Loss of parkin reduces lung tumor development by blocking p21 degradation.

Several epidemiological studies have demonstrated the reciprocal relationship between the development of cancer and Parkinson's disease (PD). However, the possible mechanisms underlying this relationship remain unclear. To identify this relationship, we first compared lung tumor growth in parkin knockout (KO) mice and wild-type (WT) mice. Parkin KO mice showed decreased lung tumor growth and increased expression of p21, a cell cycle arrester, as compared with WT mice. We also found that parkin interacts with p21, resulting in its degradation; however, parkin KO, knockdown, as well as mutation (R275W or G430D) reduced the degradation of p21. We investigated whether parkin KO increases the association of p21 with proliferating cell nuclear antigen (PCNA) or CDK2 by reducing p21 degradation, and, thus, arresting the cell cycle. The interaction between p21 and PCNA or CDK2 was also enhanced by parkin knockdown, and this increased interaction induced sub G0/G1 arrest, leading to cell death. Therefore, our data indicate that parkin KO reduces the development of lung tumors via cell cycle arrest by blocking the degradation of p21. These findings suggest that PD could be associated with lower lung cancer incidence.

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21.

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

Tumor growth suppressive effect of IL-4 through p21-mediated activation of STAT6 in IL-4Rα overexpressed melanoma models.

To evaluate the significance of interleukin 4 (IL-4) in tumor development, we compared B16F10 melanoma growth in IL-4-overespressing transgenic mice (IL-4 mice) and non-transgenic mice. In IL-4 mice, reduced tumor volume and weight were observed when compared with those of non-transgenic mice. Significant activation of DNA binding activity of STAT6, phosphorylation of STAT6 as well as IL-4, IL-4Rα and p21 expression were found in the tumor tissues of IL-4 mice compared to non-transgenic mice. Higher expression of IL-4, STAT6 and p21 in human melanoma tissue compared to normal human skin tissue was also found. Higher expression of apoptotic protein such as cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, p53 and p21, but lower expression levels of survival protein such as Bcl-2 were found in the tumor of IL-4 mice. In vitro study, we found that overexpression of IL-4 significantly inhibited SK-MEL-28 human melanoma cell and B16F10 murine melanoma cell growth via p21-mediated activation of STAT6 pathway as well as increased expression of apoptotic cell death proteins. Moreover, p21 knockdown with siRNA abolished IL-4 induced activation of STAT6 and expression of p53 and p21 accompanied with reduced IL-4 expression as well as melanoma cell growth inhibition. Therefore, these results showed that IL-4 overexpression suppressed tumor development through p21-mediated activation of STAT6 pathways in melanoma models.

Deficiency of C-C chemokine receptor 5 suppresses tumor development via inactivation of NF-κB and inhibition of monocyte chemoattractant protein-1 in urethane-induced lung tumor model.

To evaluate the significance of C-C chemokine receptor type 5 (CCR5) in lung tumor development, we compared carcinogen-induced tumor growth in CCR5 knockout (CCR5(-/-)) mice and wild-type (CCR5(+/+)) mice. CCR5(-/-) mice showed reduced urethane (1g/kg)-induced tumor incidence when compared with those of CCR5(+/+) mice. We investigated the activation of nuclear factor-kappaB/STAT3 since these are implicated transcription factors in the regulation of genes involving tumor growth. Significant inhibition of DNA-binding activity of nuclear factor-kappaB and STAT3, and the translocation of p50 and p65 into the nucleus and the phosphorylation of IĸB were found in the lungs of CCR5(-/-) mice compared with the lungs of CCR5(+/+) mice. Expression of apoptotic protein such as cleaved caspase-3, cleaved PARP and Bax was elevated, whereas the expression levels of survival protein such as Bcl-2 and cIAP1 was decreased in the lungs of CCR5(-/-) mice. Interestingly, we found that the level of monocyte chemoattractant protein-1 (MCP-1), a tumor growth-promoting cytokine, was significantly reduced in the lung tumor tissue and blood of CCR5(-/-) mice compared with the level in CCR5(+/+) mice. In addition, CCR5 small interfering RNA (siRNA) and inhibitor of MCP-1 blocked lung cancer cell growth, which was abolished by the addition of MCP-1 protein in cultured lung cancer cells. Moreover, inactivation of CD8(+) cytotoxic T cell and dendritic cells was significantly increased in the blood, lung tumors and spleens of CCR5(-/-) mice compared with that of CCR5(+/+) mice. Therefore, these results showed that CCR5 deficiency suppressed lung tumor development through the inhibition of nuclear factor-kappaB/STAT3 pathways and the downregulation of MCP-1 in the carcinogen-induced lung tumor model.

Cell growth inhibition and induction of apoptosis by snake venom toxin in ovarian cancer cell via inactivation of nuclear factor κB and signal transducer and activator of transcription 3.

Snake venom toxin from Vipera lebetina turanica induces apoptosis in many cancer cell lines, but there is no study about the apoptotic effect of snake venom toxin on human ovarian cancer cells. In this study, we investigated the apoptotic effect of snake venom toxin in human ovarian cancer PA-1 and SK-OV3 cells. Snake venom toxin dose dependently (0∼10 µg/mL) inhibited ovarian cancer cell growth with IC(50) values 4.5 µg/mL in PA-1 cells, and 6.5 µg/mL in SK-OV3 cells. Our results also showed that apoptotic cell death increased by snake venom toxin in a dose dependent manner (0∼10 µg/mL). Consistent with increased cell death, snake venom toxin increased the expression of pro-apoptotic protein Bax and caspase-3, but down-regulated anti-apoptotic protein Bcl-2. Untreated ovarian cancer cells showed a high DNA binding activity of nuclear factor B (NF-κB), but it was inhibited by snake venom toxin accompanied by inhibition of p50 and p65 translocation into the nucleus as well as phosphorylation of inhibitory κB. Snake venom toxin also inhibited DNA binding activity of the signal transducer and activator of transcription 3 (STAT3). Moreover, the combination treatment of NF-κB (salicylic acid, 1 or 5 µM) and STAT3 (stattic, 1 µM) with snake venom toxin (1 µg/mL) further enhanced cell growth inhibitory effects of snake venom toxin. These results showed that snake venom toxin from Vipera lebetina turanica caused apoptotic cell death of ovarian cancer cells through the inhibition of NF-κB and STAT3 signal, and suggested that snake venom toxin may be applicable as an anticancer agent for ovarian cancer.

Deficiency of C-C chemokine receptor 5 suppresses tumor development via inactivation of NF-κB and upregulation of IL-1Ra in melanoma model.

To evaluate the relevance of C-C chemokine receptor type 5 (CCR5) expression and tumor development, we compared melanoma growth in CCR5 knockout (CCR5(-/-)) mice and wild type (CCR5(+/+)) mice. CCR5(-/-) mice showed reduced tumor volume, tumor weight, and increased survival rate when compared to CCR5(+/+) mice. We investigated the activation of NF-κB since it is an implicated transcription factor in the regulation of genes involving cell growth, apoptosis, and tumor growth. Significant inhibition of DNA binding activity of NF-κB, and translocation of p50 and p65 into the nucleus through the inhibition of phosphorylation of IκB was found in the melanoma tissues of CCR5(-/-) mice compared to melanoma tissues of CCR5(+/+) mice. NF-κB target apoptotic protein expression, such as cleaved caspase-3, cleaved PARP, and Bax, was elevated, whereas the survival protein expression levels, such as Bcl-2, C-IAP1, was decreased in the melanoma tissues of CCR5(-/-) mice. Interestingly, we found that the level of IL-1Ra, a tumor growth suppressive cytokine, was significantly elevated in tumor tissue and spleen of CCR5(-/-) mice compared to the level in CCR5(+/+) mice. Moreover, infiltration of CD8(+) cytotoxic T cell and CD57(+) natural killer cells was significantly increased in melanoma tumor and spleen tissue of CCR5(-/-) mice compared to that of CCR5(+/+) mice. Therefore, these results showed that CCR5 deficiency caused apoptotic cell death of melanoma through inhibition of NF-κB and upregulation of IL-1Ra.

4-O-Methylhonokiol attenuates memory impairment in presenilin 2 mutant mice through reduction of oxidative damage and inactivation of astrocytes and the ERK pathway.

Presenilin 2 (PS2) mutation increases Aß generation and neuronal cell death in the brains of Alzheimer disease (AD) patients. In a previous study, we showed that increased oxidative damage and activation of extracellular signal-regulated kinase (ERK) were associated with Aß generation and neuronal cell death in neuronal cells expressing mutant PS2. In this study, we show that oral treatment with 4-O-methylhonokiol, a novel compound isolated from Magnolia officinalis, for 3 months (1.0mg/kg) prevented PS2 mutation-induced memory impairment and neuronal cell death accompanied by a reduction in Aß(1-42) accumulation. We also found that 4-O-methylhonokiol inhibited PS2 mutation-induced activation of ERK and ß-secretase, and oxidative protein and lipid damage, but recovered glutathione levels in the cortex and hippocampus of PS2 mutant mice. Additionally, 4-O-methylhonokiol prevented PS2 mutation-induced activation of astrocytes as well as production of TNF-α, IL-1ß, reactive oxygen species (ROS), and nitric oxide (NO) in neurons. Generation of TNF-α, IL-1ß, ROS, and NO and ERK activation in cultured astrocytes treated with lipopolysaccharide (1µg/ml) were also prevented by 4-O-methylhonokiol in a dose-dependent manner. These results suggest that the improving effects of 4-O-methylhonokiol on memory function may be associated with a suppression of the activation of ERK and astrocytes as well as a reduction in oxidative damage. Thus, 4-O-methylhonokiol may be useful in the prevention and treatment of AD.

Neurotrophic activity of obovatol on the cultured embryonic rat neuronal cells by increase of neurotrophin release through activation of ERK pathway.

Previously, we found that obovatol, a lignan compound isolated from Magnolia officinalis, has anti-cancer, anti-inflammatory, and anxiolytic effects. Recent studies showed that honokiol, magnolol, and 4-O-methylhonokiol, lignin compounds isolated from the Magnolia family have neurotrophic activity. In this study, we examined whether or not obovatol also exhibits neurite-promoting effects on rat embryonic neuronal cells. Obovatol increased neurite outgrowth in a concentration-dependent manner. Consistent with the neurite outgrowth effect, the expression of neurite differentiation markers also increased in response to obovatol. We also found that obovatol increased levels of NGF and BDNF released into the culture medium. In addition, the combination of low concentrations of obovatol (1 and 2 µM) with NGF (50 ng/ml) or with BDNF (10 ng/ml) greatly enhanced neurite outgrowth. Subsequently, we found that obovatol increased phosphorylation of ERK. However, the neurite outgrowth, and NGF and BDNF release induced by obovatol were prevented by an ERK-specific inhibitor. These results suggest that obovatol promotes neurite outgrowth due to the increased release of neurotrophic factors via activation of the ERK pathway.

Two cases of eosinophilic vasculitis with thrombosis.

Vasculitides are characterized by vessel wall inflammation of unknown etiology. We report two cases of eosinophilic vasculitis and hypereosinophilia with thrombosis. They have been treated with a high-dose glucocorticoid and anticoagulation. These cases emphasize that thrombosis should be anticipated in patients with eosinophilic vasculitis.

Associations between interferon regulatory factor-1 polymorphisms and Behçet's disease.

Interferon regulatory factor-1 (IRF-1) is a transcription factor that regulates the functions of type I and II interferons and plays a role in host protection. Behçet's disease (BD) is an idiopathic systemic vasculitis that is often complicated with thrombotic features, and infectious agents have long been postulated to be a disease-triggering factor in its pathogenesis. The authors investigated the distributions of IRF-1 promoter -415 C/A, -410 A/G, and -300 A/G, and 3'-untranslated region (UTR) A/G polymorphisms in 105 BD patients (mean age 41.7 +/- SEM 1.1 years, 44 male and 61 female) and in 105 gender- and age-matched healthy controls. The frequencies of individual alleles and genotypes were not different between the control and BD groups. However, the frequency of AGGG haplotype was significantly higher (73.5% vs 60.2%, odds ratio [OR] = 1.842, 95% confidence interval [95% CI] = 1.219-2.783, p(c) = 0.036) and that of the CAAG haplotype was significantly lower (2.2% vs 9.5%, OR = 0.195, 95% CI = 0.068-0.559, p(c) = 0.02) in BD patients than in healthy controls. In addition, the frequency of the AGGG haplotype was significantly higher (80.3% vs 57.4%, OR = 3.033, 95% CI = 1.716-5.360, p(c) = 0.001) and that of the CAAG haplotype was significantly lower (0.8% vs 12.3%; OR = 0.059, 95% CI = 0.010-0.357, p(c) = 0.005) in female BD patients than female controls. By subgroup analyses, the CAAA haplotype tended to be more common in BD patients with moderate or severe disease than in those with mild disease (25.4% vs 13.6%, OR = 2.158, 95% CI = 1.046-4.440, p = 0.037 before Bonferroni correction). When BD patients were subclassified by a history of deep vein thrombosis (DVT), the CAAA haplotype was found to be significantly increased the risk of DVT (42.1% vs 15.7%, OR = 3.906, 95% CI = 1.836-8.324, p(c) = 0.0015) and the AGGG haplotype tended to reduce this risk (57.9% vs 77.3%, OR = 0.403, 95% CI = 0.195-0.834, p(c) = 0.0685). Furthermore, the frequency of the CAAA haplotype was significantly higher in BD patients that had experienced a thrombotic event than in those that had not (40.5% vs 15.5%, OR = 3.7147, 95% CI = 1.778-7.770, p(c) = 0.0015). These results suggest that IRF-1 is a novel susceptibility gene in BD, especially in women, and furthermore, that IRF-1 polymorphisms may be related to thrombosis in BD patients.