Anticancer Agents Vinblastine Research Articles

Resin Glycosides from Operculina hamiltonii and Their Synergism with Vinblastine in Cancer Cells.

Operculina hamiltonii is a vine native to the north and northeast region of Brazil, where its roots are traded as a depurative and laxative remedy with the name of Brazilian jalap in traditional medicine. Procedures for the isolation, purification by recycling HPLC, and structure elucidation of three undescribed resin glycosides are presented herein. Hamiltonin I (1) represents a macrocyclic structure of a tetrasaccharide of (11S)-hydroxyhexadecanoic acid. Additionally, two acyclic pentasaccharides, named hamiltoniosides I (2) and II (3), were also isolated, which are related structurally to the known compounds 4 and 5, macrocyclic lactone-type batatinosides. The tetrasaccharide core of 1 was diacylated by n-decanoic acid and the unusual n-hexadecanoic acid moiety, while the pentasaccharides 2-5 were esterified by one unit of n-decanoic or n-dodecanoic acid. All the isolated compounds were found to be inactive as cytotoxic agents. However, when they were evaluated (1-25 μM) in combination with a sublethal concentration of the anticancer agent vinblastine (0.003 μM), a significant enhancement of the resultant cytotoxicity was produced, especially for multidrug-resistant breast carcinoma epithelial cells. Such combined synergistic potency may be beneficial for chemotherapy, making resin glycosides potential candidates for drug repurposing of conventional chemotherapeutic drugs to reduce their side effects.

Cytochrome P450 Enzymes as Key Drivers of Alkaloid Chemical Diversification in Plants.

Plants produce more than 20,000 nitrogen-containing heterocyclic metabolites called alkaloids. These chemicals serve numerous eco-physiological functions in the plants as well as medicines and psychedelic drugs for human for thousands of years, with the anti-cancer agent vinblastine and the painkiller morphine as the best-known examples. Cytochrome P450 monooxygenases (P450s) play a key role in generating the structural variety that underlies this functional diversity of alkaloids. Most alkaloid molecules are heavily oxygenated thanks to P450 enzymes’ activities. Moreover, the formation and re-arrangement of alkaloid scaffolds such as ring formation, expansion, and breakage that contribute to their structural diversity and bioactivity are mainly catalyzed by P450s. The fast-expanding genomics and transcriptomics databases of plants have accelerated the investigation of alkaloid metabolism and many players behind the complexity and uniqueness of alkaloid biosynthetic pathways. Here we discuss recent discoveries of P450s involved in the chemical diversification of alkaloids and how these inform our approaches in understanding plant evolution and producing plant-derived drugs.

Targeted efflux transporter inhibitors – A solution to improve poor cellular accumulation of anti-cancer agents

Efflux transporters function as vacuum cleaners of xenobiotics and therefore they hinder drugs to reach their targets at effective enough concentrations. Efflux pump inhibitors can be used to improve the cell accumulation of drugs, however all the current inhibitors lack selectivity towards cancer cells. l-Type amino acid transporter 1 (LAT1), which is expressed in many types of cancer cells can be utilized to target inhibitors of efflux transporters to these cells by converting the inhibitors into LAT1-utilizing prodrugs. In this study, we prepared 5 LAT1-utilizing prodrugs of an efflux pump inhibitor, probenecid (PRB). All novel compounds were transported into human breast cancer cells (MCF-7) mainly via LAT1. The compounds also interacted with either multiresistant proteins (MRPs), P-glycoprotein (P-gp) or breast cancer resistant protein (BCRP) and increased significantly (3–4-fold) the cellular accumulation of anti-cancer agent vinblastine (VBL). Consequently, this improved the anti-proliferative efficacy of VBL by decreasing the cell growth after 72 h from 100% (VBL treatment alone) to 48–75% (combination treatment). However, the same phenomenon was not seen with other chemotherapeutic, methotrexate (MTX). Therefore, the chemotherapeutics need to be selected carefully based on their uptake mechanism to the combinations with LAT1-utilizing prodrugs of efflux pump inhibitors to defeat effectively the multidrug resistance (MDR) of chemotherapy.

Identification, characterization and distribution of terpene indole alkaloids in ethanolic extracts of Catharanthus roseus using high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry and the study of their geographical variation.

Catharanthus roseus is a well-known dicotyledonous medicinal plant containing diverse classes of bioactive terpene indole alkaloids (TIAs), in particular the anticancer agents vinblastine and vincristine. In view of the commercial importance of these compounds there is an urgent need to develop an accurate and reliable method for the screening of TIAs from C. roseus. A method for the separation and characterization of these compounds was developed using high-performance liquid chromatography coupled with positive electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). Chromatographic separation of TIAs was carried out using a Thermo Betasil C8 column (250mm × 4.5mm, 5μm) at 25°C using 0.1% formic acid in water and acetonitrile. Diagnostic fragmentation pathways for vinpocetine, vindesine, catharanthine, vinblastine, vindoline and vincristine were established on the basis of their product ions. A total of 72 TIAs were detected of which 11 were unambiguously identified by comparison with their standards, and the remaining 61 were tentatively identified. The geographical distribution of the TIAs in ethanolic extracts of 30 samples of C. roseus collected from five states of India was studied using principal component analysis (PCA). The developed analytical method together with diagnostic fragment patterns were used to rapidly and effectively identify targeted and untargeted TIAs in C. roseus. A PCA study of the results obtained was used to discriminate among the C. roseus samples.

A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate

Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism—an alcohol dehydrogenase and a cytochrome P450—produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-β-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products.

Conversion of the Enzymatically Derived (1S,2S)-3-Bromocyclohexa-3,5-diene-1,2-diol into Enantiomerically Pure Compounds Embodying the Pentacyclic Framework of Vindoline

The enzymatically derived and enantiomerically pure (1S,2S)-3-bromocyclohexa-3,5-diene-1,2-diol (7) has been elaborated over 17 steps into compounds 8 and 32, each of which embodies the pentacyclic framework and much of the functionality associated with the alkaloid vindoline (3). This work sets the stage for effecting the conversion of the related metabolite (1S,6R)-5-ethyl-1,6-dihydroxycyclohexa-2,4-diene-1-carboxylic acid (4) into compound 3, the latter being a biogenetic precursor to the clinically significant anticancer agents vinblastine and vincristine.

Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells: Involvement of positive feedback mechanism

DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.

Local Drug Delivery Strategy for Cancer Treatment: Use of Biocompatible Sol-Gel-Derived Porous Materials

Porous silica xerogel materials have been developed to use as drug-release agents to be implanted directly in or near cancerous tissues. In order to test the capacity of the materials to absorb and then to release medicinal substances, a battery of examinations (UV and visible micro-Raman, porosity measurements, UV-visible absorption spectra) have been made using test drug molecules (clotrimazole, primaquine diphosphate and the anti-cancer agent vinblastine sulphate). Results show that the molecules can be post-doped into the gels and the Raman data provide indications of the best conditions for detecting the substances absorbed in the gels. Spectroscopic results show that the drug molecules are released by the xerogel over a period of 10 days. These results are promising for the development of these materials as drug-release agents.

3′-O, 4′-O-aromatic acyl substituted 7,8-pyranocoumarins: a new class of P-glycoprotein modulators

P-glycoprotein (Pgp) overexpression in tumour cells leads to multidrug resistance (MDR) and causes failure in cancer chemotherapy. We have previously identified (±)-praeruptorin A (PA) as a potential lead compound for Pgp modulators. In this study we investigated the MDR-reversing activities of PA derivatives. Series 7,8-pyranocoumarins with various C-3' and C-4' side chains had been semi-synthesized and their MDR-reversing activity was investigated in Pgp-overexpressing MDR tumour cell line HepG2/Dox and in a KB V1 xenograft animal model. All 7,8-pyranocoumarins exhibited equal or higher activity in modulating Pgp. DCK (12), DMDCK (15), 16, 21, 23 and 24 at 4 µm achieved 91%∼99% decrease in IC50 value (concentration inhibiting cell growth by 50%) of anticancer agents vinblastine, doxorubicin, puromycin and paclitaxel, and were more active than others. DMDCK also remarkably enhanced the growth inhibitory effect of paclitaxel on KB V1 xenografts (P < 0.05), showing a potency required for clinical usage. Mechanistic studies suggested that these 7,8-pyranocoumarins might reverse Pgp-MDR through directly binding to substrate binding site(s) or allosteric site(s) on Pgp therefore impairing Pgp-mediated drug transport. Results from the study suggested that 3'-O, 4'-O-aromatic acyl substituted 7,8-pyranocoumarins could serve as a new class of Pgp modulator. Acyls play an important role in maintaining and enhancing the Pgp-modulating ability of pyranocoumarins. 3,4-Dimethoxyl substituted aromatic acyls, bearing a methoxy that might interact with Pgp as hydrogen bond accepter, were shown to be the most potent for reversing MDR.

Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don.

Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size. The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology. The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp. The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle ( Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.

Rapid Identification of Enzyme Variants for Reengineered Alkaloid Biosynthesis in Periwinkle

Monoterpene indole alkaloids from Catharanthus roseus (Madagascar periwinkle), such as the anticancer agents vinblastine and vincristine, have important pharmacological activities. Metabolic engineering of alkaloid biosynthesis can provide an efficient and environmentally friendly route to analogs of these synthetically challenging and pharmaceutically valuable natural products. However, the narrow substrate scope of strictosidine synthase, the enzyme at the entry point of the pathway, limits a pathway engineering approach. We demonstrate that with a different expression system and screening method it is possible to rapidly identify strictosidine synthase variants that accept tryptamine analogs not turned over by the wild-type enzyme. The variants are used in stereoselective synthesis of beta-carboline analogs and are assessed for biosynthetic competence within the terpene indole alkaloid pathway. These results present an opportunity to explore metabolic engineering of "unnatural" product production in the plant periwinkle.

OV-A-4Induction of cytochrome P450 3A4 (CYP3A4) by vinblastine: Role of the nuclear receptor NR1I2

BACKGROUND Metabolism of the anticancer agent vinblastine is mediated by CYP3A4. It has been demonstrated that other microtubule stabilizing agents such as paclitaxel are capable of inducing this enzyme via activation of NR1I2 (hPXR). Therefore, we evaluated the CYP3A4 induction potential of vinblastine both clinically and in vitro. METHODS The pharmacokinetics of the CYP3A4 phenotyping probe midazolam (i.v. bolus, 0.0145 mg/kg) were determined in 6 patients with renal cell cancer undergoing treatment with vinblastine (72-h i.v. q4w) at baseline and in cycle 3. Midazolam plasma concentrations were measured by LC/MS and clearance (CL) was derived using non-compartmental analysis. Protein expression of CYP3A4 and activation of hPXR by vinblastine were measured by Western blotting and a transactivation assay using transiently transfected HepG2 cells, respectively (Mani et al, Clin Cancer Res 11:6359, 2005). RESULTS Vinblastine increased midazolam CL on average by 60% (range, 2–317%; P=0.0156, Wilcoxon test). In vitro, vinblastine at clinically-relevant concentrations induced CYP3A4 and the hPXR-ligand binding domain, but had only weak to no effect on full length hPXR, suggesting it is only a weak activator of hPXR. CONCLUSIONS Vinblastine is able to induce CYP3A4 in vivo and thus has the potential to facilitate its own elimination and cause interactions with other CYP3A4 substrates. The mechanism of protein induction is likely to be at the level of RNA stabilization or decreased protein turnover. Clinical Pharmacology & Therapeutics (2005) 79, P35–P35; doi: 10.1016/j.clpt.2005.12.125

Application of the Palladium(0)-Catalyzed Ullmann Cross-Coupling Reaction in a Total Synthesis of (±)-Aspidospermidine and thus Representing an Approach to the Lower Hemisphere of the Binary Indole - Indoline Alkaloid Vinblastine

As part of ongoing studies directed towards the construction of the anti-cancer agent vinblastine (1), the related but structurally less complex natural product aspidospermidine (3) has been synthesized. Two approaches to target 3 were pursued. In the first, which was unsuccessful, the amine-tethered enone 6 was prepared but this failed to engage in the pivotal intramolecular conjugate addition reaction to give the bicyclic system 5. In contrast, the related compound 46, incorporating tethered enone and azide moieties, engaged in an intramolecular 1,3-dipolar cycloaddition reaction to give, presumably via an intermediate triazoline, the isolable and ring-fused aziridine 47. This was then converted, over two steps, into the previously reported tetrahydrocarbazole 4. Application of established protocols to this last compound allowed for the installation of the E-ring of the title alkaloid 3 and completion of the total synthesis.

Whole-Cell Biotransformation of m-Ethyltoluene into 1S,6R-5-Ethyl-1,6-dihydroxycyclohexa-2,4-diene-1-carboxylic Acid as an Approach to the C-Ring of the Binary Indole - Indoline Alkaloid Vinblastine

The title compound 3, a potential building block for the construction of analogues of the clinically important anti-cancer agent vinblastine (1), has been prepared in an efficient manner through a whole-cell biotransformation of m-ethyltoluene (4) using the microorganism Pseudomonas putida BGXM1 which expresses the enzyme toluate dioxygenase (TADO). Metabolite 3 was readily converted into derivatives 5–8 using conventional chemical techniques and the structure, including absolute stereochemistry, of the last of these was established by single-crystal X-ray analysis.

The proto-oncogene c-myc and apoptosis.

Deregulated expression of c-Myc not only promotes proliferation, but also can either induce or sensitize cells to apoptosis. Inappropriate expression of c-Myc under conditions which inhibit growth and down-regulate endogenous c-Myc expression, including serum deprivation and exposure to cytotoxic agents including the anticancer agents vinblastine, etoposide, Ara-C, and nocodazole, usually results in programmed cell death in many different cell types. Also, inappropriate Myc expression is associated with an apoptotic response elicited by induction of differentiation. The proapoptotic property of c-Myc requires an intact N-terminal transactivation domain and bHLHZip domain, as well as interaction with Max, thereby implicating c-Myc target genes in this apoptotic process. Although some target genes, namely cdc25A and ODC, have been shown to participate in Myc-mediated apoptosis, no target gene has yet been identified which is essential for this apoptotic response. It is possible that the response of cells inappropriately expressing c-Myc is due not only to the growth arrest signals per se, but also to signals elicited by specific growth inhibitors in the context of a particular biological setting. Also regulating the response of the cells is expression of other oncogenes and tumor suppressor genes, as well as paracrine and autocrine survival factors. Apoptosis associated with inappropriate Myc expression limits the tumorigenic effect of the c-myc proto-oncogene. Mechanisms which inhibit apoptosis should enhance or promote tumorigenesis.

5-N-acetylardeemin, a novel heterocyclic compound which reverses multiple drug resistance in tumor cells. I. Taxonomy and fermentation of the producing organism and biological activity.

The ardeemins are a new family of secondary metabolites produced by submerged fermentation of a fungus which was isolated from a soil sample collected in Brazil. Based on taxonomic studies, the producing culture was identified as Aspergillus fischeri var. brasiliensis strain AB 1826M-35. 5-N-Acetylardeemin potentiated the cytotoxicity of the anticancer agent vinblastine in multidrug resistant human tumor cells.

Models for a hypothetical mechanism of action of the anticancer agent vinblastine

Models for a hypothetical mechanism of action of the anticancer agent vinblastine