Roles of residues in mammalian mitochondrial elongation factor Ts in the interaction with mitochondrial and bacterial elongation factor Tu

The crystal structure of the complex between Escherichia coli elongation factors Tu and Ts (EF-Tu.Ts) and subsequent mutagenesis work have provided insights into the roles of a number of residues in E. coli EF-Ts in its interaction with EF-Tu. The corresponding residues in bovine mitochondrial EF-Ts (EF-Tsmt) have been mutated. The abilities of the resulting EF-Tsmt derivatives to stimulate the activities of both E. coli and mitochondrial EF-Tu have been tested. Mutation of several residues in EF-Tsmt corresponding to amino acids important for the activity of E. coli EF-Ts has little or no effect on the activity of the mitochondrial factor, suggesting that these factors may use somewhat different mechanisms to promote guanine nucleotide exchange. In general, mutations that reduce the strength of the interaction between EF-Tsmt and E. coli EF-Tu increase the ability of EF-Tsmt to stimulate the activity of the bacterial factor. In contrast, these mutations tend to reduce the ability of EF-Tsmt to stimulate the activity of EF-Tumt. For example, F19A/I20A and H176A derivatives of EF-Tsmt are as active as E. coli EF-Ts in simulating E. coli EF-Tu. However, these mutations significantly decrease the ability of EF-Tsmt to stimulate EF-Tumt.     

Doxoform and Daunoform: anthracycline-formaldehyde conjugates toxic to resistant tumor cells

The recent discovery that the clinically important antitumor drugs doxorubicin and daunorubicin alkylate DNA via catalytic production of formaldehyde prompted the synthesis of derivatives bearing formaldehyde. Reaction of the parent drugs with aqueous formaldehyde at pH 6 produced in 40-50% yield conjugates consisting of two molecules of the parent drug as oxazolidine derivatives bound together at their 3'-nitrogens by a methylene group. The structures were established as bis(3'-N-(3'-N,4'-O-methylenedoxorubicinyl)) methane (Doxoform) and bis(3'-N-(3'-N,4'-O-methylenedaunorubicinyl))methane (Daunoform) from spectroscopic data. Both derivatives are labile with respect to hydrolysis to the parent drugs. 3'-N,4'-O-Methylenedoxorubicin and 3'-N,4'-O-methylenedaunorubicin are intermediates in the hydrolysis. Daunoform reacts with the self-complementary deoxyoligonucleotide (GC)4 faster than the combination of daunorubicin and formaldehyde at an equivalent concentration to given drug-DNA adducts. In spite of hydrolytic instability, Doxoform is 150-fold more toxic to MCF-7 human breast cancer cells and 10000-fold more toxic to MCF-7/ADR resistant cells. Toxicity to resistant cancer cells is interpreted in terms of higher lipophilicity of the derivatives and circumvention of catalytic formaldehyde production.     

Chemopreventive agents: protease inhibitors

Certain protease inhibitors, called the anticarcinogenic protease inhibitors in this review, are capable of preventing carcinogenesis in a wide variety of in vivo and in vitro model systems. The anticarcinogenic protease inhibitors are extremely potent agents with the ability to prevent cancer, with some unique characteristics as anticarcinogenic agents. The anticarcinogenic protease inhibitors have the ability to irreversibly suppress the carcinogenic process. They do not have to be continuously present to suppress carcinogenesis. They can be effective when applied in both in vivo and in vitro carcinogenesis assay systems at long time periods after carcinogen exposure, and are effective as anticarcinogenic agents at extremely low molar concentrations. While several different types of protease inhibitors can prevent the carcinogenic process, the most potent of the anticarcinogenic protease inhibitors on a molar basis are those with the ability to inhibit chymotrypsin or chymotrypsin-like proteases. The soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), is a potent chymotrypsin inhibitor that has been extensively studied for its ability to prevent carcinogenesis in many different model systems. Much of this review is focused on the characteristics of BBI as the anticarcinogenic protease inhibitor, as this is the protease inhibitor that has risen to the human trial stage as a human cancer chemopreventive agent. Part of this review hypothesizes that the Bowman-Birk family of protease inhibitors plays a role in plants similar to that of alpha1-antichymotrypsin in people. Both BBI and alpha1-antichymotrypsin are potent inhibitors of chymotrypsin and chymotrypsin-like enzymes, are highly anti-inflammatory, and are thought to play important roles in the defense of their respective organisms. It is believed that BBI will be shown to play a major role in the prevention and/or treatment of several different diseases, in addition to cancer.     

Anticonvulsant activity of novel derivatives of 2- and 3-piperidinecarboxylic acid in mice and rats

The relative ability of derivatives of 2-piperidinecarboxylic acid (2-PC; pipecolic acid) and 3-piperidinecarboxylic acid (3-PC; nipecotic acid) to block maximal electroshock (MES)-induced seizures, elevate the threshold for electroshock-induced seizures and be neurotoxic in mice was investigated. Protective index (PI) values, based on the MES test and rotorod performance, ranged from 1.3 to 4.5 for 2-PC benzylamides and from < 1 to > 7.2 for 3-PC derivatives. PI values based on elevation of threshold for electroshock-induced seizures and rotorod performance ranged from > 1.6 to > 20 for both types of derivatives. Since preliminary data indicated that benzylamide derivatives of 2-PC displace [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine (TCP) binding to the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor in the micromolar range and such low affinity uncompetitive antagonists of the NMDA receptor-associated ionophore have been shown to be effective anticonvulsants with low neurological toxicity, the 2-PC derivatives were evaluated in rat brain homogenates for binding affinity to the PCP site. Although all compounds inhibited [3H]TCP binding, a clear correlation between pharmacological activity and binding affinity was not apparent. Select compounds demonstrated minimal ability to protect against pentylenetetrazol-, 4-aminopyridine- and NMDA-induced seizures in mice. Corneal and amygdala kindled rats exhibited different sensitivities to both valproic acid and the nonsubstituted 2-PC benzylamide, suggesting a difference in these two models. Enantiomers of the alpha-methyl substituted benzylamide of 2-PC showed some ability to reduce seizure severity in amygdala kindled rats.     

Regulative interactions in zebrafish neural crest

Zebrafish trunk neural crest cells that migrate at different times have different fates: early-migrating crest cells produce dorsal root ganglion neurons as well as glia and pigment cells, while late-migrating crest cells produce only non-neuronal derivatives. When presumptive early-migrating crest cells were individually transplanted into hosts such that they migrated late, they retained the ability to generate neurons. In contrast, late-migrating crest cells transplanted under the same conditions never generated neurons. These results suggest that, prior to migration, neural crest cells have intrinsic biases in the types of derivatives they will produce. Transplantation of presumptive early-migrating crest cells does not result in production of dorsal root ganglion neurons under all conditions suggesting that these cells require appropriate environmental factors to express these intrinsic biases. When early-migrating crest cells are ablated, late-migrating crest cells gain the ability to produce neurons, even when they migrate on their normal schedule. Interactions among neural crest cells may thus regulate the types of derivatives neural crest cells produce, by establishing or maintaining intrinsic differences between individual cells.     

Influence of structural modifications at the 3' and 4' positions of doxorubicin on the drug ability to trap topoisomerase II and to overcome multidrug resistance

To better define the role of the amino sugar in the pharmacological and biochemical properties of anthracyclines related to doxorubicin and daunorubicin, we have investigated the effects of various substituents at the 3'- and 4'-positions of the drug on cytotoxic activity and ability to stimulate DNA cleavage mediated by DNA topoisomerase II. The study shows that the nature of the substituent at the 3'-position but not the 4'-position is critical for drug ability to form cleavable complexes. The amino group at the 3'-position is not essential for cytotoxic and topoisomerase II-targeting activities, because it can be replaced by a hydroxyl group without reduction of activity. However, the presence of bulky substituents at this position (i.e., morpholinyl derivatives) totally inhibited the effects on the enzyme, thus supporting previous observations indicating that the cytotoxic potencies of these particular derivatives are not related to topoisomerase II inhibition. This conclusion is also supported by the observation that 3'-morpholinyl and 3'-methoxymorpholinyl derivatives are able to overcome atypical (i.e., topoisomerase II-mediated) multidrug resistance. Because a bulky substituent at the 4'-position did not reduce the ability to stimulate DNA cleavage, these results support a critical role of the 3'-position in the drug interaction with topoisomerase II in the ternary complex. An analysis of patterns of cross-resistance to the studied derivatives in resistant human tumor cell lines expressing different resistance mechanisms indicated that chemical modifications at the 3'-position of the sugar may have a relevant influence on the ability of the drugs to overcome specific mechanisms of resistance.     

Synthesis and antihypertensive activity of some thienylethanolamines

Synthesis of a series of thienylethanolamines having varying substituents on the thiophene ring and on the nitrogen atom is described using the general procedure reported earlier. In the determination of their pharmacological profile, some of the derivatives showed marked antihypertensive activity in the spontaneously hypertensive rat model. Tests are also reported which demonstrated that some of these derivatives antagonized alpha- and/or beta-adrenoreceptor activities. The ability of this class of compounds to inhibit catecholamine-induced release of free fatty acids by adipose tissue was demonstrated. Structure-activity relationships in different tests were also determined.     

Synthesis and properties of acrylamide-substituted base pair specific dyes for deoxyribonucleic acid template mediated synthesis of dye polymers

We have tried to construct synthetic polymers for sequence-specific recognition and complexation of longer deoxynucleotide sequences. For this purpose, we developed a method of template-directed polymerization of basic pair specific DNA ligands such as basic dyes. The template-directed polymerization consists in a copolymerization of various dyes of different specificities staying simultaneously in a binding equilibrium with DNA. In the present paper, we describe the synthesis and the properties of base pair specific monomers especially designed for performance of radical chain polymerization reactions in aqueous medium at room temperature. Different acrylamide derivatives of well-known dyes, such as AT-specific malachite green and GC-specific phenyl neutral red, were synthesized and studied for their ability of base pair specific complex formation with DNA of different base composition. Partition equilibrium dialysis and dye titration agarose gel electrophoresis were used to ensure for several dyes that they may be incorporated into different polymers via copolymerization of their acrylamido derivatives with various small base-unspecific monomers without substantial change of their binding parameters. Furthermore, we demonstrate that acrylamide and other small acrylamide derivatives can be used as building blocks for the synthesis of polymeric links between base pair specific monomers. The results and their consequences for template polymerization reactions are discussed.     

Clinical pharmacokinetics of camptothecin topoisomerase I inhibitors

In this review the clinical pharmacokinetics of camptothecin topoisomerase I inhibitors, an important new class of anticancer drugs, is discussed. Two prototypes, topotecan and irinotecan, are currently marketed in many European countries and the USA for the treatment of patients with ovarian and colorectal cancer, respectively. Other camptothecin derivatives, including lurtotecan, 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC), are at different stages of clinical development. The common property of camptothecin analogues is their action against DNA topoisomerase I, but beyond this similarity the compounds differ widely in terms of antitumour efficacy, pharmacology, pharmacokinetics and metabolism. We review chemistry, mechanism of action, stability and bioanalysis of the camptothecins. Dosage and administration, status of clinical application, pharmacokinetics, pharmacodynamics and drug interactions are discussed.     

Influence of some quaternised 1,4-dihydropyridine derivatives on liposomes and erythrocyte membranes

1,4-Dihydropyridine derivatives possess various physiological activities but their mechanism of membranotropic action is not completely investigated yet. We have examined the membranotropic effects of 4-beta-pyridyl-1,4-Dihydropyridine derivatives containing different length alkyl chain substituent at N-quaternised 4-beta-pyridyl moiety. The results show the relation between incorporation of 1,4-dihydropyridine derivatives in the liposomal membranes and influence on bilayer fluidity. The compound with hexadecyl (C16H33) substituent in the 4-beta-pyridyl moiety possess the most pronounced incorporation ability and fluidizing effect. This compound causes the remarkable release of fluorescent probe calcein from liposomes and induces the hemolysis of human erythrocytes as well. The obtained results suggest that the length of alkyl chain at quaternized 4-beta-pyridyl moiety is significant for the expression of membranotropic effects of tested compounds.     

Biotin reagents for antibody pretargeting. 2. Synthesis and in vitro evaluation of biotin dimers and trimers for cross-linking of streptavidin

Polymerization and/or cross-linking of recombinant streptavidin (r-SAv) with biotin derivatives containing two biotin moieties (biotin dimers) or three biotin moieties (biotin trimers) has been investigated as a model for reagents to be used to increase the amount of radioactivity on cancer cells in tumor pretargeting protocols. In the investigation, six biotin dimers and three biotin trimers were synthesized. Most biotin derivatives synthesized had ether containing linker molecules incorporated to improve their aqueous solubility. The synthesized biotin dimers contained linker moieties which provided distances (when fully extended) of 13-49 A between biotin carboxylate carbon atoms, and the biotin trimers contained linker moieties which provided distances of 31-53 A between any two biotin carboxylate atoms. All of the biotin derivatives were evaluated for their ability to polymerize r-SAv in solution. When the biotin derivatives were mixed with r-SAv, none of the biotin dimers caused polymerization, but all of the biotin trimers resulted in complete polymerization. Some of the biotin dimers did cross-link r-SAv (to form r-SAv dimers, trimers, etc.), but the percentage of cross-linking was low (< or = 40%). The length of the linker molecule was important in cross-linking of biotin dimers. While linkers which provided distances of 13 and 19 A between biotin carboxylate carbon atoms did not result in cross-linking, a linker which provided a 17 A distance resulted in a small (< or = 10%) amount of cross-linking. Also cross-linking was increased in biotin dimers with linkers which provided distances between biotin carboxylate carbon atoms of > or = 23 A. Cross-linking of streptavidin bound in polystyrene wells with biotin dimers and trimers was also examined. In those experiments, an excess of each biotin derivative was incubated at 37 degrees C for 10-30 min in polystyrene wells containing bound SAv. After the excess biotin derivative was rinsed from the wells, an excess of r-[125I]SAv was incubated for another 10-30 min. The amount of r-[125I]SAv bound after rinsing the excess from the wells was an indicator of the extent of cross-linking that occurred. The process of alternating additions of reagents was repeated four times to demonstrate that bound radioactivity could be increased with each addition of [125I]SAv. The results of cross-linking r-SAv in polystyrene wells paralleled results from cross-linking in solution.     

Isoquinoline derivatives as endogenous neurotoxins in the aetiology of Parkinson's disease

The cause of neurodegeneration in Parkinson's disease (PD) remains unknown. However, isoquinoline derivatives structurally related to the selective dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite, 1-methyl-4-phenylpyridinim (MPP+), have emerged as candidate endogenous neurotoxins causing nigral cell death in Parkinson's disease. Isoquinoline derivatives are widely distributed in the environment, being present in many plants and foodstuffs, and readily cross the blood-brain barrier. These compounds occur naturally in human brain where they are synthesized by non-enzymatic condensation of biogenic amines (e.g. catecholamines and phenylethylamine) with aldehydes, and are metabolized by cytochrome P450s and N-methyltransferases. In addition, isoquinoline derivatives are oxidized by monoamine oxidases to produce isoquinolinium cations with the concomitant generation of reactive oxygen species. Neutral and quaternary isoquinoline derivatives accumulate in dopaminergic nerve terminals via the dopamine re-uptake system, for which they have moderate to poor affinity as substrates. Several isoquinoline derivatives are selective and more potent inhibitors of NADH ubiquinone reductase (complex I) and alpha-ketoglutarate dehydrogenase activity in mitochondrial fragments than MPP+, and lipophilicity appears to be important for complex I inhibition by isoquinoline derivatives. However, compared with MPP+, isoquinoline derivatives are selective but less potent inhibitors of NADH-linked respiration in intact mitochondria, and this appears to be a consequence of their rate-limiting ability to cross mitochondrial membranes. Although both active and passive processes are involved in the accumulation of isoquinoline derivatives in mitochondria, inhibition of respiration is determined by steric rather than electrostatic properties. Compared with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or MPP+, isoquinoline derivatives show selective but relatively weak toxicity to dopamine-containing cells in culture and following systemic or intracerebral administration to experimental animals, which appears to be a consequence of poor sequestration of isoquinoline derivatives by mitochondria and by dopamine-containing neurones. In conclusion, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-like cytotoxic characteristics of isoquinoline derivatives and the endogenous/environmental presence of these compounds make it conceivable that high concentrations of and/or prolonged exposure to isoquinoline derivatives might cause neurodegeneration and Parkinson's disease in humans.     

Personal experience in the diagnosis and treatment of hepatic echinococcosis

A series of newly developed paclitaxel analogues have been tested for their growth inhibitory activity on two human breast cancer cell lines, one of which expresses the MDR (multidrug resistance) phenotype. Paclitaxel (taxol) was used as a reference compound. Three new classes of taxanes were analyzed: the cephalomannine compounds, the pyrazoline derivatives and the seco-derivatives. Our results demonstrated an increased antiproliferative activity of pyrazoline derivatives on drug-resistant cancer cells with respect to paclitaxel. These compounds were able to block MDR-bearing MCF-7 ADRr cells in the G2/M phase of cell cycle and, consequently, induce programmed cell death. In keeping with the antiproliferative effects, cells treated with paclitaxel derivatives showed a more pronounced cell cycle arrest than the parent compound paclitaxel. Also, apoptotic cell death, calculated as a percent of DNA fragmentation, occurred to a greater extent in cells exposed to pyrazoline derivatives. The development of new paclitaxel analogues with greater antitumour activity on MDR-positive cells may be useful in selecting new taxanes effective on resistant tumors.     

Structure-activity relations of S-adenosylmethionine decarboxylase inhibitors on the growth of MCF-7 breast cancer cells

SAMDC is a key enzyme in the biosynthesis of spermidine and spermine, 2 polyamines that are essential for cell proliferation. Inhibition of polyamine biosynthesis is often targeted as a therapeutic strategy to suppress cancer cell growth as these cells contain elevated levels of polyamines. We examined the effect of a new group of SAMDC inhibitors, CGP33829, CGP35753, CGP36958, CGP39937, and CGP48664, (obtained from Ciba-Geigy, Basel, Switzerland), and their parent compound, MGBG, on the proliferation of MCF-7 breast cancer cells. MGBG had minimal effects on the proliferation of MCF-7 cells up to 6 microM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the pi electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 microM concentration. Other CGP compounds were less effective in inhibiting cell growth. The ability of CGP48664 to inhibit MCF-7 cell proliferation was related to its ability to inhibit SAMDC and to consequently deplete spermidine and spermine levels in the cell. Exogenous spermidine and spermine could reverse the growth inhibitory effects of this compound. CGP compounds also increased the activity of ODC, another enzyme involved in polyamine biosynthesis. Northern blot analysis of mRNA from MCF-7 cells progressing in cell cycle after G1 synchronization did not show an increase in ODC mRNA level by CGP48664. These data demonstrate structure-activity relationships of a series of MGBG derivatives on cell growth, enzyme activities, and polyamine biosynthesis in a hormone-responsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.     

Does hepatitis C virus co-infection accelerate clinical and immunological evolution of HIV-infected patients?

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both alpha2beta1 and alpha3beta1 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequence intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhesion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients.     

Newly synthesized dihydropyridine derivatives as modulators of P-glycoprotein-mediated multidrug resistance

Newly synthesized 1,4-dihydropyridine derivatives possessing alkyl chains at the 4-position screened whether they could overcome P-glycoprotein-mediated multidrug resistance in cultured cancer cells and also leukemia-bearing animals. Of these derivatives, some could overcome drug resistance to doxorubicin and vincristine in multidrug resistant human cancer cell lines. Combined administration of vincristine and some of the derivatives significantly increased the life span of P-glycoprotein overexpressing multidrug-resistant P388 leukemia-bearing mice. The calcium antagonistic activities, an undesirable effects, were weaker than that of verapamil. These results suggested that the introduction of alkyl groups at the 4-position were effective for both overcoming multidrug resistance and reducing the calcium antagonistic activity.     

Suppression of matrilysin inhibits colon cancer cell invasion in vitro

Matrilysin is a member of the matrix metalloproteinase gene family, which is believed to play an important role in tumor invasion and metastasis. We examined the effects of over- and under-expression of matrilysin on the ability of colon cancer cells to migrate across an artificial membrane in vitro. Introduction of matrilysin caused colon cancer cells to become more invasive as assessed by an in vitro invasion assay. In contrast, expression of matrilysin was down-regulated by all trans-retinoic acid or by introduction of anti-sense matrilysin in BM314 colon cancer cells. This down-regulation caused these cells to become less invasive. We demonstrated a correlation between matrilysin level and the invasive potential of human colon cancer cells, implying an important role for matrilysin in the control of tumor invasion in vitro.     

Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer

BACKGROUND: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo. METHODS: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis. RESULTS: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells. CONCLUSIONS: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.     

Allosteric interactions of quaternary strychnine and brucine derivatives with muscarinic acetylcholine receptors

The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer's disease.     

In vitro and in vivo growth inhibition of cancer cells by adamantylmaleimide derivatives

We have previously found that adamantylmaleimide derivatives inhibited the growth of several cancer cell lines in vitro. In this study we examined the effect of adamantylmaleimide derivatives on the in vivo and in vitro growth of human gastric cancer cells. Experimental results showed that N-1-adamantylmaleimide (AMI) and N-1-(3,5-dimethyladamantyl)maleimide (DMAMI) exert modest growth inhibitory activities in vitro against five different cancer cell lines. In contrast, N-1-(3,5-dimethyl-adamantyl)maleamic acid (DMAMA), N-1-adamantylmaleamic acid (AMA) and N-1-adamantylsuccinimide (ASI) were virtually inactive. These results suggest that the double bond of N-substituted maleimide plays a prominent role in their antitumor activities. Further analysis with flow cytometry showed an accumulation of apoptotic SC-M1 cells after treatment with 3-10 microM AMI or 5-20 microM DMAMI for up to 72 h. DNA fragmentation by gel electrophoresis confirmed that AMI- and DMAMI- induced cytotoxicity led to cell apoptosis. In addition, scanning electron microscopy (SEM) showed that treating cells with AMI (> or = 10 microM) for 24 h, significantly changed the morphology of SC-M1 cells, i.e. they had an irregular flat shape and the cell membrane was porous. The AMI-induced morphological changes of the cell membrane may lead to apoptosis of SC-M1 cells. AMI-induced growth inhibition was observed in vivo using SCID mice bearing SC-M1 tumors. The AMI-induced growth inhibition of SC-M1 tumor was dose-dependent.