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Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Amelogenin, an enamel matrix protein has been considered to be exclusively expressed by ameloblasts during odontogenesis. However, burgeoning evidence indicates that amelogenin is also expressed in non‐mineralizing tissues. Under the hypothesis that amelogenin may be a functional molecule in developing hair follicles which share developmental features with odontogenesis, this study for the first time elucidated the presence and functional changes of amelogenin and its receptors during rat hair follicle development. Amelogenin was specifically localized in the outer epithelial root sheath of hair follicles. Its expression appeared in the deeper portion of hair follicles, i.e. the bulbar and suprabulbar regions rather than the superficial region. Lamp‐1, an amelogenin receptor, was localized in either follicular cells or outer epithelial sheath cells, reflecting functional changes during development. The expression of amelogenin splicing variants increased in a time‐dependent manner during postnatal development of hair follicles. Amelogenin expression was increased by treatment with cyclosporin A, which is an inducer of anagen in the hair follicle, whereas the level of Lamp‐1 and ‐2 was decreased by cyclosporin A treatment. These results suggest that amelogenin may be a functional molecule involved in the development of the hair follicle rather than an inert hair shaft matrix protein.

Relaxin (Rln) is an ovarian hormone that stimulates osteoclastic and osteoblastic activities and connective tissue turnover. To investigate the expression of Rln during orthodontic tooth movement, rats were implanted with orthodontic appliances that connected a spring from the upper incisors to the first molar with a 70 cN force. Rats in each group were killed 6, 48, and 144 h after activating the appliance, and the levels of Rln1 and Rln3 expression in the ovary were determined by real-time RT-PCR, northern blots, western blots, and immunofluorescence analyses. The amount of tooth movement induced by the orthodontic force increased in a time-dependent manner. The levels of Rln1 mRNA increased by 12-, 41-, and 263-fold at 6, 48, and 144 h, respectively, after orthodontic tooth movement. The time-dependent increase in the concentration of Rln 1 protein in the ovary was also confirmed by western blotting. Rln 1 was localized in the granulosa cells of the ovarian follicles, and the immunoreactivity against Rln 1 was increased by the movement. In contrast, the concentration of Rln 3 was below the level of detection. The results of this study suggest that local changes in periodontal tissues induced by orthodontic tooth movement may affect Rln1 expression in the ovary. However, further studies are needed to decipher the mechanisms involved and the possible contribution of the increased level of expression of Rln 1 to the tooth movement.

Objectives: Cyclosporin A (CsA) is known to elicit fibrous gingival overgrowth with changes of blood vessel profiles. In this study, we examined the expression of several angiogenic and angiostatic genes during the development of CsA-induced gingival overgrowth. Methods: For the development of gingival overgrowth, Sprague-Dawley rats received subcutaneous injections of CsA in daily doses of 5, 10, 15 mg/kg body weight for 6 weeks, and another group received 10 mg/kg of CsA for 3, 6, and 12 weeks. Human gingival tissues were obtained from three CsA-treated patients following the gingivectomy procedure and from three healthy patients following the crown-lengthening procedure as a control. Gingival fibroblasts were isolated from the healthy gingival tissues of the rat or the human, and cultured with 250–1000 ng/ml of CsA. Results: Reverse transcription–polymerase chain reaction (RT–PCR) analyses showed that expressions of some angiogenic genes such as angiopoietin 1, basic fibroblast growth factor, and vascular endothelial growth factor, and angiostatic genes such as angiopoietin 2, brain-specific angiogenesis inhibitor 1 and 2, and thrombospondin 1 were not changed significantly in both gingival tissues and cultured fibroblast cells under the CsA treatments. However, expression of thrombospondin 2 (TSP2) decreased dose- and time-dependently in rat and human gingival tissues. Western blot analyses showed that the expression of TSP2 protein was dose-dependently reduced by the CsA treatments in human cultured gingival fibroblasts. Conclusions: These results indicate that the decrease in angiostatic TSP2 expression may be attributed to the CsA-induced gingival vascularization rather than to the increased expression of angiogenic genes. It suggests that TSP2 is involved in the development of CsA-induced gingival overgrowth with the gingival vascularization.