Synthesis and cytotoxicity assay of four ganglioside GM3 analogues.

A concise and efficient synthetic route for preparation of four ganglioside GM3 analogues was described. The key step is a highly regioselective and stereoselective α-sialylation from a suitably protected glycoside acceptor with a sialyl xanthate to provide the sialo-oligosaccharide in good yield. The cytotoxic properties of the synthetic gangliosides were evaluated against normal human keratinocytes and human HCT116 and K562 cancer cells. Two of them exhibited good antiproliferative activity and displayed a better cytotoxicity against cancer cell than HaCaT normal cell.

Polymersomes with PEG corona: structural changes and controlled release induced by temperature variation.

Thermoresponsive behavior of different kinds of polymersomes was studied using small angle neutron scattering (SANS), transmission electron microscopy (TEM), and proton nuclear magnetic resonance ((1)H NMR). The polymersomes were made of block copolymers containing a 2000 Da polyethylene glycol (PEG) as a hydrophilic block and either a liquidlike polymer (e.g., PBA: polybutylacrylate), a solidlike polymer (PS: polystyrene), or a liquid crystalline (LC) polymer as a hydrophobic block. Structural changes in polymersomes are driven in all cases by the critical dehydration temperature of PEG corona, which is closely related to the chemical structure and chain mobility of the hydrophobic block. No structural changes occur upon heating from 25 to 75 °C in the liquidlike polymersomes where the critical dehydration temperature of PEG should be higher than 75 °C. In contrast, glassy PEG-b-PS polymersomes and LC polymersomes show structural changes around 55 °C, which corresponds to the critical dehydration temperature of PEG in those block copolymers. Furthermore, the structural changes depend on the properties of the hydrophobic layer. Glassy PEG-b-PS polymersomes aggregate together above 55 °C, but the bilayer membrane is robust enough to remain intact. This aggregation is reversible, and rather separate polymersomes are recovered upon cooling. However, LC polymersomes display drastic and irreversible structural changes when heated above ∼55 °C. These changes are dependent on the LC structures of the hydrophobic layer. Nematic LC polymersomes turn into thick-walled capsules, whereas smectic LC polymersomes collapse into dense aggregates. As these drastic and irreversible changes decrease or remove the inner compartment volume of the vesicle, LC polymersomes can be used for thermal-responsive controlled release, as shown by a study of calcein release. Finally, toxicity studies proved that LC polymersomes were noncytotoxic and had no effect on cell morphology.

B-ring-modified isocombretastatin A-4 analogues endowed with interesting anticancer activities.

A novel class of isocombretastatin A-4 (isoCA-4) analogues with modifications at the 3'-position of the B-ring by replacement with C-linked substituents was studied. Exploration of the structure-activity relationships of theses analogues led to the identification of several compounds that exhibit excellent antiproliferative activities in the nanomolar concentration range against H1299, MDA-MB231, HCT116, and K562 cancer cell lines; they also inhibit tubulin polymerization with potency similar to that of isoCA-4. 1,1-Diarylethylenes 8 and 17, respectively with (E)-propen-3-ol and propyn-3-ol substituents at the 3'-position of the B-ring, proved to be the most active in this series. Both compounds led to the arrest of various cancer cell lines at the G(2) /M phase of the cell cycle and strongly induced apoptosis. Docking of compounds 8 and 17 in the colchicine binding site indicated that their C3' substituents guide the positioning of the B-ring in a manner different from that observed for isoCA-4.

Discovery of isoerianin analogues as promising anticancer agents.

The cytotoxic activity of a series of 23 new isoerianin derivatives with modifications on both the A and B rings was studied. Several compounds exhibited excellent antiproliferative activity at nanomolar concentrations against a panel of human cancer cell lines. The most cytotoxic compound, isoerianin (3), strongly inhibits tubulin polymerization in the micromolar range. Moreover, isoerianin leads to G2/M phase cell-cycle arrest in H1299 and K562 cancer cells, and strongly induces apoptosis. Isoerianin also disrupts the vessel-like structures formed by human umbilical vein endothelial cells (HUVECs) in vitro, suggesting that this compound may act as a vascular disrupting agent. It clearly appears that in this compound series, the 1,1-ethane bridge encountered in isoerianin derivatives can replace the 1,2-ethane bridge of natural erianin with no loss of activity. This reinforces the bioisosteric replacement approach in the combretastatin series previously reported by our research group.

Overexpression of the natural tetrapeptide acetyl-N-ser-asp-lys-pro derived from thymosin beta4 in neoplastic diseases.

The natural tetrapeptide acetyl-ser-asp-lys-pro (AcSDKP) is formed in vivo by enzymatic cleavage of the N terminus of thymosin beta4 by prolyl oligopeptidase (POP). Recently, AcSDKP was shown to promote angiogenesis. Because of the critical role of neovascularization in cancer development, the levels of AcSDKP and POP activity in a number of different malignant tissues were investigated. Our studies revealed that AcSDKP levels were markedly elevated in neoplastic diseases including hematologic malignancies and solid neoplasms. Consistent with this finding, the enhanced activity of POP was also detected in all analyzed specimens of cancer tissues. Both these novel findings are in concert with the previously reported overexpression of thymosin beta4 in a large variety of malignant tumors and with its potential role in cancerogenesis. The physiological relevance of these findings awaits further studies; however, our first results strongly suggest a key role for AcSDKP in the pathogenesis of cancer.

Cyclin K and cyclin D1b are oncogenic in myeloma cells.

BACKGROUND: Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive. RESULTS: To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis. CONCLUSIONS: Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.

Synthesis, biological evaluation of 1,1-diarylethylenes as a novel class of antimitotic agents.

The cytotoxic activities of 23 new isocombretastatin A derivatives with modifications on the B-ring were investigated. Several compounds exhibited excellent antiproliferative activity at nanomolar concentrations against a panel of human cancer cell lines. Compounds isoFCA-4 (2 e), isoCA-4 (2 k) and isoNH(2)CA-4 (2 s) were the most cytotoxic, and strongly inhibited tubulin polymerization with IC(50) values of 4, 2 and 1.5 microM, respectively. These derivatives were found to be 10-fold more active than phenstatin and colchicine with respect to growth inhibition but displayed similar activities as tubulin polymerization inhibitors. In addition, cell cycle arrest in the G(2)/M phase and subsequent apoptosis was observed in three cancer cell lines when treated with these compounds. The disruptive effect of 2 e, 2 k and 2 s on the vessel-like structures formed by human umbilical vein endothelial cells (HUVEC) suggest that these compounds may act as vascular disrupting agents. Both compounds 2 k and 2 s have the potential for further prodrug modification and development as vascular disrupting agents for treatment of solid tumors.

Isocombretastatins a versus combretastatins a: the forgotten isoCA-4 isomer as a highly promising cytotoxic and antitubulin agent.

Herein is reported a convergent synthesis of isocombretastatins A, a novel class of potent antitubulin agents. These compounds having a 1,1-diarylethylene scaffold constitute the simplest isomers of natural Z-combretastatins A that are easy to synthesize without need to control the Z-olefin geometry. The discovery of isoCA-4 with biological activities comparable to that of CA-4 represents a major progress in this field.

A novel iodomethylene-dimethyl-dihydropyranone induces G2/M arrest and apoptosis in human cancer cells.

BACKGROUND: The putative pharmacophore of a naturally cytotoxic limonoid haperforin B1, E-5-iodomethylene-6,6-dimethyl-5,6-dihydropyran-2-one (IDDP) was synthesized and its biological activity was investigated. MATERIALS AND METHODS: The cytotoxicity of IDDP was assessed using human breast, lung, colorectal and epidermal carcinomas, chronic myeloid leukemia and glioblastoma cell lines. Cell cycle analysis was performed by flow cytometry. The induction of apoptosis was studied by a caspase assay and by annexin V-propidium iodide double staining. The organization of actin and tubulin microfilaments was analysed by immunocytochemical labeling. RESULTS: IDDP was shown to inhibit the growth of a panel of human cancer cell lines independently of their p53 status with IC(50) ranging from 0.07 to 0.50 microM. All the treated cells were arrested in the G(2)/M phase in a time-dependent manner before cell death occurred through an apoptotic pathway. Immunocytochemical studies revealed that the normal organization of microfilaments and microtubules was disrupted in IDDP exposed cells. CONCLUSION: IDDP can be considered as a promising anticancer agent.

Overexpression of the angiogenic tetrapeptide AcSDKP in human malignant tumors.

BACKGROUND: The natural tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), generated from thymosin beta4 following its cleavage by prolyl oligopeptidase (POP), is a physiological stimulator of angiogenesis. Because of the critical role of neovascularisation in tumor development, the expression of AcSDKP and the activity of POP were examined in different human solid malignancies. MATERIALS AND METHODS: The expression of AcSDKP and the activity of POP were evaluated in human blood samples and tissue specimens of thyroid goiter and thyroid papillary carcinoma as well as in commercial cancer tissue microarray. RESULTS: A significantly increased concentration of AcSDKP in intratumoral blood and enhanced tissular activity of POP were detected in cancer patients. The expression of AcSDKP in human breast, colon, head and neck, kidney, lung, skin, ovary and prostate cancer tissues was shown to be greater than that in normal tissues. CONCLUSION: AcSDKP and POP contribute to the malignant phenotype and these molecules are potentiel markers of cancer.

Synthesis of anti-microtubule biaryls and preliminary evaluation as vascular-disrupting agents.

A series of new dibenzoxepines were synthesized in a straightforward and efficient manner through diastereoselective biaryl Suzuki-Miyaura coupling and Brønsted-acid-mediated cyclodehydration as key steps. The vascular-disrupting potential of these molecules was evaluated with various in vitro assays: inhibition of microtubule assembly, antiproliferative activity against cancer cell lines and normal endothelial cells, modification of endothelial cell morphology, and disruption of endothelial cell cords. Two of these compounds showed promising activities in these assays, with profiles similar to that of the reference drug NAC and markedly different from that of colchicine. Altogether, these results show that dibenzoxepines represent promising new leads for the development of more selective vascular-disrupting agents.

Synthesis and antitumor activity of benzils related to combretastatin A-4.

A series of benzil derivatives related to combretastatin A-4 (CA-4) have been synthesized by oxidation of diarylalkynes promoted by PdI(2) in DMSO. Using this new protocol, 14 benzils were prepared in good to excellent yields and their biological activity has been delineated. Several benzils exhibited excellent antiproliferative activity: for example, 4j and 4k bearing the greatest resemblance to CA-4 and AVE-8062, respectively, were found to inhibit cell growth at the nanomolar level (20-50nM) on four human tumor cell lines. Flow cytometric analysis indicates that these compounds act as antimitotics and arrest the cell cycle in G(2)/M phase. A cell-based assay indicated that compounds 4j and 4k displayed a similar inhibition of tubulin assembly with an IC(50) value similar to CA-4. These results clearly demonstrated that the Z-double bond of CA-4 can be replaced by a 1,2-diketone unit without significant loss of cytotoxicity and inhibition of tubulin assembly potency.

New C5-alkylated indolobenzazepinones acting as inhibitors of tubulin polymerization: cytotoxic and antitumor activities.

A series of 5-alkylindolobenzazepin-7-ones was synthesized by Suzuki coupling between 3-iodoindole-2-carboxylates and the appropriate alpha-alkylbenzylamino o-boronic acids followed by cyclization to the lactam. Derivatives having a linear alkyl chain at C5 were found to be highly cytotoxic to KB cells with IC50 values in the 30-80 nM range. These compounds also inhibited the polymerization of tubulin with IC50's of 1-2 microM. Compound 4f (( S)-5-ethyl) showed comparable antiproliferative activities (IC50's of 30-70 nM) in a variety of cancer cell lines, cell growth being arrested at the G2/M phase. Compound 4f induced apoptosis in a dose-dependent manner in three different cancer cell lines and was shown to affect cell morphology in a manner consistent with its inhibitory action on tubulin polymerization. Using the experimental model of glioma grafted on the chick chorio-allantoic membrane, local treatment with compound 4f markedly reduced tumor progression.

Sodium phenylacetate (NaPa) improves the TAM effect on glioblastoma experimental tumors by inducing cell growth arrest and apoptosis.

BACKGROUND: Multiform glioblastomas represent the most aggressive brain tumors. Here, the cooperative effects of sodium phenylacetate (NaPa) and/or tamoxifen (TAM) on CNS1 and 9L glioblastoma cell lines in vitro and in an experimental animal tumor model were investigated. MATERIALS AND METHODS: The drug effects on cell cycle and apoptosis were investigated by flow cytometry. CNS1 cells were implanted subcutaneously in nude mice to form tumors which were then treated with NaPa, TAM or NaPa/TAM. RESULTS: A significant inhibitory effect of NaPa on the two glioma cell lines (LD50 of 10 mM) was observed. 10(-5) M of TAM inhibited approximately 35% of 9L cell growth, and 90% of CNS1 cell growth. When a combination of both drugs included 10(-9) M of TAM, inhibition of about 50% of 9L cell growth and 75% of CNS1 cell growth occurred. The NaPa/TAM combined treatment increased the number of G0/G1 arrested cells and apoptotic cells as compared to treatments with NaPa or TAM alone. Inhibition of CNS1 tumor growth were observed after a two week treatment with NaPa (32 mg/kg/day) or TAM (6 mg/kg/day). CONCLUSION: These results showed a synergistic effect between these two drugs on tumor cell proliferation, caused by cell cycle arrest in the G0/G1 phase and by induction of apoptosis.

Asymmetric synthesis of antimicrotubule biaryl hybrids of allocolchicine and steganacin.

The asymmetric synthesis of novel axially chiral biaryl compounds 5 a-f containing a seven- or eight-membered heterocyclic medium ring is described. These molecules can be considered to be structural hybrids of allocolchicine- and steganacin-type natural products. The synthesis featured an atropo-diastereoselective biaryl Suzuki coupling in which a benzylic stereocenter efficiently transferred its stereochemical information to the biaryl axis. The coupling conditions were optimized, and two biphenylphosphane ligands (DavePhos and S-Phos) were found to give the highest yields and diastereoselectivities. A three-element stereochemical model was proposed to explain the observed diastereoselectivities. In a second key step, the medium ring of the target molecules was formed by a stereoselective S(N)1-type cyclodehydration that probably involved a configurationally stable carbocationic intermediate, as supported by calculations. Alternatively, S(N)2-type cyclizations were employed on the same Suzuki coupling products to give the target molecules in a stereodivergent or stereoconvergent manner. These cyclization methods furnished the target hybrid analogues 5 a-f with ee values above 94 %. All analogues were evaluated as antimicrotubule agents and against a panel of cancer-cell lines using colchicine (1) and N-acetylcolchinol (3) as references. Promising activities were found for R,aR-configured compounds 5 a, b and 5 f; in particular, ethyl analogue 5 b showed a twofold antimicrotubule activity relative to colchicine.

Evidence for an association of high levels of endogenous Acetyl-Ser-Asp-Lys-Pro, a potent mediator of angiogenesis, with acute myeloid leukemia development.

Evidence from clinical and laboratory studies suggests that angiogenesis is important in the progression of solid tumours and hematologic malignancies. We have shown that the naturally occurring tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent angiogenic factor normally present at nanomolar concentrations in the blood. A murine leukemia model was used to assess whether there was a correlation between levels of endogenous AcSDKP and the development of disease. Levels of AcSDKP in the plasma and bone marrow (BM) cells from mice bearing an acute myeloid leukemia (AML) were five- to ten-fold greater than those in non-leukemic mice. Furthermore, a strong correlation between the concentration of endogenous AcSDKP and the progression of AML was demonstrated. These results are consistent with the marked increase in BM vascularity observed in leukemic mice. The physiologic relevance of these findings awaits further studies and the contribution of AcSDKP to the pathogenesis of leukemia is under investigation.

The tetrapeptide acetyl-serine-aspartyl-lysine-proline improves skin flap survival and accelerates wound healing.

The tetrapeptide acetyl-serine-aspartyl-lysine-proline (AcSDKP) has recently been recognized as a potent angiogenic factor. Given the importance of vascular blood supply in the process of tissue repair we have investigated the ability of AcSDKP to contribute to the repair of cutaneous injuries by using dorsal and abdominal skin flap models. Postoperative subdermal AcSDKP injections (5 microg/kg/injection twice a day for 3 days following flap elevation) prevented marginally perfused areas from undergoing necrosis. Mean skin survival area of abdominal and dorsal flaps ranged, respectively, from 50.9+/-19.3 and 53.4+/-4.2% in the control groups to 66.4+/-7.5 and 74.7+/-6.6% in AcSDKP-treated groups. Furthermore, in an ex vivo assay, AcSDKP applied locally to skin explants at doses from 10(-8) to 10(-5) M improved survival of the explanted skin exposed to UVB irradiation at 10 J/cm2. Increased reepithelialization, as well as higher levels of expression of basal keratin 14 and increased expression of fibronectin was observed after topical application of AcSDKP. These data provide experimental evidence that AcSDKP can improve the viability of ischemic skin flaps in the rat by promoting angiogenesis. Moreover, it enhances wound healing of injured avascular skin explants. Thus, these findings identify AcSDKP as a new tissue-repair agent and suggest its potential clinical use for the management of skin wounds.

Tetrapeptide AcSDKP induces postischemic neovascularization through monocyte chemoattractant protein-1 signaling.

BACKGROUND: We investigated the putative proangiogenic activity and molecular pathway(s) of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in a model of surgically induced hindlimb ischemia. METHODS AND RESULTS: Hindlimb ischemia was induced by femoral artery ligature and an osmotic minipump was implanted subcutaneously to deliver low (0.12 mg/kg per day) or high (1.2 mg/kg per day) doses of AcSDKP, for 7 or 21 days. Angiography scores, arteriole density, capillary number, and foot perfusion were increased at day 21 in the high-dose AcSDKP-treated mice (by 1.9-, 1.8-, 1.3-, and 1.6-fold, respectively) compared with control animals (P<0.05, P<0.01, P<0.01, respectively). AcSDKP treatment for 24 hours upregulated the monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels by 1.5-fold in cultured endothelial cells (P<0.01). In the ischemic hindlimb model, administration of AcSDKP also enhanced MCP-1 mRNA levels by 90-fold in ischemic leg (P<0.001) and MCP-1 plasma levels by 3-fold (P<0.001 versus untreated ischemic control mice). MCP-1 levels upregulation were associated with a 2.3-fold increase in the number of Mac3-positive cells in ischemic area of AcSDKP-treated mice (P<0.001 versus untreated animals). Interestingly, AcSDKP-induced monocyte/macrophage infiltration and postischemic neovascularization was fully blunted in MCP-1-deficient animals. CONCLUSIONS: AcSDKP stimulates postischemic neovascularization through activation of a proinflammatory MCP-1-related pathway.

AcSDKP: a new potential marker of malignancy of the thyroid gland.

BACKGROUND AND AIMS: The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) a physiologic inhibitor of stem-cell proliferation is also known for it's strong angiogenic activity. It has been shown that blood levels of this peptide are increased in some hematological malignancies. However, no data on the concentration of AcSDKP present in solid tumor tissue are available. The aim of our study was to measure tissue concentration of AcSDKP in benign and malignant lesions of the thyroid gland. PATIENTS AND METHODS: We assessed AcSDKP level in thyroid tissue specimens using enzyme immunoassay kit. The specimens were taken intraoperatively from 20 patients (17 women and 3 men aged 21-68 years): 10 patients with benign nodular goiter and 10 patients with papillary carcinoma of the thyroid gland. RESULTS: The obtained results show that tissue concentration of AcSDKP in malignant thyroid tumors is five times higher when compared to benign lessions. CONCLUSION: We conclude that AcSDKP may play a role in the development of the thyroid gland lesions. However, the further investigations concerning the tetrapeptide concentration in other thyroid malignancies, toxic nodular, and Grave's goiter are required to conclude on the eventual use of AcSDKP as a marker of malignancy.