n-Butylidenephthalide Modulates Autophagy to Ameliorate Neuropathological Progress of Spinocerebellar Ataxia Type 3 through mTOR Pathway.

Spinocerebellar ataxia type 3 (SCA3), a hereditary and lethal neurodegenerative disease, is attributed to the abnormal accumulation of undegradable polyglutamine (polyQ), which is encoded by mutated ataxin-3 gene (ATXN3). The toxic fragments processed from mutant ATXN3 can induce neuronal death, leading to the muscular incoordination of the human body. Some treatment strategies of SCA3 are preferentially focused on depleting the abnormal aggregates, which led to the discovery of small molecule n-butylidenephthalide (n-BP). n-BP-promoted autophagy protected the loss of Purkinje cell in the cerebellum that regulates the network associated with motor functions. We report that the n-BP treatment may be effective in treating SCA3 disease. n-BP treatment led to the depletion of mutant ATXN3 with the expanded polyQ chain and the toxic fragments resulting in increased metabolic activity and alleviated atrophy of SCA3 murine cerebellum. Furthermore, n-BP treated animal and HEK-293GFP-ATXN3-84Q cell models could consistently show the depletion of aggregates through mTOR inhibition. With its unique mechanism, the two autophagic inhibitors Bafilomycin A1 and wortmannin could halt the n-BP-induced elimination of aggregates. Collectively, n-BP shows promising results for the treatment of SCA3.

Encapsulated n-Butylidenephthalide Efficiently Crosses the Blood-Brain Barrier and Suppresses Growth of Glioblastoma.

BACKGROUND: n-Butylidenephthalide (BP) has anti-tumor effects on glioblastoma. However, the limitation of BP for clinical application is its unstable structure. A polycationic liposomal polyethylenimine (PEI) and polyethylene glycol (PEG) complex (LPPC) has been developed to encapsulate BP for drug structure protection. The purpose of this study was to investigate the anti-cancer effects of the BP/LPPC complex on glioblastoma in vitro and in vivo. METHODS: DBTRG-05MG tumor bearing xenograft mice were treated with BP and BP/LPPC and then their tumor sizes, survival, drug biodistribution were measured. RG2 tumor bearing F344 rats also treated with BP and BP/LPPC and then their tumor sizes by magnetic resonance imaging for evaluation blood-brain barrier (BBB) across and drug therapeutic effects. After treated with BP/LPPC in vitro, cell uptake, cell cycle and apoptotic regulators were analyzed for evaluation the therapeutic mechanism. RESULTS: In athymic mice, BP/LPPC could efficiently suppress tumor growth and prolong survival. In F334 rats, BP/LPPC crossed the BBB and led to tumor shrinkage. BP/LPPC promoted cell cycle arrest at the G0/G1 phase and triggered the extrinsic and intrinsic cell apoptosis pathways resulting cell death. BP/LPPC also efficiently suppressed VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 expression. CONCLUSION: BP/LPPC was rapidly and efficiently transported to the tumor area across the BBB and induced cell apoptosis, anti-angiogenetic and anti-metastatic effects in vitro and in vivo.

Epigenetic targeting DNMT1 of pancreatic ductal adenocarcinoma using interstitial control release biodegrading polymer reduced tumor growth through hedgehog pathway inhibition.

Annually, 48,000 people die from pancreatic ductal adenocarcinoma (PDAC), ranking it the fourth among cancer-related deaths in the United States. Currently, anti-cancer drugs are not effective against PDAC, and only extends survival by 3 months. Aberrant DNA methylation has been shown to play an important role during carcinogenesis in PDAC, with approximately 80% of tumor overexpressing the DNA methyltransferase 1 (DNMT1) protein. In the present study, we used DNMTs as a screening platform to find a new DNMT inhibitor, n-butylidenephthalide (n-BP), which is identified from a Chinese herbal drug. n-BP could inhibit DNMT1 expression in both dose-dependent and time-dependent manner. It also displays an effect in suppressing growth of PDAC cells and inducing cell cycle arrest at G0/G1 phase leading apoptosis. Growth suppression can be restored by the overexpression of DNMT1 in PDAC cells. Furthermore, we found n-BP-mediated DNMT1 suppression influenced the protein stability rather than changing the RNA expression. Through microarray studies, we found that the patched domain contained 4 (PTCHD4) is the potential downstream gene of DNMT1. Following silencing of PTCHD4 expression by siRNA, n-BP decreased tumor growth inhibition. Finally, in vivo, two animal models were used to evaluate the efficacy and survival after n-BP treatment by interstitial control release polymer delivery. The results show that n-BP could effectively inhibit PDAC tumor volume growth and extend animal survival. In summary, n-BP may inhibit the growth of human PDAC cells though reducing DNMT1 and increasing the expression of PTCHD4 both in vitro and in vivo.

Non-Canonical Regulation of Type I Collagen through Promoter Binding of SOX2 and Its Contribution to Ameliorating Pulmonary Fibrosis by Butylidenephthalide.

Pulmonary fibrosis is a fatal respiratory disease that gradually leads to dyspnea, mainly accompanied by excessive collagen production in the fibroblast and myofibroblast through mechanisms such as abnormal alveolar epithelial cells remodeling and stimulation of the extracellular matrix (ECM). Our results show that a small molecule, butylidenephthalide (BP), reduces type I collagen (COL1) expression in Transforming Growth Factor beta (TGF-ß)-induced lung fibroblast without altering downstream pathways of TGF-ß, such as Smad phosphorylation. Treatment of BP also reduces the expression of transcription factor Sex Determining Region Y-box 2 (SOX2), and the ectopic expression of SOX2 overcomes the inhibitory actions of BP on COL1 expression. We also found that serial deletion of the SOX2 binding site on 3'COL1 promoter results in a marked reduction in luciferase activity. Moreover, chromatin immunoprecipitation, which was found on the SOX2 binding site of the COL1 promoter, decreases in BP-treated cells. In an in vivo study using a bleomycin-induced pulmonary fibrosis C57BL/6 mice model, mice treated with BP displayed reduced lung fibrosis and collagen deposition, recovering in their pulmonary ventilation function. The reduction of SOX2 expression in BP-treated lung tissues is consistent with our findings in the fibroblast. This is the first report that reveals a non-canonical regulation of COL1 promoter via SOX2 binding, and contributes to the amelioration of pulmonary fibrosis by BP treatment.

Anti-Cancer Effects of Radix Angelica Sinensis (Danggui) and N-Butylidenephthalide on Gastric Cancer: Implications for REDD1 Activation and mTOR Inhibition.

BACKGROUND/AIMS: Radix Angelica Sinensis (danggui in Chinese) is widely used in traditional chinese medicine (TCM). N-butylidenephthalide (BP), a bioactive compound in danggui, is a potential antitumor agent for various cancer types. However, its clinical effect and mechanism in the treatment of gastric cancer remain undetermined. METHODS: The in vivo protective effect of danggui in patients with gastric cancer were validated using data from Taiwan's National Health Insurance Research Database (NHIRD). The genes induced by BP-treatment were analyzed by whole transcriptome RNA sequencing (RNA-seq) and validated by real-time PCR, western blot and siRNA transfection. The effect of BP on AGS cell migration and invasion was evaluated in transwell assays. The antitumor effects of BP were evaluated in vivo in an AGS xenograft animal model. RESULTS: Danggui users were found to have an increased survival rate when compared with danggui nonusers (log-rank test p = 0.002) . The use of danggui highly associated with decreased mortality (the adjusted hazard ratio (HR) of danggui user was 0.72 [95 % CI, 0.57-0.92] (p = 0.009). The in vitro results showed that BP inhibited gastric cancer cell proliferation, and triggered cellular apoptosis depending on the activation of mitochondrial apoptotic pathway. Using RNA-seq analysis we found that REDD1 was the highest transcript induced by BP in gastric cancer cells. BP induce an increase of REDD1 expression that inhibits mTOR signaling, thus inhibiting gastric cancer growth. We used RNA interference to demonstrate that the knock-down of REDD1 attenuated the BP-induced mTORC1 activation and growth inhibition. BP suppressed the growth of AGS xenografts tumor in vivo. CONCLUSION: Danggui can prolong the survival rate of gastric cancer patients in Taiwan. BP caused gastric cancer cell death through the activation of mitochondria-intrinsic pathway and induced the REDD1 expression leading to mTOR signal pathway inhibition in gastric cancer cells. BP inhibited the in vivo growth of AGS xenograft tumors. These results may provide the basis for a new therapeutic approach toward the treatment of gastric cancer progression.

Sodium ferulate and n-butylidenephthalate combined with bone marrow stromal cells (BMSCs) improve the therapeutic effects of angiogenesis and neurogenesis after rat focal cerebral ischemia.

BACKGROUND: Studies have indicated that bone marrow stromal cell (BMSC) administration is a promising approach for stroke treatment. For our study, we chose sodium ferulate (SF) and n-butylidenephthalide (BP) combined with BMSC, and observed if the combination treatment possessed more significant effects on angiogenesis and neurogenesis post-stroke. METHODS: We established rat permanent middle cerebral artery occlusion (MCAo) model and evaluated ischemic volumes of MCAo, BMSC, SF + BP, Simvastatin + BMSC and SF + BP + BMSC groups with TTC staining on the 7th day after ischemia. Immunofluorescence staining of vascular endothelial growth factor (VEGF) and brain derived neurotrophic factor (BDNF), as well as immunohistochemistry staining of von Willebrand factor (vWF) and neuronal class III ß-tubulin (Tuj1) were performed in ischemic boundary zone (IBZ), furthermore, to understand the mechanism, western blot was used to investigate AKT/mammalian target of rapamycin (mTOR) signal pathway in ischemic cortex. We also tested BMSC derived-VEGF and BDNF expressions by western blot assay in vitro. RESULTS: SF + BP + BMSC group obviously decreased infarction zone, and elevated the expression of VEGF and the density and perimeter of vWF-vessels as same as Simvastatin + BMSC administration; moreover, its effects on BDNF and Tuj1 expressions were superior to Simvastatin + BMSC treatment in IBZ. Meanwhile, it showed that SF and BP combined with BMSC treatment notably up-regulated AKT/mTOR signal pathway compared with SF + BP group and BMSC alone post-stroke. Western blot results showed that SF and BP treatment could promote BMSCs to synthesize VEGF and BDNF in vitro. CONCLUSIONS: We firstly demonstrate that SF and BP combined with BMSC can significantly improve angiogenesis and neurogenesis in IBZ following stroke. The therapeutic effects are associated with the enhancement of VEGF and BDNF expressions via activation of AKT/mTOR signal pathway. Furthermore, triggering BMSC paracrine function of SF and BP might contribute to amplifying the synergic effects of the combination treatment.

Autophagic down-regulation in motor neurons remarkably prolongs the survival of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a lethal degenerating disease, characterized by progressive muscular atrophy without any effective treatment. Here, we demonstrated the efficacy of abrograting autophagy in motor neurons (MN) by treatment with n-butylidenephthalide (n-BP) in ALS transgenic mice (SOD1(G93A)). Pre-symptomatic oral administration of 250 mg/kg/bid n-BP significantly prolonged the survival period (203.9 ± 18.3 days), improved motor function, and attenuated MN loss compared to vehicle control (126.4 ± 7.2 days). This prolonged survival of ALS mice is much more robust than that reported with riluzole (140 days), which is an approved clinical therapy for ALS. The therapeutic mechanism targeted by n-BP involved the autophagic pathway as evidenced by decreased LC3-II expression (a biomarker of autophagy), enhanced mTOR levels, and attenuated autophagic activity, altogether increasing MN survival in a dose-dependent manner. This result was also confirmed by double transgenic mice (SOD1(G93A):LC3-GFP) which showed that oral administration of n-BP reduced GFP density and decreased caspase-3 expression. In addition, electron microscopy revealed that n-BP administration not only decreased autophagosome number but also reduced morphological dysfunction of mitochondria. In summary, these results indicate that down-regulation of autophagy activation via n-BP may pose as a therapeutic regimen for ALS and relevant neurodegenerative diseases.

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.

The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo.

The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G(0)/G(1) phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.

Relaxant effects of butylidenephthalide in isolated dog blood vessels.

We investigated the reason why butylidenephthalide (Bdph) can have an antianginal effect without changing blood pressure in conscious rats. Isolated dog coronary artery (CA), femoral vein (FV), femoral artery (FA), and mesenteric artery (MA) were used to evaluate the relaxant effects of Bdph. Bdph concentration-dependently relaxed isolated CA, FV, FA, and MA precontracted by KCl (60 mM) and phenylephrine (phe, 5 microM) with the exception that CA was precontracted by prostaglandin F 2 alpha (PGF 2 alpha, 2 microM) instead of phe. The potency order of Bdph to these blood vessels was FV > CA > FA > or = MA. Bdph also concentration-dependently and non-competitively inhibited cumulative KCl (5 - 120 mM)- and phe (0.1 - 100 microM)-induced contractions in normal, and inhibited cumulative Ca 2+-induced contractions in depolarized blood vessels. The potency order of Bdph to these blood vessels was FV congruent with CA > FA congruent with MA. Bdph (0.02 - 0.04 mM) concentration-dependently and leftward-shifted the log concentration-response curves in parallel and significantly increased the pD 2 value of forskolin, but not nitroprusside in FV. Bdph (0.1 mM) did both in CA. Bdph (0.225 - 0.45 mM) did the opposite to that of nitroprusside, but not forskolin, in FA. Bdph (0.45 - 0.9 mM) did neither in MA. Bdph (0.1 - 1 mM) significantly inhibited cAMP- but not cGMP-PDE activities in these four blood vessels, suggesting that Bdph more selectively inhibited the former in these tissues. The above results suggest that the higher potencies of Bdph on FV and CA than on FA and MA, may be interpreted as the reason why Bdph is useful in the treatment of angina pectoris without changing blood pressure, after Bdph administration in vivo, because the venoreturn may be reduced and the coronary flow may be increased without affecting the arterioles, such as MA, the main determinant of blood pressure. Abbreviations. Bdph:butylidenephthalide Phe:phenylephrine PGF 2alpha :prostaglandin F 2alpha CA:coronary artery FV:femoral vein FA:femoral artery MA:mesenteric artery cAMP:adenosine 3',5'-cyclic monophosphate cGMP:guanosine 3',5'-cyclic monophosphate PDE:phosphodiesterase