Role of Smac in cephalostatin-induced cell death.

Cephalostatin 1 is a natural compound isolated from a marine worm that induces apoptosis in tumor cells via an apoptosome-independent but caspase-9-dependent pathway and through an endoplasmic reticulum stress response that is accompanied by caspase-4 activation. Here, we show that cephalostatin evokes mitochondrial Smac (second mitochondria-derived activator of caspases) but not cytochrome c release in various carcinoma cell lines. We also show that Smac is critically involved in caspase-9 activation as evidenced by gene silencing experiments. Remarkably, caspase-2 appears to be a major target for cephalostatin-induced cytosolic Smac. Using biochemical and genetic inhibition experiments, we demonstrate that caspase-2 participates in the apoptotic machinery induced by cephalostatin. Cephalostatin-activated caspase-2 appears to act as initiator caspase and is not involved in the activation of caspase-9. Importantly, experiments immunoprecipitating PIDD (p53-induced protein with a DD), RAIDD (RIP-associated ICH-1/CED-3-homologous protein with DD) and caspase-2 identify cephalostatin as an experimental drug that induces the formation of the PIDDosome. The bis-steroid cephalostatin proves to be both a helpful tool to investigate apoptotic signaling and a promising chemotherapeutic agent.

Spongistatin 1: a new chemosensitizing marine compound that degrades XIAP.

Spongistatin 1 is a new experimental chemotherapeutic agent isolated from marine sponges. Here we show that spongistatin 1 potently induces cell death in patient primary acute leukemic cells with higher efficiency than 8/10 clinically used cytotoxic drugs and prevents long-term survival of leukemic cell lines. Spongistatin 1 triggers caspase-dependent apoptosis in Jurkat T cells by the release of cytochrome c, Smac/DIABLO and Omi/HtrA2. As caspase-9 acts as an initiator caspase and Bcl-2 and Bcl-xL overexpression suppress spongistatin 1-induced apoptosis, cell death is mediated through the mitochondrial apoptosis pathway. Importantly, spongistatin 1 leads to the degradation of the antiapoptotic X-linked inhibitor of apoptosis protein. In apoptosis-resistant leukemic tumor cells overexpressing XIAP, spongistatin 1 effectively causes cell death and potentiates cell death induction by other apoptosis-promoting factors that might be caused by spongistatin 1-mediated degradation of XIAP. Our data show that spongistatin 1 represents a promising novel therapeutic agent for the treatment of leukemic tumor cells especially in the clinically highly relevant situation of chemoresistance due to overexpression of XIAP.

Vibrational spectra and ab initio molecular orbital calculations of the novel anti-cancer drug combretastatin A-4 prodrug.

The NIR-FT Raman and FT-IR spectral studies of the novel antineoplastic and antiangiogenesis substance comprestatin A-4 prodrug (CA4P) were carried out. The equilibrium geometry, various bonding features and harmonic vibrational frequencies of CA4P have been investigated with the help of B3LYP density functional theory (DFT) method. The most preferred cis-configuration for its bioactivity has been demonstrated on the basis of torsional potential energy surface (PES) scan studies. Stability of the molecule arising from hyperconjugative interactions leading to its bioactivity, charge delocalization and mesomeric effects have been analyzed using natural bond orbital (NBO) analysis. Detailed assignments of the vibrational spectra have been made with the aid of theoretically predicted vibrational frequencies. The optimized geometry shows near-planarity of phenyl rings and perpendicular conformation of meta substituted methoxy group. The vibrational analysis confirms the differently acting ring modes, steric repulsion, pi conjugation and back-donation.

Combretastatin A-1 phosphate potentiates the antitumour activity of cisplatin in a murine adenocarcinoma model.

BACKGROUND: The tubulin depolymerizing drug Combretastatin A-1 phosphate (CA1P), a water-soluble derivative of combretastatin A-1, has been recently shown to have a better efficacy in experimental models than the clinically active, close structural analogue, combretastatin A-4 phosphate (CA4P). Previous studies with CA4P in combination with standard anti-cancer agents have demonstrated improved efficacy relative to the standard agents. MATERIALS AND METHODS: In this study the synergistic effects of administering CA1P in combination with cisplatin (CPL) in a well-differentiated transplantable murine colon model (MAC 29) was evaluated. RESULTS: CA1P at 100 mgkg-1 significantly potentiated the anti-tumour effects of CPL. The effect with CPL was similar to that seen for CA1P at its maximum tolerated dose (MTD) alone. CONCLUSION: These data demonstrate that the combination of CA1P and CPL has significant preclinical antitumour activity against a transplantable murine adenocarcinoma model that is related to the antivascular effects of CA1P.

Effects of the antimitotic natural product dolastatin 10, and related peptides, on the human malarial parasite Plasmodium falciparum.

Microtubule inhibitors from several chemical classes can block the growth and development of malarial parasites, reflecting the importance of microtubules in various essential parasite functions. With the spread of antimalarial drug resistance, there is an urgent need for new approaches to the chemotherapy of this devastating disease. We investigated the effects of two naturally occurring marine peptides, dolastatin 10 and dolastatin 15, and 10 synthetic dolastatin 10-based compounds (auristatins), on cultured malarial parasites of the species most lethal to humans, Plasmodium falciparum. Dolastatin 10 was a more potent inhibitor of P. falciparum than any other previously described microtubule inhibitor, with a median inhibitory concentration (IC50) of 10-10 M. Dolastatin 15 was less active, and compounds of the auristatin series had various potencies. Comparison of the concentrations required to inhibit P. falciparum and mammalian cell proliferation showed that the orders of potency were not the same. Dolastatin 10 and auristatin PE caused arrested nuclear division and apparent disassembly of mitotic microtubular structures in the parasite. The effects of these agents were, superficially at least, similar to those of vinblastine but different from those of paclitaxel. These studies indicate that compounds binding in the 'Vinca domain' of tubulin can be highly potent antimalarial agents.

Combretastatin A-1 phosphate a novel tubulin-binding agent with in vivo anti vascular effects in experimental tumours.

In view of the clinical potential of a number of natural products, Combretastatin A-1 phosphate was developed as a water-soluble derivative of combretastatin A-1. This study examined the anti-tumour activity of this compound against an experimental colon tumour (MAC29) in mice. A comparison was made with the clinically active combretastatin A-4 phosphate. The new compound was well-tolerated up to a dose of 250 mg/kg and was more effective at producing tumour growth delays than the A-4 analogue. Significant growth delays were seen at a dose of 50 mg/kg whereas the A-4 phosphate produced no measurable growth delay until a dose of 150 mg/kg was administered. Histological examination of treated tumours indicated that combretastatin A-1 phosphate caused very severe haemorrhagic necrosis in the tumour tissue and analysis of the sections indicated that almost 94 percent of the tumour was dead within 24 hours of treatment. The mechanism of action of combretastatin A-1 phosphate appears to be similar to the A-4 phosphate in that tumour vascular shutdown occurs within 4 hours of treatment. In summary combretastatin A-1 phosphate, the water-soluble analogue of combretastatin A-1, is more potent against a well-vascularised murine colon tumour than its predecessor, combretastatin A-4 phosphate. These data suggest this compound may have potential for clinical development.

Antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene.

The antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene (TMPN) were examined. TMPN was fungicidal for the majority of 132 reference strains and clinical isolates tested, including those resistant to fluconazole, ketoconazole, amphotericin B or flucytosine. Minimum fungicidal concentration/minimum inhibitory concentration (MFC/MIC) ratios were < or = 2 for 96% of Cryptococcus neoformans clinical isolates and 71% of Candida albicans clinical isolates. TMPN was fungicidal for a variety of other basidiomycetes, endomycetes and hyphomycetes, and its activity was unaffected by alterations in media pH. The frequency of occurrence of fungal spontaneous mutations to resistance was <10(-6). Kill-curve analyses confirmed the fungicidal action of TMPN, and demonstrated that killing was concentration- and time-dependent. At sub-MIC exposure to TMPN, C. albicans did not exhibit yeast/hyphae switching. TMPN was slightly cytotoxic for murine and human cancer cell lines (GI50=1-4 microg ml(-1)), and weakly inhibited mammalian tubulin polymerization (IC50=0.60 microg ml(-1)).

Anti-tumor and anti-vascular effects of the novel tubulin-binding agent combretastatin A-1 phosphate.

The combretastatins are derived from an African medicinal plant Combretum caffrum (Combretaceae). They have previously been shown to be potent inhibitors of microtubule assembly that cause marked haemorrhagic necrosis in murine subcutaneous tumors. Promising clinical trial results with combretastatin A-4 phosphate led to this investigation of the anti-tumor and anti-vascular effects of a close structural analog, combretastatin A-1 phosphate. This compound caused identical disruption of the tubulin cytoskeleton in HUVECs in vitro at similar concentrations and duration of exposure as combretastatin A-4 phosphate. Treatment of a well-vascularised murine colon adenocarcinoma (MAC 29) with an effective dose (150 mg/kg) of combretastatin A-1 phosphate resulted in a dramatic decrease in functional vascular volume 2 hours after administration. Vascular shutdown was complete within 4 hours after treatment apart from in small areas of the tumor periphery. Morphological examination of hepatic deposits of HT29 and DLD-1 human colon tumors in nude mice demonstrated that combretastatin A-1 phosphate displays greater anti-tumor effects than the A-4 analog at the same dose and this order of activity (A-1 > A-4) is mirrored in the subcutaneous site with the same tumor type. In summary, combretastatin A-1 phosphate can exert its anti-tumor action via an anti-vascular mechanism. The results indicate that, despite having similar in vitro anti-tubulin properties, combretastatin A-1 phosphate seems to have greater in vivo anti-tumor activity than combretastatin A-4 phosphate at the same doses and may therefore be more successful in the clinic.

First generation design, synthesis, and evaluation of azepine-based cryptophycin analogues.

[structure: see text] Azepine-based cryptophycin mimics (+)-4 and (+)-5 have been designed and synthesized. Biological evaluation revealed modest in vitro activity against several human tumor cell lines, thereby supporting the utility of novel scaffolds for the design and synthesis of cryptophycin analogues.

In vitro activities of two antimitotic compounds, pancratistatin and 7-deoxynarciclasine, against Encephalitozoon intestinalis, a microsporidium causing infections in humans.

The antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with Encephalitozoon intestinalis, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC(50)s) of pancratistatin and 7-deoxynarciclasine for E. intestinalis were 0.18 microM and 0.2 microM, respectively, approximately eightfold lower than the IC(50)s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.

In vitro activities and postantifungal effects of the potent dolastatin 10 derivative auristatin PHE.

The pentapeptide dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester (auristatin PHE) is a derivative of the anticancer drug dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine). Broth microdilution assays with a wide variety of yeast and filamentous fungal species demonstrated the specificity of auristatin PHE for Cryptococcus neoformans and several species of Trichosporon. The duration of the postantifungal effect (PAFE) for C. neoformans was determined for exposure times ranging from 30 min to 2 h. For the derivative, a PAFE was detectable after 45 min of exposure. The effect plateaued after 1 h of exposure, with a PAFE of approximately 6.5 h at four or eight times the auristatin PHE MIC. In contrast, there was no measurable PAFE after 1 h of exposure to dolastatin 10. Human serum greatly prolonged the PAFE of auristatin PHE at eight times the MIC. Auristatin PHE arrested C. neoformans in the budding stage, possibly due to a tubulin-inhibitory action. Auristatin PHE has potential as a narrow-spectrum fungicidal agent and as a probe that can be used to study cryptococcal cell division.

Bryostatin 1 induces differentiation and potentiates the antitumor effect of Auristatin PE in a human pancreatic tumor (PANC-1) xenograft model.

Pancreatic cancer has the worst prognosis of all cancers with a dismal 5-year survival rate. Hence, there is a tremendous need for development of new and effective therapy for this tumor. In an earlier study we reported a potent antitumor activity of Auristatin PE (AuriPE) against pancreatic tumor. In addition, we have also reported that bryostatin 1 (bryo1) induces differentiation of leukemia cells, but the effect of bryo1 has not been investigated in pancreatic tumors. This is the first report where we demonstrate that bryo1 induces differentiation and potentiates the antitumor effect of AuriPE in a human pancreatic tumor (PANC-1) xenograft model. A xenograft model was established by injecting the PANC-1 cells s.c. in severe combined immune deficient (SCID) mice. After development of the s.c. tumors, tumors were dissected and small fragments were transplanted in vivo to new SCID mice, with a success rate of 100% and a doubling time of 4.8 days. The SCID mouse xenograft model was used to test the in vivo differentiation effect of bryo1 and its efficacy when given alone or in combination with AuriPE. Sections from paraffin-embedded tumors excised from untreated (control) SCID mice revealed typical poorly differentiated adenocarcinoma of the pancreas. Interestingly, sections of s.c. tumors taken from bryo1-treated mice revealed carcinomas that were much lower grade and less aggressive, and displayed prominent squamous and glandular differentiation. In this study, the tumor growth inhibition (T/C), activity score and cure rate for bryo1, AuriPE and bryo1+AuriPE were 80%, (+) and 0/4; 0.0%, (++++) and 3/5; and 0.0%, (++++) and 3/4, respectively. Mice treated with either AuriPE or bryo1+AuriPE were free of tumors for more than 150 days and were considered cured. The use of bryo1 as a novel differentiating agent and its combination with AuriPE should be further explored for the treatment of adenocarcinoma of the pancreas.

Isolation and structure of five new cancer cell growth inhibitory bufadienolides from the Chinese traditional drug Ch'an Su.

Five new bufadienolides, 3beta-formyloxyresibufogenin (1), 19-oxobufalin (2), 19-oxodesacetylcinobufagin (3), 6alpha-hydroxycinobufagin (4), and 1beta-hydroxybufalin (5), have been isolated together with the previously known bufadienolides 6-20 from the Chinese traditional drug "Ch'an Su". The structures were elucidated employing spectroscopic methods. Bufadienolides 1-5 provided significant inhibitory activity against the KB and HL-60 cancer cell lines. In addition, bufadienolide 1 was found active against the MH-60 cancer cell line.

Synthesis of phakellistatin 11: a micronesia (Chuuk) marine sponge cyclooctapeptide.

The cyclic octapeptide phakellistatin 11 (1), a constituent of The Federated States of Micronesia (Chuuk) marine sponge Phakellia sp., was synthesized using solid-phase techniques. An initial solution-phase synthesis proved to be inadequate owing to spontaneous deprotection of the Fmoc group at the heptapeptide stage. Using the PAL resin attachment and proceeding from Fmoc-Glu-alpha-allyl ester, linear elongation of the octapeptide was performed until the final unit Pro was added. The allyl ester was removed using Pd(0)[P(C(6)H(5))(3)](4). Cleavage of the final Fmoc group and cyclization with PyAOP provided phakellistatin 11 (1) in 17% overall yield. The synthetic specimen of phakellistatin 11 (1) was found to be chemically but not biologically (cancer cell lines) identical to the natural product. The result suggested a conformational difference or more likely the presence of a trace amount of a highly active antineoplastic agent that binds noncovalently to the natural cyclic octapeptide 1.

Synthesis of (7S,15S)- and (7R,15S)-dolatrienoic acid.

The stereospecific synthesis of (7S,15S)- and (7R,15S)-dolatrienoic acids (2) was achieved using an approach consisting of 16 linear steps. The C-11--C-16 unit was prepared in seven steps from ethyl (S)-lactate and coupled using a trans-selective Wittig--Schlosser reaction to the C-7--C-10 fragment. Chirality at the C-7 position was introduced using an Evan's-type chiral auxiliary in a cobalt-mediated Reformatsky reaction to give the (3S,11S)-aldehyde 24. Subsequent Wittig reaction with a phosphonium salt derived in three steps from tiglic acid gave (7S,15S)-dolatrienoic acid, one of the four possible diastereoisomers of the nonpeptide portion of the strong cancer cell growth inhibitory cyclodepsipeptide dolastatin 14 (1). A second diastereoisomer, (7R,15S)-dolatrienoic acid, was synthesized employing chiral oxazolidinone 21 by an analogous synthetic route.

Antineoplastic agents. 450. Synthesis of (+)-pancratistatin from (+)-narciclasine as relay(1a).

(+)-Narciclasine (2) available in quantity from certain Amaryllidaceae species or by total synthesis was employed as a precursor for a 10-step synthetic conversion (3.6% overall yield) to natural (+)-pancratistatin (1a). The key procedures involved epoxidation of natural (+)-narciclasine (2) to epoxide 6, reduction to diol 8, and formation of cyclic sulfate 12 and its ring opening with cesium benzoate followed by saponification of the benzoate to afford (+)-pancratistatin (1a).

Evaluation of combretastatin A-4 prodrug in a non-Hodgkin's lymphoma xenograft model: preclinical efficacy.

Combretastatin A-4 prodrug (CA4P) is a new antitubulin agent currently in phase I/II clinical trials against solid tumors. We have previously reported on the in vitro activity of CA4P against a panel of malignant human B-lymphoid cell lines. In this study, we investigated the antitumor and the antiangiogenic activity of CA4P in our diffuse large cell lymphoma WSU-DLCL2-SCID mouse model. WSU-DLCL2 cells (10(7)) were injected s.c. into 5-week-old female ICR-SCID mice. Tumor-bearing mice were treated at the CA4P maximum tolerated dose (MTD) of 800 mg/kg in different dose/schedules. CA4P showed significant antitumor activity against this lymphoma model. Best results were seen when MTD was given in two and four divided doses (400 and 200 mg/kg, respectively). CA4P given in four divided doses (4 x 200 mg/kg) showed a log10 kill of 1.01, T/C of 11.7% and T-C of 12 days. Immunohistochemical staining using anti-CD31 antibody after 6, 24, 48 and 120 h treatment revealed a significant decrease in the number of tumor blood vessels after 24 h (about 80%). Only the periphery of treated tumors revealed the presence of blood vessels. Morphological examination of the tumors after tetrachrome staining showed a necrotic center in tumors of CA4P-treated animals. New blood vessel formation was noted to emerge in tumor tissues as early as 48 h following a single dose of CA4P. The G2/M arrest observed in vitro was not detected in vivo indicating predominance of the antiangiogenic effects with regard to antitumor efficacy in vivo. We conclude that CA4P has antiangiogenic activity in this lymphoma model and the use of this agent should be explored clinically in the treatment of non-Hodgkin's lymphoma.

Dolastatin 11, a marine depsipeptide, arrests cells at cytokinesis and induces hyperpolymerization of purified actin.

The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.

Studies directed towards the refinement of the pancratistatin cytotoxic pharmacophore.

Two deoxy-analogues of the anticancer/antiviral agent pancratistatin containing functionality complementary to the minimum structural pharmacophore were synthesized and subjected to anticancer screening. One of the analogues exhibited selective inhibition of certain tumor cell lines but was significantly less potent than the natural products. The minimum structural pharmacophore has now been refined from eight to three possible structures.