Trimellitic anhydride facilitates transepithelial permeability disrupting tight junctions in sinonasal epithelial cells.

Trimellitic anhydride (TMA) is a chemical agent classified as a low molecular weight (LMW) agent causing occupational rhinitis (OR) or asthma. Although TMA is recognized as a respiratory sensitizer, the direct and non-immunologic effects of TMA remain unclear. Air- liquid interface (ALI) cultured human nasal epithelial cells (HNECs) derived from control subjects were treated with TMA, followed by measurement of the transepithelial electrical resistance (TEER), paracellular permeability of fluorescein isothiocyanate (FITC)-dextran and immunofluorescence of tight junction proteins claudin-1 and zonula occludens-1 (ZO-1). The cytotoxicity of TMA was evaluated by lactate dehydrogenase (LDH) assay. TMA at concentrations of 2 and 4 mg/mL significantly reduced the TEER within 10 min (p = 0.0177 on 2 mg/mL; p < 0.0001 on 4 mg/mL). The paracellular permeability of FITC-dextran was significantly increased upon challenge with 4 mg/mL TMA for 3 h (p = 0.0088) and 6 h (p = 0.0004). TMA treatment induced a reduction in the fluorescence intensity of claudin-1 and ZO-1 in a dose-dependent manner. LDH assay revealed 4 mg/mL TMA induced cytotoxicity only after 6 h incubation, while 1 or 2 mg/mL TMA caused no cytotoxicity. Our results suggest that TMA has a potential to penetrate the epithelial barrier by disrupting claudin-1 and ZO-1, indicating an important role for sensitization and OR development.

Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells.

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G0/G1 phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

Enhanced anticancer activity and endocytic mechanisms by polymeric nanocarriers of n-butylidenephthalide in leukemia cells

Purpose The purpose of this study was to investigate the antitumor mechanisms of n-butylidenephthalide (BP) and to further examine the delivery efficacy of polycationic liposome containing PEI and polyethylene glycol complex (LPPC)-encapsulated BP in leukemia cells. Method MTS, flow cytometric and TUNEL assays were performed to assess cell viability and apoptosis. BP and BP/LPPC complex delivery efficiency was analyzed by full-wavelength fluorescent scanner and fluorescence microscope. The expressions of cell cycle- and apoptosis-related proteins were conducted by Western blotting. Results The results showed that BP inhibited leukemia cell growth by inducing cell cycle arrest and cell apoptosis. LPPC-encapsulated BP rapidly induced endocytic pathway activation, resulting in the internalization of BP into leukemia cells, causing cell apoptosis within 1 h. Conclusions LPPC encapsulation enhanced the cytotoxic activity of BP and did not influence the effects of BP induction that suggested LPPC-encapsulated BP might be developed as anti-leukemia drugs in future (AU)

Enhanced anticancer activity and endocytic mechanisms by polymeric nanocarriers of n-butylidenephthalide in leukemia cells.

PURPOSE: The purpose of this study was to investigate the antitumor mechanisms of n-butylidenephthalide (BP) and to further examine the delivery efficacy of polycationic liposome containing PEI and polyethylene glycol complex (LPPC)-encapsulated BP in leukemia cells. METHODS: MTS, flow cytometric and TUNEL assays were performed to assess cell viability and apoptosis. BP and BP/LPPC complex delivery efficiency was analyzed by full-wavelength fluorescent scanner and fluorescence microscope. The expressions of cell cycle- and apoptosis-related proteins were conducted by Western blotting. RESULTS: The results showed that BP inhibited leukemia cell growth by inducing cell cycle arrest and cell apoptosis. LPPC-encapsulated BP rapidly induced endocytic pathway activation, resulting in the internalization of BP into leukemia cells, causing cell apoptosis within 1 h. CONCLUSIONS: LPPC encapsulation enhanced the cytotoxic activity of BP and did not influence the effects of BP induction that suggested LPPC-encapsulated BP might be developed as anti-leukemia drugs in future.

Liposome Consolidated with Cyclodextrin Provides Prolonged Drug Retention Resulting in Increased Drug Bioavailability in Brain.

Although butylidenephthalide (BP) is an efficient anticancer drug, its poor bioavailability renders it ineffective for treating drug-resistant brain tumors. However, this problem is overcome through the use of noninvasive delivery systems, including intranasal administration. Herein, the bioavailability, drug stability, and encapsulation efficiency (EE, up to 95%) of BP were improved by using cyclodextrin-encapsulated BP in liposomal formulations (CDD1). The physical properties and EE of the CDD1 system were investigated via dynamic light scattering, transmission electron microscopy, UV-Vis spectroscopy, and nuclear magnetic resonance spectroscopy. The cytotoxicity was examined via MTT assay, and the cellular uptake was observed using fluorescence microscopy. The CDD1 system persisted for over 8 h in tumor cells, which was a considerable improvement in the retention of the BP-containing cyclodextrin or the BP-containing liposomes, thereby indicating a higher BP content in CDD1. Nanoscale CDD1 formulations were administered intranasally to nude mice that had been intracranially implanted with temozolomide-resistant glioblastoma multiforme cells, resulting in increased median survival time. Liquid chromatography-mass spectrometry revealed that drug biodistribution via intranasal delivery increased the accumulation of BP 10-fold compared to oral delivery methods. Therefore, BP/cyclodextrin/liposomal formulations have potential clinical applications for treating drug-resistant brain tumors.

Antitumor Effect of n-Butylidenephthalide Encapsulated on B16/F10 Melanoma Cells In Vitro with a Polycationic Liposome Containing PEI and Polyethylene Glycol Complex.

Advanced melanoma can metastasize to distal organs from the skin and yield an aggressive disease and poor prognosis even after treatment with chemotherapeutic agents. The compound n-Butylidenephthalide (BP) is isolated from Angelica sinensis, which is used to treat anemia and gynecological dysfunction in traditional Chinese medicine. Studies have indicated that BP can inhibit cancers, including brain, lung, prostate, liver, and colon cancers. However, because BP is a natural hydrophobic compound, it is quickly metabolized by the liver within 24 h, and thus has limited potential for development in cancer therapy. This study investigated the anticancer mechanisms of BP through encapsulation with a novel polycationic liposome containing polyethylenimine (PEI) and polyethylene glycol complex (LPPC) in melanoma cells. The results demonstrated that BP/LPPC had higher cytotoxicity than BP alone and induced cell cycle arrest at the G0/G1 phase in B16/F10 melanoma cells. The BP/LPPC-treated cell indicated an increase in subG1 percentage and TUNEL positive apoptotic morphology through induction of extrinsic and intrinsic apoptosis pathways. The combination of BP and LPPC and clinical drug 5-Fluorouracil had a greater synergistic inhibition effect than did a single drug. Moreover, LPPC encapsulation improved the uptake of BP values through enhancement of cell endocytosis and maintained BP cytotoxicity activity within 24 h. In conclusion, BP/LPPC can inhibit growth of melanoma cells and induce cell arrest and apoptosis, indicating that BP/LPPC has great potential for development of melanoma therapy agents.

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.

Biodegradable interstitial release polymer loading a novel small molecule targeting Axl receptor tyrosine kinase and reducing brain tumour migration and invasion.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour. The neoplasms are difficult to resect entirely because of their highly infiltration property and leading to the tumour edge is unclear. Gliadel wafer has been used as an intracerebral drug delivery system to eliminate the residual tumour. However, because of its local low concentration and short diffusion distance, patient survival improves non-significantly. Axl is an essential regulator in cancer metastasis and patient survival. In this study, we developed a controlled-release polyanhydride polymer loading a novel small molecule, n-butylidenephthalide (BP), which is not only increasing local drug concentration and extending its diffusion distance but also reducing tumour invasion, mediated by reducing Axl expression. First, we determined that BP inhibited the expression of Axl in a dose- and time-dependent manner and reduced the migratory and invasive capabilities of GBM cells. In addition, BP downregulated matrix metalloproteinase activity, which is involved in cancer cell invasion. Furthermore, we demonstrated that BP regulated Axl via the extracellular signal-regulated kinases pathway. Epithelial-to-mesenchymal transition (EMT) is related to epithelial cells in the invasive migratory mesenchymal cells that underlie cancer progression; we demonstrated that BP reduced the expression of EMT-related genes. Furthermore, we used the overexpression of Axl in GBM cells to prove that Axl is a crucial target in the inhibition of GBM EMT, migration and invasion. In an in vivo study, we demonstrated that BP inhibited tumour growth and suppressed Axl expression in a dose-dependent manner according to a subcutaneous tumour model. Most importantly, in an intracranial tumour model with BP wafer in situ treatment, we demonstrated that the BP wafer not only significantly increased the survival rate but also decreased Axl expression, and inhibited tumour invasion. These results contribute to the development of a BP wafer for a novel therapeutic strategy for treating GBM invasion and increasing survival in clinical subjects.

Liquid chromatography-tandem mass spectrometry evaluation of the pharmacokinetics of a diacid metabolite of norcantharidin loaded in folic acid-targeted liposomes in mice.

A previous study has reported diacid metabolite (DM) as the stable form of norcantharidin (NCTD), which is almost 100% metabolized to DM-NCTD. However, the unreliable pharmacokinetic characteristics of DM-NCTD could result in low bioavailability, hindering the clinical use of DM-NCTD in the treatment of diseases. A liposomal drug delivery system could overcome the shortcomings of DM-NCTD by improving the relative bioavailability (Fr), reducing drug toxicity, and increasing the therapeutic efficacy. However, there are no data concerning the pharmacokinetics of a DM-NCTD-loaded liposomal drug delivery system in animals, which is required for assessing its safety profile. Therefore, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of DM-NCTD in mouse plasma. Standard curves were linear (r=0.9966) over the range 10.0-1.00×10(4)ng/ml in mouse plasma with a lower limit of quantification (LLOQ) of 10ng/ml. This study successfully investigated the pharmacokinetics of DM-NCTD and DM-NCTD encapsulated in polyethylene glycol (PEG)-Liposomes (DM-NCTD/PEG-Liposome) or folic acid (FA)-PEG-Liposomes (DM-NCTD/FA-PEG-Liposome) in Kunming mice after a single intravenous dose of 2mg/kg. The plasma profile data of the three groups adhered to a two-compartment model. Compared with the DM-NCTD group, the Liposome groups had longer circulation times following intravenous administration in mice, and the Fr of DM-NCTD increased significantly (P<0.05). Furthermore, the area under the concentration-time curve (AUC) declined with an increase in the volume of distribution (Vd) from the PEG-Liposome to the FA-PEG-Liposome groups, which indicates a more efficient removal of the drug from the plasma of the FA-PEG-Liposome group. This result suggests a possible increased risk of DM-NCTD intoxication in normal tissues with FA-PEG-Liposomes. Based on this study, further investigation of the biodistribution of DM-NCTD/FA-PEG-Liposomes in healthy animals is warranted. In addition, the plausibility of formulating a safe DM-NCTD-loaded system without increasing toxicity against normal tissues needs to be determined.

Liposomal n-butylidenephthalide protects the drug from oxidation and enhances its antitumor effects in glioblastoma multiforme.

BACKGROUND: The natural compound n-butylidenephthalide (BP) can pass through the blood-brain barrier to inhibit the growth of glioblastoma multiforme tumors. However, BP has an unstable structure that reduces its antitumor activity and half-life in vivo. OBJECTIVE: The aim of this study is to design a drug delivery system to encapsulate BP to enhance its efficacy by improving its protection and delivery. METHODS: To protect its structural stability against protein-rich and peroxide solutions, BP was encapsulated into a lipo-PEG-PEI complex (LPPC). Then, the cytotoxicity of BP/LPPC following preincubation in protein-rich, acid/alkaline, and peroxide solutions was analyzed by MTT. Cell uptake of BP/LPPC was also measured by confocal microscopy. The therapeutic effects of BP/LPPC were analyzed in xenograft mice following intratumoral and intravenous injections. RESULTS: When BP was encapsulated in LPPC, its cytotoxicity was maintained following preincubation in protein-rich, acid/alkaline, and peroxide solutions. The cytotoxic activity of encapsulated BP was higher than that of free BP (~4.5- to 8.5-fold). This increased cytotoxic activity of BP/LPPC is attributable to its rapid transport across the cell membrane. In an animal study, a subcutaneously xenografted glioblastoma multiforme mouse that was treated with BP by intratumoral and intravenous administration showed inhibited tumor growth. The same dose of BP/LPPC was significantly more effective in terms of tumor inhibition. CONCLUSION: LPPC encapsulation technology is able to protect BP's structural stability and enhance its antitumor effects, thus providing a better tool for use in cancer therapy.

Butylidenephthalide antagonizes cromakalim-induced systolic pressure reduction in conscious normotensive rats.

BACKGROUND: Butylidenephthalide (Bdph), a main constituent of Ligusticum chuanxiong Hort., was reported to have selective antianginal effect without changing blood pressure in conscious rat. Recently, we have observed that Bdph antagonized cromakalim, an ATP-dependent K(+) channel opener, in guinea-pig trachea. Thus, we were interested in investigating whether Bdph at the dose without changing blood pressure antagonized cromakalim-induced systolic pressure reduction in conscious rats. METHODS: Systolic arterial pressures of conscious rats were determined by using the indirect tail-cuff method. RESULTS: Bdph (30 mg/kg, i.p.) did not affect baseline systolic pressure in conscious normotensive and spontaneous hypertensive rats. Bdph (30 mg/kg, i.p.) also did not affect log dose-response curves of prazosin, clonidine and Bay K 8644, a Ca(2+) channel activator, in normotensive rats. However, Bdph (30 mg/kg, i.p.) similar to 4-aminopyridine (4-AP, 0.4 mg/kg, i.p.), a K(+) channel blocker, non-parallelly but surmountably, and partially similar to glibenclamide (GBC, 10 mg/kg, i.v.), an ATP-sensitive K(+) channel blocker, surmountably but not parallelly rightward shifted the log dose-systolic pressure reduction curve of cromakalim, an ATP-sensitive K(+) channel opener, in normotensive rats, respectively. DISCUSSION: The antagonistic effect of Bdph against cromakalim was similar to that of 4-AP, a K+ channel blocker of Kv1 family, and partially similar to that of GBC, an ATP-sensitive K+ channel blocker. Thus, Bdph may be a kind of K+ channel blockers, which have been reviewed to have a potential clinical use for Alzheimer disease. Indeed, Bdph has also been reported to reverse the deficits of inhibitory avoidance performance and improve memory in rats. Recently, 4-AP was reported to treat Episodic ataxia type 2 (EA2) which is a form of hereditary neurological disorder. Consistently, Bdph was recently reported to have antihyperglycemic activity in mice, since GBC is a powerful oral hypoglycemic drug. CONCLUSIONS: Bdph similar to 4-AP and partially similar to GBC may block Kv1 family and ATP-sensitive K(+) channels in conscious normotensive rats.

Eosinophil-dependent skin innervation and itching following contact toxicant exposure in mice.

BACKGROUND: Contact toxicant reactions are accompanied by localized skin inflammation and concomitant increases in site-specific itch responses. The role(s) of eosinophils in these reactions is poorly understood. However, previous studies have suggested that localized eosinophil-nerve interactions at sites of inflammation significantly alter tissue innervation. OBJECTIVE: To define a potential mechanistic link between eosinophils and neurosensory responses in the skin leading to itching. METHODS: BALB/cJ mice were exposed to different contact toxicants, identifying trimellitic anhydride (TMA) for further study on the basis of inducing a robust eosinophilia accompanied by degranulation. Subsequent studies using TMA were performed with wild type versus eosinophil-deficient PHIL mice, assessing edematous responses and remodeling events such as sensory nerve innervation of the skin and induced pathophysiological responses (ie, itching). RESULTS: Exposure to TMA, but not dinitrofluorobenzene, resulted in a robust eosinophil skin infiltrate accompanied by significant levels of degranulation. Follow-up studies using TMA with wild type versus eosinophil-deficient PHIL mice showed that the induced edematous responses and histopathology were, in part, causatively linked with the presence of eosinophils. Significantly, these data also demonstrated that eosinophil-mediated events correlated with a significant increase in substance P content of the cutaneous nerves and an accompanying increase in itching, both of which were abolished in the absence of eosinophils. CONCLUSIONS: Eosinophil-mediated events following TMA contact toxicant reactions increase skin sensory nerve substance P and, in turn, increase itching responses. Thus, eosinophil-nerve interactions provide a potential mechanistic link between eosinophil-mediated events and neurosensory responses following exposure to some contact toxicants.

Inhaled multiwalled carbon nanotubes modulate the immune response of trimellitic anhydride-induced chemical respiratory allergy in brown Norway rats.

The interaction between exposure to nanomaterials and existing inflammatory conditions has not been fully established. Multiwalled carbon nanotubes (MWCNT; Nanocyl NC 7000 CAS no. 7782-42-5; count median diameter in atmosphere 61 ± 5 nm) were tested by inhalation in high Immunoglobulin E (IgE)-responding Brown Norway (BN) rats with trimellitic anhydride (TMA)-induced respiratory allergy. The rats were exposed 2 days/week over a 3.5-week period to a low (11 mg/m(3)) or a high (22 mg/m(3)) concentration of MWCNT. Nonallergic animals exposed to MWCNT and unexposed allergic and nonallergic rats served as controls. At the end of the exposure period, the allergic animals were rechallenged with TMA. Histopathological examination of the respiratory tract showed agglomerated/aggregated MWCNT in the lungs and in the lung-draining lymph nodes. Frustrated phagocytosis was observed as incomplete uptake of MWCNT by the alveolar macrophages and clustering of cells around MWCNT. Large MWCNT agglomerates/aggregates were found in granulomas in the allergic rats, suggesting decreased macrophage clearance in allergic rats. In allergic rats, MWCNT exposure decreased serum IgE levels and the number of lymphocytes in bronchoalveolar lavage. In conclusion, MWCNT did not aggravate the acute allergic reaction but modulated the allergy-associated immune response.

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.

IL-6, VEGF, KC and RANTES are a major cause of a high irritant dermatitis to phthalic anhydride in C57BL/6 inbred mice.

BACKGROUND: In previous studies, several strains of mice were used as chemical-induced skin irritation models to identify immunological hazards and elucidate the molecular and cellular mechanisms by which irritant dermatitis disease occur. BALB/c and C57BL/6 mice have been used for most of these experiments. Although there are some differences in the immune response to chemical allergens between these strains, few studies have been conducted to determine what regulatory factors contribute to these variations. METHODS: To investigate the cause of high responses to skin irritation in C57BL/6 mice that are widely used to study atopic dermatitis, changes in various immune-related factors such as ear thickness, myeloperoxidase activity, lymph node weight, IgE concentration and cytokine concentration were measured in C57BL/6 and BALB/c mice following phthalic anhydride (PA) treatment. RESULTS: Based on analysis of the skin irritation, C57BL/6 mice showed a greater skin irritation to PA than BALB/c mice, although the IgE concentration and auricular lymph node weight did not contribute to this difference in the response. However, the concentration of several cytokines and chemokines (interleukin [IL]-6 and vascular endothelial growth factor [VEGF], keratinocyte-derived chemokine [KC] and regulated on activation normal T cell expressed and secreted [RANTES]) were significantly higher in C57BL/6 mice than BALB/c mice following treatment with PA. CONCLUSIONS: Our results suggest that several of the cytokines and chemokines secreted from irritant site could contribute to the regulation mechanism responsible for the difference in the skin irritation among various strains of mice following exposure to PA.

Extended O6-methylguanine methyltransferase promoter hypermethylation following n-butylidenephthalide combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on inhibition of human hepatocellular carcinoma cell growth.

Epigenetic alteration of DNA methylation plays an important role in the regulation of gene expression associated with chemosensitivity of human hepatocellular (HCC) carcinoma cells. With the aim of improving the chemotherapeutic efficacy for HCC, the effect of the naturally occurring compound n-butylidenephthalide (BP), which is isolated from a chloroform extract of Angelica sinensis, was investigated. In both HepG2 and J5 HCC cell lines, a synergistic antiproliferative effect was observed when a low dosage of BP was combined with the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is an alkylating agent, and it prompts us to examine one of DNA repair genes, O(6)-methylguanine methyltransferase (MGMT). It was evident from methylation-specific polymerase chain reaction (PCR) analysis that BP/BCNU combined treatment caused a time- and concentration-dependent enhancement of MGMT promoter methylation. Overexpression of MGMT could abolish BP-induced growth inhibition in the J5 tumor cell line as measured by colony formation assay. When BP was combined with BCNU and administered, it showed significant antitumor effects in both HepG2 and J5 xenograft tumors as compared with the use of only one of these drugs. The BCNU-induced apoptosis and inhibited MGMT protein expression in HCC cells, both in vitro and in vivo, resulting from the combination treatment of BP and BCNU suggest a potential clinical use of this compound for improving the prognosis for HCCs.

Smad3 regulates dermal cytokine and chemokine expression and specific antibody production in murine responses to a respiratory chemical sensitizer.

BACKGROUND: The cytokine transforming growth factor-beta(TGF-beta) has important regulatory roles in the immune system. To investigate the role of intact TGF-beta signaling during the contact hypersensitivity (CHS) response to a respiratory allergen, we exposed Smad3-/- mice to topical trimellitic anhydride (TMA). METHODS: CHS was induced by topical application of TMA. The swelling of the TMA-exposed ears was analyzed, and lymph node, ear tissue and skin biopsies were collected for RNA isolation, histology and histochemical analyses. Lymph node cell proliferation was measured and blood samples were collected for analysis of TMA-specific immunoglobulin. RESULTS: Topical TMA exposure resulted in increased mRNA expression of proinflammatory and suppressive cytokines (IL-1beta, TNF-alpha, IL-6, IFN-gamma, IL-4, IL-10, IL-17, IL-23, TGF-beta), chemokines (CXCL9, CXCL10, CCL24) and chemokine receptors (CCR7, CCR8, CXCR2), increased numbers of CD3+ T cells in ear tissue, and lymphadenopathy in the Smad3-/- mice. The IL-10 result was confirmed at the protein level by immunohistochemistry. However, the ear-swelling response and infiltration of eosinophils, F4/80+ cells, CD11c+ cells and mast cells were similar in the Smad3-/- mice compared to their wild-type (WT) siblings. While TMA-specific IgE was induced equally in the WT and Smad3-/- mice, the concentration of TMA-specific IgG2a was significantly lower in the Smad3-/- mice. CONCLUSIONS: The Smad3 molecule contributes significantly to the regulation of the cytokine and chemokine network during the CHS response to TMA. The lack of Smad3 resulted in a potent Th2 shift, confirmed by strongly impaired IgG2a levels.

Investigation of the chemical-induced selective type II (T(H)2) allergic response in mice: effect of the length of the sensitizing phase.

Allergies are immune system disorders characterized by abnormal, acquired sensitivity to various environmental chemicals. We investigated the mechanism of chemical-induced selective type II (T(H)2) allergy by using three different sensitization protocols and the well-known respiratory sensitizer trimellitic anhydride (TMA). Mice were sensitized for either 1, 2, or 3 weeks. For each sensitization schedule, the mice were allocated into 3 or 4 groups: -/- group, both sensitized and challenged with vehicle; -/+ group, sensitized with vehicle and challenged with 0.1% TMA; +/- group, sensitized with 1% TMA and challenged with vehicle; and +/+ group, both sensitized and challenged with 0.1% TMA. After challenge, we assayed the auricular lymph nodes of all mice for number of lymphocytes, surface antigen expression of B-cells, and local cytokine production, and we measured TMA-specific serum IgE levels. Some parameters in mice sensitized for 1 or 2 wk showed, at most, mild changes. In contrast, all parameters in animals receiving 3-wk sensitization showed marked increases, as well as marked increases in the IgE/major histocompatibility complex (MHC) Class II-positive B-cell population and T(H)2 cell production of IL-10 and IL-13. These results indicate that 3 wk of sensitization according to our protocol led to overt respiratory allergic reactions. While these studies showed that using the approach here, positive reactions were elicited using a typical allergen; whether the same events occur after sensitization by other chemicals that are found in the environment remains uncertain. These findings here should be regarded moreover as preliminary in scope and that additional studies with irritants, dermal sensitizers and other respiratory sensitizers are needed to further evaluate the overall sensitivity and selectivity of this novel protocol.

Comparative studies of lymph node cell subpopulations and cytokine expression in murine model for testing the potentials of chemicals to induce respiratory sensitization.

OBJECTIVES: To investigate immunological changes in lymph nodes based on expression of cell-specific receptors and cytokine expression profile and accompanying inflammatory reactions in lungs of mice treated with chemicals of known potentials to induce respiratory sensitization and those in which activity in this regard is unclear. MATERIALS AND METHODS: On day 1 and 7, Balb/c mice received toluene-2,4-diisocyanate (TDI), trimellitic anhydride (TMA), 1-chloro-2,4-dinitrobenzene (DNCB), glutaraldehyde (GA), formaldehyde (FA), benzalkonium chloride (ChB) or vehicle. On day 14, they received a single intranasal instillation with the same chemical or vehicle. On day 15, auricular lymph nodes (LN) were excised and used for analyzes of T-, B-cells, expression of CD44 and for the estimation of IL-4 and IFN-gamma production after in vitro stimulation with concanavalin A (ConA) and also for IL-4 and IFN-gamma mRNA expression analyses using Real-Time PCR. Inflammatory changes in lungs were observed by estimation of TNF-alpha and MIP-2 concentrations and cell numbers and their type in BAL. RESULTS: There were no significant changes in cell subpopulations of T helper cells in LN. The percent of B cells was significantly increased after treatment with DNCB, TDI, and GA. Increased expression of CD44 on T cells was also observed. Both IL-4 and IFN-gamma were found increased in TDI- and FA-treated mice, while only IL-4 was increased in TMA-treated mice. Real-Time PCR analyses, however, showed increased IL-4 mRNA expression for TDI- and TMA-, and IFN-gamma mRNA expression for DNCB-treated mice. We haven't observed significant changes in inflammatory reactions in the lungs of exposed animals. CONCLUSIONS: Studying immunological changes with first determining the activation status of T cells followed by analyzes of expression of mRNA for Th1 and Th2 cytokines in murine model could be a useful method for assessment of the potentials of chemicals to induce respiratory sensitization but is not sufficient. Addition of ventilatory measurements, but not necessarily inflammatory reactions, could complete the model.

Orphan nuclear receptor, Nurr-77 was a possible target gene of butylidenephthalide chemotherapy on glioblastoma multiform brain tumor.

The natural compound n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis, has been investigated for its antitumoral effects on glioblastoma multiform (GBM) brain tumors both in vitro and in vivo. To determine the mechanism of BP-induced growth arrest and apoptosis, we examined BP-induced changes in gene expression by microarray screening using human GBM brain tumor cells. This analysis identified several BP-inducible genes, including the nuclear receptors NOR-1, Nurr1, and Nur77. Among these genes, Nur77 is particularly interesting because it plays an important role in the apoptotic processes in various tumor cell lines. BP was able to increase Nur77 mRNA and protein expression in a time-dependent manner. After BP treatment in GBM 8401 cells, Nur77 translocated from the nucleus to the cytoplasm, the cytochrome c was released from the mitochondria, and caspase 3 became activated. Furthermore, using Nur77 promoter-luciferase assay, BP increased Nur77 was AP1 related. Inhibition of BP-induced Nur77 expression by Nur77 short interfering RNA blocked BP-induced apoptosis in GBM 8401 cells, suggesting that the induction of Nur77 negatively affected GBM 8401 cell survival. In summary, our results suggest that up-regulation of Nur77 may explain the antitumoral activity of BP in brain tumor cells.