Inhibition Mechanism of Novel Pyrazolo[1,5-a]pyrazin-4(5H)-one Derivatives Against Proliferation of A549 and H322 Cancer Cells.

OBJECTIVE: To explore the inhibition mechanism and safety of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives against proliferation of human lung cancer A549 cells, H322 cells, and human umbilical vein endothelial cell (HUVEC). METHODS: Cells were treated with 40 Μmol/L of the ppo3a, ppo3b, ppo3i, and 0.1% DMSO (control) for 48 hours, respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II, p53, and heat shock protein (HSP) 70 protein levels were detected by Western blotting in A549 cells treated with ppo3b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B (SRB) assay. RESULTS: Ppo3a, ppo3b, and ppo3i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3b effectively elevated the p53 protein level, but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3a, ppo3b, and ppo3i. CONCLUSIONS: ppo3a, ppo3b, and ppo3i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.

Detection of mutations by fill-in ligation reaction with enzyme-linked immunosorbent assay for rapid medical diagnosis.

Several approaches for parallel genotyping have been developed with increasingly available information on DNA variation. However, these methods require either complex laboratory procedures or expensive instrumentation. None of these procedures is readily performed in local clinical laboratories. In this study, we developed a flexible genotyping method involving fill-in ligation reaction with enzyme-linked immunosorbent assay successfully applied to detect important single-nucleotide polymorphisms (SNPs) for EGFR c.2573T > G (L858R), EGFR c.2582T > A (L861Q), and EGFR c.2155G > T (G719C). This assay exhibited excellent specificity, with a sensitivity as low as 0.5%. Eight out of 62 clinical samples were identified as heterozygotes for the SNP site of L858R, whereas only two samples were identified as heterozygotes by direct sequencing. The developed method enabled accurate identification of SNP in a simple and cost-effective manner adapted to routine analysis.

Synthesis of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives and their inhibition against growth of A549 and H322 lung cancer cells.

A series of substituted pyrazolo[1,5-a]pyrazin-4(5H)-one was synthesized by the reaction of ethyl 1-(2-oxo-2-phenylethyl)-3-phenyl-1H-pyrazole-5-carboxylate derivatives and 2-(2-aminoethoxy)ethanol or 2-morpholinoethanamine in the condition of microwave-assisted one-step and solvent-free in a good yield. The structures of the compounds were determined by IR, (1)H NMR and mass spectroscopy. In addition, a representative single-crystal structure was characterized by using X-ray diffraction analysis. Preliminary biological evaluation showed that the compounds could inhibit the growth of A549 and H322 cells in dosage-dependent manners.

20(S)-Protopanaxdiol Suppresses the Abnormal Granule-Monocyte Differentiation of Hematopoietic Stem Cells in 4T1 Breast Cancer-Bearing Mouse.

Panax notoginseng (PN) has been used as a qi- and blood-activating (Huoxue) drug for thousands of years in China. It has also been widely used as an anticancer drug at present. As a Huoxue drug, the effect of PN on hematopoietic differentiation in tumor-bearing body has been paid more and more attention. Our research found that panax notoginseng saponins (PNS), especially panaxadiol saponins (PDS) and its aglucon 20(S)-Protopanaxdiol (PPD), could improve the immunosuppressive state by regulating the abnormal hematopoietic differentiation in a tumor-bearing body by multiple ways. An interesting phenomenon is that PDS reduced the neutrophil-lymphocyte ratio (NLR) via its inhibition effect on the granule-monocyte differentiation of spleen cells, which is associated with a decrease in the secretion of tumor MPO, G-CSF, PU.1, and C/EBPα. Otherwise, PDS increased the proportion of both hematopoietic stem cells and erythroid progenitor cells in the bone marrow, but inhibited spleen erythroid differentiation via inhibiting secretion of tumor EPO, GATA-1, and GATA-2. This study suggests that PNS regulated the tumor-induced abnormal granule-monocyte differentiation of hematopoietic stem cells, affecting the distribution and function of haemocytes in tumor-bearing mice.

[Expression of B-glucosidase gene of Sacchromycopsis fibuligera in Pichia pastoris].

A beta-Glucosidase gene (BGL 1) was amplified with PCR from the total DNA of Sacchromycopsis fibuligera, and was linked with pGEM-T vector. After cut down by restriction enzyme from pGEM-T vector, BGL 1 was inserted into the expression vector pPIC9K of Pichia pastoris in reading frame with alpha-factor secreting signal peptide sequence to construct the recombinant plasmid pSHL9K. The recombinant plasmid pSHL9K was transformed into Pichia pastoris GS115 with electroporation. The recombinant Pichia pastoris strains which could efficiently secret recombinant beta-Glucosidase were selected. The optimum temperature of the recombinant beta-Glucosidase was 50degreesC, and the optimum pH was 5.4. The activity of beta-Glucosidase could reach to 47U/mL in the culture medium.

Percutaneous transhepatic biliary drainage and stenting for malignant obstructive jaundice: A report of two cases.

Malignant obstructive jaundice comprises a group of diseases that can be caused by primary biliary and extra-biliary carcinomas. Generally, surgical resection is the primary treatment for malignant obstructive jaundice; however, for the patients that are unable to undergo surgery, urgent treatment is required to improve hepatic function. Percutaneous transhepatic biliary drainage (PTBD) and stenting are emerging alternative treatments for malignant obstructive jaundice. PTBD and stenting have exhibited good efficacy for the treatment of malignant obstructive jaundice, with few complications and reduced associated pain.

Characterisation of free and bound volatile compounds from six different varieties of citrus fruits.

Free volatile compounds in six varieties of citrus juices were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry. Bound fractions were isolated and extracted with methanol and Amberlite XAD-2 resin and then hydrolyzed by almond ß-glucosidase. A total of 43 free and 17 bound volatile compounds were identified in citrus. Free volatile contents in sweet orange were the most abundant, followed by those in grapefruits and mandarins. Among free volatiles, terpenes were the most abundant in citrus juice. Sensory analysis results showed that the flavor of the same citrus cultivars was similar, but the flavor of different cultivars varied. Among bound volatiles, benzenic compounds were the most abundant in these citrus juices. Bound volatiles also significantly differed among cultivars. In addition, only p-vinylguaiacol were detected in all of the samples.

Novel chiral ferrocenylpyrazolo[1,5-a][1,4]diazepin-4-one derivatives--synthesis, characterization and inhibition against lung cancer cells.

A series of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one derivatives with optical activity (2) was synthesized in the microwave-assisted condition and characterized by means of IR, (1)H NMR and mass spectroscopy, and furthermore confirmed by X-ray analysis of a representative compound (R)-2a. Preliminary biological evaluation showed that some compounds could suppress the growth of A549, H322 and H1299 lung cancer cells. Among the tested compounds, 2b-d were more effective and might perform their action through cell cycle arrest for A549 cell. Whereas these compounds inhibited growth of H1299 and H322 cells by inducing apoptosis. The anti-tumor activities of these compounds were related to the nature of substituents in benzene moiety. In addition, the results indicated also that compounds 2b-d possessed notable cytotoxicity and selectivity for A549 vs H1299 and H322 lung cancer cells.