X-ray irradiation promotes apoptosis of hippocampal neurons through up-regulation of Cdk5 and p25.

BACKGROUND: Cranial radiation therapy has been used for the treatment of primary and metastatic brain tumors. A prominent feature of brain injury induced by the radiation therapy is hippocampal dysfunction, characterized by a decline in memory. Cdk5 plays an important role in memory formation. Abnormal Cdk5 activity is associated with neuronal apoptosis induced by neurotoxic stimuli. However, the roles of Cdk5 in hippocampal apoptosis in response to X-ray irradiation have not been explored. METHODS: The expression of Cdk5 activators, p35 and p25, in hippocampal neurons was tested in both in vivo animal and in vitro couture after X-ray irradiation. RESULTS: After X-ray irradiation at 20 Gy and 30 Gy in rats, the number of hippocampal neuronal pyknosis was increased, but the number of hippocampal neuron was decreased, in the hippocampal CA1 region of rats. In these animals undergone with X-ray irradiation, the expression of p35 was significantly down-regulated, but it was up-regulated in p25. These opposite expressions were also shown in the primary cultured hippocampal neurons with 30 Gy irradiation. The apoptosis induced by X-ray irradiation were significantly prevented by the pretreatment of Cdk5 inhibitor, roscovitine, in both in vivo and in vitro settings. CONCLUSIONS: X-ray irradiation resulted in a hippocampal neuronal apoptosis through up-regulation of p25, the Cdk5 activator. Hyperactivity of Cdk5 was involved in the pathogenesis of X-ray irradiation-induced hippocampal neuronal apoptosis. Blockade of Cdk5 signal pathway effectively protected neurons from the irradiation-induced brain injury.

3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-one (CHP) inhibits human ovarian cell proliferation by inducing apoptosis.

Coumarins induce apoptosis by activating mitochondrial pathway and caspase-3-dependent apoptotic pathway. In the present study, we first time investigated the effect of 3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-one (CHP) on induction of apoptosis in human ovarian carcinoma cells. The data from MTT assay revealed a significant inhibitory effect on cell viability at 30 (87%) and 50 µM (74%) concentration of CHP in OVCAR-3 and OVCAR-420 cells, respectively after 72 h. Apoptosis analysis using annexin V/PI double staining followed by flow cytometry showed 59 and 52% binding to annexin V-FITC in OVCAR-3 and OVCAR-420 cells respectively. propidium iodide (PI) staining and flow cytometry examination indicated a significant increase in percentage of cells in G2/M phase after treatment with CHP compared to DMSO control group. Reactive oxygen species (ROS) assay kit showed increase in levels of ROS. We used rhodamine-123 (Rh-123) staining and flow cytometry assay to determine changes in mitochondrial membrane potential (ΛΨm). The results revealed that CHP significantly decreased MMP to 85.65 ± 1.2443% & 49.78 ± 1.6554% at 10 and 30 µM respectively in OVCAR-3 compared to 95.97 ± 2.1243% in control group. Western blot analysis clearly indicated a significant increase in the expression of Caspase-3, Bax, and release of Cytochrome c and decrease in Bcl-2, CDK1 and Cyclin B1 expression on treatment with CHP. Therefore, CHP may become a potential candidate for the treatment of human ovarian cancers.

[p35 and p25 expressions and Cdk5 kinase activity in primary cultured rat hippocampal neurons with X-ray exposure].

OBJECTIVE: To study the expressions of p35 and p25 and Cdk5 kinase activity in cultured rats hippocampal neurons following X-ray exposure to provide experimental evidence for prevention and treatment of radiation encephalopathy. METHODS: The hippocampal neurons cultured for 12 days were subjected to a single-dose X-ray exposure of 30 Gy. Western blotting was used to detect the p35 and p25 protein levels, and the effect of pretreatment with roscovitine, a Cdk5 inhibitor, on the apoptosis of the hippocampal neurons following the exposure was examined with 4',6-diamidino-2-phenylindole (DAPI) staining. RESULTS: The protein level of p35 increased significantly 3.5 and 4 h after the irradiation by 1.51-/+0.13 and 1.45-/+0.14 folds in comparison with the control level, respectively (P<0.01), and the p25 level increased significantly 6 h after irradiation by 1.62-/+0.28 folds (P<0.05). Nuclear condensation occurred in (24.8-/+3.97)% of the neurons 24 h after 30 Gy X-ray exposure, a rate significantly higher than that in the nonexposed cells [(1.82-/+1.08)%, P<0.01) and that in roscovitine-pretreated neurons [(7.74-/+2.27)%, P<0.01). CONCLUSION: X-ray exposure activates Cdk5 by increasing the p35 and p25 expressions in rat hippocampal neurons, and inhibition of Cdk5 activity with roscovitine can significantly protect the neurons from apoptosis.