prepared by C-11 and -15 esterification of 1. Antiproliferative effects of the natural parent compound (1) and all synthesized kobusine derivatives against human cancer cell lines, including a triple-negative breast cancer (TNBC) cell line as well as a P-glycoprotein overexpressing multidrug-resistant subline, were assessed. The structure-based design strategy resulted in the lead derivative 11,15-dibenzoylkobusine (3; average IC50 7.3 μM). Several newly synthesized kobusine derivatives (particularly, 5–8, 10, 13, 15–26) exhibited substantial suppressive effects against all tested human cancer cell lines. In contrast, kobusine (1), 11,15-O-diacetylkobusine (2), 11-acylkobusine derivatives (3a, 6a, 9a, 11a, 12a, 15a, 27a), and 15-acylkobusine derivatives (2b, 3b, 7b, 9b, 10b, 15b) showed no effect. The most active kobusine derivatives primarily had two specific substitution patterns, C-11,15 and C-11. Notably, 11,15-diacylkobusine derivatives (3, 6–10, 13, 15, 16, 18, 23) were more potent compared with 11- and 15-acylkobusine derivatives (3a, 3b, 6a–10a, 7b, 9b, 10b, 13a, 15a, 15b, 16a, 18a, 23a). Derivatives 13 and 25 induced MDA-MB-231 cells to the sub-G1 phase within 12 h. 11,15-Diacylation of kobusine (1) appears to be crucial for inducing antiproliferative activity in this alkaloid class and could introduce a new avenue to overcome TNBC using natural product derivatives.

Establishing and characterizing a novel doxorubicin-resistant acute myeloid leukaemia cell line

Drug resistance is a major setback in cancer treatment, thus models to study its mechanisms are needed. Our work aimed to establish and characterize a resistant cell line from a sensitive acute myeloid leukaemia (AML) cell line – HL60 – by treating the sensitive cells with increasing concentrations of doxorubicin. We confirmed (cell viability assays) that the established subline, HL60-CDR, was resistant to doxorubicin for at least 30 days without drug treatment. The HL60-CDR cells were also resistant to three other drugs (cisplatin, etoposide and daunorubicin), exhibiting a multidrug resistant (MDR) profile. We verified (Western Blotting) that the MDR cells do not express drug efflux pumps, nor present altered expression of apoptotic proteins, when compared with the parental cell line. HL60-CDR cells presented alterations in the cell cycle profile, and in the expression levels of proteins involved in DNA repair mechanisms and drug metabolism, when compared with their drug sensitive counterpart. Proteomic analysis revealed that HL60-CDR cells presented an upregulation of proteins involved in oncogenic pathways, such as TSC2, PDPK1, Annexin A2, among others. Overall, we established an AML MDR subline – HL60-CDR – which presents several resistance mechanisms, providing an in vitro model to test new compounds to circumvent MDR in AML.

Peptaibol-Containing Extracts of Trichoderma atroviride and the Fight against Resistant Microorganisms and Cancer Cells.

Fighting resistance to antibiotics and chemotherapeutics has brought bioactive peptides to the fore. Peptaibols are short α-aminoisobutyric acid-containing peptides produced by Trichoderma species. Here, we studied the production of peptaibols by Trichoderma atroviride O1 and evaluated their antibacterial and anticancer activity against drug-sensitive and multidrug-resistant bacterium and cancer cell lines. This was substantiated by an analysis of the activity of the peptaibol synthetase-encoding gene. Atroviridins, 20-residue peptaibols were detected using MALDI-TOF mass spectrometry. Gram-positive bacteria were susceptible to peptaibol-containing extracts of T. atroviride O1. A synergic effect of extract constituents was possible, and the biolo-gical activity of extracts was pronounced in/after the peak of peptaibol synthetase activity. The growth of methicillin-resistant Staphylococcus aureus was reduced to just under 10% compared to the control. The effect of peptaibol-containing extracts was strongly modulated by the lipoteichoic acid and only slightly by the horse blood serum present in the cultivation medium. Peptaibol-containing extracts affected the proliferation of human breast cancer and human ovarian cancer cell lines in a 2D model, including the multidrug-resistant sublines. The peptaibols influenced the size and compactness of the cell lines in a 3D model. Our findings indicate the molecular basis of peptaibol production in T. atroviride O1 and the potential of its peptaibol-containing extracts as antimicrobial/anticancer agents.

Fatty acid synthase, a novel poor prognostic factor for acute lymphoblastic leukemia which can be targeted by ginger extract

Altered metabolism of fatty acid synthesis is considered a hallmark characteristic of several malignancies, including acute lymphoblastic leukemia (ALL). To evaluate the impact of fatty acid synthase (FASN) on drug resistant ALL, bone marrow samples were collected from 65 pediatric ALLs, including 40 de novo and 25 relapsed patients. 22 non-cancer individuals were chosen as controls. Quantitative RT-PCR showed increased expression levels of FASN in drug resistant patients compared with the therapy responders. Single and combined treatment of malignant cells were analyzed using Annexin-V/PI double staining and MTT assays. Incubation of resistant primary cells with ginger showed simultaneous increased apoptosis rates and reduced FASN expression levels. Furthermore, docking studies demonstrated high affinity bindings between ginger derivatives and FASN thioesterase and ketosynthase domains, compared with their known inhibitors, fenofibrate and morin, respectively. Finally, combined treatment of in-house multidrug resistant T-ALL subline with ginger and dexamethasone induced drug sensitivity and down regulation of FASN expression, accordingly. To the best of our knowledge, this is the first study that introduces FASN upregulation as a poor prognostic factor for drug resistant childhood ALL. Moreover, it was revealed that FASN inhibition may be applied by ginger phytochemicals and overcome dexamethasone resistance, subsequently.

Changes in the Surface Expression of Intercellular Adhesion Molecule 3, the Induction of Apoptosis, and the Inhibition of Cell-Cycle Progression of Human Multidrug-Resistant Jurkat/A4 Cells Exposed to a Random Positioning Machine.

Experiments from flight- and ground-based model systems suggest that unexpected alterations of the human lymphoblastoid cell line Jurkat, as well as effects on cell growth, metabolism, and apoptosis, can occur in altered gravity conditions. Using a desktop random positioning machine (RPM), we investigated the effects of simulated microgravity on Jurkat cells and their multidrug-resistant subline, Jurkat/A4 cells. The viability of Jurkat/A4 cells decreased after simulated microgravity in contrast with the Jurkat cells. At the same time, the viability between the experimental Jurkat cells and control Jurkat cells was not significantly different. Of note, Jurkat cells appeared as less susceptible to apoptosis than their multidrug-resistant clone Jurkat/A4 cells, whereas cell-cycle analysis showed that the percentage of Jurkat/A4 cells in the S-phase was increased after 72 and 96 h of RPM-simulated microgravity relative to their static counterparts. The differences in Jurkat cells at all phases between static and simulated microgravity were not significant. The surface expression of the intercellular adhesion molecule 3 (ICAM-3)—also known as cluster of differentiation (CD)50—protein was changed for Jurkat/A4 cells following exposure to the RPM. Changes in cell morphology were observed in the Jurkat/A4 cells after 96 h of RPM-simulated microgravity. Thus, we concluded that Jurkat/A4 cells are more sensitive to RPM-simulated microgravity as compared with the parental Jurkat cell line. We also suggest that intercellular adhesion molecule 3 may be an important adhesion molecule involved in the induction of leukocyte apoptosis. The Jurkat/A4 cells with an acquired multidrug resistance phenotype could be a useful model for studying the effects of simulated microgravity and testing anticancer drugs.

Cytotoxic diterpenoid alkaloid from Aconitum japonicum subsp. subcuneatum.

We explored new cytotoxic C19-diterpenoid alkaloid, lipojesaconitine (1), from rhizoma of Aconitum japonicum THUNB. subsp. subcuneatum (NAKAI) KADOTA. Two additional non-cytotoxic new C19-diterpenoid alkaloids, 10-hydroxychasmanine (2) and 3-hydroxykaracoline (3), together with eight known C19- and C20-diterpenoid alkaloids (4-11) were also isolated. Their structures were elucidated by extensive spectroscopic methods including NMR (1D and 2D), IR, and MS (HRMS). Six known diterpenoid alkaloids, foresticine (5), neolinine (6), aconicarchamine A (7), 9-hydroxynominine (8), kobusine (9), and torokonine (10), were isolated for the first time from A. japonicum subsp. subcuneatum. Alkaloid 1 showed cytotoxicity with IC50 values ranging from 6.0 to 7.3µM against four human tumor cell lines, except a multidrug-resistant subline, suggesting that 1 was likely exported by P-glycoprotein.

Cytotoxic Effects of Diterpenoid Alkaloids Against Human Cancer Cells.

Diterpenoid alkaloids are isolated from plants of the genera Aconitum, Delphinium, and Garrya (Ranunculaceae) and classified according to their chemical structures as C18-, C19- or C20-diterpenoid alkaloids. The extreme toxicity of certain compounds, e.g., aconitine, has prompted a thorough investigation of how structural features affect their bioactivities. Therefore, natural diterpenoid alkaloids and semi-synthetic alkaloid derivatives were evaluated for cytotoxic effects against human tumor cells [A549 (lung carcinoma), DU145 (prostate carcinoma), MDA-MB-231 (triple-negative breast cancer), MCF-7 (estrogen receptor-positive, HER2-negative breast cancer), KB (identical to cervical carcinoma HeLa derived AV-3 cell line), and multidrug-resistant (MDR) subline KB-VIN]. Among the tested alkaloids, C19-diterpenoid (e.g., lipojesaconitine, delcosine and delpheline derivatives) and C20-diterpenoid (e.g., kobusine and pseudokobusine derivatives) alkaloids exhibited significant cytotoxic activity and, thus, provide promising new leads for further development as antitumor agents. Notably, several diterpenoid alkaloids were more potent against MDR subline KB-VIN cells than the parental drug-sensitive KB cells.

Structure-activity relationships and evaluation of esterified diterpenoid alkaloid derivatives as antiproliferative agents.

Diterpenoid alkaloids with remarkable chemical properties and biological activities are frequently found in plants of the genera Aconitum, Delphinium, and Garrya. However, little information has been reported on the antiproliferative effects of the diterpenoid alkaloid constituents of Aconitum and Delphinium plants. C-1 and 14 esterifications of delcosine (1) were carried out to provide 39 new diterpenoid alkaloid derivatives (3-14, 16-29, 3a-7a, 9a, 13a, 13b, 14a, 14b, 16a, 17a, 24a, 35a). Selected compounds (3-14, 16-29, 3a-7a, 9a, 13a, 13b, 14a, 14b, 16a, 17a, 24a, 35a) were evaluated for antiproliferative activity against three to five human tumor cell lines including triple-negative breast cancer (TNBC) and P-glycoprotein (P-gp)overexpressing multidrug-resistant (MDR)subline. Several newly synthesized delcosine derivatives (6, 7, 13, 13a, 13b) showed substantial suppressive effects against all human tumor cell lines tested. In contrast, the natural alkaloids (1, 31, 33) showed no effect. Most of the active compounds were delcosine derivatives with two specific substitution patterns-C-1 and C-1,14. In particular, 1-acyldelcosine derivative (5-7) displayed more potency than 1,14-diacyldelcosine derivatives (5a-7a). These acylated alkaloid derivatives caused accumulation of TNBC cells at sub-G1 within 24h. 1-Acylation of 1 appears to be critical for producing antiproliferative activity in this alkaloid class and a means to provide promising new leads for further development into antitumor agents.

Synthesis, antimicrobial and antiproliferative properties of epi-oligomycin A, the (33S)-diastereomer of oligomycin A

abstractWe describe the synthesis of epi-oligomycin A, a (33S)-diastereomer of the antibiotic oligomycin A. The structure of (33S)-oligomycin A was determined by elemental analysis, spectroscopic studies, including 1D and 2D NMR spectroscopy, and mass spectrometry. Isomerization of C33 hydroxyl group led to minor changes in the potency against Aspergillus niger, Candida spp., and filamentous fungi whereas the activity against Streptomyces fradiae decreased by approximately 20-fold compared to oligomycin A. We observed that 33-epi-oligomycin A had the same activity on the human leukemia cell line K562 as oligomycin A but was more potent for the multidrug resistant subline K562/4. Non-malignant cells were less sensitive to both oligomycin isomers. Finally, our results pointed at the dependence of the cytotoxicity of oligomycins on oxygen supply.

Design and synthesis of novel 7-[(N-substituted-thioureidopiperazinyl)-methyl]-camptothecin derivatives as potential cytotoxic agents

As part of continuing our research on diverse C-7 derivatives of camptothecin (CPT), 16 CPT derivatives bearing piperazinyl-thiourea chemical scaffold and different substituent groups have been designed, synthesized and evaluated in vitro for cytotoxicity against five tumor cell lines (A-549, MDA-MB-231, MCF-7, KB and KBvin). As a result, all the synthesized compounds showed promising in vitro cytotoxic activity against the five tumor cell lines tested, and were more potent than irinotecan. Importantly, compounds 13 g (IC50 = 0.514 μM) and 13o (IC50 = 0.275 μM) possessed similar or better antiproliferative activity against the multidrug-resistant (MDR) KBvin subline than that of topotecan (IC50 = 0.511 μM) and merit further development as anticancer candidates for clinical trail. With these results in hand, we have a reason to conclude that incorporating piperazinyl-thiourea motifs into position-7 of camptothecin confers well cytotoxic activity against cancer cell lines, probably resulting in new anticancer drugs.

Discovery and synthesis of sulfur-containing 6-substituted 5,8-dimethoxy-1,4-naphthoquinone oxime derivatives as new and potential anti-MDR cancer agents

Multi-drug resistance (MDR) to anticancer drugs is the primary impediment to successful treatment of cancer. Hunting for new compounds with potent anti-MDR activity is an effectual approach to conquer cancer drug resistance. In this work, 33 new sulfur-containing 1,4-naphthoquinone oxime derivatives were prepared and investigated for their cytotoxicity against a panel of tumor cell lines and fibroblast normal cell line. Cell-based assay showed that most of target compounds displayed more potent cytotoxic potency than positive controls. Meanwhile, all of compounds were non-toxic to normal cells. More importantly, the cytotoxic activity of these oxime derivatives toward drug-resistant cancer cell lines was found to be much stronger than that toward drug-susceptible cell lines (anti-drug resistance coefficient (ADRC) > 1). Of these, compound 12 m was identified as the most effective molecule with IC50 values in the range of 0.29 ± 0.01 to 1.33 ± 0.05 μM toward MDR sublines. Further mechanism studies demonstrated that 12 m could inhibit colony formation, cause G1 phase arrest and promote cell apoptosis mediated by augmenting Bax/Bcl-2 ratio of Bel7402/5-FU cells. Our findings provide promising start points for development of sulfur-containing 1,4-naphthoquinone oxime derivatives as potential anti-MDR agents.

Ancistrobreveines A-D and related dehydrogenated naphthylisoquinoline alkaloids with antiproliferative activities against leukemia cells, from the West African liana Ancistrocladus abbreviatus.

A unique series of six biaryl natural products displaying four different coupling types (5,1′, 7,1′, 7,8′, and 5,8′) were isolated from the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae). Although at first sight structurally diverse, these secondary metabolites all have in common that they belong to the rare group of naphthylisoquinoline alkaloids with a fully dehydrogenated isoquinoline portion. Among the African Ancistrocladus species, A. abbreviatus is so far only the second one that was found to produce compounds with such a molecular entity. Here, we report on four new representatives, named ancistrobreveines A–D (12–14, and 6). They were identified along with the two known alkaloids 6-O-methylhamateine (4) and ent-dioncophylleine A (10). The two latter naphthylisoquinolines had so far only been detected in Ancistrocladus species from Southeast Asia. All of these fully dehydrogenated alkaloids have in common being optically active despite the absence of stereogenic centers, due to the presence of the rotationally hindered biaryl axis as the only element of chirality. Except for ent-dioncophylleine A (10), which lacks an oxygen function at C-6, the ancistrobreveines A–D (12–14, and 6) and 6-O-methylhamateine (4) are 6-oxygenated alkaloids, and are, thus, typical ‘Ancistrocladaceae-type’ compounds. Ancistrobreveine C (14), is the first – and so far only – example of a 7,8′-linked fully dehydrogenated naphthylisoquinoline discovered in nature that is configurationally stable at the biaryl axis. The stereostructures of the new alkaloids were established by spectroscopic (in particular HRESIMS, 1D and 2D NMR) and chiroptical (electronic circular dichroism) methods. Ancistrobreveine C (14) and 6-O-methylhamateine (4) exhibited strong antiproliferative activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000.

5′-Chloro-2,2′-dihydroxychalcone and related flavanoids as treatments for prostate cancer

Several flavonoids and their biosynthetic precursor chalcones were designed and synthesized to improve the biological effects of the lead compound 2′-hydroxyflavonone against androgen receptor (AR)-dependent transcriptional stimulation. Newly synthesized chalcones 19 and 26 suppressed AR-dependent transcription as well as DHT-dependent growth stimulation at a low micromolar level. These compounds were also effective against ligand-independent constitutively active mutant AR derived from castration-resistant PCa (CRPC). Compounds 19 and 26 showed broad spectrum antiproliferative activity at 5–10 μM against multiple tumor cell lines including androgen-independent and taxane-resistant prostate cancer as well as a multidrug-resistant subline. Mode of action studies suggested that 19 induced sub-G1 accumulation in PC-3 cells by disrupting the microtubule network without affecting cell cycle progression. Furthermore, the in vivo effectiveness of chalcone 19 was confirmed in a xenograft model antitumor assay. Thus, chalcone 19 has the potential to be a bifunctional lead for treatment of AR-dependent PCa at lower doses as well as AR-independent PCa, including CRPC, at higher doses.

Abstract LB-066: A high-throughput screen of an annotated small molecule library identifies substrates of P-glycoprotein

Abstract A major difficulty in treating cancer is the development of cellular resistance to chemotherapeutics, a phenomenon known as multidrug resistance (MDR). MDR is primarily conferred by an upregulation of ATP-binding cassette (ABC) transporters, most notably P-glycoprotein (P-gp, MDR1, ABCB1), which are responsible for the active transport of drugs out of the cells thereby minimizing drug-target interactions. While numerous strategies to overcome transporter-mediated MDR have been explored, none have proved successful. An alternative approach is to exploit resistance by identifying drugs that target MDR cells over the nonresistant parental cells from which they were selected. As such, the development of MDR1-selective agents represents a novel and relatively unexplored strategy for resolving multidrug resistance. To identify such agents, we developed a series of robust, high-throughput assays to examine differential toxicity between drug-naïve cancer cell lines and their drug-selected, multidrug-resistant sublines. We performed high-throughput screens against several libraries of annotated compounds including the NCATS Pharmaceutical Collection (NPC), a comprehensive collection of all clinically approved drugs - to identify P-gp substrates, and classes of small molecules that exhibit selective toxicity against MDR tumor lines. Further mechanistic evaluation of these compounds has provided valuable insight into targets or pathways that appear to have been rendered more sensitive to inhibition in the course of multidrug resistance development. Citation Format: Tobie D. Lee, Olivia W. Lee, Kyle R. Brimacombe, Min Shen, Lu Chen, Rajarshi Guha, Bethelihem Tebase, Robert W. Robey, Suresh V. Ambudkar, Michael M. Gottesman, Matthew D. Hall. A high-throughput screen of an annotated small molecule library identifies substrates of P-glycoprotein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-066.

Ancistrolikokine I and further 5,8′-coupled naphthylisoquinoline alkaloids from the Congolese liana Ancistrocladus likoko and their cytotoxic activities against drug-sensitive and multidrug resistant human leukemia cells

The Congolese liana Ancistrocladus likoko (Ancistrocladaceae) produces naphthylisoquinoline alkaloids that are, chemotaxonomically remarkable, all based on the same coupling type, with the biaryl axis located between C-5 and C-8′. About 20 alkaloids, belonging to the subclass of 5,8′-linked naphthylisoquinolines, have so far been discovered in this plant species. Here, we report on the isolation and structure elucidation of six further such 5,8′-coupled monomeric alkaloids, named ancistrolikokines I (9), C3 (10), F2 (11), J (12), J2 (13), and J3 (14). They were identified in the twigs of A. likoko, along with the two new atropo-diastereomeric dimers michellamines A8 (15a) and B8 (15b) and the naphthalene-devoid dihydroisoquinoline ent-ealaine D (19). The latter had previously only been known from total synthesis and has now been identified for the first time as an authentic natural product. Three of the new alkaloids, 12–14, are the only fully dehydrogenated naphthylisoquinolines with a 5,8'-biaryl linkage, apart from one single known other example previously likewise found in A. likoko. The stereostructures of the new alkaloids were established by spectroscopic (in particular HRESIMS, 1D and 2D NMR), chemical (oxidative degradation), and chiroptical (electronic circular dichroism) methods. The new ancistrolikokines exhibited moderate to strong cytotoxic activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and against cells of their multidrug-resistant subline, CEM/ADR5000. A first structure-activity relationship (SAR) study on a small library of 5,8'-coupled naphthylisoquinolines from the twigs of A. likoko suggests that the oxygenation patterns in the isoquinoline portion at C-6 and C-8 play a crucial role for the antileukemic activities within this group of alkaloids.