Berberine inhibits metastasis of nasopharyngeal carcinoma 5-8F cells by targeting Rho kinase-mediated Ezrin phosphorylation at threonine 567.

Ezrin is highly expressed in metastatic tumors and is involved in filopodia formation as well as promotion of tumor metastasis. Thus, Ezrin may serve as a potential target for anti-metastatic therapy. This study demonstrates that berberine reduces filopodia formation of a nasopharyngeal carcinoma (NPC) cell line, 5-8F, at non-cytotoxic concentrations. Furthermore, invasion and motility of 5-8F cells are decreased in a dose- and time-dependent manner, resulting in 73.0% invasion and 67.0% motility inhibition at 20 mum. The inhibitory effects of berberine on 5-8F cell metastasis were further confirmed in a mouse model of metastasis. Berberine treatment in vivo resulted in a 51.1% inhibition of tumor metastasis to the lymph nodes and decreased Ezrin phosphorylation at threonine 567 in metastatic samples. Berberine suppressed the presence of phosphorylated Ezrin (phospho-Ezrin) in a dose- and time-dependent manner but had no effect on total Ezrin protein expression at non-cytotoxic concentrations. Furthermore, the inhibitory effects of berberine on phospho-Ezrin were dependent on the suppression of Rho kinase activity. Reduction of Ezrin phosphorylation at Thr(567) by berberine was associated with its inhibitory effect on filopodia formation in 5-8F cells. However, berberine did not effectively inhibit the motility and invasion of NPC cells containing Ezrin Thr(567) mutants. These results confirm that berberine inhibits Ezrin phosphorylation at Thr(567). Nonetheless, berberine reduces motility and invasion of cells and inhibits tumor metastasis. The reduction of Rho kinase-mediated Ezrin phosphorylation mediated by berberine may be a novel anti-metastatic pathway in NPC 5-8F cells.

HSP70 and mucin 5B: novel protein targets of N,N'-dinitrosopiperazine-induced nasopharyngeal tumorigenesis.

N,N'-Dinitrosopiperazine (DNP) induces nasopharyngeal carcinoma (NPC) and shows organ specificity to the nasopharyngeal epithelium. To investigate its mechanism, the rat NPC model was induced using DNP. Rat NPC and normal nasopharyngeal cells were obtained from the NPC model using laser capture. The total proteins from these cell samples were separated with two-dimension polyacrylamide gel electrophoresis techniques, and highly expressed proteins (> five-fold) were analyzed using matrix-assisted laser desorption/ionization time of flight and bioinformatics. The results showed that HSP70 and mucin 5B expression increased not only in rat NPC but also in atypical hyperplasia nasopharyngeal tissues, a precancer stage of NPC. High-expression of heat shock protein 70 (HSP70) and mucin 5B was further supported by western blot analysis. The immunofluorescence and western-blotting studies further showed that DNP induced the expression of HSP70 and mucin 5B in a dosage-dependent manner in normal nasopharyngeal epithelia cells. Our data indicate that DNP triggers over-expression of HSP70 and mucin 5B, and is involved in nasopharyngeal tumorigenesis. HSP70 and mucin 5B may be important targets in nasopharyngeal tumorigenesis induced by DNP.

Quantitative determination of erythromycylamine in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study of dirithromycin.

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 degrees C, and the residue was dissolved in 100 microL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm x 2.1 mm I.D., 5 microm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6-9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.

Quantitative determination of azithromycin in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study.

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of azithromycin in human plasma was developed and validated. Azithromycin in plasma (0.2mL) was extracted with methyl tert-butyl ether-hexane (50:50, v/v), organic phase was transferred to another clear 1.5mL Eppendorf tube and evaporated to dryness at 40 degrees C and dissolved in mobile phase, samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150mmx2.1mm i.d., 5microm), together with a mobile phase containing of 20mM ammonium acetate (pH 5.2)-acetonitrile-methanol (50:40:10, v/v/v) and was isocratically eluted at a flow rate of 0.2mL/min. Azithromycin and its internal standard, clarithromycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 2 to 1000ng/mL with r=0.9977. The limit of quantification for azithromycin in plasma was 2ng/mL with good accuracy and precision. The higher mean extraction recovery, say 81.2% and 75.5% for azithromycin and internal standard (IS), respectively, was obtained in this work. The intra-day and inter-day precision ranged from 4.8% to 8.6% and 6.4% to 10.7% (R.S.D.), respectively. The established method has been successfully applied to bioequivalence study of 2 azithromycin formulations for 24 healthy volunteers.

[Effect of yiqijiedu granule on the implanted-tumor protein expression in nasopharyngeal carcinoma].

OBJECTIVE: To investigate the inhibitory mechanism of Yiqijiedu granule on the implanted-tumor growth of nasopharyngeal carcinoma in nude mice. METHODS: Twenty nude mice were injected nasopharyngeal carcinoma cells (HNE1), with 5 x 10(6) cells for a nude mouse. Implanted-tumors grew for 20 d, whose volume reached 1 cm x 1 cm x 1 cm. These nude mice were divided into 2 groups: Yiqijiedu group and control group. The Yiqijiedu group was given Yiqijiedu granule, and the control was given normal saline for 30 d, and then were killed. The volume and weight of implanted-tumors were measured. A 100-mg tissue from implanted-tumors was used to extract total protein by current methods, in which the proteins were separated by two-dimension electrophoresis and stained by silver, and protein profiles of implanted-tumors were obtained. Different proteins in the profiles were analyzed by ImageMaster 2D Elite 4.01. Nineteen different proteins, in which 4 expressed in the Yiqijiedu group, 4 expressed in the control, and the other 11 were differently expressed at 5 folds, were identified by MALDI-TOF-MS. RESULTS: The Yiqijiedu granule could inhibit the growth of implanted-tumors. The volume and weight of implanted-tumors in the Yiqijiedu group were (0.207 +/- 0.023) cm3 and (0.132 +/- 0.021) g respectively, and that of the control was (1.342 +/- 0.298) cm3 and (1.017 +/- 0.015) g ( P < 0.05). The inhibitory rate was 84.58%. The analyses of two-dimension electrophoresis and ImageMaster 2D Elite 4.01 showed that there were 567 +/- 49 protein dots in the Yiqijiedu group and 679 +/- 59 in the control. We found 243 proteins were dys-regulated, of which 112 proteins were observed, up-regulated and the other 131 proteins were down-regulated. MALDI-TOF-MS and Database analysis showed that the high expression proteins were HKR2 protein, 10 Phosphoribosyl pyrophosphate synthetase, TNFR superfamily member, and Apoptosis regulator. The lower expression proteins were Fibulin-3, Zinc finger protein 266, Carboxy terminus of HSP70-interacting protein, et al. CONCLUSION: Yiqijiedu granule could inhibit the growth of nasopharyngeal carcinoma, which may be associated with those proteins regulated by itself.