Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents

A series of novel curcumin analogs were synthesized and screened for anti-cancer and anti-angiogenesis activities at Emory University and at the National Cancer Institute (NCI). These compounds are symmetrical alpha,beta-unsaturated and saturated ketones. The majority of the analogs demonstrated a moderate degree of anti-cancer activity. Compounds 10, 11, and 14 exhibited a high degree of cytotoxicity in the NCI in vitro anti-cancer cell line screen. In addition, this screen revealed that these compounds inhibit tumor cell growth with a higher potency than the commonly used chemotherapeutic drug, cisplatin. In independent in vitro screens conducted at Emory, the same compounds plus 4, 5, 8, 9, and 13 exhibited a high degree of cytotoxicity to tumor cells. Analogs that were effective in the anti-cancer screens were also effective in in vitro anti-angiogenesis assays. Compounds 4, 9, 11, and 14 were most effective in the anti-angiogenesis assays run at Emory. In the assays conducted by the NCI, compound 14 was almost as potent as the anti-angiogenic drug TNP-470, which has undergone clinical trials. Based on the favorable in vitro anti-cancer and anti-angiogenesis results with 14, further in vivo tests were conducted. This compound effectively reduced the size of human breast tumors grown in female athymic nude mice and showed little toxicity. This data, coupled with the remarkable in vitro data, suggests that compound 14 may potentially be an effective chemotherapeutic agent. As a follow-up, a 3D quantitative structure relationship based on 14 has been developed. It shows a cross-validated r2(q2) and a predictive r2(p2) = 0.71. COMPARE analysis suggests the compound to be a possible RNA/DNA antimetabolite, but also implies that the compound's cytotoxicity may arise from a presently unknown mechanism.     

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C10 alkylamines to produce C10-PEAA. Incubation of C10-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C10-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C10-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C10-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C10-PEAA-LUV. It is concluded that C10-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.     

Induction of an interferon-gamma Stat3 response in nerve cells by pre-treatment with gp130 cytokines

Many cytokines mediate their effects through Jak/STAT signaling pathways providing many opportunities for cross-talk between different cytokines. We examined the interaction between two cytokine families, gp130-related cytokines and interferon-gamma (IFN-gamma), which are coexpressed in the nervous system during acute trauma and pathological conditions. Typical nerve cells show an IFN-gamma response that is restricted to activating STAT1, with minor activation of STAT3. IFN-gamma elicited a pronounced STAT3 response in cells pre-treated for 5-7 h with ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or interleukin-6. CNTF or interleukin-6 induced an IFN-gamma STAT3 response in a variety of cells including SH-SY5Y human neuroblastoma, HMN-1 murine motor neuron hybrid cells, rat sympathetic neurons and human hepatoma HepG2 cells. The enhancement was measured as an increase in tyrosine phosphorylated STAT3, in STAT3-DNA binding and in STAT-luciferase reporter gene activity. The enhanced STAT3 response was not due to an increase in overall STAT3 levels but was dependent upon ongoing protein synthesis. The induction by CNTF was inhibited by the protein kinase C inhibitor, BIM, and the MAPK-kinase inhibitor, U0126. Further, H-35 hepatoma cells expressing gp130 receptor chimeras lacking either the SHP-2 docking site or the Box 3 STAT binding sites failed to enhance the IFN-gamma STAT3 response. These results provide evidence for an interaction between gp130 and IFN-gamma cytokines that can significantly alter the final cellular response to IFN-gamma.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Changes in Sugar Content and Activities of Sucrose Metabolizing Enzymes in Roots and Nodules of Lentil

Activities of acid and alkaline invertases and sucrose synthase were determined in roots and nodules of lentil at various stages of development. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodule cytosol, but there was only a small amount of acid invertase present. Activity of sucrose metabolizing enzymes in roots was significantly less than that observed in the nodules. Amongst sugars, sucrose was found to be the main component in the host cytosol. Lentil neutral invertase (LNI) was partially purified from nodules at 50 days after sowing (DAS). Two forms of invertase were identified, i.e., a major form of 71 kDa which was taken for enzyme characterization and a minor form of 270 kDa which was not used for further studies. The purified enzyme exhibited typical hyperbolic saturation kinetics for sucrose hydrolysis. It had a Km of 11.0 to 14.0 mM for sucrose depending upon the temperature, a pH optimum of 6.8 and an optimum temperature of 40 °C. Compared with raffinose and stachyose, sucrose was better substrate for LNI. The enzyme showed no significant hydrolysis of maltose and p-nitrophenyl--D-glucopyranoside, showing its true -fructosidase nature. LNI is completely inhibited by HgCl₂, MnCl₂ and iodoacetamide but not by CaCl₂, MgCl₂ or BaCl₂.     

Biochemical and molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis thaliana

12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with K(m) values of 50 and 10 microm, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring in A. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.     

Sequential opening of IP(3)-sensitive Ca(2+) channels and SOC during alpha-adrenergic activation of rabbit vena cava

Alpha(1)-aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca(2+) oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release channels (IP(3)R channels) can prevent the initiation and abolish ongoing alpha(1)-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP(3)R channels, not to its slight inhibition of the L-type voltage-gated Ca(2+) channel and the sarco(endo)plasmic reticulum Ca(2+) ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca(2+) release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP(3)R-channel-mediated SR Ca(2+) release or store depletion. This is in accordance with its sensitivity to Ni(2+) and La(3+) (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP(3)R channels and the SOC are required for PE-mediated [Ca(2+)](i) oscillations and constriction of the rabbit IVC.     

Plasticity in structure and interactions is critical for the action of indolicidin,an antibacterial peptide of innate immune origin

The comparative analysis of two cationic antibacterial peptides of the cathelicidin family-indolicidin and tritrypticin-enabled addressing the structural features critical for the mechanism of indolicidin activity. Functional behavior of retro-indolicidin was found to be identical to that of native indolicidin. It is apparent that the gross conformational propensities associated with retro-peptides resemble those of the native sequences, suggesting that native and retro-peptides can have similar structures. Both the native and the retro-indolicidin show identical affinities while binding to endotoxin, the initial event associated with the antibacterial activity of cationic peptide antibiotics. The indolicidin-endotoxin binding was modeled by docking the indolicidin molecule in the endotoxin structure. The conformational flexibility associated with the indolicidin residues, as well as that of the fatty acid chains of endotoxin combined with the relatively strong structural interactions, such as ionic and hydrophobic, provide the basis for the endotoxin-peptide recognition. Thus, the key feature of the recognition between the cationic antibacterial peptides and endotoxin is the plasticity of molecular interactions, which may have been designed for the purpose of maintaining activity against a broad range of organisms, a hallmark of primitive host defense.