Anticancer drugs from nature--natural products as a unique source of new microtubule-stabilizing agents

This review article provides an overview on the current state of research in the area of microtubule-stabilizing agents from natural sources, with a primary focus on the biochemistry, biology, and pharmacology associated with these compounds. A variety of natural products have been discovered over the last decade to inhibit human cancer cell proliferation through a taxol-like mechanism. These compounds represent a whole new range of structurally diverse lead structures for anticancer drug discovery.

New cytotoxic saturated and unsaturated cyclohexanones from Anthemis maritima

Two new cyclohexenones (antheminones A and B) and a new cyclohexanone, (antheminone C) along with five known compounds were isolated from the leaves of Anthemis maritima L. The structures were mainly deduced from extensive 1D and 2D NMR spectroscopy and mass spectrometry. The new compounds were tested in vitro for their cytotoxic activity against adherent and non-adherent cancer cell lines. Antheminones A and C exhibited significant antiproliferative activity against leukemia cells with IC(50) values ranging from 3.2 to 14 microM.

Epothilones as lead structures for the synthesis-based discovery of new chemotypes for microtubule stabilization

Epothilones are macrocyclic bacterial natural products with potent microtubule-stabilizing and antiproliferative activity. They have served as successful lead structures for the development of several clinical candidates for anticancer therapy. However, the structural diversity of this group of clinical compounds is rather limited, as their structures show little divergence from the original natural product leads. Our own research has explored the question of whether epothilones can serve as a basis for the development of new structural scaffolds, or chemotypes, for microtubule stabilization that might serve as a basis for the discovery of new generations of anticancer drugs. We have elaborated a series of epothilone-derived macrolactones whose overall structural features significantly deviate from those of the natural epothilone scaffold and thus define new structural families of microtubule-stabilizing agents. Key elements of our hypermodification strategy are the change of the natural epoxide geometry from cis to trans, the incorporation of a conformationally constrained side chain, the removal of the C3-hydroxyl group, and the replacement of C12 with nitrogen. So far, this approach has yielded analogs 30 and 40 that are the most advanced, the most rigorously modified, structures, both of which are potent antiproliferative agents with low nanomolar activity against several human cancer cell lines in vitro. The synthesis was achieved through a macrolactone-based strategy or a high-yielding RCM reaction. The 12-aza-epothilone ("azathilone" 40) may be considered a "non-natural" natural product that still retains most of the overall structural characteristics of a true natural product but is structurally unique, because it lies outside of the general scope of Nature's biosynthetic machinery for polyketide synthesis. Like natural epothilones, both 30 and 40 promote tubulin polymerization in vitro and at the cellular level induce cell cycle arrest in mitosis. These facts indicate that cancer cell growth inhibition by these compounds is based on the same mechanistic underpinnings as those for natural epothilones. Interestingly, the 9,10-dehydro analog of 40 is significantly less active than the saturated parent compound, which is contrary to observations for natural epothilones B or D. This may point to differences in the bioactive conformations of N-acyl-12-aza-epothilones like 40 and natural epothilones. In light of their distinct structural features, combined with an epothilone-like (and taxol-like) in vitro biological profile, 30 and 40 can be considered as representative examples of new chemotypes for microtubule stabilization. As such, they may offer the same potential for pharmacological differentiation from the original epothilone leads as various newly discovered microtubule-stabilizing natural products with macrolactone structures, such as laulimalide, peloruside, or dictyostatin.

DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYMERIC MICELLES FOR GENE-DIRECTED ENZYME PRODRUG THERAPY

Gene-directed enzyme prodrug therapy is a form of cancer therapy in which delivery of a gene that encodes an enzyme is able to convert a prodrug, a pharmacologically inactive molecule, into a potent cytotoxin. Currently delivery of gene and prodrug is a two-step process. Here, we propose a one-step method using polymer nanocarriers to deliver prodrug, gene and cytotoxic drug simultaneously to malignant cells. Prodrugs acyclovir, ganciclovir and 5-doxifluridine were used to directly to initiate ring-opening polymerization of epsilon-caprolactone, forming a hydrophobic prodrug-tagged poly(epsilon-caprolactone) which was further grafted with hydrophilic polymers (methoxy poly(ethylene glycol), chitosan or polyethylenemine) to form amphiphilic copolymers for micelle formation. Successful synthesis of copolymers and micelle formation was confirmed by standard analytical means. Conversion of prodrugs to their cytotoxic forms was analyzed by both two-step and one-step means i.e. by first delivering gene plasmid into cell line HT29 and then challenging the cells with the prodrug-tagged micelle carriers and secondly by complexing gene plasmid onto micelle nanocarriers and delivery gene and prodrug simultaneously to parental HT29 cells. Anticancer effectiveness of prodrug-tagged micelles was further enhanced by encapsulating chemotherapy drugs doxorubicin or SN-38. Viability of colon cancer cell line HT29 was significantly reduced. Furthermore, in an effort to develop a stealth and targeted carrier, CD47-streptavidin fusion protein was attached onto the micelle surface utilizing biotin-streptavidin affinity. CD47, a marker of self on the red blood cell surface, was used for its antiphagocytic efficacy, results showed that micelles bound with CD47 showed antiphagocytic efficacy when exposed to J774A.1 macrophages. Since CD47 is not only an antiphagocytic ligand but also an integrin associated protein, it was used to target integrin alpha(v)beta(3), which is overexpressed on tumor-activated neovascular endothelial cells. Results showed that CD47-tagged micelles had enhanced uptake when treated to PC3 cells which have high expression of alpha(v)beta(3). The synthesized multifunctional polymeric micelle carriers developed could offer a new platform for an innovative cancer therapy regime.

Activation of the unfolded protein response is associated with favorable prognosis in acute myeloid leukemia

PURPOSE: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. EXPERIMENTAL DESIGN: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box-binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. RESULTS: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). CONCLUSIONS: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.

Synthesis and biological evaluation of epothilone A dimeric compounds

The preparation and biological evaluation of a novel series of dimeric epothilone A derivatives (1-6) are described. Two types of diacyl spacers were introduced to establish the various dimeric epothilone A constructs. The effect of these compounds on tubulin polymerization and their cytotoxicity against four different cancer cell lines are reported. Several of the newly synthesized compounds inhibit endothelial cell differentiation and endothelial cell migration that are key steps of the angiogenic process.

Epothilone analogues with benzimidazole and quinoline side chains: chemical synthesis, antiproliferative activity, and interactions with tubulin

A series of epothilone B and D analogues bearing isomeric quinoline or functionalized benzimidazole side chains has been prepared by chemical synthesis in a highly convergent manner. All analogues have been found to interact with the tubulin/microtubule system and to inhibit human cancer cell proliferation in vitro, albeit with different potencies (IC(50) values between 1 and 150 nM). The affinity of quinoline-based epothilone B and D analogues for stabilized microtubules clearly depends on the position of the N-atom in the quinoline system, while the induction of tubulin polymerization in vitro appears to be less sensitive to N-positioning. The potent inhibition of human cancer cell growth by epothilone analogues bearing functionalized benzimidazole side chains suggests that these systems might be conjugated with tumor-targeting moieties to form tumor-targeted prodrugs.

Cannabinoid receptor ligands as potential anticancer agents--high hopes for new therapies?

OBJECTIVES: The endocannabinoid system is an endogenous lipid signalling network comprising arachidonic-acid-derived ligands, cannabinoid (CB) receptors, transporters and endocannabinoid degrading enzymes. The CB(1) receptor is predominantly expressed in neurons but is also co-expressed with the CB(2) receptor in peripheral tissues. In recent years, CB receptor ligands, including Delta(9)-tetrahydrocannabinol, have been proposed as potential anticancer agents. KEY FINDINGS: This review critically discusses the pharmacology of CB receptor activation as a novel therapeutic anticancer strategy in terms of ligand selectivity, tissue specificity and potency. Intriguingly, antitumour effects mediated by cannabinoids are not confined to inhibition of cancer cell proliferation; cannabinoids also reduce angiogenesis, cell migration and metastasis, inhibit carcinogenesis and attenuate inflammatory processes. In the last decade several new selective CB(1) and CB(2) receptor agents have been described, but most studies in the area of cancer research have used non-selective CB ligands. Moreover, many of these ligands exert prominent CB receptor-independent pharmacological effects, such as activation of the G-protein-coupled receptor GPR55, peroxisome proliferator-activated receptor gamma and the transient receptor potential vanilloid channels. SUMMARY: The role of the endocannabinoid system in tumourigenesis is still poorly understood and the molecular mechanisms of cannabinoid anticancer action need to be elucidated. The development of CB(2)-selective anticancer agents could be advantageous in light of the unwanted central effects exerted by CB(1) receptor ligands. Probably the most interesting question is whether cannabinoids could be useful in chemoprevention or in combination with established chemotherapeutic agents.

Synthesis and SAR of C12-C13-oxazoline derivatives of epothilone A

The SAR of a series of new epothilone A derivatives with a 2-substituted-1,3-oxazoline moiety trans-fused to the C12-C13 bond of the deoxy macrocycle have been investigated with regard to tubulin polymerization induction and cancer cell growth inhibition. Significant differences in antiproliferative activity were observed between different analogs, depending on the nature of the substituent at the 2-position of the oxazoline ring. The most potent compounds showed comparable activity with the natural product epothilone A. Modeling studies provide a preliminary rationale for the observed SAR.

Making epothilones fluoresce: design, synthesis, and biological characterization of a fluorescent N12-aza-epothilone (azathilone)

A green fluorescent 12-aza-epothilone (azathilone) derivative has been prepared through the attachment of the 4-nitro-2,1,3-benzoxadiazole (NBD) fluorophore to the 12-nitrogen atom of the azamacrolide core structure. While less potent than natural epothilones or different N12-acylated azathilone derivatives, NBD-azathilone (3) promotes tubulin assembly, inhibits cancer cell proliferation in vitro and arrests the cell cycle at the G2/M transition. Most significantly, the binding of 3 to cellular microtubules (MTs) could be directly visualized by confocal fluorescence microscopy. Based on competition binding experiments with laulimalide-stabilized MTs in vitro, the N12-Boc substituted azathilone 1, Epo A, and NBD-azathilone (3) all interact with the same tubulin-binding site. Computational studies provided a structural model of the complexes between beta-tubulin and 1 or 3, respectively, in which the NBD moiety of 3 or the BOC moiety of 1 directly and specifically contribute to MT binding. Collectively, these data demonstrate that the cellular effects of 3 and, by inference, also of other azathilones are the result of their interactions with the cellular MT network.

Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3beta

N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

New cassane butenolide hemiketal diterpenes from the marine creeper Caesalpinia honduc and their antiproliferative activity

Two new cassane butenolides, caesalpinolide A (1) and B (2), epimeric at the hemiketal position were isolated from the marine creeper Caesalpinia bonduc. The structures were elucidated by the analysis of spectroscopic data and relative stereochemistry was assigned on the basis of ROESY correlations. Compounds 1 and 2 were found to inhibit MCF-7 breast cancer cell lines with ICso values of (IlM)12.8 and 6.1, respectively, along with the inhibition of endometrial and cervical cancer cell lines

Thioureides of 2-(phenoxymethyl)benzoic acid 4-R substituted: a novel class of anti-parasitic compounds

Fifty members of a novel class of antimicrobial compounds, 2-(4-R-phenoxymethyl)benzoic acid thioureides, were synthesized and characterized with respect to their activities against three parasites of human relevance, namely the protozoa Giardia lamblia and Toxoplasma gondii, and the larval (metacestode) stage of the tapeworm Echinococcus multilocularis. To determine the selective toxicity of these compounds, the human colon cancer cell line Caco2 and primary cultures of human foreskin fibroblasts (HFF) were also investigated. The new thioureides were obtained in a three-step-reaction process and subsequently characterized by their physical constants (melting point, solubility). The chemical structures were elucidated by (1)H NMR, (13)C NMR, IR spectral methods and elemental analysis. The analyses confirmed the final and intermediate compound structures and the synthesis. The compounds were then tested on the parasites in vitro. All thioureides, except two compounds with a nitro group, were totally ineffective against Giardia lamblia. 23 compounds inhibited the proliferation of T. gondii, three of them with an IC(50) of approximately 1 microM. The structural integrity of E. multilocularis metacestodes was affected by 22 compounds. In contrast, HFF were not susceptible to any of these thioureides, while Caco2 cells were affected by 17 compounds, two of them inhibiting proliferation with an IC(50) in the micromolar range. Thioureides may thus present a promising class of anti-infective agents.

Synthesis and Biological Activity of 7,8,9-Trideoxy- and 7 R DesTHP-Peloruside A

The stereoselective syntheses of 7,8,9-trideoxypeloruside A (4) and a monocyclic peloruside A analogue lacking the entire tetrahydropyran moiety (3) are described. The syntheses proceeded through the PMB-ether of an ω-hydroxy β-keto aldehyde as a common intermediate which was elaborated into a pair of diastereomeric 1,3-syn and -anti diols by stereoselective Duthaler–Hafner allylations and subsequent 1,3-syn or anti reduction. One of these isomers was further converted into a tetrahydropyran derivative in a high-yielding Prins reaction, to provide the precursor for bicyclic analogue 4. Downstream steps for both syntheses included the substrate-controlled addition of a vinyl lithium intermediate to an aldehyde, thus connecting the peloruside side chain to C15 (C13) of the macrocyclic core structure in a fully stereoselective fashion. In the case of monocyclic 3 macrocyclization was based on ring-closing olefin metathesis (RCM), while bicyclic 4 was cyclized through Yamaguchi-type macrolactonization. The macrolactonization step was surprisingly difficult and was accompanied by extensive cyclic dimer formation. Peloruside A analogues 3 and 4 inhibited the proliferation of human cancer cell lines in vitro with micromolar and sub-micromolar IC50 values, respectively. The higher potency of 4 highlights the importance of the bicyclic core structure of peloruside A for nM biological activity.

Loss of p53 in enterocytes generates an inflammatory microenvironment enabling invasion and lymph node metastasis of carcinogen-induced colorectal tumors

Loss of p53 is considered to allow progression of colorectal tumors from the adenoma to the carcinoma stage. Using mice with an intestinal epithelial cell (IEC)-specific p53 deletion, we demonstrate that loss of p53 alone is insufficient to initiate intestinal tumorigenesis but markedly enhances carcinogen-induced tumor incidence and leads to invasive cancer and lymph node metastasis. Whereas p53 controls DNA damage and IEC survival during the initiation stage, loss of p53 during tumor progression is associated with increased intestinal permeability, causing formation of an NF-κB-dependent inflammatory microenvironment and the induction of epithelial-mesenchymal transition. Thus, we propose a p53-controlled tumor-suppressive function that is independent of its well-established role in cell-cycle regulation, apoptosis, and senescence.