Tumour necrosis factor induced an early release of superoxide and a late mitochondrial membrane depolarization in L929 cells. Increase in the production of superoxide is not sufficient to mimic the action of TNF.

Tumour necrosis factor alpha (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O2-*). In the present study, we attempted to examine the role of O2-* in the regulation of mitochondrial membrane potential (Delta(Psi)m) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O2-*. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O2-* and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the Delta(Psi)m. Reduction in the release of O2-* by rotenone (50 microM) or thenoyltrifluoroacetone (250 microM) suppressed both the TNF-mediated Delta(Psi)m depolarization and cyto c release. However, increase in the production of O2-* by antimycin A (25 microM) only slightly enhanced the TNF effect in altering the Delta(Psi)m and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in Delta(Psi)m nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O2-* was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.

The anti-tumour effect of Klebsiella pneumoniae capsular polysaccharides.

K24 capsular polysaccharide (K24-CPS), with a known structure of a repeating unit, was isolated from the capsule of Klebsiella pneumoniae serotype K24. The polysaccharide was found to suppress the proliferation of Ehrlich ascites tumour (EAT) cells in vitro, but did not alter the cell cycle distribution of cells. K24-CPS treatment reduced the tyrosine phosphorylation of some proteins in EAT cells. Furthermore, the treatment also decreased the expression of c-JUN, but had no effect on the levels of c-FOS and c-MYC. It is speculated that the growth suppression effect of K24-CPS may be related to its effect in down-regulating c-JUN expression.

Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells.

Green tea catechins (GTCs) including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) were shown to suppress cell growth and induce apoptosis in various cell systems in addition to their chemo-preventive effect. In this study, except EC which was inactive, green tea extract (TE) and other 3 GTCs were found to suppress the growth and induce apoptosis in human prostate cancer DU145 cells largely through an increase in reactive oxygen species formation and mitochondrial depolarization. The conclusion was supported by the fact that the profiles for different GTCs in growth suppression, apoptosis induction, ROS formation and mitochondrial depolarization are in a similar order, i.e. ECG > EGCG > EGC > EC. Although the molecular mechanisms are still not clear, apoptosis induced by GTCs is not related to the members of BCL-2 family as EGCG did not alter the expression of BCL-2, BCL-X(L) and BAD in DU145 cells.

Reversal of TNF-alpha resistance by hyperthermia: role of mitochondria.

The aim of this study is to examine the effect of hyperthermia on tumour necrosis factor-alpha (TNF-alpha) resistance in L929-11E cells. L929-11E is a TNF-alpha resistant variant derived from L929 cells, a commonly used model for TNF-alpha study. Based on the results from flow cytometry and Western blotting, hyperthermia (43 degrees C, 3 h) was found to induce apoptosis, mitochondrial potential (delta psi(m)) depolarization and release of cytochrome c in L929-11E cells. Similar responses were found in L929 cells when treated with TNF-alpha. Heating at 43 degrees C for 1 h did not significantly damage the mitochondria of L929-11E cells but partially reversed their resistance to TNF-alpha. When L929-11E cells were sequentially treated with heating (43 degrees C, 1 h) and TNF-alpha, a more severe damage in mitochondria was observed. Taken together, our results indicate (1) hyperthermia induced apoptosis in L929-11E cells via mitochondrial damages in a way very similar to the action of TNF-alpha in L929 cells, (2) hyperthermia could be used to overcome TNF-alpha resistance by altering mitochondrial activities and (3) L929-11E and its parental cells provide a useful model in elucidating the signalling linkage between TNF-alpha receptor and mitochondria.

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-alpha-mediated apoptosis.

Tumour necrosis factor-alpha (TNF-alpha) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-alpha increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-alpha induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-alpha (50 ng/ml), a slow rise in intracellular free Ca2+ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3 h after the addition of TNF-alpha) were observed in fluo-3 and fura-red or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-alpha in the presence of BAPTA/AM, an intracellular Ca2+ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-alpha-mediated Ca2+ rise. Moreover, when cells were exposed to TNF-alpha with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca2+ are two cross-talk messengers important in TNF-alpha-mediated apoptosis.

The immunostimulating activities of anti-tumor polysaccharide from K1 capsular (polysaccharide) antigen isolated from Klebsiella pneumoniae.

We have previously shown that K1 capsular polysaccharide antigen (K1CPS) of Klebsiella exhibits anti-tumor activities. In the present study, we examined the effect of K1CPS on cytotoxic effector cells. We found that K1CPS could activate many cytotoxic effector cells including alloreactive cytotoxic T cells and tumor-infiltrating lymphocytes (TILs). Moreover, K1CPS could increase the anti-tumor activity of lymphokine-activated killer (LAK) cells, both in vitro and in vivo. The i.p. injection of K1CPS in low dose could enhance the LAK cytotoxicity and the effect was further potentiated by coculture of LAK cells with K1CPS and low concentration of murine rIL-2 in vitro. The phenotypic characterization revealed that K1CPS might contribute to the increase in CD3+ LAK cell subpopulation by its in vivo priming effect. In addition, the K1CPS-treated LAK cells were able to inhibit the growth of WEHI-164 tumor cells in vivo in Winn-type inhibition assay. Subcutaneous (s.c.) and intraperitoneal (i.p.) adoptive infusion of LAK cells (splenocytes from K1CPS-treated WEHI-164-bearing mice cultured with K1CPS-plus-rIL-2) into WEHI-164 sarcoma-bearing mice could slightly cause regression in terms of tumor diameter, and more significantly in sarcoma weight.

Hyperthermia and tumour necrosis factor-alpha induced apoptosis via mitochondrial damage.

Hyperthermia is a potential anti-cancer regimen but the mode of action is far from clear. Based on the flow cytometric analysis with FITC-annexin V and propidium iodide, apoptosis was found to be the major form of cell death after the treatment with hyperthermia (43 degrees C, 3 h) and/or recombinant murine tumour necrosis factor-alpha (TNF-alpha, 50 ng/ml) in L929 cells. Since mitochondria are thought to play a key role in apoptosis, experiments were done to assess their role in the hyperthermia-mediated apoptosis. Our results indicate that hyperthermia was able to depolarize the mitochondrial membrane potential (delta psi m) and release cytochrome c to the cytoplasm, in a way very similar to the action of TNF-alpha. With the use of cyclosporin A to inhibit the delta psi m dissipation, the cytotoxicity mediated by hyperthermia or TNF-alpha was suppressed. Taken together, our results indicate that hyperthermia and TNF-alpha can induce apoptosis in L929 cells and the mitochondrial dysfunction plays a key role in the cell death process.

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c.

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypodiploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by alpha-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha.

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.

Enhanced cytotoxicity and suppression of glucose transport rate by combined treatment of recombinant human tumour necrosis factor-alpha and hyperthermia on L929 cells.

Combined treatment with human recombinant TNF-alpha (rhTNF-alpha) and hyperthermia at 43 degrees C arrested the growth of mouse fibrosarcoma L929 cells in vitro. The cytotoxic effect was enhanced in combined treatment compared with that following administration of rhTNF-alpha or hyperthermia alone. When the cells were subjected to hyperthermia at 43 degrees C for 3 hours and then incubated with 0.4 ng/ml rhTNF-alpha at 37 degrees C for 24 hours, a statistically significant 65% decrease in the rate of cellular glucose uptake was observed. This suppressive effect was synergistic in terms of effect achieved by rhTNF-alpha or hyperthermia individually. Since the growth of tumour cells depends mainly on catabolism of glucose, our findings indicate that one manner by which combined rhTNF-alpha and hyperthermia treatment inhibits L929 cell growth may be by reducing the supply of glucose to the cells.

Inhibition of glucose transporter gene expression by antisense nucleic acids in HL-60 leukemia cells.

Glucose is the basic source of energy for mammalian cells. The energy-independent transport of glucose down its concentration gradient is mediated by the facilitative glucose transporter family (GLUT). It has long been recognised that glucose transporter genes are overexpressed in many human cancer cells, to help provide extra energy for the rapid growth of cancer cells. In the present study, antisense oligonucleotides and plasmid-derived antisense RNA against GLUT-1 gene were synthesized and transfected into human leukemia HL-60 cells to investigate the effect of these antisense nucleic acids on tumour growth. Our results show that antisense nucleic acids inhibited the proliferation of HL-60 cells by 50-60% and the mRNA expression of GLUT-1 gene was suppressed as detected by Northern hybridization.

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development.

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but not GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs.

The isolation and characterization of an immunomodulatory and anti-tumor polysaccharide preparation from Flammulina velutipes.

Alkaline-soluble antitumor polysaccharide was prepared from the cell wall of the mushroom Flammulina velutipes. The backbones) of the polysaccharide is mainly composed of beta-(1-->3)-D-linked glucose and its molecular weight was estimated to be about 200 kD. The polysaccharide was found to be non-toxic by brine shrimp assay. When injected into mice intraperitoneally, the polysaccharide triggered proliferation of splenic lymphocytes and also vascular dilation and hemorrhage (VDH) response. The polysaccharide exhibited potent anti-tumor activity against sarcoma SC-180 in vivo but not in vitro.

Slow increase in intranuclear and cytosolic free calcium concentrations in L929 cells is important in tumour necrosis factor-alpha-mediated cell death.

The potential involvement of cellular calcium in the signalling pathway of tumour necrosis factor-alpha (TNF) was assessed in L929 cells using 45Ca2+ and confocal laser scanning microscopy with fluorescence calcium indicators. Our data indicate that the effect of TNF on intracellular Ca2+ mobilization is a slow process with no discernible increase in the cytosolic free Ca2+ concentration ([Ca2+]c) and intranuclear Ca2+ level ([Ca2+]n) within the 1st min of TNF (25 ng/ml) administration. However, prolonged exposure (2 h) of L929 cells to TNF brought about pronounced increase in cytosolic and intranuclear [Ca2+] even in the absence of external Ca2+. The increase in intracellular [Ca2+] was more apparent when cells were treated with thapsigargin, an inhibitor of microsomal Ca2+-ATPase. Interestingly, most of the Ca2+ released was around and confined to the nucleus. Following the pretreatment of cells with thapsigargin, a synergistic killing effect was obtained when cells were cultured with TNF. The use of 45Ca2+ also revealed that TNF enhanced the 45Ca2+ uptake in a time-dependent manner. Calcium channel blockers, verapamil and diltiazem, could alleviate both the TNF-mediated 45Ca2+-uptake and killing activity. Our results therefore suggest that an increase in cellular Ca2+ is a crucial factor in the TNF cytotoxicity.

Concanavalin A-induced apoptosis in murine macrophages through a Ca(2+)- independent pathway.

Concanavalin A (ConA), normally a mitogen of T lymphocytes, was found to induce apoptosis or programmed cell death in murine peritoneal macrophages. The following observations support this assertion: 1) incubation of peritoneal macrophages or cultured PU5-1.8 macrophage cells with ConA caused a dose- and time-dependent reduction of mitochondrial dehy-drogenase activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 2) treatment of cells with ConA induced formation of apoptotic bodies as seen under the confocal laser scanning microscope, 3) challenge of cells with ConA produced a considerable amount of cell debris with DNA content next to G0 phase as revealed by flow cytometry and 4) ConA was able to elicit DNA fragmentation in these cells. The involvement of Ca(2+) in mediating the apoptosis was studied in single cells by confocal laser scanning microscope using the Ca(2+) fluorescence dye, fluo-3. Our results show that ConA induced an immediate rise of intracellular free Ca(2+) concentration as well as opening of Ca(2+) channels on cell surface. But when the cells were treated with 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM), a Ca(2+) chelator, to buffer the rise of internal Ca(2+), ConA still caused DNA fragmentation. Furthermore, injection of Ca(2+) into the cell with ionomycin had no stimulatory effect on DNA fragmentation. These results suggest that Ca(2+) changes induced by ConA are not a prerequisite for apoptosis in macrophages.

K1 and K3 capsular antigens of Klebsiella induce tumor necrosis factor activities.

Capsular polysaccharide antigens isolated from Klebsiella pneumoniae sero-type 1 (K1) and sero-type 3 (K3) could induce tumor necrosis factor-alpha in ICR mice. K1 and K3 capsular antigens were found to be non-toxic by brine shrimp bioassay. When injected into Ehrlich ascites tumor-bearing mice, both K1 and K3 capsular antigens exhibited significant suppression in the growth of tumor cells. The significance of these observations is discussed.

Effect of methotrexate on the intracellular phosphoribosyl pyrophosphate level and glucose transport of Ehrlich ascites tumor cells in vitro.

Methotrexate (MTX) suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. The intracellular level of phosphoribosyl 5-pyrophosphate (PRPP) of EAT cells increased in a dose-dependent manner in response to MTX treatment. At the same time, the rate of glucose transport was lowered. Hypoxanthine reversed both these effects of MTX and partially rescued EAT cell growth. Under all conditions tested, changes in rate of glucose transport were shown to be the result of alterations in the number of glucose transporter (Vmax) rather than ligand affinity (Km). The dual action of MTX as a chemotherapeutic agent is discussed in this light.

Effect of hyperthermia on intracellular pH in human U-87 MG glioblastoma cells.

The effect of hyperthermia at 43 degrees C on intracellular pH (pHi) in human U-87 MG glioblastoma cells was studied by using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-pentaacetoxymethyl ester. The presence of Na+/H+ antiporter activity in the cells were demonstrated by the Na(+)-dependent increase in intracellular pH (pHi) after cellular acidification in the absence of HCO3-. Hyperthermia at 43 degrees C caused significant decrease in pHi. The acidification was readily reversible by cooling the cells back down to 37 degrees C. The pHi change was inhibited by the addition of 1 mM amiloride in the incubation medium. Amiloride and hyperthermia exhibited a synergistic effect in suppressing thymidine incorporation into the cells.

Endogenous production of tumor necrosis factor alpha in combination with hyperthermia for the treatment of mice bearing Ehrlich ascites tumor.

Multiple hyperthermia was found to exert an additive antitumor effect when combined with the in vivo production of tumor necrosis factor alpha (TNF-alpha) in mice bearing Ehrlich ascites tumor (EAT). TNF-alpha was produced in EAT-bearing mice by priming the animals with zymosan and subsequently challenging with lipopolysaccharide (LPS). Mice were pretreated with sulindac and D-mannoheptulose to alleviate the toxic side effects of LPS. While the ability of these tumor-bearing mice to produce TNF-alpha remained unchanged under hyperthermia, the EAT cell number was suppressed in the combined-treatment group compared with groups treated with TNF-alpha or hyperthermia alone. In the same comparison, the life span of EAT-bearing mice in the combined-treatment group was prolonged.

Activation of the anti-tumor effector cells by Radix bupleuri.

Radix bupleuri, the root of Bupleuri spp., Chinese medicinal herbs used for the treatment of influenza, malaria and menstrual disorders, were extracted with hot water and separated into five different fractions (RB, RBI, RBII, RBIII and RBIV) by stepwise alcohol precipitation. One of these fractions, RBI, was then fractionated into RBIa and RBIb by gel filtration using G-100 Sephadex. These two fractions were further purified into RBIai, RBIaii and RBIbi, RBIbii fractions respectively by ion-exchange chromatography using DEAE-Sephadex. Each of these fractions is a heteropolymer consisting mainly of carbohydrate and varying proportions of protein and uronic acid. RBIaii was found to show strong anti-tumor activities in sarcoma-bearing mice. Mechanistic studies showed that RBIaii exhibited a potent activating effect on the cytotoxic activity of macrophages, NK and LAK cells against tumor cells. In addition, RBIaii could increase the number of tumor infiltrating lymphocytes (TILs) in the tumor site of WEHI-164-bearing mice. Furthermore, RBIaii could induce the release of interferon-gamma by lymphocytes in vitro.