Cirsium japonicum extract induces apoptosis and anti-proliferation in the human breast cancer cell line MCF-7.

Cirsium japonicum is a wild perennial herb that has been used as an anti-hemorrhagic, anti-hypertensive and uretic agent in traditional Chinese medicine. Recently, it was reported that C. japonicum inhibits the growth of implanted cancer cells. However, the molecular mechanisms underlying the anti-cancer properties of C. japonicum are not fully understood. In this study, we investigated the effect of a methanol extract of C. japonicum on cell growth in the human breast cancer cell line MCF-7. C. japonicum extract inhibited the cell proliferation of MCF-7 cells in a time- and dose-dependent manner, as evaluated by the MTT assay. Furthermore, C. japonicum extract induced an anti-proliferative effect by causing G1 phase cell cycle arrest and also induced apoptosis by affecting mitochondrial apoptotic events, as determined by nuclear derangement, flow cytometry and Western blot analysis. Taken together, our findings indicate that C. japonicum extract induces the inhibition of MCF-7 cell growth at both the proliferation and apoptosis levels.

A Torilis japonica extract exerts anti-proliferative activities on the U87MG human glioblastoma cell line.

Torilis japonica is a wild biennial herb and has been used as a traditional medicine for the treatment of inflammation, skin disease and impotence. Here, we studied the effects of a T. japonica extract on the proliferation of the U87MG human glioblastoma cell line. The extract inhibited cell proliferation in a dose- and time-dependent manner, as determined using the MTT assay. We next investigated the molecular mechanisms underlying its anti-proliferation properties by examining cell cycle progression and cell death. T. japonica extract induced S-phase cell cycle arrest and inhibited the expression of cell cycle-regulatory proteins, including cyclin A, cyclin-dependent protein kinase 2 and E2F1. The extract also induced apoptotic cell death as evaluated by nuclear morphology and flow cytometry using Annexin-V/PI dual staining. Furthermore, Western blot analysis showed that apoptotic cell death was mediated by both mitochondria-independent and caspase-dependent pathways. Together, our findings indicate that the T. japonica extract contains bioactive compounds with anti-cancer effects. These materials may be useful in the chemotherapy of human glioblastoma.

Long-term cultured skeletal muscle-derived neural precursor cells and their neurogenic potentials.

For cell-based therapy, it is necessary to obtain sufficient cell quantities for cell transplantation to the diseased or injured site. However, a given tissue has only a limited number of stem cells, making it necessary to expand stem cell source through long-term culture. In this study, we evaluated whether our recently described skeletal muscle-derived neural precursor (SMNP) cells can be cultured long-term without alteration of their neural precursor characteristics. Our results showed that SMNP cells can be cultured over approximately 16 months, but their growth rate and neurogenic potential gradually decrease in a culture time-dependent manner. Importantly, approximately 120-day cultured SMNP cells retain their self-renewal ability, neural precursor characteristics, and high neurogenic potential. These results suggest that SMNP cells may be an appropriate cell source for regenerative or reparative therapies against neuronal disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits neurite outgrowth in differentiating human SH-SY5Y neuroblastoma cells.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental pollutant, is known to induce neurodevelopmental and neurobehavioral deficits. However, the underlying mechanism of TCDD-mediated neurotoxicity has remained unclear. We have studied TCDD inhibition of neurite outgrowth using human SH-SY5Y neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). TCDD, at concentrations of 3 nM or 5 nM, had no significant effect on the viability of either undifferentiating or differentiated SH-SY5Y cells. However, differentiating SH-SY5Y cells exhibited a distinct decrease of neurite outgrowth 48 h after TCDD treatment in a dose-dependent manner. TCDD treatment 12h or 24h after RA stimulation did not elicit a significant inhibition of neurite outgrowth, whereas TCDD cotreatment with RA or TCDD treatment at 6h after RA stimulation significantly inhibited neurite outgrowth. Western blot analysis of cell extracts of RA-stimulated differentiating SH-SY5Y cells showed increased level of cross reactivities with tissue glutaminase (TGase) antibody compared to control extracts, in a time-dependent manner. By contrast, treatment of differentiating SH-SY5Y cells with 1-5 nM TCDD resulted in decreased level of cross-reactivities with TGase antibody in a dose-dependent manner. The results indicate that TCDD is able to inhibit neurite outgrowth by differentiating SH-SY5Y cells and that this effect might result from reduced levels of TGase.

The GTPase domain of Galphao contributes to the functional interaction of Galphao with the promyelocytic leukemia zinc finger protein.

Go, one of the most abundant heterotrimeric G proteins in the brain, is classified as a member of the Gi/Go family based on its homology to Gi proteins. Recently, we identified promyelocytic leukemia zinc finger protein (PLZF) as a candidate downstream effector for the alpha subunit of Go (Galphao). Activated Galphao interacts with PLZF and augments its function as a repressor of transcription and cell growth. G protein-coupled receptor-mediated Galphao activation also enhanced PLZF function. In this study, we determined that the GTPase domain of Galphao contributes to Galphao:PLZF interaction. We also showed that the Galphao GTPase domain is important in modulating the function of PLZF. This data indicates that the GTPase domain of Galphao may be necessary for the functional interaction of Galphao with PLZF.

Rit contributes to neurite outgrowth triggered by the alpha subunit of Go.

Heterotrimeric GTP-binding protein transduce signals initiated by a variety of hormones and neurotransmitters. Go, a member of the Go/Gi family, is the most abundant heterotrimeric GTP-binding protein in nervous tissues and has been implicated in neuronal differentiation. The mechanism by which Go modulates neuronal differentiation has not been, however, fully elucidated. Here, we identified small GTPase Rit as an interacting partner of the alpha-subunit of Go (Goalpha). The biochemical characterizations of Goalpha::Rit interaction revealed that Rit is a candidate downstream effector for Goalpha. Furthermore, dominant negative Rit inhibited Goalpha-induced neurite outgrowth and Erk phosphorylation in Neuro2a cells. These results suggest that Rit may be involved in the signaling pathway for Goalpha-mediated neuronal differentiation.

The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions.

Heterotrimeric GTP-binding proteins (G proteins) mediate signal transduction generated by neurotransmitters and hormones. Go, a member of the Go/Gi family, is the most abundant heterotrimeric G protein in the brain. Most mechanistic analyses on Go activation demonstrate that its action is mediated by the Gbetagamma dimer; downstream effectors for its alpha subunit (Goalpha) have not been clearly defined. Here, we employ the yeast two-hybrid system to screen for Goalpha-interacting partners in a cDNA library from human fetal brain. The transcription factor promyelocytic leukemia zinc finger protein (PLZF) specifically bound to Goalpha. Interactions between PLZF and Goalpha were confirmed using in vitro and in vivo affinity binding assays. Activated Goalpha interacted directly with PLZF, and enhanced its function as a transcriptional and cell growth suppressor. Notably, PLZF activity was additionally promoted by the Go/ialpha-coupled cannabinoid receptor (CB) in HL60 cells endogenously expressing CB and PLZF. These results collectively suggest that Goalpha modulates the function of PLZF via direct interactions. Our novel findings provide insights into the diverse cellular roles of Goalpha and its coupled receptor.

Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells.

Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.

Therapeutic effect of passive mobilization exercise on improvement of muscle regeneration and prevention of fibrosis after laceration injury of rat.

OBJECTIVE: To evaluate the muscle healing effect of passive mobilization exercises after a laceration injury. DESIGN: Randomized controlled trial. SETTING: Basic science laboratory. ANIMALS: Male Sprague-Dawley rats (N=36), age ranging from 8 to 10 weeks and weight ranging from 300 to 400 g. INTERVENTION: The bilateral gastrocnemius muscles were lacerated. The left leg muscles were used as the study groups and the right side was used as the control (lacerated muscles without any treatment, n=8). In the exercise group (n=24), passive mobilization exercise (15 min/d) was performed for 5 days starting from different time points (2, 7, and 14d postlaceration). The decorin group (n=8) was injected with decorin (50 microg at 14d postlaceration), which is a well-known antifibrotic agent. Four animals were used as the normal controls, in which only the muscle strength was evaluated. All the animals were killed 4 weeks after the laceration. MAIN OUTCOME MEASURES: The histologic characterization of muscle regeneration (hematoxylin and eosin staining, number and diameter of the centronucleated, regenerating myofibers), muscle fibrosis (vimentin-positive area, Masson modified trichrome staining positive area), and muscle strength (analysis of fast twitch strength). RESULTS: The level of fibrosis was more than 50% lower in the exercise and decorin groups than in the control (P<.05). The decorin group showed the highest number of regenerated, new myofibers and the highest muscle strength. All of the exercise groups, regardless of the starting time of exercise, also showed significant improvement in regeneration and strength. However, the exercise group starting 14 days after the laceration showed the best results. CONCLUSIONS: Stretching exercises after a muscle laceration injury has a strong antifibrotic effect, as much as a well-known antifibrotic agent, decorin. According to the results, the best time to begin stretching exercises is 14 days after laceration for antifibrosis and muscle regeneration.

PCL microparticle-dispersed PLGA solution as a potential injectable urethral bulking agent.

The PCL microparticle-dispersed PLGA solutions were prepared as a potential injectable urethral bulking agent. The mixture solutions were prepared by mixing polycarprolactone (PCL) microparticles (diameter, 100 approximately 200mum; fabricated by a temperature-induced phase transition method) and poly(dl-lactic-co-glycolic acid) (PLGA) solution (dissolved in tetraglycol to 10wt%) with different PCL microparticle to PLGA solution ratio. The mixture solution was solidified by the precipitation of PLGA when the solution was contact with water. In contact with water, the PCL microparticles exhibited a well-packed structure entrapped in a solidified porous PLGA matrix, which can effectively prevent the microparticle migration in the body and retain its initial volume even after PLGA matrix degradation. The PCL microparticle-dispersed PLGA solution (particle to solution ratio, 45/55 (w/v)) was easily injected through 18G needle into back of hairless mouse (subcutaneously) and stably located at the apply site. The surrounding tissue including blood vessel were gradually infiltrated into the implant up to 8 weeks without the initial injected volume change and with little inflammatory response. The PCL microparticle-dispersed PLGA solution may be a good candidate as an injectable bulking agent for the treatment of urinary incontinence owing to its good injectability, volume retention potential as well as biocompatibility.

cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK.

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.

Prevention of postsurgical tissue adhesion by anti-inflammatory drug-loaded pluronic mixtures with sol-gel transition behavior.

Sol-gel transition temperature-controllable Pluronic F127/F68 mixtures including mildly crosslinked alginate and nonsteroidal anti-inflammatory drug (ibuprofen) were prepared to evaluate their potential as tissue adhesion barrier gels. The sol-gel transition temperatures of the Pluronic mixtures could be controlled by adjusting F127/F68 ratio and polymer concentration. The mildly crosslinked alginate with still flow property provided the residence stability of Pluronic mixture gels in the body. Ibuprofen was loaded in Pluronic mixtures to reduce inflammatory response in the body and, thus, to prevent tissue adhesion. The gelation temperatures of the Pluronic mixtures were not affected by the alginate but lowered by the addition of ibuprofen. The in vitro drug release behavior and in vivo peritoneal tissue adhesion of the Pluronic mixtures with the sol-gel transition just below body temperatures were investigated. The drug release behavior from the ibuprofen (1 wt%)-loaded Pluronic mixture gels at 37 degrees C was examined using a membrane-less dissolution model. The drug in the mixture gels was released continuously up to about 45-65% of the total loading amount during the first 7 days. For in vivo evaluation of tissue anti-adhesion potential, the Pluronic mixtures with/without drug were coated on the peritoneal wall defects of rats and their tissue adhesion extents and tissue reactions (inflammatory response, granulation tissue formation, and toxicity in organs) were compared. It was observed that ibuprofen has a positive effect for the peritoneal tissue anti-adhesion. The Pluronic F127/F68/alginate/ibuprofen mixture gel (25 wt% of F127/F68 [7/3], 1 wt% ibuprofen) was highly effective for the prevention of peritoneal tissue adhesion and showed a relatively low inflammatory response and non-toxicity, and thus can be a good candidate material as a coatable or injectable tissue adhesion barrier gel.

Isolation of muscle derived stem cells from rat and its smooth muscle differentiation [corrected].

We investigated whether stem cells (MDSC) from primary cultures of rat skeletal muscle can differentiate into the smooth muscle lineage in response to vascular endothelial growth factor (VEGF) and coculture with bladder smooth muscle cells. The MDSC were isolated from gastrocnemius muscle biopsies of normal 3-6 week-old Sprague-Dawley rats and purified by the preplate technique. Cells that took approximately 6 days to adhere to the collagen-coated flasks were termed late preplate (LP) cells, and were used in all the experiments. The early plate (EP) cells (pp1-pp4) contained some myogenic cells but were mostly fibroblasts (< 15% desmin+ cells) whereas the LP cells (pp5-pp6) were highly purified muscle-derived cells (pp6) (> 90% desmin+ cells). The muscle-derived stem cells (LP cells) were CD34+ or Sca-1+, CD45- and desmin+ by immunohistochemical staining. After two days of co-culture with bladder smooth muscle cells, about 25% of the muscle-derived stem cells were positive for alpha-smooth muscle actin (alpha-SMA)+. RT-PCR for alpha-SMA was positive in the VEGF stimulated MDSC, but negative in the absence of VEGF. In conclusion, rat muscle-derived stem cells exhibited stem cell properties (CD34+ or Sca-1+), and were not of hematogeous (CD45-) but of myogenic origin (desmin+). RT-PCR of alpha-SMA was positive in the VEGF stimulated muscle-derived stem cells.

Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves.

The survival of muscle-derived cells injected into the urethra and bladder wall was described recently. In this study, we tested whether injections of periurethral muscle-derived stem cells (MDSC) and bovine collagen (BC) after denervation of the pudendal nerve could increase leak point pressure (LPP) and closing pressure (CP) in female rats over the long term. S-D rats were anesthetized with halothane and the pudendal nerves transected bilaterally via a dorsal incision in order to denervate the external urethral sphincter. In the collagen and MDSC groups (C & M), injection of collagen or MDSC was made into the proximal urethra after pudendal nerve transection. At 4 and 12 week, visually identified LPP and CP measurements were made using the vertical tilt/intravesical pressure clamp model of stress urinary incontinence. The rats were then sacrificed and urethra harvested for histology. Both LPP and CP were significantly lower in the denervated (D) group at each time compared with the normal (N), C, and M groups, and both LPP and CP in the C and M groups were significantly higher than in the D group at both 4 and 12 weeks. The persistence of MDSC over the period of study was verified by histology. Thus pudendal nerve denervation led to a progressive decline in LPP and CP that was evident at 4 week and persisted to 12 week, and injection of MDSC into the denervated rats led to a long term increase in LPP and CP.

Modulation of the N-type calcium channel gene expression by the alpha subunit of Go.

Go, a heterotrimeric G-protein, is enriched in brain and neuronal growth cones. Although several reports suggest that Go may be involved in modulation of neuronal differentiation, the precise role of Go is not clear. To investigate the function of Go in neuronal differentiation, we determined the effect of Goalpha, the alpha subunit of Go, on the expression of Ca(v)2.2, the pore-forming unit of N-type calcium channels, at the transcription level. Treatment with cyclic AMP (cAMP), which triggers neurite outgrowth in neuroblastoma F11 cells, increased the mRNA level and the promoter activity of the Ca(v)2.2 gene. Overexpression of Goalpha inhibited neurite extension in F11 cells and simultaneously repressed the stimulatory effect of cAMP on the Ca(v)2.2 gene expression to the basal level. Targeted mutation of the Goalpha gene also increased the level of Ca(v)2.2 in the brain. These results suggest that Go may regulate neuronal differentiation through modulation of gene expression of target genes such as N-type calcium channels.

Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells.

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.

Inhibition of BETA2/NeuroD by Id2.

Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.