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.

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.

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 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.

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.

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.

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.

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.

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.

Re-evaluation of tumor-specific cytotoxicity of mitomycin C, bleomycin and peplomycin.

Three antitumor antibiotics, mitomycin C, bleomycin sulfate and peplomycin sulfate, were compared for their tumor-specific cytotoxicity, using human oral squamous cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and NA), human promyelocytic leukemic cell line HL-60 and human normal oral cell types (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Among these three compounds, mitomycin C showed the highest tumor-specificity, due to its higher cytotoxic activity against human oral tumor cell lines than bleomycin and peplomycin. However, there was considerable variation of drug sensitivity among the six tumor cell lines. Mitomycin C induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in HL-60 cells only after 24 h. On the other hand, mitomycin C induced no clear-cut DNA fragmentation in HCS-2 cells, although it activated caspase-3, -8 and -9 to a slightly higher extent. Western blot analysis demonstrated that mitomycin C did not induce any apparent change in the intracellular concentration of anti-apoptotic protein (Bcl-2) and pro-apoptotic proteins (Bax, Bad). Electron microscopy of mitomycin C-treated HL-60 cells showed intact mitochondria (as regards to integrity and size) and cell surface microvilli, without production of an apoptotic body or autophagosome, at an early stage after treatment. The present study suggests the incomplete induction of apoptosis or the induction of another type of cell death by mitomycin C treatment.

Cytotoxic activity of selected trifluoromethyl ketones against oral tumor cells.

Several trifluoromethyl ketones (TF1-4) and related non-fluorinated ketones (TF5 and 6) were tested for their relative cytotoxicity on four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4 and promyelocytic leukemia HL-60) and three normal human cells [gingival fibroblasts (HGF), pulp cells (HPC) and periodontal ligament fibroblasts (HPLF)]. Trifluoromethylated a-diketone (TF1, CF3COCOPh) and alpha-hydroxy ketones (TF2, CF3CH(OH)COPh; TF3, CF3CH(OH)COCH2Ph) showed higher tumor-specific cytotoxic activity than the corresponding non-fluorinated analogs (TF5, CH3COCOPh; TF6, CH3CH(OH)COPh), while the anti-tumor potency of trifluoromethyl ketone (TF4, CF3COCH2Ph) was lower. Among four tumor cell lines, HL-60 cells were the most sensitive to TF1-4, followed by HSC-2 and HSC-3 cells. HSC-4 cells were the most resistant in most cases. Agarose gel electrophoresis showed that TF1-3 did not induce intemucleosomal DNA fragmentation nor activated caspase-3. The cytotoxic activities of TF1-3 were not significantly affected by FeCl3. Electron microscopy of TF2- or 3-treated HL-60 cells showed the development of autophagosomes in HL-60 cells, without the production of an apoptotic body, or affecting the mitochondria and cell surface microvilli. The autophagy inhibitor, 3-methyladenine (3-MA), partially inhibited the TF2- or 3-induced cytotoxicity. These data suggest the induction of non-apoptotic cell death by TF2 or 3.

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.

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.

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.

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.

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.

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.