Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies

Cadmium represents a major environmental pollutant that may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. Owing to their contractile properties, glomerular mesangial cells play a major role in the control of glomerular hemodynamics and influence the ultrafiltration coefficient. Cell cultures provide alternative and fruitful models for study of in vitro toxicology. However, the use of primary human mesangial cell cultures is hampered by their limited survival span and their rapid dedifferentiation during passages. This study presents a human stable immortalized mesangial cell line, designated IP15. Cell characteristics were investigated by the detection of known mesangial markers, as well as their ability to contract in response to angiotensin II. IP15 cells were used to investigate cadmium uptake and morphological changes such as cell contraction and cytoskeleton protein expression. The IC₅₀ cytotoxicity index was obtained with 3.55 μmol/L using neutral red assay for 24 h. After cadmium exposure (1 μmol/L, determined as nonlethal concentration), 0.38 μg Cd/mg protein was internalized by the cells as evaluated by inductively coupled plasma optical emission spectrometry (ICP/OES). Cadmium induced a significant cell surface reduction that correlated with smooth-muscle α-actin disorganization. Thus, the IP15 cell line is a suitable model for study of in vitro cadmium cytotoxicity in mesangial cells and allows sufficient material to be obtained for future studies of the intracellular effects of cadmium exposure.     

Novel achiral titanocene anti-cancer drugs synthesised from bis-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylamino fulvene and lithiated heterocyclic compounds

Abstract From the carbolithiation of 6-bis-N,N-dimethylamino fulvene (3a) and different ortho-lithiated heterocycles (furan, thiophene and N-methylpyrrole), the corresponding lithium cyclopentadienide intermediate (4a–c) was formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl₄ resulting in bis-N,N-dimethylamino-functionalised titanocenes 5a–c. When these titanocenes were tested against LLC-PK cells, the IC₅₀-values obtained were of 240, and 270 μM for titanocenes 5b and 5c, respectively. The most cytotoxic titanocene in this paper, 5a with an IC₅₀-value of 36 μM was found to be approximately six times less cytotoxic than its mono-N,N-dimethylamino substituted analogue Titanocene C (IC₅₀ = 5.5 μM) and almost ten times less cytotoxic than cisplatin, which showed an IC₅₀-value of 3.3 μM, when tested on the LLC-PK cell line. Graphical abstract Bis-(bis- (N,N-dimethylamino)-2-(N′-methylpyrrolyl)methylcyclopentadienyl) titanium (IV) dichloride, {η⁵-C₅H₄-CH[N(CH₃)₂]₂[C₅H₃NCH₃]}₂TiCl₂ was synthesised starting from 6-bis-(N,N-dimethylamino) fulvene and 2-N-methylpyrrolyl lithium. Herein, we present the synthesis and DFT structure of the titanocene and two further derivatives followed by MTT-based cytotoxicity tests on pig kidney epithelial (LLC-PK) cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK1 cells

We have previously demonstrated that a cultured porcine kidney cell, LLC-PK(1), maintains the characteristics of a polar renal epithelial cell in culture, and responds to salmon calcitonin and [arginine]vasopressin by increasing cyclic AMP content. To demonstrate the usefulness of this cell line as a model for the study of the biochemical events distal to cyclic AMP production, the activation of cyclic AMP-dependent protein kinase was examined. Intact cells in monolayer demonstrated progressive increases in cyclic AMP content and activation of protein kinase in response to [arginine]vasopressin (2-200nm) and salmon calcitonin (0.03-30nm) with both hormones fully activating the enzyme at a cell cyclic AMP content of 35pmol/mg of protein. Of the total cyclic AMP-dependent protein kinase activity, 80% was found in the 27000g supernatant fraction of sonicated cell material, and this soluble protein kinase could be fully activated by hormone. Conversely, the 27000g pellet contained a significant proportion of cyclic AMP-independent protein kinase and only 20% of total cell cyclic AMP-dependent protein kinase; the latter showed little response to hormone. On the basis of DEAE-cellulose chromatography, type II protein kinase was the predominant isoenzyme in both soluble and particulate fractions of the LLC-PK(1) cells and the soluble fractions of rat and guinea-pig renal medulla. Thus, the LLC-PK(1) cell line can serve as a model for hormonal modulation of protein kinase and as a potential source for defining the endogenous substrates for these enzymes.     

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

 We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Δ34–220), which had most of the Ia domain deleted but retained amino acid residues 1–33 and 221–252 of this domain, was constructed. EGF-PE(Δ34–220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Δ34–220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(Δ1–252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(Δ34–220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(Δ34–220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Δ34–220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells. Received : 20 May 1994 / Received last revision : 9 September 1994 / Accepted : 28 September 1994     

人SDCT2蛋白亚细胞定位信号分析

为了确定人高亲和力钠离子依赖性二羧酸共转运蛋白（high-affinity sodium-dependent dicarboxylate co-transporter, SDCT2,NaDC3）在细胞内的定位,构建了SDCT2与增强型绿色荧光蛋白（EGFP）的融合蛋白表达载体,并转染肾小管上皮细胞LLC-PK1,激光共聚焦显微镜观察显示,SDCT2蛋白主要定位于细胞的基底侧膜上.同时将SDCT2-EGFP融合基因mRNA显微注射到爪蟾卵母细胞中表达,可见融合蛋白的绿色荧光仅分布在细胞膜上.为了进一步确定该蛋白质的亚细胞定位信号序列,将SDCT2基因的N端及C端分别缺失,并构建缺失突变体与EGFP的融合蛋白表达载体,将它们转染到LLC-PK1中,观察SDCT2 缺失体在细胞内的分布情况.结果显示,N端缺失的SDCT2蛋白主要位于细胞质中,顶膜和基底侧膜上也有表达;C端缺失的SDCT2蛋白主要位于基底侧膜上,顶膜几乎没有表达,细胞质中表达很少.免疫组化结果也显示,SDCT2只表达于人近端肾小管上皮细胞的基底侧膜.这表明SDCT2蛋白的N端序列对其亚细胞定位是必需的,人SDCT2蛋白的基底膜定位信号位于N端序列中.     

Cytotoxin production by a merineLyngbya strain (cyanobacterium) in a large-scale laboratory bioreactor

A cytotoxic compound was produced by the marine cyanobacteriumLyngbya sp. Pearl strain in large laboratory-scale batch cultures. Adsorption and fractionation of methanol extracts with reverse phase (C-18) cartridges provided a rapid method for removal of bioassay interference from salts, biopolymers and pigments and concentration of the cytotoxic principles. Cytotoxicity to the murine leukemia cell line P-388 was produced in two cycles coinciding with the initiation of exponential growth and again during the late exponential growth phase. Antiviral activity against influenza virus PR8 was found in extracts prepared from early exponential growth phase cells but antiviral activity was not detected in extracts of mid-log or late-log growth phase cells. These differences in bioactivity suggests that the cytotoxic principles produced during early and late exponential growth may be different compounds. Cytotoxicity assays using murine P-388 leukemia indicates that the semi-pure compound has an IC₅₀ of < 0.25 μg ml⁻¹ to this cell line. P-388 cytotoxicity in cell extracts increased during the late exponential growth phase and the specific yield was estimated at approximately 0.14 mg g⁻¹ (dry cells).     

Tumour-expressed tissue factor inhibits cellular cytotoxicity

Aims: The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. Materials and methods: A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Results: Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Δ=15–40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. Conclusions: We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells.     

Immunochemotherapy of malignant glioma: synergistic activity of CD95 ligand and chemotherapeutics

 Malignant glioma cells are susceptible to CD95(Fas/APO-1)-mediated apoptosis triggered by agonistic antibody. Here we examined the proapoptotic effects of the natural CD95 ligand, a cytotoxic cytokine homologous to tumor necrosis factor, on malignant glioma cell lines LN-229, LN-308 and T98G. We assessed whether glioma cell killing is synergistically enhanced by cotreatment with CD95 ligand and chemotherapeutic agents, including doxorubicin, carmustine, vincristine, etoposide, teniposide, 5-fluorouracil and cytarabine. Synergy was examined at low concentrations of cytotoxic drugs and CD95 ligand with a defined effect level (IC₁₅). Short-term-cytotoxicity assays showed prominent killing of the glioma cells by CD95 ligand but not by the drugs at relevant concentrations. CD95 ligand-induced apoptosis in the acute toxicity paradigm was augmented by doxorubicin and vincristine. Growth-inhibition assays revealed prominent synergy between CD95 ligand and all drugs examined. The best synergy was obtained with CD95 ligand and doxorubicin, vincristine or teniposide. The strong synergistic antiproliferative effects were observed at much lower concentrations of CD95 ligand and cytotoxic drugs than the moderate synergistic acute cytotoxic effects. All cell lines examined express the Bcl-2 protein. LN-229 has partial wild-type p53 activity. T98G has mutant p53. LN-308 has a deleted p53 gene and lacks p53 protein expression. Thus, synergistic effects of CD95 ligand and cytotoxic drugs were observed in cell lines exhibiting two features thought to play a role in the chemoresistance of human malignant glioma cells: loss of wild-type p53 activity and acquisition of bcl-2 expression. Ectopic expression of murine bcl-2 conferred partial protection from CD95 ligand and drugs when administered alone but did not interfere with the mechanisms underlying the synergistic effects of CD95 ligand and chemotherapeutic drugs. Received: 31 October 1996 / Accepted: 4 January 1997     

Cytotoxic activity induced by crude extracts of Ganoderma lucidum (W. Curt.: Fr.) P. Karst. on mouse myeloma cancer cell-line

Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC₅₀ of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml. After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after 72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei. The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as 45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further confirmed the occurrence of various apoptotic and necrotic features.     

In vitro cytotoxicity of the organophosphorus insecticide methylparathion to FG-9307, the gill cell line of flounder (Paralichthys olivaceus)

FG-9307, a cell line derived from the gill of flounder Paralichthys olivaceus, was used to determine the acute cytotoxic effects of the organophosphorus insecticide methylparathion. The cytotoxic effects of methylparathion were initially measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay, and cell protein assay. Results indicated that concentrations of methylparathion ranging from 5 μg/ml to 60 μg/ml were toxic, and there was no significant difference in cytotoxic effects between the three test systems. Thus, the FG-9307 cell line is one of several choices for evaluating the acute toxicities of organophosphorus insecticides such as methylparathion. The ultrastructure of the cells was also studied. It was found that the ultrastructure of the cells was markedly altered by methylparathion, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear necrosis, and production of numerous lysosomes and lipid vacuoles. This appears to be the first report that a marine fish cell line can be used for acute in vitro cytotoxicity evaluation of methylparathion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin–Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction.The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.     

Development of a bispecific F(ab′)2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity

 A bispecific F(ab′)₂ antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and a variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab′ fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5′-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of “clump-like” cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively. Received: 2 May 1996 / Accepted: 21 May 1996     

Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion

Increased blood polyamine levels, often observed in cancer patients, have negative impacts on patient prognosis and are associated with tumor progression. The purpose of our study was to examine the effects of polyamines on cellular immune function. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured with the human natural polyamines spermine, spermidine, or putrescine, and the effects on immune cell function were examined. The correlation between post-operative changes in blood polyamine levels and lymphokine-activated killer (LAK) activity was also examined in cancer patients. Spermine decreased the adhesion of non-stimulated PBMCs to tissue culture plastic in a dose- and a time-dependent manner without affecting cell viability or activity. This decrease in adhesion capacity was accompanied by a decrease in the number of CD11a bright-positive and CD56 bright-positive cells. Upon stimulation with interleukin 2 to activate LAK cytotoxicity, PBMCs cultured overnight with 100 or 500 μM spermine showed decreased cytotoxic activity against Daudi cells (91.5 ± 1.7 and 84.9 ± 3.0%, respectively (n = 6) compared to PBMC cultured without polyamines). In a group of 25 cancer patients, changes in blood spermine levels after surgery were negatively correlated with changes in LAK cytotoxicity after surgery (r = −0.510, P = 0.008: n = 25). Increased blood spermine levels may be an important factor in the suppression of anti-tumor immune cell function.     

Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observed.     

Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects

Some Bence Jones proteins (BJPs) can display catalytic activity. Although the catalytic activity of BJPs might be associated with the pathogenesis of disease, this relationship has not yet been established. We tested the effects of seven BJPs on LLC-PK1 cells to assess their pathogenicity. Two out of the seven BJPs showed cytotoxic activity, as assessed by microscopic analysis, the WST method and TUNEL staining. Moreover, the cytotoxic BJPs were excreted by patients who presented with renal impairment. The cytotoxic BJPs displayed 20- to 40-fold higher catalytic activities (kcat of 3.5-2.2 min(-1)) in hydrolyzing a chromogenic substrate compared to the other BJPs. By treating the cytotoxic BJPs with diisopropylfluorophosphate, they lost not only their catalytic activity, but also the cytotoxic effects. These results indicate a direct link between cytotoxicity and the catalytic activity of the BJPs. The catalytic activity of BJPs contributes to the pathogenesis, as well as to development, of symptoms of multiple myeloma. Inhibition of the catalytic activity of BJPs may form the basis of a novel treatment for multiple myeloma patients with renal dysfunction.     

Effects of cucurbitacins on cell morphology are associated with sensitization of renal carcinoma cells to TRAIL-induced apoptosis

Cucurbitacins B and D were among the compounds identified as sensitizers of cancer cells to TRAIL-mediated apoptosis in a high-throughput screen. Therefore a series of cucurbitacins was further investigated for TRAIL sensitization and possible mechanisms of action. A total of six cucurbitacins promoted TRAIL-induced apoptosis (B, I, E, C, D, and K) and one (P) was inactive. Sensitization of renal adenocarcinoma cells to TRAIL was apparent after as little as 1–4 h pretreatment and did not require continued presence of cucurbitacin. Active cucurbitacins induced caspase-8 activation only after subsequent TRAIL addition and caspase activation was required for apoptosis suggesting amplified proximal signaling from TRAIL death receptors. Cucurbitacin-sensitized TRAIL-induced cytotoxicity was inhibited by N-acetyl cysteine. Structure–activity relationship analysis in comparison to published studies suggests that TRAIL-sensitizing and general cytotoxic activities of cucurbitacins may be decoupled. Cucurbitacins are reported to be inhibitors of STAT3 activation. However, their TRAIL-sensitizing activity is STAT3-independent. Treatment of renal carcinoma cells with active cucurbitacins produced rapid and dramatic changes in cell morphology and cytoskeletal organization (also prevented by NAC). Therefore, cucurbitacins may be useful as tools for investigating the molecular mechanism(s) of action of TRAIL sensitizers, particularly with regard to temporal aspects of sensitization and modulation of TRAIL signaling by cell morphology, and could form the basis for future therapeutic development in combination with TRAIL death receptor agonists.     

Antimicrobial peptides derived from different animals: comparative studies of antimicrobial properties,cytotoxicity and mechanism of action

Animals posses a large variety of antimicrobial peptides (AMPs) that serve as effective components in innate host defenses against microbial infections. These antimicrobial peptides differ in amino acid composition, range of antimicrobial specificities, hemolysis, cytotoxicity and mechanisms of action. This study was designed to evaluate their therapeutic potential of the following six antimicrobial peptides initially found from animals: cecropin P1, indolicidin, LL-37, palustrin-OG1, LFP-20 and LFB-11. Our results indicated that cecropin P1 possessed the most desired biological activity, with fast and potent antimicrobial activity but only slight hemolytic or cytotoxic activity against human cells. Indolicidin was more effective against gram-positive bacteria but with higher hemolytic and cytotoxic activity on human peripheral blood mononuclear cell (PBMCs) (P < 0.05). Although LFP-20 and LFB-11 had moderate activity against tested strains and need 30 min to kill E. coli, they showed almost no hemolytic and cytotoxic activity towards PBMCs (P < 0.01). Indolicidin could form pores of well-defined structure in bacterial membranes whereas lysis of E. coli cells was observed after addition LFB-11 and LL-37 at 1 × MIC for 1 h. LL-37 treatment could lead to the leakage of entire bacterial cytoplasmic contents. The most obvious phenomenon was protuberant structures on the E. coli cell surface after incubation with LFP-20, cecropin P1 and palustrin-OG1. The results presented here illustrate that AMPs derived from different animals exhibited different antimicrobial characteristics. Because of their potent and broad-spectrum antimicrobial activity, low cytotoxicity towards normal cells, and the unique mechanism of action, these peptides may provide the impetus for the development of novel strategies for the prevention of bacterial infections in animals.     

Regulation of intracellular calcium release and PP1α in a mechanism for 4-hydroxytamoxifen-induced cytotoxicity

Treatment with tamoxifen, or its metabolite 4-hydroxytamoxifen (4OHT), has cytostatic and cytotoxic effects on breast cancer cells in vivo and in culture. Although the effectiveness of 4OHT as an anti-breast cancer agent is due to its action as an estrogen receptor-alpha (ERα) antagonist, evidences show that 4OHT is also cytotoxic for ERα-negative breast cancer cells and can be effective therapy against tumors that lack estrogen receptors. These findings underscore 4OHT signaling complexities and belie the most basic understandings of 4OHT action and resistance. Here, we have investigated the effects of 4OHT on Ca²⁺ homeostasis and cell death in breast cancer cells in culture. Measurement of Ca²⁺ signaling in breast cancer cells showed that 4OHT treatment altered Ca²⁺ homeostasis and was cytotoxic for both an ERα+ and an ERα- cell line, MCF-7 and MDA-MB-231, respectively. Further investigation lead us to the novel discovery that 4OHT-induced increase of ATP-dependent Ca²⁺ release from the endoplasmic reticulum correlated with 4OHT-induced upregulation of protein phosphatase 1α (PP1α) and the inositol 1,4,5-trisphosphate receptor (IP3R). Blocking 4OHT-induced PP1α upregulation by siRNA strategy reduced the effects of 4OHT on both Ca²⁺ signaling and cytotoxicity. Results from these investigations strongly suggest a role for PP1α upregulation in a mechanism for 4OHT-induced changes to Ca²⁺ signaling that ultimately contribute to the cytotoxic effects of 4OHT. Aliccia Bollig, Liping Xu- contributed equally to this work     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Lonely killers

The majority of the most effective monoclonal antibodies (mAbs) currently in the clinics bind to cancer or immune cells. Classic mechanisms of cell killing by therapeutic mAbs include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis by engagement of specific cell ligands. A few reports have described mAbs whose cytotoxic activity is Fc-independent and that do not induce the morphological and biochemical changes associated with the apoptosis-type of cell death. Even fewer works describe mAbs able to directly induce membrane lesions. Here, we discuss the available data on those molecules and their cell killing activity, with particular attention to the case of a mAb specific for the tumor-associated N-glycolyl (Neu5Gc)-GM3 ganglioside (GM3(Neu5Gc)). Some similarities are found in the cell death pathways triggered by these mAbs, but data are not abundant. We conclude that the usefulness of mAbs with a direct cytotoxic activity for immunotherapeutic strategies deserves deeper research.