Combined inhibition of Chk1 and Wee1: In vitro synergistic effect translates to tumor growth inhibition in vivo

Targeting Chk1 protein kinase can enhance the antitumor effects of radio- and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. To identify genes in synthetic lethality with Chk1, we did a high-throughput screening using a siRNA library directed against 719 human protein kinases in the human ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibitors. Wee1 tyrosine kinase was the most significant gene in synthetic lethality with Chk1. Treatment with non-toxic concentrations of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) confirmed the marked synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate), independently of the p53 status. Detailed molecular analysis showed that the combination caused cancer cells to undergo premature mitosis before the end of DNA replication, with damaged DNA leading to cell death partly by apoptosis. In vivo treatment of mice bearing OVCAR-5 xenografts with the combination of Chk1 and Wee1 inhibitors led to greater tumor growth inhibition than with the inhibitors used as single agents with no toxicity. These data provide a strong rationale for the clinical investigation of the combination of a Chk1 and a Wee1 inhibitor.     

Starch-based microspheres for sustained-release of curcumin: Preparation and cytotoxic effect on tumor cells

Curcumin (CUR) has been proved to be highly cytotoxic against different tumor cell lines. However, its poor solubility in aqueous medium and fast degradation in physiological pH are the common drawbacks preventing its efficient practical use. Herein, we report the development of original microspheres based on the biopolymer starch crosslinked with N,N-methylenebisacrylamide (MBA) to be applied as an efficient delivering system for CUR. The starch-based microspheres showed high loading efficiency even in loading solution with different CUR concentrations. In vitro release assays data showed that the CUR release is governed by anomalous transport (n = 0.73) and it is pH-dependent. Cytotoxicity assays showed that starch microspheres could improve the cytotoxicity of CUR toward Caco-2 and HCT-116 tumor cell lines up to 40 times than that found for pure CUR. This behavior was attributed to the slowly and sustained release of CUR from the microspheres.     

Synthesis and cytotoxic activity of boswellic acid analogues

Fifteen stereoisomers of boswellic acid analogues bearing 2-OH, 24-OH, 3-keto or 2-OH, 3-OH, 24-OH groups were synthesised and their structures were confirmed using ¹H NMR, ¹³C NMR, 2D NMR and HRMS. The cytotoxic activities of these compounds toward three human tumor cell lines, K562, PC3 and A549, were evaluated. Preliminary biological evaluation indicated most of these compounds exhibited cytotoxic activity comparable to that of 3-O-acetyl-11-keto-β-boswellic acid (AKBA). Notably, several analogues exhibited relatively stronger cytotoxicity, with IC₅₀ values less than 10 μM against A549 and PC3 cell lines. For the 24-OH series of BAs analogues, structure-activity relationship (SAR) analysis indicated that the stereochemical configurations of compounds incorporating 2-OH, 3-keto or 2-OH, 3-OH group pairs could not predictably or markedly impact cytotoxic activity, except when 2β-OH and/or 3β-OH were present. Esterification of 2-OH, 3-OH and 24-OH groups tended to decrease cytotoxicity.     

Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16-to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

Peptide mapping of the epitope on target cells recognized by nonspecific cytotoxic cells (NCC) from channel catfish

Nonspecific cytotoxic cells (NCC) may comprise an important effector population specific for recognition of aberrant (tumour) cells, regulation of cell interactions including antibacterial action and lysis of protozoan parasites. In the present study, peptides were synthesized based on the amino acid sequence of a novel protein (Natural Killer cell Target Antigen, NK Tag) found on the protozoan parasite Tetrahymena pyriformis and on NCC-sensitive tumour target cells. Partially purified NK Tag was obtained from Tetrahymena. It inhibited NCC lysis of a large variety of mammalian tumour target cells. Synthetic peptides composed of short 20 mer sequences obtained from the N-terminal and midregion portions of NK Tag were tested for their ability to inhibit NCC cytotoxicity. Synthetic peptide comprised of aa # 55-74 significantly inhibited NCC lysis of IM-9 target cells. A monoclonal antibody generated against an N-terminal dodecapeptide of NK Tag bound to Tetrahymena and to several mammalian NK-sensitive target cells including K562, YAC-1, U937, NC-37, EL-4, IM-9, HL-60 and MOLT-4. NK Tag sequence comparisons using Swisspro database revealed no significant homologies except in a restricted domain region of several glycolytic pathway enzymes. A supergene family relationship was indicated because of these similarities.     

Synthesis of shorter active analogues of Bactenecin7: The effect of change of N-terminal configuration on antimicrobial activity

Summary Bactenecin7, a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa=hydrophobic residues). A series of peptides, Xaa-Pro-Arg-Pro (Xaa=D-Ala, D-Leu, D-Val, D-Phe and D-Lys) were synthesized to investigate the effect of change ofN-terminal configuration on antimicrobial activity. The conformational preferences of these peptides in water and TFE were examined by circular dichroism. All the synthetic peptides with D-amino acid substitution atN-terminal showed potent antifungal activity againstAspergillus niger, Aspergillus flavus andFusarium moniliforme at the concentration level of 8–10 μg ml⁻¹. But the same tetrapeptides were unable to kill or suppress the growth of gram-negative and gram-positive bacteria such asEscherichia coli HB101,Pseudomonas aeruginosa, Klebsiella pneumoniae andStaphylococcus aureus even at the concentration level of 400 μg ml⁻¹. The present study reveals that the change of configuration at theN-terminal of tetrapeptide has negative impact on antibacterial activity but enhanced antifungal activity.     

Synthesis of shorter active analogues of Bactenecin7: The effect of change of N-terminal configuration on antimicrobial activity

Bactenecin7, a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residues). A series of peptides, Xaa-Pro-Arg-Pro (Xaa = D-Ala, D-Leu, D-Val, D-Phe and D-Lys) were synthesized to investigate the effect of change of N-terminal configuration on antimicrobial activity. The conformationalpreferences of these peptides in water and TFE were examined by circular dichroism. All the synthetic peptides with D-amino acid substitution at N-terminal showed potent antifungal activity against Aspergillus niger, Aspergillus flavus and Fusarium moniliforme at the concentration level of 8–10 g ml^-1. But the same tetrapeptides were unable to kill or suppress the growth of gram-negativeand gram-positive bacteria such as Escherichia coli HB101, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus even at the concentration level of 400 g ml^-1. The present study reveals that the change of configuration at the N-terminal of tetrapeptide has negative impact on antibacterial activity but enhanced antifungal activity.     

Selective cytotoxic effect of an immunotoxin on human erythroid tumor cells]

The possibility of efficient directed elimination of human erythroblastoid cells by the conjugate of IgM-monoclonal antibody HAE9 directed against the erythroblast antigen and the A-chain of a plant toxin ricin has been demonstrated. The conjugate contained 2 molecules of A-chain per one antibody molecule. The efficiencies of the cytotoxic effect of native ricin and the conjugate were compared according to the number of binding sites on the surface of K562 cells as well as to the internalization rate of these molecules. As was shown, that the number of binding sites for the antibody approaches 2.7.10(4) molecules/cell, K a being equal to 1.7.10(8) M-1 while for ricin these indices constitute 2.4.10(5) and 4.6.10(8) M-1. Almost 100% of antibodies and 36% of ricin are internalized within 10 min at 37 degrees C. At a concentration 10(-11) of native ricin and 10(-10) of immunotoxin the 50% inhibition of growth of K562 cells carrying the erythroblast antigen on their surface is observed.     

Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro

Green tea extract and its polyphenolic components have been found to possess anticarcinogenic, antimutagenic, antihypertensive and antihepatotoxic effects, and several mechanisms have been proposed for these effects. In this study, the effects of five tea polyphenols, (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−) epicatechin-3-gallate (ECG), (−) epicatechin (EC) and (+)-catechin (C), were examined on the viability of Ehrlich ascites tumor cells in vitro and a possible relationship with tyrosine phosphorylation was determined. Proteins extracted from the cells treated with the tea polyphenols were separated by SDS-PAGE, and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibody and the extent of phosphorylation determined. EGC (100 μM) caused a significant decrease in cell viability to 4.1±0.2% of the control value, and this correlated with a stimulation of protein tyrosine kinase (PTK) activity. EGCG (100 μM) also caused a slight decrease in cell viability (70% of the control value) but this and the other polyphenols, which had no effect on cell viability likewise, had no effect on tyrosine phosphorylation. Tyrosine phosphorylations of 42 and 45 kDa proteins were also observed for EGC. Further evaluation of the effect of EGC showed that the activity of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis in cells, decreased significantly as well. A significant correlation has therefore been observed between a cellular event, namely, a reduction in the viability of Ehrlich ascites tumor cells and an association with a tyrosine phosphorylation of 42 and 45 kDa proteins by the polyphenol EGC.     

Are laminin binding sites on tumor cell surface involved in the indomethacin-induced sensitivity to natural cytotoxic cells?

Prostaglandins are secreted by a variety of tumor cell lines. The prostaglandin synthesis inhibitor indomethacin (IND) inhibits 3LL tumor growth after both intramuscular or intrasplenic transplantation (45 and 72%, respectively). Moreover, when tumor cells were cultured with IND, the sensitivity of 3LL cells to natural cytotoxic (NC) effector cells was increased (30%) and a higher cytotoxicity was reached when both target and effector cells were treated. This effect was reversed partially or totally when the assay was performed in the presence of laminin or an octapeptide from the laminin B1 chain. In addition, we correlate the increased cytotoxicity mediated by IND with an enhanced ability of 3LL tumor cells to bind labeled laminin (55%). In summary, our results show that the blockage or modulation of cell surface laminin binding components could be directly correlated with the sensitivity of tumor target cells to be eliminated by way of natural cytotoxicity.     

抗肿瘤小分子多肽的研究进展

抗肿瘤小分子多肽具有分子量小、低毒性、高活性、易于穿透肿瘤细胞等特点,一些抗肿瘤小分子多肽已进入临床研究,成为肿瘤药物研发的新热点。本文从抗肿瘤小分子多肽的不同来源途径、结构改造及生物活性等方面,对其近年来的研究进展作简要概述。     

In situ cytotoxic T cells in a methylcholanthrene-induced tumor.

The in situ localization of cytotoxic T-cells was examined in a methylcholanthrene-induced fibrosarcoma. The MCA 2 tumor was initiated in this laboratory and utilized before extensive in vivo passage. Tumor cell suspensions were separated by unit velocity sedimentation. The T cell-enriched tumor-derived fractions and spleen cells from MCA 2-bearing mice were cytotoxic for in vivo isolated MCA 2 and in vitro cultured MCA 2 tumor cells, but not for SAD2 tumor cells. The cytotoxic activity of effector cells isolated from MCA 2 tumors was abrogated by treatment with anti-theta serum plus complement. The significance of cytotoxic T cells with in a progressing tumor is discussed.     

Effect of the microbial lipopeptide on tumor cell lines: apoptosis induced by disturbing the fatty acid composition of cell membrane

Microbial lipopeptides play an important role in apoptosis induction of tumor cells. However, there is little knowledge about the relationship between apoptosis induction and membrane fatty acids. The present study focused on the effects of lipopeptides produced by Bacillus subtilis HSO121 on Bcap-37 cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl (MTT) colorimetric assay and surface tension measurements, showed that the critical micelle concentration (CMC) was a critical level for the inhibitory activity of lipopeptides on the growth of Bcap-37 cells. Under the CMC, the order of least to greatest cytotoxicity effect on cancer cell lines by lipopeptides is C₁₃-lipopeptide<C₁₄-lipopepitde<C₁₅-lipopeptide. Above CMC, all lipopeptides directly exert cytolytic activity. The flow cytometric analysis and Hoechst33258 staining experiments confirmed the apoptosis of Bcap-37 cell lines induced by lipopeptides in a dose-dependent manner. This apoptosis was associated with a significant decrease of the unsaturated degree of the cellular fatty acids of Bcap-37 cell lines due to the changes in the cellular fatty acids composition induced by the lipopeptide treatment. These results indicated that disturbance of the cellular fatty acid composition of breast cancer cell lines were related to in the cell apoptosis. Furthermore, significant difference in IC₅₀ values of tumor cells and normal cell showed that the lipopeptide exerted selective cytotoxicity on the cancer cells. Thus HSO121 lipopeptides may have potential applications as an anticancer leads.     

Cytotoxic and cytostatic effect of avermectines on tumor cells in vitro]

Effect of natural avermectin complex (aversectin C) and separate avermectins A1, A2, B1 and B2 in the cell culture of murine myeloma Ns/o, Erlich carcinoma ascites and human larynx carcinoma Hep-2 was investigated. It was shown that aversectin C within the concentrations of 0.1 to 1.0 mcg/ml inhibited proliferation of tumor cells and induced their death. Proliferation inhibition was due to the delay of the cells cycle start (lag-phase prolongation) and blocking of mitotic cycle. Ns/o cells death had apoptosis signs: chromatin condensation and fragmentation, DNA fragmentation. It was demonstrated that only avermectin A1 has cytotoxic activity within the concentrations used, avermectins A2 and B2 had cytostatic activity, avermectin B1 showed no activity under the experimental conditions.     

姜黄素类似物的合成及体外抗氧化活性研究

利用不同的芳香醛和乙酰丙酮缩合反应,合成了4种姜黄素类似物(A1～A4),化合物的结构经IR1、HNMR及MS等测试技术表征确证。采用邻苯三酚法研究化合物的体外抗氧化活性,台盼蓝细胞计数法研究体外抗肿瘤活性。结果表明,化合物A1、A2、A3的抗氧化活性和对K562细胞增殖的抑制活性均高于姜黄素,其活性与酚羟基密切相关。     

Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. Protein A (PA) of Staphylococcal aureus has been found to have diverse biological response modifying properties and to possess antitumor, antitoxic and antiparasitic effects. In this study we examined the anti-tumor effect of these two superantigens used separately as well as in combination in mice carrying the Ehrlich ascites tumor. With combined treatment, DNA cell cycle analysis of tumor cells showed a significant (P < 0.05) percentage of tumor cell death. Levels of the soluble mediators TNF-alpha, IFN-gamma and IL-1 as well as NO were elevated. Additionally, CD4(+) and CD8(+) specific T cells in spleen, thymus and PBMC in tumor carrying mice were increased (P < 0.01). Our data altogether suggests that enhanced tumor cell death is caused by the increased CTL activity, cytokine and nitric oxide levels, in response to the combined effect of SEA + PA.     

In vitro fenoprofenyl–coenzyme a thioester formation: Interspecies variations

In vitro coenzyme A thioester formation from (?)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (?)-(R)-fenoprofen, the Lineweaver–Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar K_m values). The V_max values for the thioesterification of (?)-(R)-fenoprofen present large species variations from 2.1 ± 1.0 with sheep liver microsomes to 60.6 ± 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the V_max values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (?)-(R)-fenoprofen–CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent. © 1995 Wiley-Liss, Inc.