A phase I study of docetaxel plus synthetic lycopene in metastatic prostate cancer patients.

PURPOSE: Our preclinical studies showed that lycopene enhanced the anti-prostate cancer efficacy of docetaxel in animal models. A phase I trial (NCT0149519) was conducted to identify an optimum dose of synthetic lycopene in combination with docetaxel (and androgen blockade [androgen deprivation therapy, ADT]), and to evaluate its effect on the safety and pharmacokinetics of docetaxel in men with metastatic prostate cancer. METHODS: Subjects were treated with 21-day cycles of 75 mg/m2 docetaxel (and ADT), plus lycopene at 30, 90 or 150 mg/day. A Bayesian model averaging continual reassessment method was used to guide dose escalation. Pharmacokinetics of docetaxel and multiple correlative studies were carried out. RESULTS: Twenty-four participants were enrolled, 18 in a dose escalation cohort to define the maximum tolerated dose (MTD), and six in a pharmacokinetic cohort. Docetaxel/ADT plus 150 mg/day synthetic lycopene resulted in dose-limiting toxicity (pulmonary embolus) in one out of 12 participants with an estimated probability of .106 and thus was chosen as the MTD. Lycopene increased the AUCinf and Cmax of plasma docetaxel by 9.5% and 15.1%, respectively. Correlative studies showed dose-related changes in circulating endothelial cells and vascular endothelial growth factor A, and reduction in insulin-like growth factor 1R phosphorylation, associated with lycopene therapy. CONCLUSIONS: The combination of docetaxel/ADT and synthetic lycopene has low toxicity and favourable pharmacokinetics. The effects of lycopene on biomarkers provide additional support for the toxicity-dependent MTD definition. HIGHLIGHTS: The maximum tolerated dose was identified as 150 mg/day of lycopene in combination with docetaxel/ADT for the treatment of metastatic prostate cancer patients. Small increases in plasma exposure to docetaxel were observed with lycopene co-administration. Mechanistically significant effects were seen on angiogenesis and insulin-like growth factor 1 signalling by lycopene co-administration with docetaxel/ADT.

ATPase Copper Transporting Beta (ATP7B) Is a Novel Target for Improving the Therapeutic Efficacy of Docetaxel by Disulfiram/Copper in Human Prostate Cancer.

Docetaxel has been the standard first-line chemotherapy for lethal metastatic prostate cancer (mPCa) since 2004, but resistance to docetaxel treatment is common. The molecular mechanisms of docetaxel resistance remain largely unknown and could be amenable to interventions that mitigate resistance. We have recently discovered that several docetaxel-resistant mPCa cell lines exhibit lower uptake of cellular copper and uniquely express higher levels of a copper exporter protein ATP7B. Knockdown of ATP7B by silencing RNAs (siRNA) sensitized docetaxel-resistant mPCa cells to the growth-inhibitory and apoptotic effects of docetaxel. Importantly, deletions of ATP7B in human mPCa tissues predict significantly better survival of patients after their first chemotherapy than those with wild-type ATP7B (P = 0.0006). In addition, disulfiram (DSF), an FDA-approved drug for the treatment of alcohol dependence, in combination with copper, significantly enhanced the in vivo antitumor effects of docetaxel in a docetaxel-resistant xenograft tumor model. Our analyses also revealed that DSF and copper engaged with ATP7B to decrease protein levels of COMM domain-containing protein 1 (COMMD1), S-phase kinase-associated protein 2 (Skp2), and clusterin and markedly increase protein expression of cyclin-dependent kinase inhibitor 1 (p21/WAF1). Taken together, our results indicate a copper-dependent nutrient vulnerability through ATP7B exporter in docetaxel-resistant prostate cancer for improving the therapeutic efficacy of docetaxel.

Dysfunction of the aging female mouse urethra is associated with striated muscle loss and increased fibrosis: an initial report.

The decline of urethral function with advancing age plays a major role in urinary incontinence in women, impairing quality of life and economically burdening the health care system. However, none of the current urinary incontinence treatments address the declining urethral function with aging, and the mechanisms by which aging impacts urethra physiology remain little known or explored. Here, we have compared functional, morphometric, and global gene expression of urethral tissues between young and old female mice. Bladder leak point pressure (LPP) measurement showed that the aged female mice had 26.55% lower LPP compared to younger mice. Vectorized Scale-Invariant Pattern Recognition (VIPR) analysis of the relative abundance of different tissue components revealed that the mid-urethra of old female mice contains less striated muscle, more extracellular matrix/fibrosis, and diminished elastin fibers ratio compared to young mice. Gene expression profiling analysis (bulk RNA-seq of the whole urethra) showed more down-regulated genes in aged than young mice. Immune response and muscle-related (striated and smooth) pathways were predominantly enriched. In contrast, keratinization, skin development, and cell differentiation pathways were significantly downregulated in aged urethral tissues compared to those from young female mice. These results suggest that molecular pathways (i.e., ACVR1/FST signaling and CTGF/TGF-ß signaling) leading to a decreased striated muscle mass and an increase in fibrous extracellular matrix in the process of aging deserve further investigation for their roles in the declined urethral function.

The Potent Anti-Tumor Effects of Rhodiola Drinking Are Associated with the Inhibition of the mTOR Pathway and Modification of Tumor Metabolism in the UPII-Mutant Ha-Ras Model.

Background: SHR-5 has been used as an "adaptogen" for enhancing physical and mental performance and for fighting stress in the healthy population. The purpose of this study is to determine the chemopreventive efficacy of SHR-5 for superficial bladder cancer and to investigate the underlying mechanisms of action. Methods: UPII-mutant Ha-ras bladder-cancer-transgenic mice, that developed low-grade and noninvasive papillary transitional urothelial cell carcinoma, were fed with 1.25 and 6.25 mg/mL SHR-5 in drinking water for 6 months. The survival of the mice, obstructive uropathy, tumor burden and morphology, and proliferation were evaluated by pathological, molecular, metabolic, and statistical analyses. Results: Approximately 95% or more of the male UPII-mutant Ha-ras mice that drank SHR-5 daily survived over 6 months of age, while only 33.3% of those mice that drank normal water survived over 6 months of age (p < 0.0001); SHR-5 drinking exposure also reduced tumor-bearing bladder weight and urinary tract obstruction and inhibited mTOR signaling in neoplastic tissues. Global metabolic analysis revealed that SHR-5 resulted in increased phenolic metabolites and decreased CoA, a critical metabolic cofactor for lipid metabolism. Conclusions: Our findings highlight the potential of SHR-5 as an anti-aging agent for bladder cancer prevention through reshaping tumor metabolism via the inhibition of the mTOR signaling. Global metabolomics profiling provides a unique and efficient tool for studying the mechanisms of complex herb extracts' action.

Kawain Inhibits Urinary Bladder Carcinogenesis through Epigenetic Inhibition of LSD1 and Upregulation of H3K4 Methylation.

Epidemiological evidence suggests that kava (Piper methysticum Forst) drinks may reduce the risk of cancer in South Pacific Island smokers. However, little is known about the anti-carcinogenic effects of kava on tobacco smoking-related bladder cancer and its underlying mechanisms. Here we show that dietary feeding of kawain (a major active component in kava root extracts) to mice either before or after hydroxy butyl(butyl) nitrosamine (OH-BBN) carcinogen exposure slows down urinary bladder carcinogenesis and prolongs the survival of the OH-BBN-exposed mice. OH-BBN-induced bladder tumors exhibit significantly increased expression of lysine-specific demethylase 1 (LSD1), accompanied by decreased levels of H3K4 mono-methylation compared to normal bladder epithelium, whereas dietary kawain reverses the effects of OH-BBN on H3K4 mono-methylation. Human bladder cancer tumor tissues at different pathological grades also show significantly increased expression of LSD1 and decreased levels of H3K4 mono-methylation compared to normal urothelium. In addition, kava root extracts and the kavalactones kawain and methysticin all increase the levels of H3K4 mono- and di-methylation, leading to inhibitory effects on cell migration. Taken together, our results suggest that modification of histone lysine methylation may represent a new approach to bladder cancer prevention and treatment and that kavalactones may be promising agents for bladder cancer interception in both current and former smokers.

Kavalactone Kawain Impedes Urothelial Tumorigenesis in UPII-Mutant Ha-Ras Mice via Inhibition of mTOR Signaling and Alteration of Cancer Metabolism.

UPII-mutant Ha-ras transgenic mice develop urothelial hyperplasia and low-grade papillary carcinoma, which mimics human non-muscle invasive bladder cancer (NMIBC). We investigated the effects and mechanisms of kawain, a main kavalactone in the kava plant, on oncogenic Ha-ras-driven urothelial carcinoma in these mice. The mice were fed at six weeks of age with vehicle control or kawain (6 g/kg) formulated food for approximately five months. Seventy-eight percent of the mice or more fed with kawain food survived more than six months of age, whereas only 32% control food-fed male mice survived, (p = 0.0082). The mean wet bladder weights (a surrogate for tumor burden) of UPII-mutant Ha-ras transgenic mice with kawain diet was decreased by approximately 56% compared to those fed with the control diet (p = 0.035). The kawain diet also significantly reduced the occurrence of hydronephrosis and hematuria in UPII-mutant Ha-ras transgenic mice. Histological examination and immunohistochemistry analysis revealed that vehicle control-treated mice displayed more urothelial carcinoma and Ki67-positive cells in the bladder compared to kawain treated mice. Global metabolic profiling of bladder tumor samples from mice fed with kawain food showed significantly more enrichment of serotonin and less abundance of xylulose, prostaglandin A2, D2 and E2 compared to those from control diet-fed mice, suggesting decreased shunting of glucose to the pentose phosphate pathway (PPP) and reduced inflammation. In addition, kawain selectively inhibited the growth of human bladder cancer cell lines with a significant suppression of 4E-BP1 expression and rpS6 phosphorylation. These observations indicate a potential impact of kawain consumption on bladder cancer prevention by rewiring the metabolic programs of the tumor cells.

Prostate cancer immunotherapy: a review of recent advancements with novel treatment methods and efficacy.

Immunotherapy remains to be an appealing treatment option for prostate cancer with some documented promise. Prostate cancer is traditionally considered as an immunologically "cold" tumor with low tumor mutation burden, low expression of PD-L1, sparse T-cell infiltration, and a immunosuppressive tumor microenvironment (TME). Sipuleucel-T (Provenge) is the first FDA approved immunotherapeutic agent for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC); demonstrating a benefit in overall survival. However various clinical trials by immune checkpoint inhibitors (ICIs) and their combinations with other drugs have shown limited responses in mCRPC. Up to now, only a small subset of patients with mismatch repair deficiency/microsatellite instability high and CDK12 mutations can clinically benefit from ICIs and/or their combinations with other agents, such as DNA damage agents. The existence of a large heterogeneity in genomic alterations and a complex TME in prostate cancer suggests the need for identifying new immunotherapeutic targets. As well as designing personalized immunotherapy strategies based on patient-specific molecular signatures. There is also a need to adjust strategies to overcome histologic barriers such as tissue hypoxia and dense stroma. The racial differences of immunological responses between men of diverse ethnicities also merit further investigation to improve the efficacy of immunotherapy and better patient selection in prostate cancer.

Flavokawain A Reduces Tumor-Initiating Properties and Stemness of Prostate Cancer.

We have previously demonstrated the in vivo chemopreventive efficacy of flavokawain A (FKA), a novel chalcone from the kava plant, in prostate carcinogenesis models. However, the mechanisms of the anticarcinogenic effects of FKA remain largely unknown. We evaluated the effect of FKA on prostate tumor spheroid formation by prostate cancer stem cells, which were sorted out from CD44+/CD133+ prostate cancer cells 22Rv1 and DU145. FKA treatment significantly decreased both the size and numbers of the tumor spheroids over different generations of spheroid passages. In addition, the dietary feeding of FKA-formulated food to Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing CD44+/CD133+ 22Rv1 xenograft tumors resulted in a significant reduction of tumor growth compared to those fed with vehicle control food-fed mice. Furthermore, the expression of stem cell markers, such as Nanog, Oct4, and CD44, were markedly downregulated in both tumor spheroids and tumor tissues. We also observed that FKA inhibits Ubc12 neddylation, c-Myc, and keratin-8 expression in both CD44+/CD133+ prostate tumor spheroids and xenograft tumors. Our results suggest that FKA can reduce the tumor-initiating properties and stemness of prostate cancer, which provides a new mechanism for the chemoprevention efficacy of FKA.

Chemoprevention of Urothelial Cell Carcinoma Tumorigenesis by Dietary Flavokawain A in UPII-Mutant Ha-ras Transgenic Mice.

Non-muscle-invasive bladder cancer (NMIBC) has one of the highest recurrence rates among all solid cancers and the highest lifetime treatment cost per patient. Therefore, the development of chemoprevention strategies for reducing the occurrence and recurrence of NMIBC as well as its burdens on the healthcare system is valuable. Our aim was to determine whether flavokawain A (FKA), a kava chalcone isolated from the kava plant, can target the in vivo activated Ha-ras pathway for prevention and treatment of NMIBC. UPII-mutant Ha-ras transgenic mice that develop papillary urothelial cell carcinoma were fed orally with vehicle control or FKA-formulated food for 6 months starting at 6 weeks of age. Seventy-nine percent (15/19) of male mice fed with 6 g FKA per kilogram (kg) of food survived beyond the 6 months of treatment, while 31.6% (6/19) of control food-fed male mice survived the 6-month treatment period (p = 0.02). The mean bladder weights in FKA vs. control food-fed mice were 0.216 ± 0.033 vs. 0.342 ± 0.039 g in male mice (p = 0.0413) and 0.043 ± 0.004 vs. 0.073 ± 0.004 g in female mice (p < 0.0001); FKA reduced bladder weight by 37% and 41%, respectively. The tumor burdens, determined by the wet bladder weight, in these mice were inversely related to plasma FKA concentrations. In addition to decreased bladder weight, FKA treatment significantly reduced the incidences of hydronephrosis and hematuria. FKA-treated mice exhibited more well-differentiated tumors in the bladder and ureter. Immunohistochemical analysis of FKA-treated tumors compared to those in the control group revealed fewer Ki-67- and survivin-positive cells and an increased number of p27- and TUNEL-positive cells, indicating that FKA inhibits proliferation and induces apoptosis. Overall, the results suggest that FKA can target the in vivo activated Ha-ras pathway for the prevention and treatment of NMIBC.

Kava root extracts hinder prostate cancer development and tumorigenesis by involvement of dual inhibition of MAO-A and LSD1.

AIM: Here, we aim to evaluate the chemopreventive efficacy of kava root extracts (KRE) in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and investigate potential molecular targets of kavalactones, the main components of kava. METHODS: TRAMP mice were administrated with KRE formulated food for different periods of time, and then the incidences of high-grade prostatic intraepithelial neoplasia (HG-PIN) and adenocarcinomas and tumor burdens were compared between vehicle control and KRE food fed groups. In addition, the inhibitory effect of the KRE and kavalactones on monoamine oxidase A (MAO-A) and lysine-specific demethylase 1 (LSD1) enzyme activities were examined by commercially available inhibitor screening kits. Histone H3 lysine 9 dimethylation was also evaluated in prostate cancer cells and tumor tissues using Western blotting analysis. RESULTS: Dietary feeding of 0.3% and 0.6% KRE to TRAMP mice from ages of 6 weeks to 12 weeks inhibited HG-PIN by 43.5% and 59.7%, respectively, and prostate adenocarcinoma by 53.5% and 66.4%, respectively. In addition, 0.6% KRE fed TRAMP mice from ages of 6 weeks to 24 weeks exhibited a significant reduction of genitourinary weight (a surrogate of tumor burden) by 54.5% and reduced body weight gain. Furthermore, the KRE and kavalactones showed a significant inhibition of LSD1 and MAO-A enzyme activities. CONCLUSION: Our results suggest that consumption of kava products through diet can delay prostate cancer development and progression and that kavalactones may be a new structure model for developing a potent dual inhibitor of LSD1 and MAO-A.

Endoplasmic reticulum stress, an important factor in the development of Parkinson's disease.

Similar to other types of neuronal degeneration, Parkinson's disease (PD) is characterized by the aggregation of a pathological protein, α-synuclein. The endoplasmic reticulum (ER) is the principal site of protein synthesis, quality control and degradation. Genetic mutants, environmental insults and other factors disturb ER balance and induce the accumulation of misfolded/unfolded proteins, which initiate ER stress and disturb normal cell function. ER stress perturbs Ca2+ homeostasis and initiates the activation of autophagy and inflammasomes, which have been identified as risk factors for the development of PD. However, the mechanisms by which ER stress contributes to the processed of PD pathogenesis and development remain unclear. This review summarizes current knowledge of ER stress and highlights the principal role of ER stress in PD pathogenesis which may help reveal novel sight to illustrate the pathomechanism of PD.

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation.

BACKGROUND: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. METHODS: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. RESULTS: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. CONCLUSION: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment exerting potent antiproliferative activity through microtubule destabilization.

Novel bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment as antiproliferative agents by targeting tubulin were synthesized and their preliminary structure activity relationships (SARs) were explored. Among all these chemical agents, 2-(Benzo[d]oxazol-2-ylthio)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (4d) exhibited the potent antiproliferative activity against MGC-803 cells with an IC50 value of 0.45 µM by induction of G2/M pahse arrest and cell apoptosis. In addition, 4d could change the membrane potential (ΔΨ) of the mitochondria against MGC-803 cells. Importantly, 4d acted as a novel tubulin polymerization inhibitor binding to colchicine site with an IC50 value of 3.35 µM.

Rhodiola rosea L.: an herb with anti-stress, anti-aging, and immunostimulating properties for cancer chemoprevention.

PURPOSE OF REVIEW: Rhodiola rosea extracts have been used as a dietary supplement in healthy populations, including athletes, to non-specifically enhance the natural resistance of the body to both physical and behavior stresses for fighting fatigue and depression. We summarize the information with respect to the new pharmacological activities of Rhodiola rosea extracts and its underlying molecular mechanisms in this review article. RECENT FINDINGS: In addition to its multiplex stress-protective activity, Rhodiola rosea extracts have recently demonstrated its anti-aging, anti-inflammation, immunostimulating, DNA repair and anti-cancer effects in different model systems. Molecular mechanisms of Rhodiola rosea extracts's action have been studied mainly along with one of its bioactive compounds, salidroside. Both Rhodiola rosea extracts and salidroside have contrast molecular mechanisms on cancer and normal physiological functions. For cancer, Rhodiola rosea extracts and salidroside inhibit the mTOR pathway and reduce angiogenesis through down-regulation of the expression of HIF-1α/HIF-2α. For normal physiological functions, Rhodiola rosea extracts and salidroside activate the mTOR pathway, stimulate paracrine function and promote neovascularization by inhibiting PHD3 and stabilizing HIF-1α proteins in skeletal muscles. In contrast to many natural compounds, salidroside is water-soluble and highly bioavailable via oral administration and concentrated in urine by kidney excretion. SUMMARY: Rhodiola rosea extracts and salidroside can impose cellular and systemic benefits similar to the effect of positive lifestyle interventions to normal physiological functions and for anti-cancer. The unique pharmacological properties of Rhodiola rosea extracts or salidroside deserve further investigation for cancer chemoprevention, in particular for human urinary bladder cancer.

A new coumarin derivative, IMM-H004, attenuates okadaic acid-induced spatial memory impairment in rats.

AIM: A novel coumarin derivative 7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) has shown anti-apoptotic, anti-inflammatory and neuroprotective activities. In this study we investigated the effects of IMM-H004 on spatial memory in rats treated with okadaic acid (OKA), which was used to imitate Alzheimer's disease (AD)-like symptoms. METHODS: SD rats were administered IMM-H004 (8 mg·kg(-1)·d(-1), ig) or donepezil (positive control, 1 mg·kg(-1)·d(-1), ig) for 25 d. On d 8 and 9, OKA (200 ng) was microinjected into the right ventricle. Morris water maze test was used to evaluate the spatial memory impairments. Tau and ß-amyloid (Aß) pathology in the hippocampus was detected using Western blot and immunohistochemistry. TUNEL staining was used to detect cell apoptosis. RESULTS: OKA-treated rats showed significant impairments of spatial memory in Morris water maze test, which were largely reversed by administration of IMM-H004 or donepezil. Furthermore, OKA-treated rats exhibited significantly increased phosphorylation of tau, deposits of Aß protein and cell apoptosis in the hippocampus, which were also reversed by administration of IMM-H004 or donepezil. CONCLUSION: Administration of IMM-H004 or donepezil protects rats against OKA-induced spatial memory impairments via attenuating tau or Aß pathology. Thus, IMM-H004 may be developed as a therapeutic agent for the treatment of AD.

Environment-contact administration of rotenone: A new rodent model of Parkinson's disease.

Epidemiological studies suggest an association between pesticides and the incidence of Parkinson's disease (PD). Individuals are likely to be exposed to numerous natural or synthetic environmental agents by ingestion, inhalation, or skin contact. Here, we describe a novel environment-contact administration of rotenone model, in which male C57BL/6 mice (15 per group per time-point) were placed in one bedding-free, rotenone-applied cage for 2h every day over a period of 2-6 weeks, mimicking the common ways a person may be exposed to pesticides. Our results showed that rotenone exposure had no detrimental effect on body weights of mice during 6 weeks, nor did it cause systemic toxicity (HPLC analysis of rotenone in blood and brain, as well as complex I activity measurements in brain and muscle), but it caused significant impairments in motor function (open field test, pole test, and rotarod test) from 4 weeks that were responsive to apomorphine. Accordingly, rotenone caused significant dopamine depletion from the striatum (HPLC analysis), nigrostriatal degeneration (quantitative tyrosine hydroxylase immunohistochemistry and western blot), and accumulation of α-synuclein in the substantia nigra and striatum (α-synuclein immunohistochemistry) in a time-dependent manner. In addition, rotenone-exposed mice also developed deficits in gastrointestinal and olfactory function (fecal pellet output and buried food pellet test) prior to the motor dysfunction. Furthermore, we observed that α-synuclein accumulated in the anterior olfactory nucleus and the enteric nervous system at 2 weeks. In summary, this novel rotenone model was able to reproduce many key aspects of PD progression. Therefore, it provides new insight into how environmental factors could trigger PD and provides a useful tool for studying PD pathogenesis and testing neuroprotective strategies.

Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse.

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson's disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.

IMM-H004, a novel coumarin derivative compound, attenuates the production of inflammatory mediatory mediators in lipopolysaccharide-activated BV2 microglia.

Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. 7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) is a novel compound and has been reported exerting potent neuroprotective effects which may be related to anti-inflammation. In the present study, the anti-inflammatory effects of IMM-H004 were investigated in lipopolysaccharide (LPS)-treated BV2 microglia. Our observations indicated that treatment with IMM-H004 significantly inhibited BV2 microglia activation, protected PC12 cells and primary neurons against indirect toxicity mediated by exposure to conditioned medium (CM) from LPS-treated BV2 cells. Additionally, IMM-H004 significantly suppressed the release of TNF-α, IL-1ß and NO, and suppressed the expression of pro-inflammatory mediators and cytokines such as iNOS, COX-2, and IL-6 in LPS-stimulated BV2 microglia. The nuclear translocation of NF-κB and the phosphorylation level of JNK and p38 MAPK pathways were also inhibited by IMM-H004 in LPS-treated BV2 microglia. Moreover, IMM-H004 also was a strong selective OH scavenger whose effect was similar with vitamin C. Overall, our findings suggested that IMM-H004 might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.

α-Synuclein is prone to interaction with the GC-box-like sequence in vitro.

α-Synuclein (α-syn) is a presynaptic protein that is widely implicated in the pathophysiology of Parkinson's disease, a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons and the formation of Lewy bodies. Evidence suggests that α-syn could be imported into the nucleus and subsequently disrupt normal neuronal function. The existence of α-syn in the nucleus provides the possibility of interaction with DNA leading to gene transcript regulation. Thus, CD spectra were used to determine the specific DNA sequence with which α-syn is most likely to interact. Our results indicated that α-syn was prone to preferentially interact with the GC-box-like sequence in vitro at a ratio of 2:1 or less (α-syn: the GC-box-like sequence).

The nuclear accumulation of alpha-synuclein is mediated by importin alpha and promotes neurotoxicity by accelerating the cell cycle.

α-Synuclein (α-syn), a 14 kDa pre-synaptic protein, is widely involved in the Parkinson's disease (PD) pathogenesis. Recent studies have shown that the nuclear accumulation of α-syn might have a toxic effect. The main purpose of the present study was to explore which amino acid residues in α-syn are associated with its nuclear accumulation, the molecule(s) mediated the nuclear import of α-syn, and the role of α-syn accumulated in the nucleus. It has been noted that the nuclear import of α-syn may be mediated by importin α and that both the amino acid residues 1-60 and 103-140 of α-syn were indispensable for its nuclear import. After imported into the nucleus, the accumulated α-syn played a toxic role in both the PC12 cells and the C57 mice. Furthermore, α-syn-nuclear localization signal-injected mice showed behavioral symptoms associated with PD. Further studies performed in vitro showed that the toxicity of α-syn in the nucleus might be due to an interference of the cell cycle. Thus, it can be concluded that α-syn can accumulate in nucleus, which is mediated by importin α, and promote neurotoxicity by accelerating the cell cycle.