SCF(Fbxo22)-KDM4A targets methylated p53 for degradation and regulates senescence.

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCF(Fbxo22)-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCF(Fbxo22) ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCF(Fbxo22)-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22(-/-) mice are almost half the size of Fbxo22(+/-) mice owing to the accumulation of p53. These results indicate that SCF(Fbxo22)-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.

Neovibsanin B inhibits human malignant brain tumor cell line proliferation and induces apoptosis.

The present study was designed to examine the effect of neovibsanin B on glioma cell viability, apoptosis and on the survival time in mice bearing tumor xenografts. The results demonstrated that neovibsanin B significantly reduced the cell viability of GL261-NS and GL261-AC cells in a dose-dependent manner. However the inhibition of proliferation was more significant in GL261-NS cells. The IC50 value of neovibsanin B against GL261-NS and GL261-AC cells is 5 and 25 nM, respectively. The inhibitory effect of neovibsanin B on cell growth was more effective than that of vincristine (VCR) (P < 0.05). We also observed a significant decrease in sphere-forming ability of GL261-NS cells on treatment with neovibsanin B. The number of colonies formed by GL261-NS cells on treatment with neovibsanin B, VCR and DMSO were 3.34 ± 1.02, 12.53 ± 3.46 and 61.34 ± 9.89% respectively after 7 days. The flow cytometry revealed a marked increase in apoptotic cell death of GL261-NS cells on treatment with neovibsanin B. The western blots showed a significant decrease in the level of activated caspase-3 on treatment with neovibsanin B after 24 h. In addition, neovibsanin B increased the median survival time of glioma-bearing mice (P < 0.05). Therefore, neovibsanin B effectively inhibits glioma cell viability by inducing apoptosis, and can be a potent therapeutic agent for the treatment of malignant glioma.

Specific cellular immune response elicited by the necrotic tumor cell-stimulated macrophages.

OBJECTIVE: To determine whether the necrotic tumor cell-stimulated macrophages (NTCSM) could elicit specific immune response. METHODS: Mice were immunized with the necrotic H22 tumor cell lysate-stimulated macrophages and the specific immune responses against the same tumor challenge were examined. The morphologic characteristics were observed with the transmission electron microscope and scanning electron microscopy. The expression of CD14, CD68, CD80 and CD86 were detected with the flow cytometer. The cytotoxicity and cytokine production of splenocytes were measured with the MTT assay and ELISA assay respectively. RESULTS: Our research results reveal that NTCSMs are larger cells which generally generate spherical and elongated protrusions, folding membrane, and vesicles on their surface. Also, abundant lysosomes, secondary lysosomes, phagosomes, rough endoplasmic reticulum, and lipid bodies were found in their cytoplasm. The flow cytometry results show that the necrotic H22 tumor cell lysate could enhance the expression of CD14 and CD86 molecules and the NTCSM was characterized by the expression of CD14+/-CD68+CD80-CD86+. After the mice were vaccinated with NTCSMs, the tumor forming rate, tumor volume and weight of the NTCSM-vaccinated group were significantly lower than those of the sterile saline-injected group and untreated macrophage-vaccinated group (p<0.05). The cytotoxicity to H22 tumor cells of the splenocytes obtained from the NTCSM-immunized group was higher than that of the sterile saline-injected group and untreated macrophage-vaccinated group (p<0.05). Meanwhile, the levels of IL-2 and IFN-γ in the culture supernatant of the NTCSM-immunized group were higher significantly than those of the saline-injected group and untreated macrophage-vaccinated group. The level of IL-4 of the NTCSM-immunized group was significantly lower than those of the other two groups. CONCLUSION: Our results indicated that NTCSMs could elicit specific cellular immune responses in vivo.

Nicotinamide overload may play a role in the development of type 2 diabetes.

AIM: To investigate whether nicotinamide overload plays a role in type 2 diabetes. METHODS: Nicotinamide metabolic patterns of 14 diabetic and 14 non-diabetic subjects were compared using HPLC. Cumulative effects of nicotinamide and N(1)-methylnicotinamide on glucose metabolism, plasma H(2)O(2) levels and tissue nicotinamide adenine dinucleotide (NAD) contents of adult Sprague-Dawley rats were observed. The role of human sweat glands and rat skin in nicotinamide metabolism was investigated using sauna and burn injury, respectively. RESULTS: Diabetic subjects had significantly higher plasma N(1)-methylnicotinamide levels 5 h after a 100-mg nicotinamide load than the non-diabetic subjects (0.89 +/- 0.13 micromol/L vs 0.6 +/- 0.13 micromol/L, P < 0.001). Cumulative doses of nicotinamide (2 g/kg) significantly increased rat plasma N(1)-methylnicotinamide concentrations associated with severe insulin resistance, which was mimicked by N(1)-methylnicotinamide. Moreover, cumulative exposure to N(1)-methylnicotinamide (2 g/kg) markedly reduced rat muscle and liver NAD contents and erythrocyte NAD/NADH ratio, and increased plasma H(2)O(2) levels. Decrease in NAD/NADH ratio and increase in H(2)O(2) generation were also observed in human erythrocytes after exposure to N(1)-methylnicotinamide in vitro. Sweating eliminated excessive nicotinamide (5.3-fold increase in sweat nicotinamide concentration 1 h after a 100-mg nicotinamide load). Skin damage or aldehyde oxidase inhibition with tamoxifen or olanzapine, both being notorious for impairing glucose tolerance, delayed N(1)-methylnicotinamide clearance. CONCLUSION: These findings suggest that nicotinamide overload, which induced an increase in plasma N(1)-methylnicotinamide, associated with oxidative stress and insulin resistance, plays a role in type 2 diabetes.

[VEGF165-induced angiogenesis by regulating intracellular free Mg2+ in HUVECs].

AIM: The mechanism of vascular endothelial growth factor165 (VEGF165) on intracellular free magnesium ([Mg2+]i) in human umbilical vein endothelial cells (HUVECs) was investigated. METHODS: [Mg2+]i in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected the use of intracellular cation measurement system. RESULTS: VEGF165 significantly increased [Mg2+]i in the extracellular Mg2+ and this effect could be blocked by pretreatment with tyrosine kinase inhibitors (tyrphostin A23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, phospholipase Cgamma (PLCgamma) inhibitor analog (U73343), mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the VEGF165-induced [Mg2+]i increase. CONCLUSION: The increase of [Mg2+]i by VEGF165 originates from intracellular Mg2+ pool through tyrosine kinase/ PI3K/PLCgamma-dependent signaling pathways.

[Evidence for a major role of Mg2+ in VEGF165-mediated angiogenesis].

OBJECTIVE: The effect of vascular endothelial growth factor(165) (VEGF(165)) on intracellular free magnesium ([Mg(2+)](i)) and the relationship between Mg(2+) and angiogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. METHODS: [Mg(2+)](i) in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected with the use of intracellular cation measurement system. HUVECs were obtained from normal fetus and cultured in M199 with 0.2 fetal bovine serum. The angiogenesis effects of VEGF(165) were observed in presence of 0 mmol/L, 1 mmol/L or 2 mmol/L of extracellular Mg(2+). RESULTS: VEGF(165) significantly increased [Mg(2+)](i) in a dose-dependent manner independent of extracellular Mg(2+), Na(+) and Ca(2+) and this effect could be blocked by pretreatment with VEGF(165) receptor-2 (KDR) inhibitor (SU1498). The angiogenesis induced by VEGF(165) was significantly inhibited cells with 0 mmol/L extracellular Mg(2+), the angiogenesis effects of VEGF(165) were similar in cells with 1 mmol/L and 2 mmol/L extracellular Mg(2+) and these effects could be blocked by SU1498. CONCLUSIONS: These results suggest that the [Mg(2+)](i) increase induced by VEGF(165) originates from intracellular Mg(2+) pools and promotes angiogenesis via KDR-dependent signaling pathways.

[Labia minora skin flap vaginoplasty using tissue expansion].

OBJECTIVE: This is to introduce a method for vagina reconstruction using the expanded labia minora flap. METHODS: Two tissue expanders were implanted in the labia minora bilaterally and expanded slowly over 4 weeks. In the operation, the expanded labial tissue was advanced as a bipedicle flap to line the reconstructed vagina. Five patients were treated with this method. Postoperative stent placement and dilation resulted in a vaginal canal exceeding 8 cm in depth. RESULTS: During the follow-up of 6 months to 2 years, four of the five patients got married. The vulva exhibited almost indistinguishable appearance. The reconstructed vagina had sensory and secretary functions. Its morphology and depth well meet the physiological demand. CONCLUSIONS: The modified method of tissue expansion vaginoplasty using the labia minora bipedicle flap is a good option for vagina surgery. The reconstructed vagina possesses the anatomical and physiological resemblance.