Clinacanthus nutans Mitigates Neuronal Apoptosis and Ischemic Brain Damage Through Augmenting the C/EBPß-Driven PPAR-γ Transcription.

Clinacanthus nutans Lindau (C. nutans) is a traditional herbal medicine widely used in Asian countries for treating a number of remedies including snake and insect bites, skin rashes, viral infections, and cancer. However, the underlying molecular mechanisms for its action and whether C. nutans can offer protection on stroke damage in brain remain largely unknown. In the present study, we demonstrated protective effects of C. nutans extract to ameliorate neuronal apoptotic death in the oxygen-glucose deprivation model and to reduce infarction and mitigate functional deficits in the middle cerebral artery occlusion model, either administered before or after hypoxic/ischemic insult. Using pharmacological antagonist and siRNA knockdown approaches, we demonstrated ability for C. nutans extract to protect neurons and ameliorate ischemic injury through promoting the anti-apoptotic activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ), a stress-induced transcription factor. Reporter and chromatin immunoprecipitation promoter analysis further revealed C. nutans extract to selectively increase CCAAT/enhancer binding protein (C/EBP)ß binding to specific C/EBP binding site (-332~-325) on the PPAR-γ promoter to augment its transcription. In summary, we report a novel transcriptional activation involving C/EBPß upregulation of PPAR-γ expression to suppress ischemic neuronal apoptosis and brain infarct. Recognition of C. nutans to enhance the C/EBPߠ→ PPAR-γ neuroprotective signaling pathway paves a new way for future drug development for prevention and treatment of ischemic stroke and other neurodegenerative diseases.

Clinacanthus nutans Protects Cortical Neurons Against Hypoxia-Induced Toxicity by Downregulating HDAC1/6.

Many population-based epidemiological studies have unveiled an inverse correlation between intake of herbal plants and incidence of stroke. C. nutans is a traditional herbal medicine widely used for snake bite, viral infection and cancer in Asian countries. However, its role in protecting stroke damage remains to be studied. Despite of growing evidence to support epigenetic regulation in the pathogenesis and recovery of stroke, a clear understanding of the underlying molecular mechanisms is still lacking. In the present study, primary cortical neurons were subjected to in vitro oxygen-glucose deprivation (OGD)-reoxygenation and hypoxic neuronal death was used to investigate the interaction between C. nutans and histone deacetylases (HDACs). Using pharmacological agents (HDAC inhibitor/activator), loss-of-function (HDAC siRNA) and gain-of-function (HDAC plasmid) approaches, we demonstrated an early induction of HDAC1/2/3/8 and HDAC6 in neurons after OGD insult. C. nutans extract selectively inhibited HDAC1 and HDAC6 expression and attenuated neuronal death. Results of reporter analysis further revealed that C. nutans suppressed HDAC1 and HDAC6 transcription. Besides ameliorating neuronal death, C. nutans also protected astrocytes and endothelial cells from hypoxic-induced cell death. In summary, results support ability for C. nutans to suppress post-hypoxic HDACs activation and mitigate against OGD-induced neuronal death. This study further opens a new avenue for the use of herbal medicines to regulate epigenetic control of brain injury.

Anti-apoptotic actions of PPAR-gamma against ischemic stroke.

Stroke is a leading cause of adult disability and mortality. Diabetes is a major risk factor for stroke. Patients with diabetes have a higher incidence of stroke and a poorer prognosis after stroke. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-modulated transcriptional factor and a therapeutic target for treating type II diabetes. It is well-documented that activation of PPAR-gamma can also attenuate postischemic inflammation and damage. In this review, we focus on the newly revealed anti-apoptotic actions of PPAR-gamma against cerebral ischemia. PPAR-gamma, by increasing superoxide dismutase/catalase and decreasing nicotinamide adenine dinucleotide phosphate oxidase levels, attenuated ischemia-induced reactive oxygen species and subsequently alleviated the postischemic degradation of Bcl-2, Bcl-xl, and Akt. The preserved Akt phosphorylated Bad. Meanwhile, PPAR-gamma also promotes the transcription of 14-3-3epsilon. Elevated 14-3-3epsilon binds and sequesters p-Bad and prevents Bad translocation to neutralize the anti-apoptotic function of Bcl-2. This review further supports the notion that PPAR-gamma may serve as a potential therapeutic target for treating ischemic stroke.

Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation.

BACKGROUND: Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-dependent; however, it is unclear how PPAR-gamma activation confers resistance to ischemia-reperfusion injury. METHODS AND RESULTS: We evaluated the effects of rosiglitazone or PPAR-gamma overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR-gamma overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-gamma small interfering RNA. In mice with knock-in of a PPAR-gamma dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3epsilon was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3epsilon was abrogated by PPAR-gamma small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR-gamma binding to specific regulatory elements on the 14-3-3epsilon promoter and thereby increased 14-3-3epsilon transcription. 14-3-3epsilon Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-gamma overexpression, whereas 14-3-3epsilon recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3epsilon enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential. CONCLUSIONS: Ligand-activated PPAR-gamma confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3epsilon transcription. 14-3-3epsilon Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

The lethal effect of bis-type azridinylnaphthoquinone derivative on oral cancer cells (OEC-M1) associated with anti-apoptotic protein bcl-2.

Several drugs of aziridinylbenzoquinone analogs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill tumor cells preferentially within the hypoxic microenvironment. From our previous reported data, it was found that the synthesized 2-aziridin-1-yl-3-[(2-[2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy]ethyl)thio]naphthoquinone (AZ-1) is a bioreductive compound with potent lethal effect on oral cancer cell, OEC-M1. It was found in this study that the lethal effect of the oral cancer cell lines OEC-M1 induced by AZ-1 was mediated through the cell cycle arrest and apoptosis pathway. The LC50 values of OEC-M1 and KB cells induced by AZ-1 compound were 0.72 and 1.02 microM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50 = 5.6 microM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2 microM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50 microM on OEC-M1 cells. Comparing the cytotoxicity of AZ-1 and AZQ on OEC-M1 cells, AZ-1 is approximately 70 times more potent than AZQ. By using Western blot, both G2/M phase cell cycle arresting protein, cyclin B, and anti-apoptotic protein, bcl-2, were expressed in OEC-M1 cell when the concentrations of AZ-1 were increased from 0.125 to 0.5 microM and then decreased from 1 to 2 microM of AZ-1 treatment as compared with control for 24 h. Both proteins were expressed most abundantly at 0.5 microM AZ-1. However, the expression of bcl-2 protein in OEC-M1 was significantly decreasing in a dose-dependent manner and was only about 50% protein level at 2 microM AZ-1 for 48h as compared with control. The cell survival check protein p53 increased from 1.72- to 2.8-fold and 1.36- to 2.16-fold at concentrations of AZ-1 from 0.125 to 2.0 microM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48 h treatments, respectively. The apoptotic-related phenomena were observed, which included apoptotic body formation and the enzyme activity change of caspase-3. The apoptotic bodies and caspase-3 activity of OEC-M1 were induced only at 2 microM AZ-1 for a 24h treatment, yet apoptotic body formation was observed at as low as 0.5 microM AZ-1 and in a dose-dependently increasing manner for a 48 h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2muM concentrations of AZ-1 for a 48 h treatment as compared with control. These results indicate that AZ-1 induced the cell death of OEC-M1 through the G2/M phase arrest of cell cycle and anti-apoptosis first and then apoptosis following a 48 h treatment. All of the pathway might be associated with bcl-2 and p53 protein expression. We propose that the AZ-1 could be used as anti-oral cancer drug for future studies with animal models.

The p53-dependent apoptotic pathway of breast cancer cells (BC-M1) induced by the bis-type bioreductive compound aziridinylnaphthoquinone.

INTRODUCTION: Several aziridinylbenzoquinone drugs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill cells preferentially within the hypoxia tumor microenvironment. The bioreductive compound of bis-type naphthoquinone synthesized in our laboratory, 2-aziridin-1-yl-3-[(2-{2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy}ethyl)thio]naphthoquinone (AZ-1), had the most potent death effect on the breast cancer cells BC-M1 in our previous screening. In the present study, we determined that the mechanism of the death effect of BC-M1 cells induced by AZ-1 was mediated by the apoptosis pathway. METHODS: We evaluated the cytotoxicity of AZ-1 and the anti-breast cancer drugs tamoxifen and paclitaxel to BC-M1 cells and MCF-7 cells by the MTT assay and measured the apoptosis phenomena by Hoechst 33258 staining for apoptotic bodies. We also quantified the sub-G1 peak area and the ratio of the CH2/CH3 peak area of the cell membrane in BC-M1 cells by flow cytometry and 1H-NMR spectra, respectively. The apoptosis-related protein expressions, including p53, p21, the RNA-relating protein T-cell restricted intracellular antigen-related protein, cyclin-dependent kinase 2 (cell cycle regulating kinase) and pro-caspase 3, were detected by western blot, and the caspase-3 enzyme activity was also quantified by an assay kit. RESULTS: AZ-1 induced two of the breast cancer cell lines, with IC50 = 0.51 microM in BC-M1 cells and with IC50= 0.57 microM in MCF-7 cells, and showed less cytotoxicity to normal fibroblast cells (skin fibroblasts) with IC50= 5.6 microM. There was a 10-fold difference between two breast cancer cell lines and normal fibroblasts. Of the two anti-breast cancer drugs, tamoxifen showed IC50= 0.12 microM to BC-M1 cells and paclitaxel had much less sensitivity than AZ-1. The expression of p53 protein increased from 0.5 to 1.0 microM AZ-1 and decreased at 2.0 microM AZ-1. The p21 protein increased from 0.5 microM AZ-1, with the highest at 2 microM AZ-1. Regarding the AZ-1 compound-induced BC-M1 cells mediating the apoptosis pathway, the apoptotic body formation, the sub-G1 peak area, the ratio of CH2/CH3 of phospholipids in the cell membrane and the enzyme activity of caspase-3 were all in direct proportion with the dose-dependent increase of the concentration of AZ-1. The death effect-related proteins, including T-cell restricted intracellular antigen-related protein, cyclin-dependent kinase 2, and pro-caspase-3, all dose-dependently decreased with AZ-1 concentration. CONCLUSIONS: The AZ-1-induced cell death of BC-M1 cells mediating the apoptosis pathway might be associated with p53 protein expression, and AZ-1 could have the chance to be a candidate drug for anti-breast cancer following more experimental evidence, such as animal models.

A novel bis-aziridinylnaphthoquinone with anti-solid tumor activity in which induced apoptosis is associated with altered expression of Bcl-2 protein.

Aziridine-containing compounds have been of interest as anticancer agents since the late 1970s. The design, synthesis, and study of aziridinylnaphthoquinone analogues to obtain compounds with enhanced activity/toxicity profiles are an ongoing research effort in our group. A series of bis-aziridinylnaphthoquinone derivatives has been prepared, and the cytotoxic activities of these synthetic bis-aziridinylnaphthoquinone derivatives has been investigated. The synthetic derivatives displayed significant cytotoxicity against human carcinoma cell lines and weak cytotoxic activities against skin fibroblasts (SF). The bis-aziridinylnaphthoquinone 1 c was the most effective of the tested analogues at reducing the viability of Hep2 cells, with an LD(50) value of 5.23 microM, and also exhibited weak cytotoxic activity against SF cells, with an LD(50) value of 54.12 microM. The DNA alkylation and DNA interstrand cross-linking abilities of 1 c were also investigated. Bis-aziridinylnaphthoquinone 1 c was an effective agent for alkylation of DNA after chemical reduction in vitro, and its bifunctional alkylating moieties were able to cross-link DNA. We also report here our efforts to determine direct antitumor effects of 1 c on Hep2 cells. Growth arrest in Hep2 cells was preceded by early induction of G(2)-M cell cycle arrest at 0.75 microM of 1 c after culture for 24 h, and was then followed by apoptosis after 60 h. This was associated with decreased expression of antiapoptotic bcl2 protein (by 78 %) upon culture with 3.0 microM of 1 c after 60 h. Our results suggest that 1 c is a novel antitumor aziridinylnaphthoquinone with therapeutic potential against solid tumors.

Synthesis and biological evaluation of novel bis-aziridinylnaphthoquinone derivatives.

A series of bis-aziridinylnaphthoquinone derivatives has been prepared. The cytotoxic activities and DNA alkylation abilities of these synthetic bis-aziridinylnaphthoquinone derivatives were investigated. They displayed significant cytotoxicity against human carcinomna cell lines and weak cytotoxic activities against HL60 and skin fibroblast (SF). The bisaziridinylnaphthoquinone 1a was the most potent agent among those tested, with an LD50 value of 0.57 microM against the BC-M1 cell line. It exhibited the weakest activity against SF and HL60 with LD50 values of 5.67 and 20.1 microM, respectively, and it was able to alkylate DNA after chemical reduction in vitro. The analogues without aziridinyl moiety 2a and 3a lack DNA alkylation abilities.