Biological Properties of the Aggregated Form of Chitosan Magnetic Nanoparticle.

BACKGROUND/AIM: Chitosan-coated iron oxide nanoparticles (Chi-NP) have gained attention because of their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. In this study, we investigated various biological properties of Chi-NP. MATERIALS AND METHODS: Chi-NP was prepared by mixing magnetic NP with chitosan FL-80. Particle size was determined by scanning and transmission electron microscopes, cell viability by MTT assay, cell cycle distribution by cell sorter, synergism with anticancer drugs by combination index, PGE2 production in human gingival fibroblast was assayed by ELISA. RESULTS: The synthetic process of Chi-NP from FL-80 and magnetic NP increased the affinity to cells, up to the level attained by nanofibers. Upon contact with the culture medium, Chi-NP instantly formed aggregates and interfered with intracellular uptake. Aggregated Chi-NP did not show cytotoxicity, synergism with anticancer drugs, induce apoptosis (accumulation of subG1 cell population), protect the cells from X-ray-induced damage, nor affected both basal and IL-1ß-induced PGE2 production. CONCLUSION: Chi-NP is biologically inert and shows high affinity to cells, further confirming its superiority as a scaffold for drug delivery.

Changes in Metabolic Profiles of Human Oral Cells by Benzylidene Ascorbates and Eugenol.

Sodium-5,6-benzylidene-L-ascorbate (SBA), and its component units, benzaldehyde (BA) and sodium ascorbate (SA), are known to exert antitumor activity, while eugenol exerts anti-inflammatory activity. To narrow down their intracellular targets, metabolomic analysis was performed. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to capillary electrophoresis-mass spectrometry (CE-MS) for quantification of intracellular metabolites. Results showed that SBA was cleaved into BA and SA under acidic condition. Among these three compounds, BA showed the highest-tumor specificity in vitro against human oral squamous cell carcinoma (OSCC) cell line. BA did not induce the vacuolization in HSC-2 OSCC cells, and its cytotoxicity was not inhibited by catalase, in contrast to SBA and SA. Only BA suppressed the tricarboxylic acid (TCA) cycle at early stage of cytotoxicity induction. Eugenol more rapidly induced the vacuolization and suppressed the TCA cycle in three human normal oral cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell). Neither BA nor eugenol affected the ATP utilization, further supporting that they do not induce apoptosis. The present study demonstrated for the first time that both BA and eugenol suppressed the TCA cycle in tumor cells and normal cells, respectively. It is crucial to design methodology that enhances the antitumor potential of BA and reduces the cytotoxicity of eugenol to allow for safe clinical application.

Induction of Apoptosis in Human Oral Keratinocyte by Doxorubicin.

BACKGROUND/AIM: We have previously reported that doxorubicin (DXR) showed much higher cytotoxicity against human oral squamous cell carcinoma cell lines compared to normal human mesenchymal normal oral cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell), yielding high tumor-specificity. However, we unexpectedly found that doxorubicin showed potent cytotoxicity against human normal oral keratinocytes and primary gingival epithelial cells. In the present study, we investigated the reproducibility, underlining mechanisms and generality of this unexpected finding. MATERIALS AND METHODS: Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, fine cell structure by transmission electron microscopy and apoptosis induction by western blot analysis. RESULTS: Doxorubicin induced keratinocyte toxicity, regardless of cell density and concentration of FBS in the culture medium. Doxorubicin induced apoptosis (characterized by the loss of cell surface microvilli, chromatin condensation, nuclear fragmentation and caspase-3 activation) in keratinocytes. A total of 11 anticancer drugs showed similar keratinocyte toxicity. Alkaline extract of the leaves of Sasa senanensis Rehder partially alleviated the DXR-induced keratinocyte cytotoxicity by promoting cell growth. CONCLUSION: The present study suggested that oral keratinocyte toxicity is a novel adverse effect of most anticancer agents.

Effects of 3-styrylchromones on metabolic profiles and cell death in oral squamous cell carcinoma cells.

4H-1-benzopyran-4-ones (chromones) are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E)-3-(4-hydroxystyryl)-6-methoxy-4H-chromen-4-one (compound 11), (E)-6-methoxy-3-(4-methoxystyryl)-4H-chromen-4-one (compound 4), (E)-6-methoxy-3-(3,4,5-trimethoxystyryl)-4H-chromen-4-one (compound 6) showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC) lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

Effects of TiO2 nano glass ionomer cements against normal and cancer oral cells.

BACKGROUND/AIM: Incorporation of nanoparticles (NPs) into the glass ionomer cements (GICs) is known to improve their mechanical and antibacterial properties. The present study aimed to investigate the possible cytotoxicity and pro-inflammation effect of three different powdered GICs (base, core build and restorative) prepared with and without titanium dioxide (TiO2) nanoparticles. MATERIALS AND METHODS: Each GIC was blended with TiO2 nanopowder, anatase phase, particle size <25 nm at 3% and 5% (w/w), and the GIC blocks of cements were prepared in a metal mold. The GICs/TiO2 nanoparticles cements were smashed up with a mortar and pestle to a fine powder, and then subjected to the sterilization by autoclaving. Human oral squamous cell carcinoma cell lines (HCS-2, HSC-3, HSC-4, Ca9-22) and human normal oral cells [gingival fibroblast (HGF), pulp (HPC) and periodontal ligament fibroblast (HPLF)] were incubated with different concentrations of GICs in the presence or absence of TiO2 nanoparticles, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Prostaglandin E2 was quantified by enzyme-linked immunosorbent assay (ELISA). Changes in fine cell structure were assessed by transmission electron microscopy. RESULTS: Cancer cells exhibited moderate cytotoxicity after 48 h of incubation, regardless of the type of GIC and the presence or absence of TiO2 NPs. GICs induced much lower cytotoxicity against normal cells, but induced prostaglandin E2 production, in a synergistic wanner with interleukin-1ß. CONCLUSION: The present study shows acceptable to moderate biocompatibility of GICs impregnated with TiO2 nanoparticles, as well as its pro-inflammatory effects at higher concentrations.

3D dynamic pituitary MR imaging with CAIPIRINHA: initial experience and comparison with 2D dynamic MR imaging.

OBJECTIVES: To evaluate the validity of 3D dynamic pituitary MR imaging with controlled aliasing in parallel imaging results in higher acceleration (CAIPIRINHA), with special emphasis on demarcation of pituitary posterior lobe and stalk. METHODS: Participants comprised 32 patients who underwent dynamic pituitary MR imaging due to pituitary or parasellar lesions. 3D dynamic MR with CAIPIRINHA was performed at 3T with 20-s-interval, precontrast, 1st to 5th dynamic images. Normalized values and enhanced ratios (dynamic postcontrast image values divided by precontrast ones) were compared between 3D and 2D dynamic MR imaging for patients with visual identification of posterior lobe and stalk. RESULTS: In 3D, stalk was identified in 29 patients and unidentified in 3, and posterior lobe was identified in 28 and unidentified in 4. In 2D, stalk was identified in 26 patients and unidentified in 6 patients, and posterior lobe was identified in 15 and unidentified in 17. Normalized values of pituitary posterior lobe and stalk were higher in 3D than 2D (P<0.001). No significant difference in enhancement ratio was seen between 3D and 2D. CONCLUSIONS: 3D dynamic pituitary MR provided better identification and higher normalized values of pituitary posterior lobe and stalk than 2D.

Cytotoxicity and pro-inflammatory action of chemo-mechanical caries-removal agents against oral cells.

BACKGROUND: Chemo-mechanical caries removal eliminates the outermost portion of the infected layer, leaving behind healthy dentine surfaces, with scarce dental tissue damage; however, the safety of caries solvents has not been established. The aim of the present study was to investigate the possible cytotoxicity of two popular chemo-mechanical caries removal agents. MATERIALS AND METHODS: The cytotoxicity of Carisolv, Papacarie Duo and control vehicle solution (0.155-20% v/v) against human oral squamous cell carcinoma cells (HCS-2, HSC-3, HSC-4, Ca9-22) human gingival fibroblast (HGF), pulp (HPC) and periodontal ligament fibroblast (HPLF) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Prostaglandin E2 (PGE2) was quantified by enzyme-linked immunosorbent assay. Changes in fine cell structure were assessed by transmission electron microscopy. RESULTS: Carisolv exhibited neither cytotoxicity nor hormetic growth stimulation. Papacarie Duo significantly reduced the viable cell number within 30 min. HSC-4 exhibited the highest sensitivity, followed by HSC-2>HSC-3>HPLF>Ca9-22>HPC>HGF cells. Interleukin-1ß (3 ng/ml) stimulated HGF, but not HPC cells to produce PGE2 in the culture medium. Papacarie Duo stimulated HGF cells to produce PGE2 in synergistic fashion with interleukin-1ß. CONCLUSION: Carisolv had acceptable biocompatibility with both normal and cancerous oral cells. On the other hand, Papacarie Duo had a rapid but slight cytotoxicity and pro-inflammatory action against oral cells, suggesting the importance of careful application of this agent.

Quantitative structure-activity relationship analysis of cytotoxicity and anti-UV activity of 2-aminotropones.

BACKGROUND: We newly synthesized twenty 2-aminotropones with different lengths of methylene units, with or without introduction of isopropyl group at C-4 position of the cycloheptatriene ring, which were then subjected to quantitative structure-activity relationship (QSAR) analysis. MATERIALS AND METHODS: Viable cell number was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The tumor specificity was determined by the ratio of the mean CC50 (50% cytotoxic concentration) for the normal cells (human gingival fibroblast, HGF) to that of the human oral squamous cell carcinoma (OSCC) cell line (Ca9-22) derived from gingival tissue. Anti-UV activity (SI) was determined by the ratio of CC50 to EC50 (the concentration that increased the viability of UV-irradiated cells to 50%) using HSC-2 OSCC cells. Physico-chemical, structural, and quantum-chemical parameters were calculated based on conformations optimized by the LowModeMD method followed by the Discrete Fourier Transform (DFT) method. Fine-cell structure was observed by transmission electron microscopy. RESULTS: 2-Aminotropones induced cytotoxicity, accompanied by the production of many rough endoplasmic reticula with enlarged lacuna and vacuolated mitochondria. Their cytotoxicity was a positive function of the number of methylene units and hydrophobicity. Anti-UV activity showed a good correlation with lowest unoccupied molecular orbital (LUMO) energy, but not with the length of methylene units. All twenty 2-aminotropones induced a very low level of hormetic growth stimulation at lower concentrations. CONCLUSION: Different types of chemical descriptors may be applicable to estimating the cytotoxicity and anti-UV activity of 2-aminotropones.

Effects of TiO2 nanoparticles on cytotoxic action of chemotherapeutic drugs against a human oral squamous cell carcinoma cell line.

BACKGROUND: Despite the rapid development of nanotechnology, the biological significance of TiO2 nanoparticles (NPs), possibly released from dental materials, is not well-understood. We investigated the effect of TiO2 NPs on the sensitivity of human oral squamous cell carcinoma (OSCC) cell line (HSC-2) to five popular chemotherapeutic agents. MATERIALS AND METHODS: Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The aggregation and cellular uptake of TiO2 NPs were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. Adsorption of TiO2 NPs to anticancer drugs was assessed by the antitumor activity recovered from the TiO2 NP-free supernatant. RESULTS: When mixed with culture medium, TiO2 NPs instantly aggregated, and some particles were incorporated into the cells, exclusively in the vacuoles. TiO2 NPs showed no cytotoxicity nor hormetic growth stimulation at lower concentrations. Doxorubicin, melphalan, 5-fluorouracil and gefitinib were cytotoxic, whereas docetaxel was cytostatic with or without TiO2 NPs. TiO2 NPs, at wide concentration ranges (0.2-3.2 mM), did not significantly affect the adsorption of NPs to any of these anticancer drugs, nor affected their cytotoxic or cytostatic activity. CONCLUSION: This experimental study demonstrated for the first time that TiO2 NP do not affect the antitumor potential of chemotherapeutic agents against the HSC-2 OSCC cell line.

Induction of prostaglandin E2 production by TiO2 nanoparticles in human gingival fibroblast.

BACKGROUND: Despite recent progress in the research of nanoparticles (NPs) spanning in many scientific fields, study of NPs in dentistry is limited. This triggered us to investigate the effect of TiO2 NPs on the drug-sensitivity of oral squamous cell carcinoma and inflammation of human gingival fibroblasts (HGFs). MATERIALS AND METHODS: The number of viable HGF cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Prostaglandin E2 (PGE2) was quantified by enzyme-linked immunosorbent assay. Contamination with lipopolysaccharide (LPS) was assayed by the endotoxin assay kit. Intracellular uptake and distribution of TiO2 NPs were assessed by transmission electron microscopy. RESULTS: TiO2 NPs (0.05-3.2 mM) did not affect HGF cell viability, although TiO2 NPs clusters were dose-dependently incorporated into the vacuoles of cells. Interleukin-1ß (IL-1ß) (3 ng/ml) stimulated the secretion of PGE2 into the culture medium by HGF cells. TiO2 NPs also induced PGE2 production, in synergy with IL-1ß. Since only a minor amount of LPS was detected in TiO2 NPs, the enhanced production of PGE2 was not simply due to LPS contamination. CONCLUSION: The present study demonstrates, for the first time to our knowledge, that TiO2 NPs at concentrations higher than 0.2 mM exert an pro-inflammatory action against HGF cells, regardless of the presence or absence of IL-1ß.

Cell death induced by nutritional starvation in mouse macrophage-like RAW264.7 cells.

Amino acid utilization of mouse macrophage-like RAW264.7 cells was investigated. During the logarithmic growth stage, RAW264.7 cells grew very fast, with an approximate doubling time of 11 hours, in DMEM supplemented with 10% heat-inactivated fetal bovine serum. RAW264.7 cells consumed glutamine at the fastest rate, followed by serine, leucine, isoleucine, arginine, lysine, valine and other amino acids. When the cell density reached a critical threshold level, cells began to suffer non-apoptotic cell death characterized by mitochondrial damage (revealed by transmission electron microscopy) and a smear pattern of DNA fragmentation (revealed by agarose gel electrophoresis). At this point, glutamine, serine and glucose in the medium were almost completely exhausted, whereas other amino acids remained at more than 40% of their initial concentrations. Based on these data, it is recommended that glutamine, serine and glucose should be supplemented for the long culture of RAW264.7 cells.

Tumor-specific cytotoxicity and type of cell death induced by gefitinib in oral squamous cell carcinoma cell lines.

Gefitinib is an orally active, selective epidermal growth factor receptor-tyrosine kinase inhibitor. The present study was aimed at evaluating the antitumor activity of gefitinib alone or in combination with other antitumor agents. Gefitinib showed higher cytotoxicity against five human tumor cell lines (HSC-2, HSC-3, HSC-4, T98G and U87MG) than against three human normal oral cells (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Gefitinib showed little or no growth stimulation effects at lower concentrations (so-called hormetic effect). Non-cytotoxic concentration of gefitinib effectively enhanced the cytotoxicity of docetaxel against HSC-2 and T98G cell, but failed to enhance the cytotoxicity of other antitumor agents (mitoxantrone, doxorubicin, methotrexate, cisplatin, sodium ascorbate, sodium fluoride) or herbal extracts (Drynaria baronii, Angelica sinensis and Cornus officinalis Sieb. et Zucc). Gefitinib alone and combined with docetaxel induced internucleosomal DNA fragmentation and caspase-3 activation in human promyelocytic leukemia HL-60 cells, but not in HSC-2 or T98G cells. Combination treatment with gefitinib and docetaxel induced the formation of acidic organelles (stained with acridine orange) and mitochondrial shrinkage, vacuolization and production of autophagosome and the loss of cell surface microvilli, without destruction of cell surface and nuclear membranes in HSC-2 and T98G cells (demonstrated by transmission electron microscopy), suggesting the induction of autophagy in HSC-2 and T98G cells.

Non-apoptotic cell death induced by nutritional starvation in J774.1 mouse macrophage-like cell line.

The growth and amino acid utilization of a mouse macrophage-like cell line J774.1 was investigated in two different culture media supplemented with 10% fetal bovine serum (FBS). The J774.1 cells grew faster, and consumed glutamine and serine at higher rates in DMEM than in RPMI1640 medium. The consumption of other amino acids was much less, while considerable quantities of alanine, glutamic acid and glycine were produced by the J774.1 cells. When the cells became confluent, serine, but not glutamine, was nearly depleted from the culture medium, followed by cell death characterized by smear DNA fragmentation, slight caspase-3 activation and structural damage of the mitochondria. Serine is required for the growth of mouse macrophage-like cell lines, and DMEM is superior to RPMI1640 for long-term cell culture.

Tumor-specific cytotoxicity and type of cell death induced by peplomycin in oral squamous cell carcinoma cell lines.

The antitumor antibiotic peplomycin showed higher cytostatic antiproliferative effect on five cultured human oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and NA), as compared with three human oral normal cells (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Although the antiproliferative activity of peplomycin declined with increasing cell density, peplomycin showed tumor-specific cytotoxicity at any cell density. The five OSCC cell lines showed considerable differences in sensitivity against peplomycin; the HSC-2 cells were the most sensitive, followed by the NA, HSC-3, Ca9-22 and HSC-4 cells. Peplomycin did not induce internucleosomal DNA fragmentation in any of the five OSCC cell lines, and only slightly modified caspase-3, -8 and -9 activities in the HSC-2, Ca9-22 and NA cell lines. Electron microscopy revealed that peplomycin induced the vacuolation of mitochondria accompanying electron lucent matrices lacking cristae and the enlargement of the endoplasmic reticulum in the HSC-2 cells. These data suggest that the anti-proliferative effect of peplomycin is time-dependent, and therefore prolonged treatment with peplomycin in combination with cytotoxic chemotherapeutic agents may induce greater cytotoxic action.

Tumor-specific cytotoxicity and type of cell death induced by benzocycloheptoxazines in human tumor cell lines.

Twenty-six benzocycloheptoxazine derivatives were investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). Benzo[b]cyclohepta[e][1,4]thiazine [1] exhibited very weak cytotoxicity, whereas its 6,8,10-tribromo derivative [3] exhibited higher cytotoxicity and tumor specificity (TS = 5.6). 6H-Benzo[b]cyclohepta[e][1,4]diazine [4] and its cation [5] exhibited no tumor specificity. Among eighteen benzo[b]cyclohepta[e][1,4]oxazine derivatives [6-23], 6,8,10-triboromo- [9], 6-bromo-2-methyl- [20], and 6-bromo-2-chloro- [21] derivatives showed the highest tumor-specific cytotoxicity (TS = 12.5, 9.1 and 11.5, respectively). 14H-[1,4]Benzoxazino[3',2':3,4]cyclohepta[1, 2-b][1,4]benzoxazine [24] and its 7-bromo- [25] and 7-isopropyl- [26] derivatives had much lower cytotoxicity and tumor-specificity. Compounds [9, 20, 21] at 50% cytotoxic concentration (CC50) induced internucleosomal DNA fragmentation and caspase activation in HL-60 cells. On the other hand, these compounds induced apoptosis only at concentrations higher than CC50 in HSC-2 cells and failed to induce apoptosis in HSC-4 cells. Compounds [9, 20, 21] induced the formation of acidic organelles as measured by acridine orange staining. Transmission electron microscopy demonstrated the induction of moderate enlargement of mitochondria, the endoplasmic reticulum and nuclear membrane, and the vacuolation of the endoplasmic reticulum and the presence of a number of lamellar body-like organelles. These results indicate the diversity of the type of cell death induced by benzocycloheptoxazine derivatives in human tumor cell lines.

Tumor-specific cytotoxicity and type of cell death induced by beta-cyclodextrin benzaldehyde inclusion compound.

The cytotoxicity of beta-cyclodextrin benzaldehyde inclusion compound (CDBA) against human normal and cancer cell lines was investigated. CDBA showed slightly higher cytotoxicity against human tumor cell lines, as compared to normal cells, with a tumor-specificity index of 2.2. Human myelogenous leukemia cell lines (HL-60, ML-1, KG-1) were the most sensitive to CDBA, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human glioblastoma (T98G, U87MG). Human normal cells (gingival fibroblasts, pulp cells, periodontal ligament fibroblasts) were the most resistant. CDBA induced internucleosomal DNA fragmentation in HL-60 cells and caspase-3, -8, -9 activation, but to a much lesser extent than that attained by UV irradiation or actinomycin D. On the other hand, CDBA did not induce DNA fragmentation, nor caspase activation in HSC-2, HSC-4 or T98G cells. Electron microscopy demonstrated that CDBA induced the destruction of mitochondrial structure and digestion of broken organelles by secondary lysosomes in all of these cells. CDBA also increased the number of acidic organelles as judged by acridine orange staining. The present study suggests that CDBA induces autophagic cell death in cancer cell lines.

Rapid changes in amino acid and polyamine metabolism during copper-induced cell death of human gingival fibroblast.

There are very few studies on the interaction between dental alloys and oral tissues. The effect of direct contact with copper (Cu) on the cellular function of human gingival fibroblast (HGF) derived from the periodontal tissues was investigated. When HGF cells were inoculated onto a Cu plate, the viability of HGF cells immediately declined. This was accompanied by vacuolization and chromatin condensation near the nuclear membrane. The intracellular concentration of spermidine and spermine declined, whereas that of putrescine slightly increased. Amino acid analysis of the medium revealed that glutamine was consumed at the greatest rate, amounting to more than half of the total amino acid consumption. Contact with the Cu plate resulted in the complete elimination of glutamine utilization and a simultaneous increase in the production of most amino acids, possibly due to enhanced proteolysis. This was accompanied by a time-dependent increase in the consumption of cystine, possibly due to oxidative reactions, and the enhanced production of glycine and glutamic acid. These data suggest that the contact with the Cu plate induced non-apoptotic cell death in HGF cells, which was tightly coupled with a rapid dysfunction of amino acid and polyamine metabolism.

Tumor-specificity and type of cell death induced by trihaloacetylazulenes in human tumor cell lines.

Twenty trihaloacetylazulene derivatives with one atom of fluorine, chlorine, bromine or iodine was investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast, HGF; pulp cell, HPC; periodontal ligament fibroblast, HPLF) and four human tumor cell lines (squamous cell carcinoma, HSC-2, HSC-3, HSC-4; promyelocytic leukemia, HL-60). There was no apparent difference in the cytotoxic activity between 2-methoxyazulenes [1a-1e, 2a-2e] and 2-ethoxyazulenes [3a-3e, 4a-4e]. Trichloroacetylazulenes [2a-2e, 4a-4e] generally showed higher cytotoxicity and tumor-specificity (expressed as a TS value) as compared with the corresponding trifluoroacetylazulenes [1a-1e, 3a-3e]. Substitution of chloride [1c, 2c, 3c. 4c], bromide [1d, 2d, 3d, 4d] or iodine [1e, 2e, 3e, 4e] at the C-3 position further enhanced cytotoxic activity against four tumor cell lines, especially HL-60 cells. Among twenty trihaloacetylazulene derivatives, two compounds [2d] and [4c] showed the highest tumor specificity (TS = > 3.5 and > 2.5, respectively). Compounds [2d] and [4c] induced apoptotic cell death characterized by caspase-3, -8 and -9 activation and internucleosomal DNA fragmentation in HL-60 cells. On the other hand, compounds [2d] and [4c] induced autophagic cell death characterized by lower activation of caspases, lack of DNA fragmentation, vacuolization and autophagosome formation detected by acridine orange and LC3-GFP fluorescence, without the decline of the intracellular concentration of three major polyamines in HSC-4 cells. The cytotoxic activity of [4c], but not [2d], was slightly reduced by 3-methyladenine, an inhibitor of autophagy. These results suggest the diversity of cell death type induced in human tumor cell lines by trihaloacetylazulene derivatives.

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.