Systematic peptide engineering and structural characterization to search for the shortest antimicrobial peptide analogue of gaegurin 5

As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.     

Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --> Leu or Pro9 --> Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.     

2-Amino-4-methyl-5-phenylethyl substituted-7-N-benzyl-pyrrolo[2,3-d]pyrimidines as novel antitumor antimitotic agents that also reverse tumor resistance

Gangjee et al. recently reported a novel series of 2-amino-4-methyl-5-phenylethyl substituted-7-benzyl-pyrrolo[2,3-d]pyrimidines, some of which exhibited two digit nanomolar antitumor and antimitotic activity and were not subject to P-glycoprotein (Pgp) or multidrug resistance protein 1 (MRP1) mediated tumor resistance (unlike the Vinca alkaloids and taxanes). Some of these compounds, in addition to their antitumor activity, had the ability to reverse the Pgp-mediated resistance to clinically used antimitotic agents. This report consists of an attempt to optimize the various activities of the parent compounds by synthetic variations of the phenyl ring of the 5-phenylethyl side chain. The target compounds were synthesized via a nine-step synthesis involving a Sonogashira reaction. The substituted phenylacetylenes as coupling partners were in turn synthesized from unactivated aryl bromides or iodides. The target compounds exhibited moderate cytotoxicity against MCF-7 tumor cells. However, most of these compounds showed improved cytotoxicity against the resistant NCI/ADR and MCF-7/VP. This study afforded an analog which reversed both Pgp-mediated as well as MRP1-mediated resistance to clinically used antimitotic agents, along with its own antimitotic mediated antitumor activity. In addition, in the NCI-60 cell line panel one of the compounds inhibited the growth of MDA-MD-435 breast cancer cell line at submicromolar concentration.     

Correlation between the activities of alpha-helical antimicrobial peptides and hydrophobicities represented as RP HPLC retention times

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.     

Extraction, purification and antitumor activity of a water-soluble polysaccharide from the roots of Polygala tenuifolia

One polysaccharide PTP was isolated and purified from the roots of Polygala tenuifolia. It consisted of galactose, glucose and galactose in the ratio of 3.1:3.7:2.5, and a small amount of rhamnose, mannose and xylose. 17 general amino acids were identified to be components of the protein-bound polysaccharide analyzed by automatic amino acid analyzer. In order to test the anti-cancer activity of PTP, we investigated its effect against the growth of human ovarian cancer cells SKOV3 in vitro and in ovarian cancer rats. The intracellular reactive oxygen species (ROS) and glutathione (GSH) in SKOV3 cells following PTP treatment were also quantified to explore the possible mechanism underlying the antitumor activity of the polysaccharide. The result showed that PTP is effective on inhibiting the proliferation of SKOV3 cells in a concentration-dependent manner. Furthermore, treatment with PTP caused a rapid depletion of intracellular GSH content and accumulation of intracellular ROS, thus resulting in the apoptosis, which may prove to be a pivotal mechanism for its cancer protection action. In addition, a significant tumor growth inhibition effect was observed in nude mice after PTP administration for 7 weeks. All above indicated PTP could be beneficial towards ovarian cancer therapy.     

Membrane active antitumor activity of NK-18, a mammalian NK-lysin-derived cationic antimicrobial peptide

As the increasing emergence of multi-drug resistant tumor cells, there is an urgent need for developing new chemotherapeutic agents. NK-lysin was a novel effector of cytotoxic T cells and natural killer (NK) cells and had broad antimicrobial activity. In this study, we developed a core region of NK-lysin termed NK-18, and studied its antitumor activity and possible action mode. Our results showed that NK-18 (with 18 amino acids) possesses potent antitumor activity against bladder and prostate cancer cells by disrupting the integrity of cell membrane, but has negligible hemolysis activity against mouse erythrocytes. In addition, CD spectra was employed to study its conformation in membrane mimicking environment. NK-18 takes a standard α-helical conformation in membrane mimicking environment, which could be accounted for its more potent antitumor activity compared with its low α-helical content homologous derivatives. These findings together with its shorter amino acid sequence and lower synthesis cost suggest that NK-18 could present an alternative therapeutic strategy to cancer chemotherapy and play a promising role in fighting the multi-drug resistant tumors.     

Selective Disruption of Insulin-like Growth Factor-1 (IGF-1) Signaling via Phosphoinositide-dependent Kinase-1 Prevents the Protective Effect of IGF-1 on Human Cancer Cell Death

Insulin-like growth factor-1 (IGF-1) signaling system exerts a broad antiapoptotic function and plays a crucial role in resistance to anticancer therapies. Exposure of MCF-7 breast cancer cells to IGF-1 rapidly and transiently induced tyrosine phosphorylation and activation of phosphoinositide-dependent kinase-1 (PDK1). This was paralleled by Akt/protein kinase B and protein kinase C-ζ phosphorylation, at Thr³⁰⁸ and Thr⁴¹⁰, respectively. IGF-1 treatment also enhanced PDK1 interaction with IGF-1 receptor (IGF-1R) in intact MCF-7 cells. Pulldown assays revealed that PDK1 bound IGF-1R in vitro and that the region encompassing amino acids 51–359 of PDK1 was necessary for the interaction. Synthetic peptides corresponding to IGF-1R C terminus amino acids 1295–1337 (C43) and to PDK1 amino acids 114–141 reduced in vitro IGF-1R/PDK1 interaction in a concentration-dependent manner. Loading of fluoresceinated-C43 (fluorescein isothiocyanate (FITC)-C43) into MCF-7 cells significantly reduced IGF-1R/PDK1 interaction and phosphorylation of PDK1 substrates. Moreover, FITC-C43 intracellular loading reverted the protective effect of IGF-1 on growth factor deprivation-induced cell death. Finally, the inhibition of IGF-1R/PDK1 interaction and signaling by FITC-C43 was accompanied by 2-fold enhanced killing capacity of cetuximab in human GEO colon adenocarcinoma cells and was sufficient to restore cell death in cetuximab-resistant cell clones. Thus, disruption of PDK1 interaction with IGF-1R reduces IGF-1 survival effects in cancer cells and may enhance cell death by anticancer agents.     

PTP1B modulates the association of beta-catenin with N-cadherin through binding to an adjacent and partially overlapping target site

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of beta-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872-891 fail to bind PTP1B. This domain partially overlaps with the beta-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH(2)-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the beta-catenin target, effectively competes for binding of beta-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on beta-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the beta-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated beta-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or beta-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with beta-catenin, stable expression at the cell surface, and consequently, cadherin function.     

Synthesis and antiproliferative activity of 2,7-diamino l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives as potent telomeric G-quadruplex DNA ligands

A novel series of l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives with terminal ammonium substituents at C2 and C7 positions on the acridone ring were successfully synthesized as antiproliferation agents. The biologic activity of the acridone compounds against leukemia CCRF-CEM cells demonstrated that some of the compounds displayed good antiproliferative activity, among which compound 6a containing dimethylamine substituents at the terminal C2 and C7 positions exhibited the highest cytotoxicity with IC₅₀ at 0.3 μM. In addition compound 6a showed little toxicity against normal 293T cells proliferation with IC₅₀ more than 100 μM. Further study indicated that compound 6a had strong binding activity to human telomeric G-quadruplex DNA, as detected by mass spectrometry, CD spectroscopy, UV absorption, FRET and fluorescence quenching assays. Our data suggested that the activity of 6a might be associated with its stabilization of G-quadruplex DNA, which can be developed as potent antitumor agent.     

Design,synthesis, and cytotoxic activity of novel 7-substituted camptothecin derivatives incorporating piperazinyl-sulfonylamidine moieties

In our continuing search for camptothecin (CPT)-derived antitumor drugs, novel 7-substituted CPT derivatives incorporating piperazinyl-sulfonylamidine moieties were designed, synthesized and evaluated for cytotoxicity against five tumor cell lines (A-549, MDA-MB-231, MCF-7, KB, and KB-VIN). All of the derivatives showed promising in vitro cytotoxic activity against the tested tumor cell lines, and were more potent than irinotecan. Remarkably, most of the compounds exhibited comparable cytotoxicity against the multidrug-resistant (MDR) KB-VIN and parental KB tumor cell lines, while irinotecan lost activity completely against KB-VIN. Especially, compounds 13r and 13p (IC₅₀ 0.38 and 0.85 μM, respectively) displayed the greatest cytotoxicity against the MDR KB-VIN cell line and merit further development into preclinical and clinical drug candidates for treating cancer, including MDR phenotype.     

Synthesis and structure-activity relationship studies of 4,11-diaminonaphtho[2,3-f]indole-5,10-diones

We describe the synthesis of derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione and their cytotoxicity for human tumor cells that express major determinants of altered anticancer drug response, the efflux pump P-glycoprotein, and non-functional p53. Nucleophilic substitution of methoxy groups in 4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione with various ethylenediamines yielded the derivatives of 4,11-diaminonaphtho[2,3-f]indole-5,10-dione, the indole containing analogues of the antitumor agent ametantrone. The cytotoxicity of novel compounds for multidrug resistant, P-glycoprotein-expressing tumor cells is highly dependent on the N-substituent at the terminal amino group of the ethylenediamine moiety. Whereas p53 null colon carcinoma cells were less sensitive to the reference drug doxorubicin than their counterparts with wild type p53, the majority of novel naphthoindole derivatives were equally potent for both cell lines, regardless of the p53 status.     

Synthesis and cytotoxic properties of 4,11-bis[(aminoethyl)amino]anthra[2,3-b]thiophene-5,10-diones,novel analogues of antitumor anthracene-9,10-diones

We developed the synthesis of a series of thiophene-fused tetracyclic analogues of the antitumor drug ametantrone. The reactions included nucleophilic substitution of methoxy groups in 4,11-dimethoxyanthra[2,3-b]thiophene-5,10-diones with ethylenediamines, producing the derivatives of 4,11-diaminoanthra[2,3-b]thiophene-5,10-dione in good yields. Several compounds showed marked antiproliferative potency against doxorubicin-selected, P-glycoprotein-expressing tumor cells and p53^?/? cells. The cytotoxicity of some novel compounds for P-glycoprotein-positive cells is highly dependent on N-substituent at the terminal amino group of ethylenediamine moiety. The cytotoxic potency of selected compounds correlated with their ability to attenuate the functions of topoisomerase I and telomerase, strongly suggesting that these enzymes are the major targets of antitumor activity of anthra[2,3-b]thiophene-5,10-dione derivatives.     

The carboxyl terminal 17 amino acids within Apg7 are essential for Apg8 lipidation,but not for Apg12 conjugation

In the yeast, Saccharomyces cerevisiae, two ubiquitin-like modifications, Apg12 conjugation with Apg5 and Apg8 lipidation with phosphatidylethanolamine, are essential for autophagy and the cytoplasm-to-vacuole transport of aminopeptidase I (Cvt pathway). As a unique E1-like enzyme, Apg7 activates two modifiers (Apg12 and Apg8) in an ATP-dependent manner and, for this activity, the carboxyl terminal 40 amino acids are essential. For a better understanding of the function of the carboxyl terminus of Apg7, we performed a sequential deletion of the region. A mutant expressing Apg7DeltaC17 protein, which lacks the carboxyl 17 amino acids of Apg7, showed defects in both the Cvt pathway and autophagy. Apg8 lipidation is inhibited in the mutant, while Apg12 conjugation occurs normally. A mutant expressing Apg7DeltaC13 protein showed a defect in the Cvt pathway, but not autophagy, suggesting that the activity of Apg7 for Apg8 lipidation is more essential for the Cvt pathway than for autophagy. Mutant Apg7DeltaC17 protein is still able to interact with Apg8, Apg12 and Apg3, and forms a homodimer, indicating that the deletion of the carboxyl terminal 17 amino acids has little effect on these interactions and Apg7 dimerization. These results suggest that the carboxyl terminal 17 amino acids of Apg7 play a specific role in Apg8 lipidation indispensable for the Cvt pathway and autophagy.     

Combined effect of epirubicin and lymphokine-activated killer cells on the resistant human breast cancer cells

Accumulating evidence suggests the concept that epirubicin and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radicals generation and P-glycoprotein-positive (Pg-p⁺) cancer cells are more sensitive for LAK cells than their drug-sensitive parental lines. We tested this hypothesis further by exposing drug-sensitive (WT) and epirubicin-resistant MCF-7 human breast tumor cells to epirubicin and LAK cells. Subsequently, we monitored cell proliferation as a measure of cytotoxicity. The cytotoxicity of epirubicin, LAK, and LAK + epirubicin (1/10 of IC₅₀) was evaluated in 400-fold epirubicin resistant MCF-7 EPI^R (P-glycoprotein overexpressing) and drug-sensitive MCF-7 WT cells. IC₅₀ values were measured using the MTT cytotoxicity test. The MCF-7 EPI^R cells exhibited an increased susceptibility to LAK cells than did the MCF-7 WT cells. P-gp⁺ MCF-7 EPI^R cells were lysed by human LAK cells to a greater extend than were their drug-sensitive counterparts. LAK + epirubicin combined treatment increased susceptibility of MCF-7 WT and MCF-7 EPI^R cells to LAK cells cytotoxicity. For both cell lines, cytotoxicity was dependent upon the concentration of the epirubicin and effector cell/target cell (E/T) ratio. The resistance of MCF-7 EPI^R cells to epirubicin appears to be associated with a developed tolerance to superoxide, most likely because of a tree-fold increase in superoxide dismutase (SOD) activity and 13-fold augmented selenium dependent glutathione peroxidase (GSH-Px) activity. Acting in concert, these two enzymes would decrease the formation of hydroxyl radical from reduced molecular oxygen intermediates. The addition of SOD decreased cytotoxicity of epirubicin and LAK cells. Taken together, these observations support the role of oxygen radicals in the cytotoxicity mechanism of epirubicin and suggest further that the development of resistance to this drug by the MCF-7 EPI^R tumor cells may have a component linked to oxygen free radicals. It is proposed that production of reactive oxygen species by the treatment of epirubicin and LAK cells can cause cytotoxicity of MCF-7 WT and MCF-7 EPI^R cells. SOD, catalase, GSH-Px, GST (glutathione S-transferase), and GSH (reduced glutathione) must be considered as part of the intracellular antioxidant defense mechanism of MCF-7 WT and MCF-7 EPI^R cells against reactive oxygen species.     

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.     

Structure-activity relationships of fowlicidin-1, a cathelicidin antimicrobial peptide in chicken

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti-infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad-spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858-2867). Here we report that fowlicidin-1 mainly adopts an alpha-helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin-1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)-binding activities. The short C-terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16-23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5-7) in the N-terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin-1 analog, with deletion of the second N-terminal segment (residues 5-7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti-infective agent.     

Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

Evaluation of metabolic labeling for comparative proteomics in breast cancer cells

Protein expression patterns in the cytosol of MCF-7 cells resistant to adriamycin and to adriamycin/verapamil were compared to that of the parental MCF-7 cell line and to each other using metabolic labeling and two-dimensional gel electrophoresis. Growing the parental MCF-7 cell line in 13C6-arginine- and 13C6-lysine-enriched medium resulted in C-terminal labeling of all tryptic peptides. The culture media was optimized for the incorporation of these labeled amino acids under conditions that also supported cell growth. Protein abundances were found to be distinctive in MCF-7 cells resistant to adriamycin and those selected for resistance to both adriamycin and verapamil.