Cytotoxic C20 diterpenoid alkaloids from the Australian endemic rainforest plant Anopterus macleayanus

In order to identify new anticancer compounds from nature, a prefractionated library derived from Australian endemic plants was generated and screened against the prostate cancer cell line LNCaP using a metabolic assay. Fractions from the seeds, leaves, and wood of Anopterus macleayanus showed cytotoxic activity and were subsequently investigated using a combination of bioassay-guided fractionation and mass-directed isolation. This led to the identification of four new diterpenoid alkaloids, 6α-acetoxyanopterine (1), 4′-hydroxy-6α-acetoxyanopterine (2), 4′-hydroxyanopterine (3), and 11α-benzoylanopterine (4), along with four known compounds, anopterine (5), 7β-hydroxyanopterine (6), 7β,4′-dihydroxyanopterine (7), and 7β-hydroxy-11α-benzoylanopterine (8); all compounds were purified as their trifluoroacetate salt. The chemical structures of 1–8 were elucidated after analysis of 1D/2D NMR and MS data. Compounds 1–8 were evaluated for cytotoxic activity against a panel of human prostate cancer cells (LNCaP, C4-2B, and DuCaP) and nonmalignant cell lines (BPH-1 and WPMY-1), using a live-cell imaging system and a metabolic assay. All compounds showed potent cytotoxicity with IC50 values of <400 nM; compound 1 was the most active natural product from this series, with an IC50 value of 3.1 nM toward the LNCaP cell line. The live-cell imaging assay on 1–8 showed a concentration- and time-dependent effect on the cell morphology and proliferation of LNCaP cells.

Human Kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

Memory effect in the deposition of C20 fullerenes on a diamond surface

In this paper, the deposition of C-20 fullerenes on a diamond (001)-(2x1) surface and the fabrication of C-20 thin film at 100 K were investigated by a molecular dynamics (MD) simulation using the many-body Brenner bond order potential. First, we found that the collision dynamic of a single C-20 fullerene on a diamond surface was strongly dependent on its impact energy. Within the energy range 10-45 eV, the C-20 fullerene chemisorbed on the surface retained its free cage structure. This is consistent with the experimental observation, where it was called the memory effect in "C-20-type" films [P. Melion , Int. J. Mod. B 9, 339 (1995); P. Milani , Cluster Beam Synthesis of Nanostructured Materials (Springer, Berlin, 1999)]. Next, more than one hundred C-20 (10-25 eV) were deposited one after the other onto the surface. The initial growth stage of C-20 thin film was observed to be in the three-dimensional island mode. The randomly deposited C-20 fullerenes stacked on diamond surface and acted as building blocks forming a polymerlike structure. The assembled film was also highly porous due to cluster-cluster interaction. The bond angle distribution and the neighbor-atom-number distribution of the film presented a well-defined local order, which is of sp(3) hybridization character, the same as that of a free C-20 cage. These simulation results are again in good agreement with the experimental observation. Finally, the deposited C-20 film showed high stability even when the temperature was raised up to 1500 K.

Impact induced chemisorption of C20 isomers on diamond (001)–(2×1) surface

The adsorption of low-energy C20 isomers on diamond (0 0 1)–(2×1) surface was investigated by molecular dynamics simulation using the Brenner potential. The energy dependence of chemisorption characteristic was studied. We found that there existed an energy threshold for chemisorption of C20 to occur. Between 10 and 20 eV, the C20 fullerene has high probability of chemisorption and the adsorbed cage retains its original structure, which supports the experimental observations of memory effects. However, the structures of the adsorbed bowl and ring C20 were different from their original ones. In this case, the local order in cluster-assembled films would be different from the free clusters.

The biology of breast tumor progression : acquisition of hormone independence and resistance to cytotoxic drugs

Many breast tumors appear to follow a predictable clinical pattern, being initially responsive to endocrine therapy and to cytotoxic chemotherapy but ultimately exhibiting a phenotype resistant to both modalities. Using the MCF-7 human breast cancer cell line as an example of an 'early' phenotype (estrogen and progesterone receptor positive, steroid responsive, low metastatic potential), we have isolated and characterized a series of hormone-independent but hormone-responsive variants (MIII and MCF7/LCC1). However, these variants remain responsive to both antiestrogens and cytotoxic drugs (methotrexate and colchicine). MIII and MCF7/LCCl cells appear to mimic some of the critical aspects of the early progression to a more aggressive phenotype. An examination of the phenotype of these cells suggests that some hormone-independent breast cancer cells are derived from hormone-dependent parental cells. The development of a hormone-independent phenotype can arise independently of acquisition of a cytotoxic drug resistant phenotype.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Ecionines A and B, two new cytotoxic pyridoacridine alkaloids from the Australian marine sponge, Ecionemia geodides

Chemical investigations of the Australian marine sponge Ecionemia geodides resulted in the isolation of two new pyridoacridine alkaloids, ecionines A (1) and B (2), along with the previously isolated marine natural products, biemnadin (3) and meridine (4). Compounds 1 and 2 both contain an imine moiety, which is rare for the pyridoacridine structure class. The chemical structures of 1 and 2 were determined by extensive 1D and 2D NMR and MS data analyses. All compounds were tested against a panel of human bladder cancer cell lines, the increasingly metastatic TSU-Pr1 series (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1- B2) and the superficial bladder cancer cell line 5637. Ecionine A (1) displayed cytotoxicity against all cell lines, with IC50 values ranging from 3 to 7 mM. This is the first report of chemistry from the sponge genus Ecionemia.

Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T-lymphocyte antigen-4 (CD152)-CD80/CD86 interactions in CD4(+) T lymphocytes

The costimulatory receptors CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 and their ligands, CD80 and CD86, are expressed on T lymphocytes; however, their functional roles during T cell-T cell interactions are not well known. The consequences of blocking CTLA-4-CD80/CD86 interactions on purified mouse CD4(+) T cells were studied in the context of the strength of signal (SOS). CD4(+) T cells were activated with phorbol 12-myristate 13-acetate (PMA) and different concentrations of a Ca2+ ionophore, Ionomycin (I), or a sarcoplasmic Ca2+ ATPase inhibitor, Thapsigargin (TG). Increasing concentrations of I or TG increased the amount of interleukin (IL)-2, reflecting the conversion of a low to a high SOS. During activation with PMA and low amounts of I, intracellular concentrations of calcium ([Ca2+](i)) were greatly reduced upon CTLA-4-CD80/CD86 blockade. Further experiments demonstrated that CTLA-4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of I or TG (high SOS) but the opposite occurred with PMA and low amounts of I or TG (low SOS). These results were confirmed by surface T-cell receptor (TCR)-CD3 signalling using a low SOS, for example soluble anti-CD3, or a high SOS, for example plate-bound anti-CD3. Also, CTLA-4-CD80/CD86 interactions enhanced the generation of reactive oxygen species (ROS). Studies with catalase revealed that H2O2 was required for IL-2 production and cell cycle progression during activation with a low SOS. However, the high amounts of ROS produced during activation with a high SOS reduced cell cycle progression. Taken together, these results indicate that [Ca2+](i) and ROS play important roles in the modulation of T-cell responses by CTLA-4-CD80/CD86 interactions.

Synthesis, antimicrobial and cytotoxic activities of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles

A new class of 1,3,4-oxadiazoles were prepared from acid hydrazides on treatment with different carboxylic acids in the presence of phosphorus oxychloride. Interconversion of oxadiazoles to thiadiazoles and triazoles was carried out with appropriate reagents. The antimicrobial and cytotoxic activities of compounds 7a-d to 12a-d were tested. Compounds 10d and 12d showed pronounced antimicrobial activity. Further, compound 10d exhibited maximum cytotoxicity. (C) 2008 Elsevier Masson SAS. All rights reserved.

Synthesis and in vitro cytotoxic evaluation of novel diazaspiro bicyclo hydantoin derivatives in human leukemia cells: A SAR study

To study the structure activity relationship (SAR) on the cytotoxic activity and probe the structural requirement for the potent antitumor activity, a series of novel diazaspiro bicyclo hydantoin derivatives were designed and synthesized. Their structures were confirmed by H-1 NMR, LCMS and IR analyses. The antiproliferative effect of these compounds were determined against human leukemia, K562 (chronic myelogenous leukemia) and CEM (T-cell leukemia) cells using trypan blue and MTT assay, and the SAR associated with the position of N-terminal substituents in diazaspiro bicyclo hydantoin have also been discussed. It has been observed that these compounds displayed strong, moderate and weak cytotoxic activities. Interestingly, compounds having electron withdrawing groups at third and fourth position of the phenyl ring displayed selectively cytotoxic activities to both the cell lines tested with IC50 value lower than 50 mu M. In addition, the cytotoxic activities of the compounds 7(a-o) bearing the substituents at N-3 position of diazaspiro bicyclo hydantoin increases in the order alkene > ester > ether and plays an important role in determining their antitumor activities. The position and number of substituents in benzyl group attached to N-8 of diazaspiro bicyclo hydantoin nucleus interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.

Cytotoxic Effects and Biocompatibility of Antimicrobial Materials

The rising demand for medical implants for ageing populations and ongoing advancements in medical technology continue to drive the use of implantable devices. Higher implant usage has a consequent increased incidence of implant-related infections, and associated prolonged patient care, pain and loss of limb and other organ function. Numerous antibacterial surfaces have been designed that prevent the onset of biofilm formation, thus reducing or preventing implant-associated infections through inhibiting bacterial adhesion or by killing the organisms that successfully attach to the surface of the implant. Other surfaces have been designed to stimulate a local immune response, promoting the natural clearing of the invading pathogen. The desired antibacterial effects are typically achieved by modulating the surface chemistry and morphology of the implant material, by means of the controlled release of pharmacological agents and bioactive compounds from the surface of the material, or by a combination of both processes. An important issue for any type of antibacterial surface modification lies in balancing the non-fouling, bacteriostatic or bactericidal effects against local and systemic biocompatibility. In this chapter, we will first describe the concept of biocompatibility and its evolution, from devices that do not evoke a negative host response to those that actively drive host regeneration. We will then review the challenges associated with merging the need for an implant material to withstand a bacterial load with those associated with supporting function restoration and tissue healing.

A potent nonporphyrin class of photodynamic therapeutic agent: Cellular localisation, cytotoxic potential and influence of hypoxia

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics. © 2005 Cancer Research UK.

Synthesis and Cytotoxic Evaluation of Novel 2-(4-(2,2,2-Trifluoroethoxy)-3-methylpyridin-2-ylthio)-1H-benzo[d]imid azole Derivatives

The present work deals with the anticancer effect of benzimidazole derivatives associated with the pyridine framework. By varying the functional group at N-terminal of the benzimidazole by different L-amino acids, several 2-(4-(2,2,2-trifluoroethoxy)-3-methylpyridin-2-ylthio)-1H-benzo[d]imid azole derivatives 9(a-j) were synthesized. Their chemical structures were confirmed by H-1 NMR, IR and mass spectroscopic techniques. The synthesized compounds were examined for their antiproliferative effects against human leukemia cell lines, K562 and CEM. The preliminary results showed most of the derivatives had moderate antitumor activity. Compound 9j containing cysteine residue exhibited good inhibition compared to other amino acid resides. In addition DNA fragmentation results suggest that 9j is more cytotoxic and able to induce apoptosis.

Recognition of nonstructural protein-peptides by cytotoxic t-lymphocytes raised against japanese encephalitis-virus

We have previously reported that Lyt(2+) cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K(d)-restricted CTL and to examine their cross-recognition of West Wile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K(d) binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.