A Mixture of Baicalein, Wogonin, and Oroxylin-A Inhibits EMT in the A549 Cell Line via the PI3K/AKT-TWIST1-Glycolysis Pathway.

Non-small cell lung cancer (NSCLC) is a worldwide disease with a high morbidity and mortality rate, which is most derived from its metastasis. Some studies show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cell migration and invasion, leading to NSCLC metastasis. Total flavonoid aglycones extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds [baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)], the reconstructed TFAE (reTFAE) inhibited the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of the PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through the glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC.

Total flavonoid aglycones extract in Radix scutellariae inhibits lung carcinoma and lung metastasis by affecting cell cycle and DNA synthesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Scutellariae (Scutellaria baicalensis Georgi, RS), a traditional herbal medicine commonly used to treat inflammation, hypertension, cardiovascular disease, bacterial and viral infections, is reported to treat lung cancer by supplements of modern medicine. The total flavonoid aglycones extract (TFAE) from RS is the most important composition for the pharmacodynamic effects. The present study was designed to evaluate the anti-lung tumor effect of TFAE on A549 cells and A549 cell nude mice xenografts. The aim of the study is to investigate the effect and mechanism of TFAE treating non-small cell lung cancer both in vitro and in vivo. MATERIALS AND METHODS: The anti-tumor activity of TFAE in vitro was investigated using the MTT assay. The changes of cell invasion and migration were detected by Transwell assay and tube formation experiments were used to detect the anti-angiogenic effect. The anti-tumor effects of TFAE in vivo were evaluated in A549 cell nude mice xenografts. The mechanism of TFAE was detected by flow cytometry technology, western blot assay and immuno-histochemistry assay. RESULTS: In vitro, TFAE inhibited the proliferation, invasion and migration of A549 cells in a dose- and time-dependent manner. In vivo, TFAE by oral administration at 100mg/kg for 30 days decreased the tumor volume and tumor weight in A549 cell xenograft by 25.5% with no statistical significance (P<0.05) compared to the cis-platinum positive control group (30.0%). The cell cycle and DNA synthesis experiment illustrated that TFAE could induce A549 cell cycle to arreste in S phase and DNA synthesis in A549 cells be inhibited, while TFAE had no influence on apoptosis of A549 cells. Western Blot assay demonstrated that the treatment of TFAE could make Cyclin D1 decrease and p53 increase both in vitro and in vivo. CONCLUSION: TFAE displayed the inhibition effects of non-small cell lung cancer both in vitro and in vivo and the underlying mechanism might be related to the increased p53 protein expression and decreased Cyclin D1 expression, leading to cell cycle arrested in S phase and the decrease of DNA synthesis.

[Photochemotherapy with psoralen and ultraviolet A induced apoptosis of NB4 cells and its effects on caspase-8 and caspase-8 protein expressions].

OBJECTIVE: To study the regulatory effects of psoralen (PSO) plus ultraviolet A (UVA), which is PUVA, on cell apoptosis of human leukemia cell line NB4 and signal pathway of cell apoptosis. METHODS: Human leukemia cell line NB4 was cultured in vitro. The NB4 cells were treated with PSO extracted from Chinese medicine psoralea fruits at different concentrations (0, 5, 10, 20 and 40 microL) plus UVA of wave length 360 nm at different irradiation time points (0 and 5 min). The apoptosis ratio was detected by flow cytometry (FCM). The ultrastructure changes were observed using transmission electron microscope (TEM). The expressions of Caspase-8 and Caspase-8 protein were detected by immunocytochemical method (ICC). RESULTS: After treatment of PSO at different concentrations with a 0 and 5-min exposure of UVA, the apoptosis rate of NB4 cells increased dose-and time-dependently, and was up to peak after treatment of PSO at 40 microg/mL with 5-min exposure of UVA. An interaction was shown between the two factors (P <0. 01). There were obvious morphological apoptosis of NB4 cells under TEM after treated with PUVA. The expressions of Caspase-3 and Caspase-8 protein were up-regulated by PSO, UVA, and PUVA, but the effects of PUVA on Caspase-3 protein were stronger than PSO and UVA at 12 h time-dependently (P <0.01).An interaction was shown between the concentration of PSO and time of UVA (P <0.01). CONCLUSIONS: The optimal combination of PUVA was PSO in 40 microg/mL and 5-min exposure of UVA. PUVA could induce the apoptosis of NB4 cells and in vitro activate Caspase-3 and Caspase-8 genes.

Differences in metabolites of different tongue coatings in patients with chronic hepatitis B.

Tongue coating is one of the important foundations of tongue diagnosis in traditional Chinese medicine (TCM) and plays an important role in reflecting the occurrence, development, and prognosis of the disease. However, its material basis is still poorly understood. In this study, a urinary metabonomic method based on gas chromatography coupled to mass spectrometry (GC/MS) was developed. The distinct clustering in metabolic profile was observed from Group A (thick yellow coating in patients with chronic hepatitis B), Group B (thick white coating in patients with chronic hepatitis B), and Group C (thin white coating with healthy humans) using orthogonal projections to latent structures (OPLS). Based on the variable of importance in the project (VIP) values, some significantly changed metabolites have been identified. These changes were related to the disturbance in energy metabolism, amino acid metabolism, nucleotide metabolism, and gut microflora, which were helpful to understand the material basis leading to the formation of tongue coating. This study demonstrated that tongue coating may have an objective material basis.

Study on dissolution and absorption of four dosage forms of isosorbide mononitrate: level A in vitro-in vivo correlation.

The objective of the present study was to develop a novel in vitro system to simulate the process of dissolution and permeation of oral solid dosage forms in vivo, and to establish a correlation between in vitro permeation and in vivo absorption that could predict the bioavailability (BA) and bioequivalence (BE) of congeneric products. The in vitro dissolution and absorption kinetics of four dosage forms of isosorbide mononitrate (ISMN) were evaluated by the USP basket/paddle system and drug dissolution/absorption simulating system (DDASS). The corresponding pharmacokinetic study was performed in beagle dogs. A comparative study was carried out between the classical and the novel method to estimate the effectiveness of the modified DDASS in simulating the course of dissolution and absorption in vivo. Indeed, the correlation coefficients of in vitro dissolution and in vivo absorption obtained from DDASS and dogs were higher. Moreover, a higher level A in vitro-in vivo correlation (IVIVC) between DDASS permeation and dog absorption was established, with correlation coefficients of 0.9968, 0.9872, 0.9921, and 0.9728. The DDASS method was more accurate at modeling the process of dissolution and absorption in vivo for both immediate-release (IR) and sustained-release (SR) dosage forms of ISMN.

[Compound huangdai tablet as induction therapy for 193 patients with acute promyelocytic leukemia].

OBJECTIVE: To report the results of curative and adverse effects of compound huangdai tablet (CHDT) as induction therapy for 193 patients with acute promyelocytic leukemia (APL). METHODS: CHDT was administered 1.25 g orally three times a day after meal for three days, then the dosage was gradually increased to 7.5 g/d. RESULTS: One hundred and ninety-three patients achieved complete remission (CR), 78.8% of whom in 30 to 60 days with an average time of 44.3 d. No serious infection, bleeding or DIC occurred during the treatment course. The major adverse effects were gastrointestinal symptoms. There was no change in lanine transaminase, urea, creatinine or electrocardiographic QTc interval in 110 APL patients observed before and after the treatment. CONCLUSION: CHDT therapy is a modality of higher CR rate, good safety and tolerance without bone marrow suppression for APL patients.

[Progress in studies of mechanism of anti-fibrosis traditional Chinese drugs and effective components].

The progress in the research of pharmacological mechanism of anti-fibrosis traditional Chinese drugs and the effective components is summarized. It's demonstrated by pharmacological experiments that anti-fibrosis traditional Chinese drugs can inhibit the cell proliferation, regulate the cytokines and ECM, and intervente in the signal transduction.

Pre-deliver chitosanase to cells: a novel strategy to improve gene expression by endocellular degradation-induced vector unpacking.

A radio-labeled plasmid pTracer/Bsd/LacZ containing LacZ reporter gene was complexed with different molecular weights of chitosans (CS). Mouse myoblast cell line C2C12 was transfected by these chitosan-plasmid DNA complexes, and lipofectamine 2000 was used as control. Forty-eight hours after transfection, the activity of beta-galactosidase and radioactive count of cell lysis were determined. It was found that chitosan, especially low molecular weight species, had a surprising ability to deliver DNA into cells, since the radioactive count of cells transfected by chitosan-DNA complexes was even two times that of cells transfected by lipofectamine 2000. But the beta-galactosidase activity of chitosan/DNA complexes was much lower compared to that of lipofectamine 2000. Chitosanase which could degrade chitosan in specific mode was transported into C2C12 cells by osmotic lysis prior to gene delivery. Then these chitosanase-modified cells were transfected by CS-DNA complexes. The results indicated that beta-galactosidase activity in these cells increased markedly to 425.4 +/- 45.1 U/mg protein, nearly two-fold as that of cells transfected by liposome. This transfection protocol was also applied to 3T3 mouse fibroblast, 2T3 mouse osteoblast and MG63 human osteosarcoma cell lines, and an increased gene expression level was observed without exception. It is thought that the incorporated chitosanase could aid in chitosan degradation, which would promote gene unpacking, consequently increasing gene expression.

Self-aggregation behavior of alkylated chitosan and its effect on the release of a hydrophobic drug.

Chitosan (CS) was hydrophobically modified with butyl bromide and dodecyl bromide. The self-aggregation in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light-scattering method. The results indicate that introducing butyl and dodecyl moieties leads to the formation of self-aggregates. Along with the enhancement in the hydrophobicity of chitosan the self-association occurs at a lower concentration, and the mean size of self-aggregates increases. The loading capacity of butylated chitosan (4-CS) and dodecylated chitosan (12-CS) for vitamin B2 are markedly increased compared to that of chitosan, and the release of drug from alkylated chitosans is somewhat hindered due to its increased affinity for hydrophobic carriers.

N-alkylated chitosan as a potential nonviral vector for gene transfection.

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.