Estrogen receptor mediates simvastatin-stimulated osteogenic effects in bone marrow mesenchymal stem cells.

Simvastatin, an HMG-CoA reductase inhibitor, is known to promote osteogenic differentiation. However, the mechanism underlying simvastatin-induced osteogenesis is not well understood. In this study, we hypothesize that the estrogen receptor (ER) mediates simvastatin-induced osteogenic differentiation. ER antagonists and siRNA were used to determine the involvement of the ER in simvastatin-induced osteogenesis in mouse bone marrow mesenchymal stem cells (D1 cells). Osteogenesis was evaluated by mRNA expression, protein level/activity of osteogenic markers, and mineralization. The estrogen response element (ERE) promoter activity and the ER-simvastatin binding affinity were examined. Our results showed that the simvastatin-induced osteogenic effects were decreased by treatment with ERα antagonists and ERα siRNA but not by an antagonist specific for the G protein-coupled estrogen receptor (GPER-1). The simvastatin-induced osteogenic effects were further increased by E2 treatment and were reversed by ERα antagonists or siRNA treatment. Luciferase reporter gene assays demonstrated that simvastatin increase ERα-dependent transcriptional activity that was suppressed by ERα antagonists. Furthermore, the ERα-simvastatin binding assay showed that IC50 value of simvastatin is 7.85 µM and that of E2 is 32.8 nM, indicating that simvastatin is a weak ligand for ERα. These results suggest that simvastatin-stimulated osteogenesis is mediated by ERα but not GPER-1. Moreover, this is the first report to demonstrate that simvastatin acts as an ERα ligand and a co-activator to enhance ERα-dependent transcriptional activity and thus promotes osteogenesis. These results indicate that simvastatin-induced osteogenesis is mediated via an ERα-dependent pathway.

Taiwanin A targets non-steroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma.

Taiwanin A (α,ß-bis(piperonylidene)-γ-butyrolactone) is extracted from Taiwania cryptomerioides. Taiwanin A is extracted from tree bark and exhibits antitumor activity in breast, liver, and lung cancer cell lines. The objective of this study was to demonstrate the cytotoxicity of Taiwanin A against tumor cells by increasing the expression of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). NAG-1 has been reported to exhibit antitumor and proapoptotic activities, suggesting potential use in cancer therapy. Inhibiting NAG-1 mRNA expression in A549 reduced the cytotoxicity caused by Taiwanin A. Furthermore, the c-Jun-N-terminal kinase/Ste20-related protein proline/alanine-rich kinase (JNK/SPAK) pathway played a key role in the influence of NAG-1 on cell viability, whereas the addition of the JNK pathway inhibitor SP600125 resulted in an inhibitory effect on NAG-1 and recovery of Taiwanin-A-treated cells. A xenograft tumor model demonstrated that Taiwanin A dose-dependently significantly decreases tumor-mediated growth in nude mice by increasing the NAG-1 expression accompanying tumor apoptosis. These data supported the hypothesis that Taiwanin A inhibits lung carcinoma growth by increasing NAG-1 expression through the JNK pathway both in vivo and in vitro. This result can contribute to a compound design for increasing cytotoxicity activity in the future.

Proliferation and differentiation potential of human adipose-derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures.

Aging has less effect on adipose-derived mesenchymal stem cells (ADSCs) than on bone marrow-derived mesenchymal stem cells (BMSCs), but whether the fact holds true in stem cells from elderly patients with osteoporotic fractures is unknown. In this study, ADSCs and BMSCs of the same donor were harvested and divided into two age groups. Group A consisted of 14 young patients (36.4 ± 11.8 years old), and group B consisted of eight elderly patients (71.4 ± 3.6 years old) with osteoporotic fractures. We found that the doubling time of ADSCs from both age groups was maintained below 70 hrs, while that of BMSCs increased significantly with the number of passage. When ADSCs and BMSCs from the same patient were compared, there was a significant increase in the doubling time of BMSCs in each individual from passages 3 to 6. On osteogenic induction, the level of matrix mineralization of ADSCs from group B was comparable to that of ADSCs from group A, whereas BMSCs from group B produced least amount of mineral deposits and had a lower expression level of osteogenic genes. The p21 gene expression and senescence-associated ß-galactosidase activity were lower in ADSCs compared to BMSCs, which may be partly responsible for the greater proliferation and differentiation potential of ADSCs. It is concluded that the proliferation and osteogenic differentiation of ADSCs were less affected by age and multiple passage than BMSCs, suggesting that ADSCs may become a potentially effective therapeutic option for cell-based therapy, especially in elderly patients with osteoporosis.

Expression of Nur77 induced by an n-butylidenephthalide derivative promotes apoptosis and inhibits cell growth in oral squamous cell carcinoma.

In spite of numerous advances, the 5-year survival rate for head and neck squamous cell cancer has remained largely stagnant and few new anti-tumor drugs have been developed. PCH4, a derivative of n-butylidenephthalide, has been investigated for its anti-tumor effects on oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the anti-tumor mechanism of a potential target gene, Nur77, in OSCC cells, which can be induced by PCH4 treatment. Data show that PCH4 promoted Nur77 translocation from the nucleus to the cytoplasm and induced cell apoptosis in OSCC cells. When Nur77 translocation was blocked, the degree of tumor apoptosis caused by PCH4 was significantly inhibited (p < 0.05). Within the MAPK pathway, PCH4 only induced JNK phosphorylation. Furthermore, treatment with a JNK inhibitor significantly reduced PCH4-induced apoptosis (p < 0.05) and decreased PCH4-induced Nur77 expression (p < 0.05). In a xenograft animal model, administration of PCH4 also showed anti-tumor effects. We have demonstrated that OSCC cells are sensitive to PCH4 and that Nur77 protein translocation from the nucleus to the cytoplasm might be associated with the induction of apoptosis by PCH4. These results indicate that PCH4 may serve as a potential anti-tumor drug for OSCC therapy.

Local interstitial delivery of z-butylidenephthalide by polymer wafers against malignant human gliomas.

We have shown that the natural compound z-butylidenephthalide (Bdph), isolated from the chloroform extract of Angelica sinensis, has antitumor effects. Because of the limitation of the blood-brain barrier, the Bdph dosage required for treatment of glioma is relatively high. To solve this problem, we developed a local-release system with Bdph incorporated into a biodegradable polyanhydride material, p(CPP-SA; Bdph-Wafer), and investigated its antitumor effects. On the basis of in vitro release kinetics, we demonstrated that the Bdph-Wafer released 50% of the available Bdph by the sixth day, and the release reached a plateau phase (90% of Bdph) by the 30th day. To investigate the in situ antitumor effects of the Bdph-Wafer on glioblastoma multiforme (GBM), we used 2 xenograft animal models-F344 rats (for rat GBM) and nude mice (for human GBM)-which were injected with RG2 and DBTRG-05MG cells, respectively, for tumor formation and subsequently treated subcutaneously with Bdph-Wafers. We observed a significant inhibitory effect on tumor growth, with no significant adverse effects on the rodents. Moreover, we demonstrated that the antitumor effect of Bdph on RG2 cells was via the PKC pathway, which upregulated Nurr77 and promoted its translocation from the nucleus to the cytoplasm. Finally, to study the effect of the interstitial administration of Bdph in cranial brain tumor, Bdph-Wafers were surgically placed in FGF-SV40 transgenic mice. Our Bdph-Wafer significantly reduced tumor size in a dose-dependent manner. In summary, our study showed that p(CPP-SA) containing Bdph delivered a sufficient concentration of Bdph to the tumor site and effectively inhibited the tumor growth in the glioma.

Butylidenephthalide suppresses human telomerase reverse transcriptase (TERT) in human glioblastomas.

BACKGROUND: Telomerase is widely expressed in most human cancers, but is almost undetectable in normal somatic cells and is therefore a potential drug target. Using the human telomerase promoter platform, the naturally occurring compound butylidenephthalide (BP) was selected for subsequent investigation of antitumor activity in vitro and in vivo. METHODS: We treated human glioblastoma cells with BP and found a dose-dependent decrease in human telomerase reverse transcriptase (hTERT) mRNA expression and a concomitant increase in p16 and p21 expression. Because c-Myc and Sp1 are involved in transcriptional regulation of hTERT, the effect of BP on c-Myc and Sp1 expression was examined. RESULTS: Using electrophoretic mobility shift assays and western blotting, we showed that BP represses hTERT transcriptional activity via downregulation of Sp1 expression. Using the telomerase repeat amplification protocol, an association between BP concentration and suppression of telomerase activity, induction of human glioblastoma senescence, and inhibition of cellular proliferation was identified. This was supported by a mouse xenograft model, in which BP repressed telomerase and inhibited tumor proliferation, resulting in tumor senescence. Overexpression of hTERT restored telomerase activity in human glioblastoma cells and overcame replicative senescence. CONCLUSIONS: These findings suggest that BP inhibits proliferation and induces senescence in human glioblastomas by downregulating hTERT expression and consequently telomerase activity. This is the first study to describe regulation of telomerase activity by BP in human glioblastomas.

Inhibitory effect of n-butylidenephthalide on neointimal hyperplasia in balloon injured rat carotid artery.

This investigation was designed to determine the inhibitory effects and mechanisms of n-butylidenephthalide (BP) from Angelica sinensis on smooth muscle cell (SMC) proliferation in vitro and in balloon injured rat carotid artery. Treatment of cultured rat aorta SMC-derived A7r5 cells with 25-100 µg/mL BP significantly inhibited the proliferation and arrested the cell cycle in G(0)/G(1) phase. BP induced the expression and migration of Nur77 from the nucleus to the cytoplasm. Among signal pathways, JNK and p38 MAPK were phosphorylated after BP treatment. In vivo, the neointimal area of common carotid artery 2 weeks after balloon injury reduced significantly in Sprague-Dawley rats treated with 150-300 mg/kg BP compared with the control. The proliferative activity indicated by immunohistochemical detection of Ki-67 positive cells in the neointima was significantly decreased in the 60-300 mg/kg BP treatment groups. The apoptotic activity indicated by cleaved caspase-3 positive cells and Nur77 positive cells in the neointima was significantly increased in rats treated with 60-300 mg/kg BP. This study demonstrated BP inhibited neointimal hyperplasia in balloon injured rat carotid artery due to its dual effects of proliferative inhibition and apoptotic induction on SMCs. Up-regulation of Nur77 gene may partly explain the antihyperplasia activity of BP on the neointima.

Overexpression of the orphan receptor Nur77 and its translocation induced by PCH4 may inhibit malignant glioma cell growth and induce cell apoptosis.

BACKGROUND: In previous study, n-butylidenephthalide (BP), a natural compound from Angelica sinensis, has anti-glioblastoma multiform (GBM) cell effects. In this study, we modified BP structure to increase anti-GBM cell effects. The anti-GBM cell effects of one derivative of BP, (Z)-N-(2-(dimethylamino)ethyl)-2-(3-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)phenoxy)acetamide (PCH4) were tested in vitro and in vivo. METHODS: MTT assay and PI/Annexin V assay were performed to evaluate the anti-GBM effects of PCH4. The Nur77 expression and translocation were assayed by RT-PCR and Western blot. The Nur77 siRNA was used to downregulate the Nur77 expression. The JNK inhibitor (SP600125) was used to block the JNK pathway. RESULTS: The anti-GBM effect of PCH4 is four times more than BP. The IC(50) of PCH4 on DBTRG-05MG cells was 50 µg/ml. Nur77 expression and translocation from the nucleus to the cytoplasm were important in PCH4-induced apoptosis. Furthermore, the downregulation of PCH4-induced Nur77 expression by Nur77 siRNA reduced PCH4-induced apoptosis. In addition, PCH4-induced apoptosis was associated with the JNK pathway. The JNK inhibitor, SP600125, inhibited Nur77 mRNA expression and reduced PCH4-induced apoptosis. CONCLUSIONS: In conclusion, PCH4, a derivative of BP, induced Nur77-mediated apoptosis via the JNK pathway and this mechanism, which is different from that of BP, may explain the increase in the anti-tumor effects on GBM.

Botanical drugs and stem cells.

The potential to generate virtually any differentiated cell type from stem cells offers the possibility of creating new sources of cells for regenerative medicine. To realize this potential, it will be essential to control stem cell differentiation. Chinese herbal medicine is a major aspect of traditional Chinese medicine and is a rich source of unique chemicals. As such, individual herbs or extracts may play a role in the proliferation and differentiation of stem cells. In this review, we discuss some of the Chinese herbal medicines that are used to treat human diseases such as neuronal degenerative diseases, cardiovascular diseases, and osteoporosis. We also describe the relationship between Chinese herbal medicines and stem cell regulation.

Transplantation of human Wharton's Jelly-derived stem cells alleviates chemically induced liver fibrosis in rats.

There is currently no effective treatment method available for liver fibrosis. We therefore evaluated the use of Wharton's jelly stem cells (WJSCs; the major umbilical cord stem cell population) to treat chemically induced liver fibrosis via intraperitoneal injection of thioacetamide. WJSCs were transplanted into liver-damaged rats via the portal vein and the treatment was evaluated by assessing serum biochemistry and histopathology. Transplanted WJSCs were distributed in the fibrotic area and around blood vessels, and hepatic recovery was accelerated. Serum prothrombin time significantly recovered, and serum albumin also improved at 21 days posttransplantation; collagen accumulation also decreased at 14 days. Thus, human WJSCs promoted recovery after chronic liver damage. Using immunohistochemical analyses, we determined that transplanted WJSCs produce albumin, hepatocyte growth factor (HGF), and metalloproteinase (MMP) after transplantation to chemically injured liver, indicating that WJSC may help to decrease liver collagen and thus may be useful for treating liver fibrosis.