Cathepsin B degrades RbcL during freezing-induced programmed cell death in Arabidopsis.

KEY MESSAGE: Cathepsin B plays an important role that degrades the Rubisco large subunit RbcL in freezing stress. Programmed cell death (PCD) has been well documented in both development and in response to environmental stresses in plants, however, PCD induced by freezing stress and its molecular mechanisms remain poorly understood. In the present study, we characterized freezing-induced PCD and explored its mechanisms in Arabidopsis. PCD induced by freezing stress was similar to that induced by other stresses and senescence in Arabidopsis plants with cold acclimation. Inhibitor treatment assays and immunoblotting indicated that cathepsin B mainly contributed to increased caspase-3-like activity during freezing-induced PCD. Cathepsin B was involved in freezing-induced PCD and degraded the large subunit, RbcL, of Rubisco. Our results demonstrate an essential regulatory mechanism of cathepsin B for Rubisco degradation in freezing-induced PCD, improving our understanding of freezing-induced cell death and nitrogen and carbohydrate remobilisation in plants.

ß-elemene induces caspase-dependent apoptosis in human glioma cells in vitro through the upregulation of Bax and Fas/ FasL and downregulation of Bcl-2.

BACKGROUND: ß-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ß-elemene against glioma cells remains unclear. In the present study, we assessed effects of ß-elemene on human glioma cells and explored the underlying mechanism. MATERIALS AND METHODS: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ß-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ß-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ß-elemne treatment group. RESULTS: With increase in the concentration of ß-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability (IC50) was 48.5 µg/mL for 24h. ß-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ß-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ß-elemene in a time and does- dependent manner. CONCLUSIONS: Our results indicate that ß-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

Curcumin suppresses malignant glioma cells growth and induces apoptosis by inhibition of SHH/GLI1 signaling pathway in vitro and vivo.

AIMS: To study the role of curcumin on glioma cells via the SHH/GLI1 pathway in vitro and vivo. METHODS: The effects of curcumin on proliferation, migration, apoptosis, SHH/GLI1 signaling, and GLI1 target genes expression were evaluated in multiple glioma cell lines in vitro. A U87-implanted nude mice model was used to study the role of curcumin on tumor volume and the suppression efficacy of GLI1. RESULTS: Curcumin showed cytotoxic effects on glioma cell lines in vitro. Both mRNA and protein levels of SHH/GLI1 signaling (Shh, Smo, GLI1) were downregulated in a dose- and time-dependent manner. Several GLI1-dependent target genes (CyclinD1, Bcl-2, Foxm1) were also downregulated. Curcumin treatment prevented GLI1 translocating into the cell nucleus and reduced the concentration of its reporter. Curcumin suppressed cell proliferation, colony formation, migration, and induced apoptosis which was mediated partly through the mitochondrial pathway after an increase in the ratio of Bax to Bcl2. Intraperitoneal injection of curcumin in vivo reduced tumor volume, GLI1 expression, the number of positively stained cells, and prolonged the survival period compared with the control group. CONCLUSION: This study shows that curcumin holds a great promise for SHH/GLI1 targeted therapy against gliomas.

Study of TNF-α, IL-1ß and LPS levels in the gingival crevicular fluid of a rat model of diabetes mellitus and periodontitis.

OBJECTIVE: In this study, we sought to investigate the dynamic changes in the levels of TNF-α, IL-1ß and LPS in the gingival crevicular fluid (GCF) in a rat model of diabetes mellitus (DM) and periodontitis (PD). Additionally, we evaluated alveolar bone loss and the histopathological response associated with experimental diabetes mellitus and experimental periodontitis. METHODS: DM and PD were induced together in 15 rats (group 1) by streptozotocin injection and ligature induction. Periodontitis alone was produced by ligature induction in 15 rats (group 2), diabetes alone was produced by streptozotocin injection in 15 rats (group 3), and fifteen systemically and periodontally healthy rats were used as controls (group 4). The gingival TNF-α, IL-1ß and LPS levels were measured by using ELISA method. Periodontal destruction was assessed by measuring the alveolar bone loss. Periodontal inflammation was quantified by histopathological grading in H&E stained samples. RESULTS: Higher levels of TNF-α, IL1-ß and LPS, increased alveolar bone loss and more serve histopathology were found in group 1 compared with group 2, group 3 and group 4 (p< 0.05). The quantities of TNF-α, IL1-ß and LPS, the amount of alveolar bone loss and the severity of the histopathological finding were greater in group 2 than group 3 and group 4 (p< 0.05). Group 3 demonstrated higher levels of TNF-α, IL1-ß and LPS, increased alveolar bone loss and more serve histopathology than group 4 (p< 0.05). Statistically significant differences were noted between all of the groups. CONCLUSIONS: These data indicate that DM may lead to enhanced TNF-α, IL1-ß and LPS production in the periodontal tissues. The resorption values of alveolar bone and the histological inflammation were more severe in rats with periodontitis and diabetes mellitus than in those with periodontitis alone, diabetes mellitus alone and control rats. Our findings are consistent with the hypothesis that hyperglycemia contributes to the heightened inflammatory response associated with periodontitis.