Drug-resistant Tumor Cell Lines Research Articles

Antimicrobial spiroketal macrolides and dichloro-diketopiperazine from Micromonospora sp. FIMYZ51

Four compounds (1–4) featuring with an L-rhodinose and spiroketal, possess uncommon continuous hydroxy groups in the macrolide skeleton, and a dichloro-diketopiperazine (5) were isolated from a marine derived Micromonospora sp. FIMYZ51. The determination of the relative and absolute configurations of all isolates was achieved by extensive spectroscopic analyses, single-crystal X-ray diffraction analysis, and ECD calculations. According to structural characteristic and genomic sequences, a plausible biosynthetic pathway for compound 1-4 was proposed and a spirocyclase was inferred to be responsible for the formation of the rare spirocyclic moiety. Compounds 1-4 exhibited potent antifungal activities which is equal to itraconazole against Aspergillus niger. Compounds 1-5 exhibited different degree of inhibitory activities against opportunistic pathogenic bacteria of endocarditis (Micrococcus luteus) with MIC values ranging from 0.0625 μg/mL to 32 μg/mL. Compounds 2 and 3 showed moderate cytotoxicity against drug-resistant tumor cell lines (Namalwa and U266). The result not only provides active lead-compounds, but also reveal the potential of the spirocyclase gene resources from Micromonospora sp., which highlights the promising potential of the strain for biomedical applications.

Ligand- and structural-based discovery of potential small molecules that target the colchicine site of tubulin for cancer treatment

Small molecules targeting the colchicine site of tubulin represent an attractive cancer treatment strategy. In this study, a total of 468 models derived from 1076 diverse inhibitors binding to the tubulin colchicine site were constructed based on fingerprints using three machine learning approaches: 1) naive Bayesian (NB); 2) single tree (ST); and 3) random forest (RF). The overall predictive accuracy of the best models exceeded 85.12% for both the training and test sets. We designed an integrated virtual screening (VS) strategy for identifying new tubulin inhibitors by combining established models, molecular docking, and similarity-based analog searching. Through two rounds of VS, compound 23g was identified as a novel potent anticancer agent exhibiting activity against MDA-MB-231, HeLa, A549, HepG2, CNE2, and HCT116 tumor cell lines with IC50 values of 5.45, 8.61, 7.47, 2.29, 2.91, and 4.10 μM, respectively. Compared with taxol, colchicine, and adriamycin, 23g also displayed potent cytotoxicity against the drug-resistant tumor cell lines, HepG2/ADR, A549/CDDP, and A549/TAX cells, with IC50 values of 4.12, 6.58, and 6.38 μM, respectively. Further mechanistic studies revealed that 23g inhibited microtubule polymerization by binding to the colchicine site of tubulin, arrested the cell cycle at the G2/M phase, induced cell apoptosis, and exhibited potent in vitro anti-metastasis activity. Finally, molecular docking, molecular dynamics, and free energy analyses were employed to explore the detailed binding interaction between 23g and tubulin. Collectively, these findings indicated that 23g should be further investigated as a potential novel potent antitumor agent targeting tubulin.

In Vitro Antibacterial and Antiproliferative Potential of Echinops lanceolatus Mattf. (Asteraceae) and Identification of Potential Bioactive Compounds.

Many species belonging to the genus Echinops are widely used in traditional medicine to treat infectious diseases and cancers. The present study aimed to evaluate the antibacterial and antiproliferative properties of Echinops lanceolatus Mattf. (Asteraceae). The activity of the methanolic extract and subsequent partition fractions was investigated against drug-resistant bacteria (Gram-negative and Gram-positive) and human tumor cell lines using broth microdilution and sulforhodamine B (SRB) assay, respectively. Our findings revealed weak to moderate antibacterial activities of tested extracts, with the recorded minimal inhibitory concentrations ranging from 256 to 1024 µg/mL. The ethyl acetate fraction (EL-EA) was found to be the most effective. Likewise, that fraction displayed strong antiproliferative potential with recorded IC50 of 8.27 µg/mL and 28.27 µg/mL on A549 and HeLa cells, respectively. An analysis based on the ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS) of the EL-EA fraction allowed the identification of 32 compounds, of which quinic acid and derivatives, cinnamic acid derivatives, dihydrokaempferol, naringenin-7-O-glucoside, apigenin-7-O-d-glucoside, naringin, apigenin, rhoifolin, coniferyl aldehyde, and secoisolariciresinol are well-known compounds of biological importance. This study is first to report on the biological activity and phytochemical profile of E. lanceolatus. We provide a baseline to consider E. lanceolatus as a valuable source of anti-infective and antiproliferative agents.

Abstract 1430: Anti-CD3 x anti-EGFR bispecific antibody armed T cells (EGFR BATs) kills resistant pancreatic cancer cell lines and increases sensitivity to chemotherapy

Abstract Chemotherapy (chemoT) responses of pancreatic cancer (PC) are short and PC rapidly develop multidrug resistance (MDR) with median overall survival (OS) of locally advanced PC and metastatic PC patients of 7 months. Several MPC patients developed dramatic responses to the same chemoT after receiving multiple infusions of EGFR BATs. Novel therapeutic strategies are needed to improve clinical results. This study asks whether pre-targeting tumor cells with EGFR BATs can “sensitize” drug resistant tumor cell lines so that subsequent treatment can not only kill the MDR PC lines but also decrease the doses of chemotherapy required to kill tumor cells. Gemcitabine (Gem) and cisplatin (CIS) were used to generate drug-resistant MiaPaCa-2 and L3.6 by exposing step-wise increasing concentrations of Gem and Cis. MiaPaCa-2 and L3.6 survive to become resistant to Gem and CIS. Specific cytotoxicity was measured by 51Cr release. Flow cytometry data showed increased CD44+/CD24+/EpCAM+ cancer stem like cells and increased ABC transporter ABCG2+ cells in resistant cell lines compared to parental lines. EGFR or HER2 BATs from normal donors would kill Gem or CIS resistant MiaPaCa-2 or L3.6 (n = 6-8 normals) in 51Cr release assays and secrete Th1cytokines, IFN-γ and TNF-α, MIP-1b, and RANTES. We assessed whether pretreatment with EGFR BATs modify the effective dose of CIS activity directed at MiaPaCa-2 and PANC-1 cells using the xCelligence system. EGFR BATs at an E:T of 2:1 were added to the target cells for 24 hours, washed out, and CIS was added at concentrations of 3.5, 7, and 14 µM (IC50 by MTT) and MiaPaCa-2 at concentrations of 4.7, 9.3, and 18.6 µM (IC50 by MTT). The survival of the MiaPaCa2-2 cells was monitored continuously to track survival of the tumor cells. The mean cytotoxicity directed at MiaPaCa-2 increased with BATs priming at concentrations lower than the IC50 (4.7 and 9.3 µM). At the 4.7 µM dose, cytotoxicity increased from CIS alone (31±15%) to BATs primed followed by CIS (57±17%), and BATs together with CIS (77±12%). In PANC-1 cells, there was no specific cytotoxicity of with CIS concentrations at 3.5, 7.0, and 14 µM. BATs priming before addition of CIS increased specific cytotoxicity to 51±13%, 60±9%, and 84±5% at CIS concentrations of 3.5, 7.0, and 14 µM, respectively. Similar data have been obtained for 5-fluorouracil and preliminary data show similar trends for CIS resistant MiaPaCa-2 and PANC-1. In summary, we showed that: 1) BATs can kill chemo resistant tumor cells; and 2) pretreatment or sensitization of the tumor with BATs lowers the threshold for effective cytotoxic doses of CIS against MiaPaCa2-2 and PANC-1. This BATs priming strategy may augment clinical responses, decrease chemoT toxicities, and improve overall survival for pancreatic cancer patients. Citation Format: Lawrence G. Lum, Johnson Ung, Archana Thakur. Anti-CD3 x anti-EGFR bispecific antibody armed T cells (EGFR BATs) kills resistant pancreatic cancer cell lines and increases sensitivity to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1430.

Cytotoxicity of cucurbitacin E from Citrullus colocynthis against multidrug-resistant cancer cells

BackgroundCucurbitacin E (CuE) is an oxygenated tetracyclic triterpenoid isolated from the fruits of Citrullus colocynthis (L.) Schrad. PurposeThis study outlines CuE's cytotoxic activity against drug-resistant tumor cell lines. Three members of ABC transporters superfamily, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and ABCB5 were investigated, whose overexpression in tumors is tightly linked to multidrug resistance. Further factors of drug resistance studied were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). MethodsCytotoxicity assays (resazurin assays) were used to investigate the activity of Citrullus colocynthis and CuE towards multidrug resistant cancer cells. Molecular docking (In silico) has been carried out to explore the CuE's mode of binding to ABC transporters (P-gp, BCRP and ABCB5). The visualization of doxorubicin uptake was done by a Spinning Disc Confocal Microscope. The assessment of proteins expression was done by western blotting analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to cucurbitacins (CuA, CuB, CuE, CuD, CuI, and CuK). ResultsMultidrug-resistant cells overexpressing P-gp or BCRP were cross-resistant to CuE. By contrast, TP53 knock-out cells were sensitive to CuE. Remarkably, resistant cells transfected with oncogenic ΔEGFR or ABCB5 were hypersensitive (collateral sensitive) to CuE. In silico analyses demonstrated that CuE is a substrate for P-gp and BCRP. Immunoblot analyses highlighted that CuE targeted EGFR and silenced its downstream signaling cascades. The most striking result that emerged from the doxorubicin uptake by ABCB5 overexpressing cells is that CuE is an effective inhibitor for ABCB5 transporter when compared with verapamil. The COMPARE analyses of transcriptome-wide expression profiles of tumor cell lines of the NCI identified common genes involved in cell cycle regulation, cellular adhesion and intracellular communication for different cucurbitacins. ConclusionCuE represents a potential therapeutic candidate for the treatment of certain types of refractory tumors. To best of our knowledge, this is the first time to identify CuE and verapamil as inhibitors for ABCB5 transporter.

Engineering Anticancer Amphipathic Peptide-Dendronized Compounds for Highly-Efficient Plasma/Organelle Membrane Perturbation and Multidrug Resistance Reversal.

Discovering new strategies for combating drug-resistant tumors becomes a worldwide challenge. Thereinto, stubborn drug-resistant tumor membrane is a leading obstacle on chemotherapy. Herein, we report a novel tumor-activatable amphipathic peptide-dendronized compound, which could form nanoaggregates in aqueous solutions, for perturbing tumor plasma/organelle membrane and reversing multidrug resistance. Distinguished from classical linear amphipathic peptide drugs for membrane disturbance, dendritic lysine-based architecture is designed as a multivalent scaffold to amplify the supramolecular interactions of cationic compound with drug-resistant tumor membrane. Moreover, arginine-rich residues as terminal groups are hopeful to generate multiple hydrogen bonding and electrostatic interactions with tumor membrane. On the other hand, antitumor molecule (doxorubicin) is devised as a hydrophobic moiety to intensify the membrane-inserting ability owing to the prominent interactions with hydrophobic domains of drug-resistant tumor membrane. As expected, these amphipathic peptide-dendronized compounds within the nanoaggregates could severely disturb both the structures and functions of tumor plasma/organelle membrane system, thereby resulting in the rapid leakage of many critical biomolecules, highly efficient apoptotic activation and antiapoptotic inhibition. This strategy on tumor membrane perturbation demonstrates a bran-new antitumor activity with high contributions to cell cycle arrest (at the S phase), strong apoptosis-inducing ability and satisfying cytotoxicity to a variety of drug-resistant tumor cell lines.

Betulinic Acid Exerts Cytotoxic Activity Against Multidrug-Resistant Tumor Cells via Targeting Autocrine Motility Factor Receptor (AMFR).

Betulinic acid (BetA) is a naturally occurring pentacyclic triterpene isolated from the outer bark of white-barked birch trees and many other medicinal plants. Here, we studied betulinic acid's cytotoxic activity against drug-resistant tumor cell lines. P-glycoprotein (MDR1/ABCB1) and BCRP (ABCG2) are known ATP-binding cassette (ABC) drug transporters that mediating MDR. ABCB5 is a close relative to ABCB1, which also mediates MDR. Constitutive activation of the EGF receptor is tightly linked to the development of chemotherapeutic resistance. BetA inhibited P-gp, BCRP, ABCB5 and mutation activated EGFR overexpressing cells with similar efficacy as their drug-sensitive parental counterparts. Furthermore, the mRNA expressions of ABCB1, BCRP, ABCB5 and EGFR were not related to the 50% inhibition concentrations (IC50) for BetA in a panel of 60 cell lines of the National Cancer Institute (NCI), USA. In addition to well-established MDR mechanisms, we attempted to identify other molecular mechanisms that play a role in mediating BetA's cytotoxic activity. For this reason, we performed COMPARE and hierarchical cluster analyses of the transcriptome-wide microarray-based mRNA expression of the NCI cell lines panel. Various genes significantly correlating to BetA's activity were involved in different biological processes, e.g., cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination and proteasome degradation. Immunoblotting and in silico analyses revealed that the inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes. In conclusion, BetA may have therapeutic potential for the treatment of refractory tumors.

Cytotoxic benzylbenzofuran derivatives from Dorstenia kameruniana

Chromatographic separation of the extract of the roots of Dorstenia kameruniana (family Moraceae) led to the isolation of three new benzylbenzofuran derivatives, 2-(p-hydroxybenzyl)benzofuran-6-ol (1), 2-(p-hydroxybenzyl)-7-methoxybenzofuran-6-ol (2) and 2-(p-hydroxy)-3-(3-methylbut-2-en-1-yl)benzyl)benzofuran-6-ol(3) (named dorsmerunin A, B and C, respectively), along with the known furanocoumarin, bergapten (4). The twigs of Dorstenia kameruniana also produced compounds 1–4 as well as the known chalcone licoagrochalcone A (5). The structures were elucidated by NMR spectroscopy and mass spectrometry. The isolated compounds displayed cytotoxicity against the sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells, where compounds 4 and 5 had the highest activities (IC50 values of 7.17 μM and 5.16 μM, respectively) against CCRF-CEM leukemia cells. Compound 5 also showed cytotoxicity against 7 sensitive or drug-resistant solid tumor cell lines (breast carcinoma, colon carcinoma, glioblastoma), with IC50 below 50 μM, whilst 4 showed selective activity.

Identification of an HLA-A2-restricted CD147 epitope that can induce specific CTL cytotoxicity against drug resistant MCF-7/Adr cells.

Cluster of differentiation (CD)147 is highly expressed in drug-resistant tumor cell lines and is involved in the formation of tumor drug resistance. Therefore, immunotherapy utilizing CD147 epitope peptides is a promising approach for the elimination of drug-resistant tumor cells. However, like most tumor-associated antigens (TAAs), CD147 belongs to the autoantigen category, and T cells that recognize high affinity, immunodominant epitopes from autoantigens are deleted though thymic negative selection. Furthermore, wild-type autoantigen peptides cannot effectively activate and expand T lymphocytes with lower affinity T cell receptors in vivo. However, mutations of TAA peptides have been demonstrated to increase the affinity of major histocompatibility complex molecules and their binding to T cell receptor molecules, leading to activation of T lymphocytes in vitro. In the present study, a high-affinity point mutation peptide, CD147126-134L2, was predicted by the human leukocyte antigen (HLA) binding prediction algorithm and its affinity was testified using a T2 binding assay. In addition, when peptide-specific cytotoxic T lymphocytes (CTLs) were stimulated with dendritic cells loaded with the CD147126-134L2 peptide under HLA-A*02:01 restriction, interferon-γ release and cytotoxicity assays showed that peptide-specific CTLs effectively cross-recognized and lysed T2 target cells loaded either with the wild-type (CD147126-134) or mutated peptide (CD147126-134L2). Moreover, the CD147126-134L2 peptide-specific CTLs exerted strong cytotoxic activity against drug-resistant MCF-7/Adr cells, which express a high level of CD147 and are HLA-A*02:01-positive, but not against normal MCF-7 cells. Thus, this suggests that the wild-type peptide (CD147126-134) is naturally presented on HLA-A*02:01 of CD147-expressing MCF-7/Adr cells and is cross-recognized by CTLs. In conclusion, an HLA-A*02:01-restricted CD147-point mutant epitope peptide was identified that induces CTLs to efficiently lyse drug-resistant MCF-7 cells that highly express CD147. Therefore, this immunotherapeutic approach should be explored as a potential treatment for drug-resistant tumors.

Cytotoxic activity of medicinal plants of the Kakamega County (Kenya) against drug-sensitive and multidrug-resistant cancer cells

Ethnopharmacological relevanceThe geographical location of Kakamega County proximal to the Kakamega Rain Forest in Kenya and its rich flora represents an interesting resource of traditional medicinal plants. The medicinal plants in the present study are traditionally used to treat cancer in Kakamega County as recorded in published literature. Aim of the studyDue to multidrug resistance (MDR) and severe side effects of currently used drugs in clinical oncology, new candidate compounds are urgently required to improve treatment outcome. The present study explored the in vitro cytotoxic potential of 34 organic and 19 aqueous extracts of Kakamega medicinal plants towards sensitive and multidrug-resistant cancer cell lines. Methods and resultsThe cytotoxicity was determined using the resazurin assay. Eight organic and two aqueous plant extracts inhibited the growth of CCRF-CEM leukemia cells by more than 50%. The organic extracts were Harungana madagascariensis Lam. ex poir (6.6% of untreated control), Prunus africana (Hook.f.) Kalkman (19.4%), Entada abyssinica Steud. ex A. Rich (38.6%), Phyllanthus fischeri Pax (40.7%), Shirakiopsis elliptica (Hochst.) Esser Synonym: Sapium ellipticum (Hochst. kraus) Pax (41.8%), Bridelia micrantha (Hochst.) Baill (45.4%) and Futumia africana Benth. (45.8%) and Microglossa pyrifolia (Lam.) Kuntze (48%). The aqueous extracts were Bridelia micrantha (Hochst.) Baill (31.3%) and Shirakiopsis elliptica (Hochst.) Esser Synonym: Sapium ellipticum (Hochst. Kraus) Pax (48.2%). In addition to P-glycoprotein-expressing tumor cells, we also investigated other mechanisms of drug resistance, i.e. BCRP- or EGFR-transfected and TP53-knockout tumor cells. Some extracts also showed considerable cytotoxic activity against these drug-resistant cell lines. As demonstrated for selected examples, some extracts exhibited enhanced cytotoxicity towards cancer cells, if applied in combination with other extracts. DiscussionThe panel of medicinal plants used in the Kakamega County for cancer treatment revealed indeed cytotoxicity to various extent towards cancer cells in vitro. Hence, our results may at least in part substantiate the traditional use of these compounds to treat cancer. Even more interesting, several extracts inhibited otherwise drug-resistant tumor cell lines with similar or even better efficacy than their drug-sensitive counterparts. This provides an attractive perspective for further exploration of their anticancer potential to combat drug resistance of refractory tumors.

Targeting oncomiRNAs and mimicking tumor suppressor miRNAs: Νew trends in the development of miRNA therapeutic strategies in oncology (Review).

MicroRNA (miRNA or miR) therapeutics in cancer are based on targeting or mimicking miRNAs involved in cancer onset, progression, angiogenesis, epithelial-mesenchymal transition and metastasis. Several studies conclusively have demonstrated that miRNAs are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This review focuses on the most promising examples potentially leading to the development of anticancer, miRNA-based therapeutic protocols. The inhibition of miRNA activity can be readily achieved by the use of miRNA inhibitors and oligomers, including RNA, DNA and DNA analogues (miRNA antisense therapy), small molecule inhibitors, miRNA sponges or through miRNA masking. On the contrary, the enhancement of miRNA function (miRNA replacement therapy) can be achieved by the use of modified miRNA mimetics, such as plasmid or lentiviral vectors carrying miRNA sequences. Combination strategies have been recently developed based on the observation that i) the combined administration of different antagomiR molecules induces greater antitumor effects and ii) some anti-miR molecules can sensitize drug-resistant tumor cell lines to therapeutic drugs. In this review, we discuss two additional issues: i) the combination of miRNA replacement therapy with drug administration and ii) the combination of antagomiR and miRNA replacement therapy. One of the solid results emerging from different independent studies is that miRNA replacement therapy can enhance the antitumor effects of the antitumor drugs. The second important conclusion of the reviewed studies is that the combination of anti-miRNA and miRNA replacement strategies may lead to excellent results, in terms of antitumor effects.

Discovery of antitumor anthra[2,3-b]furan-3-carboxamides: Optimization of synthesis and evaluation of antitumor properties

Anthraquinones and their analogues, in particular heteroarene-fused anthracendiones, are prospective scaffolds for new compounds with improved antitumor characteristics. We herein report the use of a ‘scaffold hopping’ approach for the replacement of the core structure in the previously discovered hit compound naphtho[2,3-f]indole-5,10-dione 2 with an alternative anthra[2,3-b]furan-5,10-dione scaffold. Among 13 newly synthesized derivatives the majority of 4,11-dihydroxy-2-methyl-5,10-dioxoanthra[2,3-b]furan-3-carboxamides demonstrated a high antiproliferative potency against a panel of wild type and drug resistant tumor cell lines, a property superior over the reference drug doxorubicin or lead naphtho[2,3-f]indole-5,10-dione 2. At low micromolar concentrations the selected derivative of (R)-3-aminopyrrolidine 3c and its stereoisomer (S)-3-aminopyrrolidine 3d caused an apoptotic cell death preceded by an arrest in the G2/M phase. Studies of intracellular targets showed that 3c and 3d formed stable intercalative complexes with the duplex DNA as determined by spectral analysis and molecular docking. Both 3c and 3d attenuated topoisomerase 1 and 2 mediated unwinding of the supercoiled DNA via a mechanism different from conventional DNA-enzyme tertiary complex formation. Furthermore, 3d decreased the activity of selected human protein kinases in vitro, indicating multiple targeting by the new chemotype. Finally, 3d demonstrated an antitumor activity in a model of murine intraperitoneally transplanted P388 leukemia, achieving the increase of animal life span up to 262% at tolerable doses. Altogether, the ‘scaffold hopping’ demonstrated its productivity for obtaining new perspective antitumor drug candidates.

Cytotoxicity of the Sesquiterpene Lactones Neoambrosin and Damsin from Ambrosia maritima Against Multidrug-Resistant Cancer Cells.

Multidrug resistance is a prevailing phenomenon leading to chemotherapy treatment failure in cancer patients. In the current study two known cytotoxic pseudoguaianolide sesquiterpene lactones; neoambrosin (1) and damsin (2) that circumvent MDR were identified. The two cytotoxic compounds were isolated using column chromatography, characterized using 1D and 2D NMR, MS, and compared with literature values. The isolated compounds were investigated for their cytotoxic potential using resazurin assays and thereafter confirmed with immunoblotting and in silico studies. MDR cells overexpressing ABC transporters (P-glycoprotein, BCRP, ABCB5) did not confer cross-resistance toward (1) and (2), indicating that these compounds are not appropriate substrates for any of the three ABC transporters analyzed. Resistance mechanisms investigated also included; the loss of the functions of the TP53 and the mutated EGFR. The HCT116 p53-/- cells were sensitive to 1 but resistant to 2. It was interesting to note that resistant cells transfected with oncogenic ΔEGFR exhibited hypersensitivity CS toward (1) and (2) (degrees of resistances were 0.18 and 0.15 for (1) and (2), respectively). Immunoblotting and in silico analyses revealed that 1 and 2 silenced c-Src kinase activity. It was hypothesized that inhibition of c-Src kinase activity may explain CS in EGFR-transfected cells. In conclusion, the significant cytotoxicity of 1 and 2 against different drug-resistant tumor cell lines indicate that they may be promising candidates to treat refractory tumors.

Dolabrane-type diterpenes from the mangrove plant Ceriops tagal with antitumor activities

Chemical examination of the barks of mangrove plant Ceriops tagal resulted in the isolation of six new dolabrane-type diterpenes with the trivial names of tagalenes A–F (1–6), together with 10 known analogues. The structures of new compounds were elucidated by extensive spectroscopic data analyses. Tagalenes A–C (1–3) are characterized by 18-nordolabrane scaffold, while tagalene F (6) featured by a 2,3-seco dolabrane derivative. Antitumor assay revealed that seven compounds exhibited potent inhibitory effects against a panel of tumor cell lines with IC50<10μM, while tagalsin C (8) exerted the most potent activities in comparison with the IC50 values of the rest compounds. The primary structure–activity relationship is discussed. Tagalsin C also exerted the potent effects against a panel of drug-resistant human tumor cell lines, indicating it to be a promising molecule for further evaluation as an antitumor lead compound.

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells

Background: The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells.Material and methods: Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions.Results: P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling.Conclusion: The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild-type cells speak for a promising role of reserpine in cancer chemotherapy. Reserpine deserves further consideration for cancer therapy in the clinical setting.

Hybrids of phenylsulfonylfuroxan and coumarin as potent antitumor agents.

Sixteen furoxan-based nitric oxide (NO) releasing coumarin derivatives (6a-c, 8a-g, 10a, 13a,b, 15, and 17a,b) were designed, synthesized, and evaluated against the A549, HeLa, A2780, A2780/CDDP, and HUVEC cell lines. Most derivatives displayed potent antiproliferation activities. Among them, 8b exhibited the strongest antiproliferation activity on the four sensitive cell lines mentioned above and three drug resistant tumor cell lines A2780/CDDP, MDA-MB-231/Gem, and SKOV3/CDDP with IC50 values from 14 to 53 nM and from 62 to 140 nM, respectively. Furthermore, 8b inhibited the growth of A2780 in vivo and displayed lower toxicity on nontumorigenesis T29, showing good selectivity against malignant cells in vitro. Preliminary pharmacological studies showed that 8b induces apoptosis, arrests the cell cycle at the G2/M phase in the A2780 cell line, and disrupts the phosphorylation of MEK1 and ERK1. Overall, the NO-releasing capacity and the inhibition of ERK/MAPK pathway signaling may explain the potent antineoplastic activity of these compounds.

Cyclophilin A as a Target of Cisplatin Chemosensitizers

Platinum-based chemotherapeutics are the mainstay of treatment of a range of tumors achieving high response rates but limited in the course of disease by appearance of drug resistance. Tumor cells respond with reduced uptake and increased intracellular inactivation of the drugs, as well as increased DNA repair and general resistance to chemotherapyinduced cell death. Cisplatin is known to induce expression of cyclophilins, a group of proteins that have peptidyl-prolyl cis-trans isomerase (PPIase) and molecular chaperone activities, as stress response. Cyclophilin A (CypA) and other members of this family are inhibited by cyclosporin A (CsA) which sensitized diverse drug-resistant tumor cell lines in vitro to cisplatin. This effect of CsA was attributed to metabolic changes, inhibition of DNA repair, enhancement of apoptosis, altered intracellular signal transduction or increased production of reactive oxygen species (ROS), although no definitive explanation was provided so far. Several clinical trials employing cisplatin/carboplatin in combination with CsA yielded unsatisfactory results. Since viral replication was found to be dependent on cyclophilins of the host cells, effective new inhibitors, different from CsA or with low or absent immunosuppressive activity, are in development or clinical trials. Sanglifehrins are more potent than CsA and proved to increase toxicity of cisplatin against hepatocellular cancer cells in vitro. These novel cyclophilin inhibitors may offer new opportunities to achieve reversal of resistance to platinumbased drugs in refractory patients. Responsive cancer patients may be enriched in clinical trials by an identification of the downstream targets of Cyps responsible for chemoresistance.

Epigenetic regulation of the ABCB1 gene in drug-sensitive and drug-resistant lymphoid tumour cell lines obtained from canine patients

Multidrug resistance (MDR) is a major obstacle in the treatment of cancer. Overexpression of P-glycoprotein (P-gp), encoded by the ABCB1 (MDR1) gene, is an important factor in determining the MDR phenotype of a tumour. Although recent studies have revealed the epigenetic transcriptional regulation of the human ABCB1 gene, such regulation of this gene has not been examined in dogs. The aim of the current study was to evaluate differences in epigenetic regulation of the ABCB1 gene, between drug-sensitive and drug-resistant canine lymphoid tumour cell lines.In two drug-sensitive cell lines, GL-1 and CLBL-1, ABCB1 mRNA expression was significantly lower than in two drug-resistant cell lines, UL-1 and Ema, using real-time quantitative polymerase chain reaction (QPCR). Bisulphite sequencing and real-time methylation-specific PCR revealed that the CpG island present in the upstream region of exon 2 was hypermethylated in GL-1 and CLBL-1, but hypomethylated in UL-1 and Ema. Chromatin immunoprecipitation and QPCR revealed that histone H3 acetylation in the same CpG island was significantly increased in UL-1 and Ema compared to GL-1 and CLBL-1. Treatment with 5-aza 2′-deoxycytidine or trichostatin A increased ABCB1 mRNA expression in GL-1 and CLBL-1. DNA methylation and histone H3 acetylation were shown to be involved in ABCB1 gene expression and associated with an MDR phenotype in these canine lymphoid tumour cell lines.

A Novel Alkaloid from Marine-Derived Actinomycete Streptomyces xinghaiensis with Broad-Spectrum Antibacterial and Cytotoxic Activities

Due to the increasing emergence of drug-resistant bacteria and tumor cell lines, novel antibiotics with antibacterial and cytotoxic activities are urgently needed. Marine actinobacteria are rich sources of novel antibiotics, and here we report the discovery of a novel alkaloid, xinghaiamine A, from a marine-derived actinomycete Streptomyces xinghaiensis NRRL B24674T. Xinghaiamine A was purified from the fermentation broth, and its structure was elucidated based on extensive spectroscopic analysis, including 1D and 2D NMR spectrum as well as mass spectrometry. Xinghaiamine A was identified to be a novel alkaloid with highly symmetric structure on the basis of sulfoxide functional group, and sulfoxide containing compound has so far never been reported in microorganisms. Biological assays revealed that xinghaiamine A exhibited broad-spectrum antibacterial activities to both Gram-negative persistent hospital pathogens (e.g. Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli) and Gram-positive ones, which include Staphylococcus aureus and Bacillus subtilis. In addition, xinghaiamine A also exhibited potent cytotoxic activity to human cancer cell lines of MCF-7 and U-937 with the IC50 of 0.6 and 0.5 µM, respectively.

Abstract 973: Salinomycin sensitized paclitaxel-resistant human ovarian cancer cells and induced apoptosis.

Abstract Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world. Drug resistance is the main cause of treatment failure and mortality in cancer patients. The objective of this study was to establish a paclitaxel (PTX)-resistant human ovarian carcinoma cell line (OVCAR3) and to sensitize chemoresistant cells. The drug-resistant tumor cell lines were established in vitro by stepwise sequential exposure to increased concentration of PTX. For sensitization, OVCAR3/PTX cells were treated with different concentration of salinomycin alone or combined with PTX. The cell viability and cell cycle distribution was measured by MTT and flow cytometric analysis. The transcriptional change of mRNA was examined by RT-PCR. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay and western blot analysis. The cell viability was significantly reduced by combination treatment in a dose dependent manner. The flow cytometry result showed an increase in sub-G1 phase. Combination treatment enhanced the sensitivity of OVCAR3/PTX cells to PTX with down-regulation of P-glycoprotein, XIAP and survivin. Taken together, we demonstrated potential effect of salinomycin in sensitization of PTX resistant ovarian tumor growth. Our results suggest salinomycin in combination with PTX may be a beneficial chemotherapeutic strategy, especially in patients with tumors refractory to PTX alone. Citation Format: Chi-Heum Cho, Hyun-Gyo Lee, Bidur Parajuli, So-Jin Shin, Sang-Hoon Kwon, Soon-Do Cha. Salinomycin sensitized paclitaxel-resistant human ovarian cancer cells and induced apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 973. doi:10.1158/1538-7445.AM2013-973