Cytotoxic Tumour-Selective 1,5-Diaryl-3-Oxo-1,4-Pentadienes Mounted on a Piperidine Ring

A series of 3,5-bis(benzylidene)-4-piperidones 2a-u were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC), thereby demonstrating tumour-selective toxicity. A further study revealed that most of the compounds in series 2 were more toxic to the human Colo-205 adenocarcinoma cell line (Colo-205), human HT29 colorectal adenocarcinoma cells (HT-29) and human CEM lymphoid cells (CEM) neoplasms than towards non-malignant human foreskin Hs27 fibroblast line (Hs27) cells. The potency of the cytotoxins towards the six malignant cell lines increased as the sigma and sigma star values of the aryl substituents rose. Attempts to condense various aryl aldehydes with 2,2,6,6-tetramethyl-4-piperidone led to the isolation of some 1,5-diaryl-1,4-pentadien-3-ones. The highest specificity for oral cancer cells was displayed by 2e and 2r. In the case of 2r, its selective toxicity exceeded that of doxorubicin and melphalan. The enones 2k, m, o have the highest SI values towards colon cancer and leukemic cells. Both 2e,r inhibited mitosis and increased the subG1 population (with a transient increase in G2/M phase cells). Slight activation of caspase-3, based on the cleavage of poly(ADP-ribose)polymerase (PARP) and procaspase 3, was detected.

Analysis of The Cells Isolated From Epithelial Cell Rests of Malassez Through Single-cell Limiting Dilution

The epithelial cell rests of Malassez (ERM) play a pivotal role in preventing ankylosis between the alveolar bone and the tooth. Although several functions of ERM has been reported, the mechanism behind preventing dentoalveolar ankylosis remains unclear. In this study, 18 clones were isolated from ERM (CRUDE) using the single-cell limiting dilution method. Among them, ERM-2 and -3, which exhibited the highest and lowest proliferation rates, respectively, were selected. ERM-2, ERM-3, and CRUDE ERM were stained with epithelial markers, including cytokeratin-wide and cytokeratin-19, via immunofluorescence. The qRT-PCR analysis revealed increased expression levels of p75 (ameloblast marker), amelogenin, and sfrp5 (inner enamel epithelial cell marker) in the ERM-2 cells. Alternatively, ameloblastin and ck-14 (outer enamel epithelial cell marker) were highly expressed in ERM-3 cells. The mineralization of human periodontal ligament fibroblast (HPDLF) was inhibited when co-cultured with ERM-2, ERM-3, and CRUDE ERM cells. The addition of an anti-amelogenin antibody restored the mineralization of HPDLF cells. Transplanted rat molar cultured in ERM-2 (high amelogenin secretive clone) cell-derived supernatant resulted in significantly smaller bone formation than those cultured in the CRUDE ERM and ERM-3 cell-derived supernatants. These findings indicate that amelogenin produced by ERM cells might be involved in preventing dentoalveolar ankylosis.

Cytotoxic evaluation of two orthodontic silver solder materials on human periodontal ligament fibroblast cells and the effects of antioxidant and antiapoptotic reagents

ABSTRACT Objectives To evaluate the cytotoxicity effects of two different solder materials used for orthodontic appliances on human periodontal ligament fibroblast (HPLF) cells, and to determine whether the mechanism of toxicity may involve oxidative stress and apoptosis. Materials and Methods The silver solder samples (Leone and Summit) were soldered to orthodontic stainless steel bands and exposed to HPLF cells via cell culture inserts for 48 hours. Cytotoxicity effect of the soldered materials on HPLF cells was measured via tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay (n = 10/sample) and morphological observation. In addition, the mechanism of cytotoxicity of the most toxic silver solder was investigated using both a caspase inhibitor Z-VAL-Ala-Asp-flu-oromethylketone (ZVAD-fmk) and the free radical scavenger Trolox (n = 8/sample). Statistical analysis was performed using one-way analysis of variance with a Bonferroni test. P < .05 was considered statistically significant. Results Compared to the control (no treatment, cells only), both silver solders were cytotoxic (P < .001). The bands alone were significantly cytotoxic compared to the control. There was a significant difference in cytotoxicity between the stainless steel bands alone and the Summit silver solder (P < .001), but not the Leone silver solder. The Summit silver solder was more cytotoxic than the Leone silver solder (P < .05). MTT results were supported by the microscopic morphological changes of the HPLF cells. Neither ZVAD-fmk nor Trolox provided significant protection. Conclusions The two silver solder materials demonstrated different levels of cytotoxicity, and neither oxidative stress nor apoptosis is involved in the mechanism of cytotoxicity.

Oxidative Damage in Human Periodontal Ligament Fibroblast (hPLF) after Methylmercury Exposure

Human exposure to mercury (Hg) is primary associated with its organic form, methylmercury (MeHg), through the ingestion of contaminated seafood. However, Hg contamination is also positively correlated with the number of dental restorations, total surface of amalgam, and organic mercury concentration in the saliva. Among the cells existing in the oral cavity, human periodontal ligament fibroblast (hPLF) cells are important cells responsible for the production of matrix and extracellular collagen, besides sustentation, renewal, repair, and tissue regeneration. In this way, the present study is aimed at investigating the potential oxidative effects caused by MeHg on hPLF. Firstly, we analyzed the cytotoxic effects of MeHg (general metabolism status, cell viability, and mercury accumulation) followed by the parameters related to oxidative stress (total antioxidant capacity, GSH levels, and DNA damage). Our results demonstrated that MeHg toxicity increased in accordance with the rise of MeHg concentration in the exposure solutions (1-7 μM) causing 100% of cell death at 7 μM MeHg exposure. The general metabolism status was firstly affected by 2 μM MeHg exposure (43.8±1.7%), while a significant decrease of cell viability has arisen significantly only at 3 μM MeHg exposure (68.7±1.4%). The ratio among these two analyses (named fold change) demonstrated viable hPLF with compromised cellular machinery along with the range of MeHg exposure. Subsequently, two distinct MeHg concentrations (0.3 and 3 μM) were chosen based on LC50 value (4.2 μM). hPLF exposed to these two MeHg concentrations showed an intracellular Hg accumulation as a linear-type saturation curve indicating that metal accumulated diffusively in the cells, typical for metal organic forms such as methyl. The levels of total GSH decreased 50% at exposure to 3 μM MeHg when compared to control. Finally, no alteration in the DNA integrity was observed at 0.3 μM MeHg exposure, but 3 μM MeHg caused significant damage. In conclusion, it was observed that MeHg exposure affected the general metabolism status of hPLF with no necessary decrease on the cell death. Additionally, although the oxidative imbalance in the hPLF was confirmed only at 3 μM MeHg through the increase of total GSH level and DNA damage, the lower concentration of MeHg used (0.3 μM) requires attention since the intracellular mercury accumulation may be toxic at chronic exposures.

The effects of different 650 nm laser diode irradiation times on the viability and proliferation of human periodontal ligament fibroblast cells

Background: Endo-perio lesions are clinical manifestations of inflammation in the periodontal and pulp tissue. Damage to the periodontal ligament can inhibit its ability to regenerate. Therefore, laser therapy use is expected to improve the prognosis with regard to healing lesions. Unfortunately, the duration of irradiation during laser diode therapy can influence the viability and proliferation of human periodontal ligament fibroblast (hPDLF) cells. Purpose: This study aims to determine the effects of different irradiation exposure times of the 650 nm laser diode of the pulsed mode type on the viability and proliferation of human periodontal ligament fibroblast cells. Methods: This study constituted a laboratory experiment on hPDLF cells using 650 nm laser diode irradiation. Six groups formed the research subjects in this study, namely; two control groups, two radiation groups respectively subjected to irradiation exposure of 15 seconds and 35 seconds duration followed by 24-hour incubation, and two radiation groups exposed to irradiation for 15 and 35 seconds respectively followed by 72-hour incubation period. The viability and proliferation of those cells were subsequently calculated by ELISA reader, while the data was analyzed by means of one-way ANOVA and Tukey tests. Results: The significance value of the viability scores between the 15-second irradiation group and the 35-second irradiation group was less than 0.05, indicating that there was a significant difference between these treatment groups. Similarly, the significance value of proliferation scores between the 15-second irradiation group and the 35-second irradiation group was less than 0.05, again indicating a significant difference between these treatment groups. Conclusion: Irradiation using a 650 nm laser diode 15 seconds and 35 seconds in duration can induce an increase in the viability and proliferation of hPDLF cells.