Induction of non-apoptotic cell death by morphinone in human promyelocytic leukemia HL-60 cells.

As previously suggested, codeinone (oxidation product of codeine) induces non-apoptotic cell death, characterized by marginal caspase activation and the lack of DNA fragmentation in HL-60 human promyelocytic leukemia cells, which was inhibited by N-acetyl-L-cysteine. Whether, morphinone, an oxidative metabolite of morphine, also induced a similar type of cell death in HL-60 cells was investigated. Morphinone showed slightly higher cytotoxic activity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, NA, Ca9-22, promyelocytic leukemia HL-60, cervical carcinoma HeLa) than against normal oral human cells (gingival fibroblast HGF, pulp cells HPC, periodontal ligament fibroblast HPLF). Morphinone also induced an almost undetectable level of internucleosomal DNA fragmentation in the HL-60 cells. Morphinone did not activate caspase-8 or -9 in these cells. Morphinone dose-dependently activated caspase-3 in both HL-60 and HSC-2 cell lines, but to a much lesser extent than actinomycin D. Electron microscopy demonstrated that morphinone induced mitochondrial shrinkage, vacuolization and production of autophagosome and the loss of cell surface microvilli, without destruction of cell surface and nuclear membranes in the HL-60 cells. The autophagy inhibitor 3-methyladenine (0.3-10 mM) slightly inhibited the morphinone-induced cytotoxicity, when corrected for its own cytotoxicity. These data suggest that morphinone induces non-apoptotic cell death in HL-60 cells.

Biological impact of contact with metals on cells.

In order to investigate the in vivo effect of metals used in dentistry, we investigated the effect of direct contact with metal plates (20 x 20 x 0.5 mm3) made of gold (Au), silver (Ag), copper (Cu) or palladium (Pd) on human promyelocytic leukemic HL-60 cells grown in RPMI1640 medium supplemented with 10% fetal bovine serum. When 0.5 mL of cell suspension was applied to the metal plates, cells were precipitated on the surface of the metal plate within 10 min. Contact with Cu induced a rapid decline of cell viability, the smear pattern of DNA fragmentation, and only minor activation of caspase-3. These effects were accompanied by a progressive decrease in the extracellular concentration of methionine, cysteine and histidine, with a corresponding increase in the concentration of methionine sulfoxide. Electron microscopy showed that contact with Cu induced vacuolization and cytoplasmic damage, prior to nuclear damage, without affecting the cell surface microvilli or mitochondrial integrity. Contact with the other metals did not induce such changes during the 3 h incubation, nor was any hormetic response (beneficial action at lower concentration) observed in the cells with any metals. Addition of N-acetyl-L-cysteine (4-5 mM) almost completely abrogated the Cu-induced cytotoxicity, whereas sodium ascorbate (0.1-0.5 mM) and catalase (6,000(1)-30,000 units/mL) were ineffective. Numerous serum proteins were adsorbed to the Ag plate, while bovine serum albumin was the major protein adsorbed to other metal plates. The present study suggests that direct contact with Cu induced non-apoptotic cell death by an oxidation-involved mechanism. The present model system may be applicable to the study of the interaction between cells and dental restorative materials.

Enhancement of cytotoxic activity of sodium fluoride against human periodontal ligament fibroblasts by water pressure.

Whether or not water pressure enhances the cytotoxic activity of sodium fluoride (NaF) against human periodontal ligament fibroblast (HPLF) was investigated. Loading with water pressure (up to 5 g) alone did not affect the cell proliferation, but significantly enhanced the cytotoxic activity of millimolar concentrations of NaF. Cytotoxic activity of NaF was reduced by supplementation with Ca2+, whereas it was enhanced by removal of Ca2+ from the culture medium. However, the enhancement of cytotoxicity of NaF under water pressure was observed even in the Ca2+ -free medium. NaF failed to induce apoptosis markers, such as the caspase-3, -8 and -9 activation, the intemucleosomal DNA fragmentation, the loss of cell surface microvilli and the changes in intracellular concentration of polyamines. Transmission electron microscopy demonstrated that the combination of NaF and water pressure slightly increased the incidence of the formation of autophagosomes engulfing organella, suggesting the induction of non-apoptotic cell death in HPLF cells. The present study suggests that the external pressure is an additional factor that enhances the cytotoxicity of NaF against HPLF cells.

Effect of anticancer agents on codeinone-induced apoptosis in human cancer cell lines.

The possible apoptosis-inducing activity of codeinone, an oxidative metabolite of codeine, without or with anticancer drugs, was investigated. Codeinone induced internucleosomal DNA fragmentation in human promyelocytic leukemia cells (HL-60), but not in human squamous cell carcinoma cells (HSC-2). Codeinone dose-dependently activated caspase-3 in both of these cells, but to a much lesser extent than that attained by actinomycin D. This property of codeinone was similar to what we have found previously in alpha,beta-unsaturated ketones. Codeinone did not activate caspase-8 or caspase-9 in these cells. The cytotoxic activity of codeinone against HSC-2 cells was inhibited by N-acetyl-L-cysteine, but somewhat additively stimulated by sodium ascorbate, epigallocatechin gallate, hydrogen peroxide, sodium fluoride, 5-fluorouridine, cisplatin, doxorubicin and methotrexate. These data suggest that codeinone has possible antitumor potential, in addition to its action as a narcotic analgesic, even though it induces incomplete apoptosis-associated characteristics.

Tumor-specific cytotoxicity and apoptosis-inducing activity of berberines.

Berberine iodide (IK-1) and acetoneberberine (IK-2) showed higher cytotoxicity against five human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4, NA, CA9-22) and one human promyelocytic leukemia (HL-60) cell lines, than against normal human oral tissue-derived cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), producing a tumor specificity index of 4.0 and 3.6, respectively. IK-1 was more potent than IK-2 in inducing the production of apoptotic cells, internucleosomal DNA fragmentation, the activation of caspases-3, -8 and -9, and the increased expression of proapoptotic BAD protein, with a corresponding decrease in the expression of anti-apoptotic Bcl-2 protein in HL-60 cells. These compounds did not induce internucleosomal DNA fragmentation (only producing larger DNA fragment), nor increased the Bad protein expression in HSC-2 cells. The present study demonstrated the tumor-specific cytotoxicity and apoptosis-inducing activity of berberines, suggesting their possible antitumor potentiaL

Tumor-specificity and apoptosis-inducing activity of stilbenes and flavonoids.

A total of eleven stilbenes [1-6] and flavonoids [7-11] were investigated for their tumor- specific cytotoxicity and apoptosis-inducing activity, using four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG and promyelocytic leukemia HL-60) and three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF). All of the compounds, especially sophorastilbene A [1], (+)-alpha-viniferin [2], piceatannol [5], quercetin [9] and isoliquiritigenin [10], showed higher cytotoxicity against the tumor cell lines than normal cells, yielding tumor-specific indices of 3.6, 4.7, >3.5, >3.3 and 4.0, respectively. Among the seven cell lines, HSC-2 and HL-60 cells were the most sensitive to the cytotoxic action of these compounds. Sophorastilbene A [1], piceatannol [5], quercetin [9] and isoliquiritigenin [10] induced internucleosomal DNA fragmentation and activation of caspases -3, -8 and -9 dose-dependently in HL-60 cells. (+)-alpha-Viniferin [2] showed similar activity, but only at higher concentrations. All the compounds failed to induce DNA fragmentation and activated caspases to much lesser extents in HSC-2 cells. Western blot analysis showed that sophorastilbene A [1], piceatannol [5] and quercetin [9] did not induce any consistent changes in the expression of pro-apoptotic proteins (Bax, Bad) and antiapoptotic protein (Bcl-2) in HL-60 and HSC-2 cells. An undetectable expression of Bcl-2 protein in control and drug-treated HSC-2 cells may explain the relatively higher sensitivity of this cell line to stilbenes and flavonoids.

Detection of GAD65-reactive T-Cells in type 1 diabetes by immunoglobulin-free ELISPOT assays.

OBJECTIVE: To investigate the prevalence of beta-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELISPOT) assay and assessed its usefulness for diagnosing this disease. RESEARCH DESIGN AND METHODS: Cellular immune responses to beta -cell autoantigens were studied both by immunoglobulin-free proliferation assays and ELISPOT assays in 33 patients with type 1 diabetes and 15 patients with type 2 diabetes, compared with 23 healthy control subjects. Autoantibodies against GAD65 and IA-2 were measured by radioimmunoassay. RESULTS: Significant proliferative responses to GAD65 were observed in 10 of 31 (32.3%) type 1 diabetic patients (P < 0.05), whereas GAD65-reactive gamma-interferon (IFN-gamma)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay (P < 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-gamma positivity in ELISPOT and two of five (40.0%) showed significant proliferation against GAD65. CONCLUSIONS: Using a newly developed ELISPOT assay, GAD-reactive T-helper 1 cells in PBMC of type 1 diabetic patients could be identified at a higher frequency than by the proliferation assay. Therefore, the immunoglobulin-free ELISPOT assay is an excellent tool for detecting T-cell reactivity to autoantigens with greater specificity and, in combination with beta-cell autoantibody determination, will improve the diagnosis of type 1 diabetes.

Fas/Fas ligand interactions play an essential role in the initiation of murine autoimmune diabetes.

Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. To elucidate the role of Fas/FasL interactions in NOD diabetes, the effects of neutralizing anti-FasL antibody on autoimmune responses were evaluated. Islet-specific CD8(+) and CD4(+) T-cells expressed FasL upon activation and mediated FasL-dependent cytotoxicity against Fas-expressing target cells in vitro, although their cytotoxicity against islet cells was not blocked by anti-FasL antibody. Moreover, administration of anti-FasL antibody failed to inhibit diabetes in vivo in the CD8(+) T-cell adoptive transfer model. On the other hand, blockade of Fas/FasL interactions significantly inhibited CD4(+) T-cell-dependent diabetes in adoptive transfer models. These results suggest a substantial contribution of Fas/FasL interactions to CD4(+), but not CD8(+), T-cell-mediated destruction of pancreatic beta-cells. When anti-FasL antibody was administered to NOD mice between 5 and 15 weeks of age, the onset of diabetes was slightly delayed but the incidence was not decreased. However, administration of anti-FasL antibody at 2-4 weeks of age completely prevented insulitis and diabetes. These results suggest that Fas/FasL interactions contribute to CD4(+) T-cell-mediated beta-cell destruction and play an essential role in the initiation of autoimmune NOD diabetes.

Tolerance mechanisms in murine autoimmune diabetes induced by anti-ICAM-1/LFA-1 mAb and anti-CD8 mAb.

A short-term administration of antibodies against ICAM-1/LFA-1 or CD8 molecules during a critical period of younger age resulted in complete protection of autoimmune diabetes in NOD mice. In this study, we attempted to elucidate the tolerance mechanisms. Transfer of splenocytes from both antibody-treated NOD mice to NOD-SCID mice failed to develop diabetes. On the other hand, when splenocytes from diabetic mice were transferred to the antibody-treated mice, 40% of both mAb-treated recipients became diabetic. In vitro response of T cells from these protected mice exhibited strong proliferation against syngeneic islet cells or ConA. Furthermore, semiquantitative RT-PCR analysis of cytokines showed that T cells from anti-CD8-treated mice could express IFN-gamma, IL-4, IL-10 and TGF-beta1 in response to islet antigen. In contrast, T cells from anti-ICAM-1/LFA-1-treated mice expressed IFN-gamma, IL-10 and TGF-beta1 but not IL-4. These results suggest that tolerance mechanisms like clonal deletion, anergy, immunoregulatory T cells or Th1 to Th2/Th3 cytokine shifting are not responsible for the tolerance induction, indicating the presence of other unrevealed mechanism responsible for the loss of capability of autoreactive T cells to infiltrate and destroy the pancreatic beta-cells in vivo.