Realgar transforming solution-induced differentiation of NB4 cell by the degradation of PML/RARα partially through the ubiquitin-proteasome pathway.

PML/retinoic acid receptor alpha (RARα), as a hallmark of acute promyeloid leukemia (APL), is directly related to the outcome of clinical APL remedy. It is reported that arsenicals can effectively degrade PML/RARα, such as arsenic trioxide and realgar. However, the high toxicity or insolubility have hampered their clinical applications. Realgar transforming solution (RTS) was produced from realgar by bioleaching process in our lab. Previous studies demonstrated that RTS had a significant anti-cancer ability on chronic myeloid leukemia through oncoprotein degradation. The capacity of RTS on treating APL is what is focused on in this study. The results showed that RTS had a noticeable sensitivity in NB4 cell, and RTS remarkably down-regulated PML/RARα expression and induced cell differentiation. Further, RTS could accumulate PML/RARα into the nuclear bodies and then execute degradation, which could be reversed by proteasome inhibitor MG132. The results also exhibited that the reduction of RTS-induced PML/RARα expression accompanied by the elevation of ubiquitin and SUMO-1 protein expression. Finally, PML and SUMO-1 had been demonstrated to be co-localized after RTS treatment by immunofluorescence co-localization assay and immunoprecipitation assay. In conclusion, these results suggested that RTS-induced cell differentiation may attribute to the PML/RARα degradation partially through the ubiquitin-proteasome pathway.

Effects of pyrite bioleaching solution of Acidithiobacillus ferrooxidans on viability, differentiation and mineralization potentials of rat osteoblasts.

Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing.

[Chemical Constituents from Young Fruits of Citrus maxima cv. Shatian].

OBJECTIVE: To investigate the chemical constituents from the young fruits of Citrus maxima cv. Shatian. METHODS: The chemical constituents were isolated and purified by silica gel column chromatography and recrystallization, and their structures were identified on the basis of physicochemical properties and spectral analysis. RESULTS: Seven compounds were isolated and identified as naringenin (I), marmin (II), naringin (III), ß-sitosterol (IV) 5,7-dihydroxylcoumarin (V) 1, 3,5-trihydroxyhenzene (VI) and xanthotoxol (VII). CONCLUSION: Except compound III, all compounds are isolated from the young fruits of Citrus maxima cv. Shatian for the first time.

Anti-tumor effects and apoptosis induction by Realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo.

BACKGROUND: Realgar which contains arsenic components has been used in traditional Chinese medicine (TCM) as an anticancer drug. However, neither Realgar nor its formula are soluble in water. As a result, high dose of Realgar has to be administered to achieve an effective blood medicine concentration, and this is associated with adverse side effects. The objective of the present study was to increase the solubility of a formula using hydrometallurgy technology as well as investigating its effects on in vitro and in vivo cell proliferation and apoptosis in Sarcoma-180 cell line. MATERIALS AND METHODS: Antiproliferative activity of Realgar Bioleaching Solution (RBS) was evaluated by MTT assay. Further, effects of RBS on cell proliferation and apoptosis were studied using flow cytometry and transmission electron microscopy. Kunming mice were administered RBS in vivo, where arsenic specifically targeted solid tumors. RESULTS: The results indicated that RBS extract potently inhibited the tumor growth of Sarcoma-180 cell line in a dose-dependent manner. Flow cytometry and transmission electron microscopy further indicated that RBS significantly induced cell apoptosis through the inhibition of cell cycle pathway in a dose-dependent manner. Further, on RBS administration to mice, arsenic was specifically targeted to solid tumors CONCLUSIONS: RBS could substitute for traditional Realgar or its formula to work as a potent tool in cancer treatment.

[Study on the chemical constituents of Buddleja davidii].

OBJECTIVE: To study the chemical constituents of Buddleja davidii. METHODS: The constituents were isolated and purified by silica gel column chromatography, polyamide column chromatography and macroporous adsorption resin and their structures were identified by spectroscopic analysis. RESULTS: Eight compounds were elucidated as : Cranioside A (1), Eutigoside A (2), 1-O-4-Dimethoxyphenylethyl-4-O-3,4-dimethoxyphenylethy-beta-D-glucopyranoside (3), Isomartynoside (4'), 4"-O-Acetylmartynoside (5), Stigmasterol glueoside (6), beta-Sitosterol (7), Daucosterol (8). CONCLUSION: All these compounds are obtained from this plant for the first time.

Biological extraction of realgar by Acidithiobacillus ferrooxidans and its in vitro and in vivo antitumor activities.

Acidithiobacillus ferrooxidans is a Gram-negative, chemolithoautotrophic bacterium involved in metal bioleaching. It is used for the extraction of coarse medical realgar, which is converted into an aqueous solution. To prove its feasibility as an anticancer drug candidate, extracted realgar (ER/Af) was evaluated for its antitumor activities both in vitro and in vivo. In cytotoxicity tests, ER/Af displayed significant inhibition on cell proliferation of HepG2, SMMC7721, and H22 cells in a time and dose dependent manner. Remarkable tumor growth inhibition and survival time prolongation effects, along with no obvious toxicity, were observed in antitumor experiments against H22 cell-bearing mice. Apoptosis induction was also confirmed as one of the mechanisms involved in the efficacy of ER/Af both in vitro and in vivo. The most important observation is that ER/Af showed high selective affinity to tumor tissues with about eight-fold higher arsenic accumulations at the tumor site of mice than those of the arsenic trioxide (ATO)-treated group at the same dose (57.8 +/- 3.34 microg/g dry tissue vs. 7.6 +/- 0.88 microg/g dry tissue). In conclusion, A. ferrooxidans could be successfully used for the extraction of realgar and ER/Af was proved to be a promising anticancer drug candidate, which is valuable for further study and clinical trials.

[The inhibitory effects of Realgar bioleaching solution on S180 ascites-tumor and its toxicity].

OBJECTIVE: To investigate the inhibitory effect of Realgar bioleaching solution (RBS) on tumor S180 cells line and estimate its toxicity, to provide experimental evidence for the further exploit of Realgar. METHODS: 24 hours after the models of ascites-tumor bearing mice were established, the mice were injected RBS once a day. The survival rates of S180 ascites-tumor bearing mice injected in RBS was studied, and the RBS acute toxicity of mice produced by oral, intraperitoneal or intravenous was evaluated by Drug Median Lethal Dose (LD50). RESULTS: The inhibitory effect of S180 cells in vivo had a dose-dependent manner. The survival rates of mice were 10% - 60% in the different dose in 15 days. The LD50 values of RBS by oral, intraperitoneal or intravenous were 5.27 mg/kg, 3.63 mg/kg and 2.68 mg/kg, respectively. CONCLUSION: RBS has potent antitumor effect, LD50 value of RBS is lower than that of traditional medicine.

Peripheral inflammation increased the synaptic expression of NMDA receptors in spinal dorsal horn.

Considerable evidence has indicated that the aberrant, sustained enhancement of spinal NMDA receptors (NMDARs) function is closely associated with behavioral sensitization during inflammatory pain. However, the molecular mechanisms underlying inflammation-induced NMDARs hyperfunction remain poorly understood. The present study performed immunoblotting analysis to evaluate the possible changes in the protein expression of spinal NMDARs after injection of complete Freund's adjuvant (CFA) in mice. We found that CFA did not affect the total protein level of NMDARs subunit NR1 in spinal dorsal horn. However, NR1 immunoreactivity at synapses significantly increased after CFA injection, which was correlated in the time course with the development of mechanical allodynia. Inhibition of spinal NMDARs with D-APV completely eliminated the CFA-induced increase in NR1 immunoreactive density at synapses, and direct application of NMDA onto the spinal cord of naïve mice mimicked the effects of CFA, suggesting the importance of NMDARs activity in regulating the synaptic content of NR1 during inflammatory pain. Moreover, cAMP-dependent protein kinase (PKA) downstream to NMDARs was also required for NR1 synaptic expression because inhibition of PKA activity abolished the enhancement of synaptic NR1 immunoreactivity evoked by either CFA or NMDA. Thus, our data suggested that NMDARs- and PKA-dependent increase in NR1 synaptic expression represented an important mechanism for the hyperfunction of spinal NMDARs following peripheral inflammation.