Synergic carcinostatic effects of ascorbic acid and hyperthermia on Ehrlich ascites tumor cell.

AIM: In this study, we evaluated the carcinostatic effects of combined ascorbic acid (AsA) and a capacitive-resistive electric transfer (CRet) hyperthermic apparatus-induced hyperthermic treatment on Ehrlich ascites tumor (EAT) cells. MATERIALS AND METHODS: EAT cells were exposed to various AsA (0-10 mM) concentrations for 1 h; they subsequently underwent CRet treatment for 15 min at 42 °C. Cell viability was assessed by the WST-8 assay 24 h after the combined treatment. Reactive oxygen species involvement was evaluated using catalase and tempol; caspase-3/7 activation was determined by their fluorescent substrates; cell proliferation were estimated by time-lapse observation. The effect on the cell cycle was analyzed by flow cytometry. RESULTS: Combined AsA and CRet treatment synergistically suppressed cell viability compared with either treatment alone, and these synergistically carcinostatic effects were evident even at noncytotoxic concentrations of AsA alone (≤ 2 mM). The carcinostatic effects of combined AsA and CRet treatment were attenuated in a dose-dependent manner by catalase addition, but not by the superoxide anion radical scavenger tempol. Time-lapse observation revealed that combined AsA and CRet treatment activated caspase-3/7 at 10-24 h after treatment, accompanied by significant cell growth suppression. Cell cycle analysis revealed that the rate of sub-G1-phase (apoptotic) cells was drastically increased at 12 h and 24 h, and that the G2/M-phase cells gradually increased at 6-24 h after treatment. CONCLUSION: These results indicate that combined AsA and CRet treatment synergistically inhibits EAT cell growth through G2/M arrest and apoptosis induction via H2O2 generation at lower AsA concentrations; this carcinostatic effect cannot be exerted by AsA alone.

Usefulness of measuring reticulocyte hemoglobin equivalent in the management of haemodialysis patients with iron deficiency.

The evaluation of iron status in dialysis patients provides information essential to the planning of adequate recombinant human erythropoietin treatment. The cellular iron status of the patients can be determined from the recently available measurement of reticulocyte hemoglobin equivalent (RET-He). RET-He is measured on the basis of automated fluorescent flow cytometry which in the reticulocyte channel, using a polymethine dye, also measures the mean value of the forward light scatter intensity of mature red blood cells and reticulocytes. These values equate with reticulocyte hemoglobin content. In this study, to clarify the accuracy of RET-He in diagnosing iron deficiency in dialysis patients, we initially compared RET-He with such iron parameters as serum ferritin levels, transferrin saturation and content of reticulocyte hemoglobin (CHr) which has been established as indicators of functional iron deficiency. Secondly, we investigated the changes in RET-He during iron supplementation for iron-deficient patients to determine whether this marker is a prospective and reliable indicator of iron sufficiency. The participants in this study were 217 haemodialysis patients. Iron deficiency was defined as havsing a transferrin saturation (TSAT) < 20% or serum ferritin < 100 ng/ml. Conventional parameters of red blood cells and RET-He were measured by on a XE-2100 automated blood cell counter (Sysmex). CHr was measured on an ADVIA120 autoanalyser (Siemens). RET-He mean value was 32.4 pg and good correlation (r = 0.858) between RET-He and CHr is obtained in dialysis patients. Receiver operating characteristic curve analysis revealed, values of the area was 0.776 and at a cutoff value of 33.0 pg, a sensitivity of 74.3% and a specificity of 64.9%, were achieved. Iron supplements given to the patients with low TSAT or ferritin, RET-He responded within 2 weeks, and this seemed to be a potential advantage of using RET-He in the estimation of iron status. RET-He is a new parameter, equivalent value to CHr, and is easily measurable on the widely spread and popular blood cell counter and is a sensitive and specific marker of iron status in dialysis patients.

Biological safety of liposome-fullerene consisting of hydrogenated lecithin, glycine soja sterols, and fullerene-C60 upon photocytotoxicity and bacterial reverse mutagenicity.

Various water-soluble derivatives of fullerene-C60 (C60) have been developed as detoxifiers for reactive oxygen species (ROS), whereas C60 incorporated in liposome (Lpsm) has not been reported yet. We prepared the liposome-fullerene (0.2% aqueous phase, Lpsm-Flln) which was composed of hydrogenated lecithin, glycine soja (soybean) sterols, and C60 in the weight ratio of 89.7:10:0.3, then examined the photocytotoxicity and bacterial reverse mutagenicity, as comparing with the Lpsm containing no C60. Photocytoxicity of Lpsm-Flln or Lpsm was examined using Balb/3T3 fibroblastic cells at graded doses of 0.49-1000 microg/mL under the condition of UVA- or sham-irradiation. The cells were irradiated with UVA (5 J/cm2, 320-400 nm, lambda max = 360 nm) at room temperature for 50 min. The resultant cell viability (% of control) did not decrease dose-dependently to 50% or less regardless of the UVA-irradiation. These results show that Lpsm-Flln or Lpsm does not possess photocytotoxicity to Balb/3T3 fibroblasts, and Lpsm-Flln may not exert a UVA-catalytic ROS-increasing action. A possibility for the reverse mutation by Lpsm-Flln or Lpsm was examined on four histidine-demanding strains of Salmonella typhimurium and a tryptophan-demanding strain of Escherichia coli. As for the dosages of Lpsm-Flln or Lpsm (313-5000 microg/plate), the dose-dependency of the number of reverse mutation colonies of each strain did not show a twice or more difference versus the negative control regardless of the metabolic activation, and, in contrast, marked differences for five positive controls (sodium azide, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine, and 2-aminoanthracene). The growth inhibition of bacterial strains and the deposition of Lpsm-Flln or Lpsm were not found. As a result, the bacterial reverse mutagenicity of Lpsm-Flln or Lpsm was judged to be negative under the conditions of this test. Thus, Lpsm-Flln and Lpsm may not give any significant biological toxic effects, such as photocytotoxicity and bacterial reverse mutagenicity.

Promotive effects of hyperthermia on the cytostatic activity to Ehrlich ascites tumor cells by diverse delta-alkyllactones.

AIM: To evaluate promotive effects of hyperthermia on antitumor activity of new delta-alkyllactones (DALs) of low molecular weight (184-254 Da), chemically synthesized, which are different from natural macrocyclic lactones of high molecular weight (348-439 Da), such as camptothecin and sultriecin. METHODS: A suspension of Ehrlich ascites tumor (EAT) cells was mixed with a DAL in a glass tube, heated at 37 or 42 degrees C for 30 min in a water bath, and cultured at 37 degrees C for 20 or 72 h. Cell viability was measured by the mitochondrial dehydrogenase- based WST-1 assay. DALs incorporated into EAT cells was extracted and measured by gas-liquid chromatography. RESULTS: The reduction of cell viability by DALs was markedly enhanced upon the treatment at 42 degrees C compared to that at 37 degrees C. At 37 degrees C, delta-hexadecalactone (DH16:0) and delta-tetradecalactone (DTe14:0) displayed cytostatic activity (at 100 microM survival level: 20.7%, 66.1%; at 50 microM--41.2%, 82.4%, respectively). Their activity was more marked at 42 degrees C (at 100 microM 10.6%, 27.6%; at 50 microM 30.6, 37.5%, ibid). The other DALs, delta-undecalactone (DU11:0), delta-dodecalactone (DD12:0), and delta-tridecanolactone (DTr13:0) were almost ineffective. Evaluation of survival rate in the cells treated for 30 min by DALs with the next culturing of EAT cells for 72 h resulted in the enhanced carcinostatic activity of DH16:0 and DTe14:0 even at concentrations as low as 25 microM at either 37 degrees C (18.5%, 78.5%, ibid) or 42 degrees C (5.0%, 42.0%, ibid), but the others exhibited slight activity or none. DH16:0 was effective at either 37 degrees C (36.0%) or 42 degrees C (23.0%) even at a lower dose of 10 microM. At the same time only the most cytostatic DH16:0 was incorporated into EAT cells and the rate of incorporation was more at 42 degrees C than at 37 degrees C. CONCLUSION: Delta-hexadecanolactone (DH16:0) exhibited the most cytostatic effect that was significantly enhanced by hyperthermia. It allows to consider it as a potent antitumor agent, especially in combination with hyperthermia.

Abnormal cell morphology and cytotoxic effect are induced by 6-0-palmitol-ascorbate-2-0-phosphate, but not by ascorbic acid or hyperthermia alone.

A new derivative of ascorbic acid (AsA), 6-0-palmitoyl-ascorbate 2-0-phosphate (Asc2P6Pal) was developed to enhance the antitumor activity of AsA. When Ehrlich ascites tumor cells were treated with 50 microM Asc2P6Pal at 37 degrees C or 42 degrees C for 1 h and then cultured for 20 h, most of cells exhibited some morphological abnormalities, including exudation of intracellular granules together with other contents on the cell membrane surface, resulting in cell fragmentation. The abnormal features were further enhanced by a long term culture for 96 h and heat treatment at 42 degrees C. In contrast, no abnormality was detected for untreated cells or cells treated with AsA (free acid) at 37 degrees C or 42 degrees C. Cells cultured for 96 h after the treatment suffered from inhibition of DNA synthesis and proliferation. This inhibition was markedly enhanced by combination with the hyperthermic treatment at 42 degrees C, but not for a short-term culture of 20 h after the treatment. No effects were seen upon similar treatment with AsA. The abnormal cells produced during culture for 20 h after the treatment were evaluated to be viable, because they failed to be stained with trypan blue and retained most of the DNA synthesizing ability of Asc2P6Pal-untreated cells. However, they appeared and died after a continuous 76 h of culture (96 h).

Identification, characterization and mapping of the human ZIS (zinc-finger, splicing) gene.

From a human fetal brain cDNA library, we isolated two transcripts (ZIS-1 and ZIS-2) corresponding to the human ZIS gene, an ortholog of the rat Zis (zinc finger, splicing). A comparison of base sequences of the cDNA and its corresponding genomic DNA (a P1-derived artificial chromosome clone) revealed that both transcripts have an ORF of 1011bp and encodes 337 amino acids, but ZIS-1 has 10 exons and ZIS-2 contains 11 exons. Although both transcripts share the first nine exons, exon 10 of ZIS-2 is lacking in ZIS-1, and instead, exon 11 (10th exon) of ZIS-1 is larger in size, leading to the longer 3'-UTR. Thus, the two transcripts result from differential splicing. A Northern blot analysis on various adult and fetal tissues revealed that 5.2- and 3.2-kb transcripts were ubiquitously expressed, and 3.9- and 1.9-kb transcripts were highly expressed in the fetal brain and kidney, respectively. There were several other transcripts that may be alternatively processed forms of the human ZIS. Considering the ZIS gene size, the 3.2-kb transcripts most likely corresponds to ZIS-1 and may act as a major transcript of ZIS. The human ZIS has a high homology to the rat Zis for the coding DNA sequence with 91% identity and for the amino acid sequence with 87% identity. ZIS and Zis contain the same numbers of exons and introns. Both genes have unusually long 3'-UTR, and their encoding proteins contain similar components, i.e. a zinc finger domain, a nuclear localization signal, an Asp-Glu region, and a Ser-Arg-rich region. Furthermore, the expression patterns of the two genes in tissues are similar each other. Thus, the human ZIS may act as a transcriptional factor to regulate transcription and/or splicing, as does the rat Zis.

Enhanced inhibitory effects of hyperthermia combined with ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells grown at a low cell density.

Effects of hyperthermia and cell densities on inhibitory activity of ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells were studied. When cells at a low density of 5 x 10(3)/ml were treated with 75 microM ascorbic acid for 1 h, DNA synthesis was inhibited after treatment at 37 degrees C and the inhibition was significantly enhanced at 42 degrees C. At a cell density as high as 1 x 10(5)/ml, however, inhibition did not occur at 37 degrees C or 42 degrees C. In contrast, dehydroascorbic acid was inactive even at a low cell density under similar conditions. Inhibitory effects of ascorbic acid on DNA synthesis were also markedly enhanced by treatment at 40 degrees C. DNA synthesis was not inhibited in the absence of the drug. Furthermore, mice transplanted with cells treated with a combination of 75 microM ascorbic acid and hyperthermia at 42 degrees C, considerably prolonged their survival time in comparison with untreated cells. Addition of ascorbic acid to hyperthermia is suggested to be an advantageous treatment for cancer.