Tumoricidal activity of monocyte-derived dendritic cells: evidence for a caspase-8-dependent, Fas-associated death domain-independent mechanism

Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Furthermore, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer.     

Synthesis and cytotoxic activity of diosgenyl saponin analogues

Diosgenyl saponins are steroidal glycosides that are often found as major components in many traditional oriental medicines. Recently, a number of naturally occurring diosgenyl saponins have been shown to exert cytotoxic activity against several strains of human cancer cells. Use of these saponin compounds for cancer treatment is hampered due to the lack of understanding of their action mechanism as well as limited access to such structurally complicated molecules. In the present paper, we have prepared a group of diosgenyl saponin analogues which contain a beta-D-2-amino-2-deoxy-glucopyranose residue having different substituents at the amino group. Moderate cytotoxic activity is found for most analogues against neuroblastoma (SK-N-SH) cells, breast cancer (MCF-7) cells, and cervical cancer (HeLa) cells. The analogue 13 that contains an alpha-lipoic acid residue exhibits the highest potency against all three cancer cell lines with IC(50) ranging from 4.8 microM in SK-N-SH cells to 7.3 microM in HeLa cells. Preliminary mechanistic investigation with one saponin analogue (10) shows that the compound induces cell cycle arrest at G(1) phase in SK-N-SH cells, but the same compound induces cell cycle arrest at G(2) phase in MCF-7 cells. This result suggests that the cytotoxic activity of these saponin analogues may involve different action mechanisms in cell lines derived from different cancer sites.     

alpha-tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilisation

Alpha-Tocopheryl succinate (alpha-TOS), but not a-tocopherol, triggered apoptosis in Jurkat T cells. Apoptosis was induced by alpha-TOS in a time- and concentration-dependent mode, and signs of apoptosis were visible at concentrations of alpha-TOS as low as 30 microM, and within 3-5 h after addition of the ester. Employing a specific fluorogenic substrate, caspase-3 was found to be activated rapidly in response to alpha-TOS at 50 microM. We also found that Jurkat T cells challenged with alpha-TOS, when exposed to the lysosomotropic weak base acridine orange, showed decreased lysosomal uptake of the dye. This is suggestive of the involvement of lysosomal destabilisation in apoptosis of the cells. Apoptosis of Jurkat T cells induced with alpha-TOS also involved a drop in the mitochondrial membrane potential, although this phenomenon occurred after the initiation of lysosomal rupture. All apoptotic features observed with alpha-TOS were very similar to those found when cross-linking of the Fas receptor triggered apoptosis. These findings are consistent with the recent idea that vitamin E can contribute to elimination of malignant cells by the induction of apoptosis, and can be of (patho)physiological significance.     

The antioxidant 4b,5,9b,10-Tetrahydroindeno[1,2-b]indole inhibits apoptosis by preventing caspase activation following mitochondrial depolarization.

Oxidative stress appears to have a central role in the induction of apoptosis following the exposure of cells to a range of cytotoxic insults. The modulation of apoptosis by a diverse range of antioxidants has been reported in many systems. We demonstrate, for the first time, the anti-apoptotic properties of the antioxidant, 4b, 5,9b,10-tetrahydroindeno[1,2-b]indole (THII), in Jurkat T cells subjected to a number of cytotoxic insults. THII was found to inhibit the morphological features of apoptosis in cells treated with the cytotoxic agents camptothecin, actinomycin D and ultraviolet (UV) irradiation. However, THII was unable to inhibit apoptosis induced by anti-Fas IgM. Peroxide and superoxide anion production following UV treatment was monitored, and THII was found to only partially inhibit superoxide anion production. THII was unable to inhibit mitochondrial depolarization in UV, Camptothecin or anti-Fas-treated cells. Further downstream, THII exibited strong inhibition of caspase-3 activation in UV, but not in anti-Fas-treated cells. These results suggest that THII may exert its effects downstream of mitochondrial depolarization, but upstream of caspase-3 activation.     

Human monocyte activation by cleaved form of alpha-1-antitrypsin involvement of the phagocytic pathway.

Production of alpha-1-antitrypsin (AAT) by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases, of four- to eightfold, in numbers of macrophages and levels of AAT and its cleavage fragments have been found in various inflammatory loci. We have found that the C-terminal peptide (C-36) of AAT, produced by specific proteinase cleavage when added in its fibrillar form at concentrations >/=5 microM to monocytes in culture for 24 h, significantly increases low density lipoprotein (LDL) binding and uptake, up-regulates levels of LDL receptors and also induces proinflammatory cytokine (interleukin-1, interleukin-6 and tumour necrosis factor alpha) production and glutathione reductase activity. Because it is known that various cells selectively internalize surface receptors and their ligands through receptor-mediated endocytosis via clathrin-coated pits, we tested whether antibodies raised against the clathrin heavy chain would block the effects of the fibrillar form of C-36 on human monocytes in culture. Addition of excess anti-(clathrin HC) with 10 microM fibrillar C-36 diminished the stimulatory effects of the latter on LDL binding, uptake and LDL receptor levels. In contrast, however, in the presence of anti-(clathrin HC), the potentially cytotoxic effects of fibrils, such as induction of cytokines, free radicals and cytosolic activity of cathepsin D, were much greater than those observed when cells were treated with fibrils alone. These results suggest that endocytosis is the pathway by which C-36 fibrils upregulate LDL receptors, and may be the natural mechanism for fibril clearance. We infer that human monocytes clear C-36 fibrils by a clathrin-dependent pathway, presumably endocytotic, and that loss of this pathway amplifies the cytotoxic effects of the fibrils by increasing their availability to other specific or nonspecific sites through which they exert their cytotoxic effects.     

Dinitrosyl-iron triggers apoptosis in Jurkat cells despite overexpression of Bcl-2

Cells expressing the cytokine-inducible NO synthase are known to trigger apoptosis in neighboring cells. Paramagnetic dinitrosyl nonheme iron complexes (DNIC) were found in tumor tissue about 40 years ago; however, the role of these NO(+)-bearing species is not completely understood. In the human Jurkat leukemia cell line, the application of the model complex DNIC-thiosulfate (50-200 microM) induced apoptosis (defined by phosphatidylserine externalization) in a concentration- and time-dependent manner. In Jurkat cells, the pan-caspase inhibitor, zVADfmk (50 microM), and/or stable transfection of antiapoptotic protein, Bcl-2, was unable to afford protection against DNIC-induced apoptosis. The membrane-impermeable metal chelator, N-methyl-D-glucamine dithiocarbamate (MGD; 200 microM), in the presence of DNIC significantly increased apoptosis, but had no effect on its own. Electron paramagnetic resonance studies showed that MGD led to rapid transformation of the extracellular DNIC into the stable impermeable NO-Fe-MGD complex and to a burst-type release of nitrosonium (NO(+)) equivalents in the extracellular space. These results suggest that in Jurkat cells, DNIC-thiosulfate induces Bcl-2- and caspase-independent apoptosis, which is probably secondary to local nitrosative stress at the cell surface. We hypothesize that the local release of nonheme Fe-NO species by activated macrophages may play a role in the killing of malignant cells that have high Bcl-2 levels.     

Synthesis of OSW saponin analogs with modified sugar residues and their antiproliferative activities

Eight monosaccharide analogs of the potent antitumor OSW saponins (2-9) were synthesized. One analog, 2-O-acetyl-alpha-l-arabinopyranoside 3, showed antiproliferative activity against the Jurkat cells (IC(50)=0.078microM) comparable to that of the disaccharide derivative (1).     

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors.

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.     

Monochloramine inhibits etoposide-induced apoptosis with an increase in DNA aberration

Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils, and it affects apoptosis signaling. We studied the effects of NH(2)Cl on the cell death induced by etoposide, a widely used anticancer agent that is directed to DNA topoisomerase II. Jurkat T cells, a human acute T cell leukemia cell line, were pretreated with 70 microM of NH(2)Cl for 10 min. After 24 h, 5-30 microM of etoposide was added to the NH(2)Cl pretreated and control cells, and their apoptosis, caspase activity, cell morphology, and cellular DNA contents were measured. NH(2)Cl pretreatment significantly inhibited apoptosis and caspase activation induced by etoposide or camptothecin, a DNA topoisomerase I poison, but not by staurosporine or Fas stimulation. The apoptosis inhibition actually resulted in the proliferation of the survived cells and, notably, the survived cells showed more aberrant morphology, such as variation in nuclear size, nuclear fragments, and multinucleated cells. DNA content analysis of the survived cells showed an increase in aneuploid nuclei. Cell cycle analysis after 24 h of NH(2)Cl treatment showed a significant decrease in S phase cells with a concurrent increase in G(0)/G(1) phase cells, which suggested that NH(2)Cl induced G(1) arrest. Using synchronized Jurkat cells, etoposide and camptothecin were found to be particularly cytotoxic to S phase cells, whereas staurosporine and Fas stimulation were not. Thus NH(2)Cl-induced G(1) arrest was a likely cause of the observed resistance to etoposide. These observations suggested that inflammation-derived oxidants may make the tumor cells more resistant to etoposide and increase the risk of tumor progression and the development of secondary tumors by increasing the survival of DNA damage-bearing cells.     

Haemolytic activity,cytotoxicity and membrane cell permeabilization of semi-synthetic and natural lupane- and oleanane-type saponins

The haemolysis of red blood cells inducing toxicity in most animals including humans is a major drawback for the clinical development of saponins as antitumour agents. In this study, the haemolytic and cytotoxic activities as well as the membrane cell permeabilization property of a library of 31 semi-synthetic and natural lupane- and oleanane-type saponins were evaluated and the structure–activity relationships were established. It was shown that lupane-type saponins do not exhibit any haemolytic activity and membrane cell permeabilization property at the maximum concentration tested (100 μM) independently of the nature of the sugar moieties. While oleanane-type saponins such as β-hederin (25) and hederacolchiside A₁ (27) cause the death of cancer cell lines by permeabilizing the cellular membranes, lupane-type saponins seem to proceed via another mechanism, which could be related to the induction of apoptosis. Altogether, the results indicate that the cytotoxic lupane-type glycosides 10 and 22 bearing an α-l-rhamnopyranose moiety at the C-3 position represent promising antitumour agents for further studies on tumour-bearing mice since they are devoid of toxicity associated with the haemolysis of red blood cells.     

Naturally occurring triterpenoid saponins

Naturally occurring new triterpenoid saponins reported from mid-1996 to March, 2007 are reviewed including their physical constants and plant sources, and are compiled in Table 1. New saponins are arranged in Table 1 on the basis of the skeletal structures of their aglycones, e.g., oleanane type, ursane type, lupane type, hopane type, taraxastane type, cycloartane type, lanostane type, tirucallane type, dammarane type, cucurbitane type, and holostane type. The known triterpenoid saponins and prosapogenins of the new saponins, the biological and pharmacological activities of which were published during 1996-2007, are also reviewed together with their plant sources listed in Table 2 according to the skeletal structures of their aglycones in the same fashion as in Table 1. The plant and animal sources of both new and known bioactive triterpenoid saponins are collected in Table 3 in alphabetical order. The biological and pharmacological activities such as antiallergic, antiatherosclerosis and antiplatelet, antibacterial, anticomplementary, antidiabetic, contraceptive, antifungal, anti-inflammatory, antileishmanial, antimalarial/antiplasmodial, anti-obesity, anti-proliferative, antipsoriatic, antispasmodic, antisweet, antiviral, cytotoxic/antitumor, detoxication, gastroprotective, haemolytic, hepatoprotective, immunomodulatory, anti-enzyme, anti-osteoporotic, insecticidal, insulin-like, membrane-porosity, molluscicidal, neuropharmacological, anti-endothelial dysfunction, snake venom antidote, and sweet activities of these saponins or derived prosapogenins are discussed briefly after Table 3.     

Five new triterpenoid saponins from the Roots of Camellia oleifera C. Abel with cytotoxic activities

Five new triterpenoid saponins, oleiferosides P–T (1–5) were isolated from the EtOH extract of the roots of Camellia oleifera C. Abel. The structures of saponins 1–5 were elucidated on the basis of integrated spectroscopic techniques. All the compounds were characterized to be oleanane-type saponins with sugar moieties linked to the C-3 of the aglycone. By using the MTT assay, an in vitro analysis of the cytotoxic activities of these saponins on the human tumor cell lines (lung adenocarcinoma A549 cells, hepatic carcinoma SMMC-7721 cells and breast cancer MCF-7 cells). Among them, compound 4 showed a certain cytotoxic activity against all the tested cell lines.     

Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells

We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.     

Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines.

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.     

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity.     

The Ca<Superscript>2+</Superscript> channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells

Flunarizine is a Ca²⁺ channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.     

Structure activity relationships of antiproliferative rocaglamide derivatives from Aglaia species (Meliaceae)

Eleven rocaglamide derivatives (cyclopentatetrahydrobenzofurans) and one structurally related aglain congener all isolated from different Aglaia species (Meliaceae) were tested for growth inhibiting properties using the human cancer cell lines MONO-MAC-6 and MEL-JUSO. Proliferation of both cell lines was efficiently inhibited in a dose and compound dependent manner. Applying MTT-Assay, the IC50 of the most active compound didesmethyl-rocaglamide (1) was observed at 0.002 and 0.006 micrograms/ml (0.004 and 0.013 microM) depending on the cell line investigated. Bulky aminoacyl substituents at C-2, acetylation of the OH substituent at C-1 or insertion of a OH or OMe substituent at C-3 of the rocaglamide skeleton all diminished the activity of the compounds investigated. The aglain derivative 12 was inactive up to a concentration of 3 micrograms/ml (4.6 microM). This loss of activity is assumed to be mainly due to the presence of a pyran ring in the aglains vs. a furan ring as found in rocaglamide derivatives. Rocaglamide derivatives may act primarily by inhibition of cell proliferation as evidenced by the absence of a significant cytotoxic effect in long-term cultures of MONO-MAC-6 cells treated with high doses of didesmethylrocaglamide. Our data suggest that rocaglamide derivatives could exert a potential role in the treatment of malignant diseases and are worth to be investigated in further studies of experimental medicine and pharmacology.     

Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process

Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 microM) of arachidonic acid and oleic acid had little effect. To explore the mechanism of inhibition, we used several measures of apoptosis to determine whether this process was involved in DHA-induced cell death in Jurkat cells. Caspase-3, an important cytosolic downstream regulator of apoptosis, is activated by death signals through proteolytic cleavage. Incubation of Jurkat cells with 60 and 90 microM DHA caused proteolysis of caspase-3 within 48 and 24 h, respectively. DHA treatment also caused the degradation of poly-ADP-ribose polymerase and DNA fragmentation as assayed by flow cytometric TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. These results indicate that DHA induces apoptosis in Jurkat leukemic cells. DHA-induced apoptosis was effectively inhibited by tautomycin and cypermethrin at concentrations that affect protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) activities, respectively, implying a role for these phosphatases in the apoptotic pathway. Okadaic acid, an inhibitor of protein phosphatase 2A, had no effect on DHA-induced apoptosis. These results suggest that one mechanism through which DHA may control cancer cell growth is through apoptosis involving PP1/PP2B protein phosphatase activities.     

Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells

The validated therapeutic effects of dihydroartemisinin (DHA) in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line) were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF) due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease.