ABSTRACT
Objective:To explore the molecular mechanism of spleen-strengthening traditional Chinese medicine （TCM） Weichang'an granule in inhibiting the invasion and metastasis of human gastric cancer MKN45 cells. Method:MKN45 cells were cultured <italic>in vitro</italic> and incubated with different concentrations（600， 900， 1 200， 1 500 mg·L<sup>-1</sup>）of Weichang'an granule for 24， 48， 72 h. Cell counting kit-8 （CCK-8） assay was used to detect its effect on the cell proliferation. Western blot was used to detect the expression of RUN and FYVE domain containing 3（RUFY3） in normal gastric mucosa cells and different gastric cancer cell lines. The expression of RUFY3 in the gastric cancer cells after Weichang'an granule intervention （600， 900， 1 200 mg·L<sup>-1</sup>） was detected by Western blot. Lentivirus transfection technique was used to achieve the stable and silenced expression of RUFY3 in gastric cancer MKN45 cells. Transwell assay was used to evaluate the influence of Weichang'an granule and silenced RUFY3 on the metastasis and invasion ability of MKN45. E-cadherin，N-cadherin，Vimentin，Zinc-finger transcription factor （SNAIL1 and SNAIL2） protein expression levels were detected by Western blot. Result:RUFY3 expression in human gastric cancer cells was significantly higher than that in normal gastric mucosa．The protein expression of RUFY3 in MKN45 cells of silenced RUFY3 group was significantly lower than that in Lentivirus negative group （<italic>P</italic><0.01）． Weichang'an granule inhibited the expression of RUFY3 in human MKN45 gastric cancer cells （<italic>P</italic><0.05， <italic>P</italic><0.01） in a concentration-dependent manner. As compared with the blank group， both Weichang'an granule and silenced RUFY3 inhibited the metastasis and invasion ability of MKN45 （<italic>P</italic><0.01）. After Weichang'an granule and silenced RUFY3 treatment， the protein expression of epithelial marker gene E-cadherin was up-regulated （<italic>P</italic><0.01）， the protein expression of Vimentin and N-cadherin decreased， but with no statistical difference，while SNAIL1 and SNAIL2 were both significantly down-regulated （<italic>P</italic><0.01）. Conclusion:By targeting RUFY3 to regulate epithelial mesenchymal transformation， the spleen-strengthening TCM compound Weichang'an granule can inhibit the invasion and metastasis of gastric cancer.
ABSTRACT
<b>Objective::To explore the effect of Weichang' an (WCA) on the autophagy of human gastric cancer MKN45 cells and its possible anti-cancer mechanism. <b>Method::MKN45 cells were cultured <italic>in vitro</italic> and incubated with different concentrations (250, 500, 1 000, 2 000 mg·L<sup>-1</sup>) of WCA for 24, 48, 72 h. Cell counting kit-8 (CCK-8) assay was used to detect the cell proliferation. AO/EB Dyeing (AO) staining and monodansylcadaverin (MDC) staining were used to observe the changes of the effect of WCA on autophagosome and autophagic vesicles in gastric cancer MKN45 cells at 48 h. Real-time polymerase chain reaction (Real-time PCR) and Western blot were used to detect microtubule-associated protein 1 light chain 3 (LC3), Beclin1, sequestosome 1 (p62), human autophagy-related gene 5 (ATG5), human autophagy-related gene 7 (ATG7) mRNA and protein expression levels. <b>Result::WCA showed a dose-and-time-dependent growth inhibition at the concentration above 1 000 mg·L<sup>-1</sup>. Compared with the blank group, WCA (500, 1 000, 2 000 mg·L<sup>-1</sup>) significantly inhibited the proliferation of MKN45 cells (<italic>P</italic><0.05, <italic>P</italic><0.01). AO staining and MDC staining showed that autophagosomes and autophagic sacs increased with the rise of WCA concentration compared with the blank group. Real-time PCR and Western blot showed that the expressions of autophagy-related proteins LC3-Ⅱ, Beclin1, ATG5, ATG7, mRNA and protein increased gradually after WCA (1 000 mg·L<sup>-1</sup>) intervention, while p62 expression decreased (<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion::WCA induces the autophagy of human gastric cancer MKN45 cells in a time-and dose-dependent manner <italic>in vitro</italic>, which may be related to the up-regulation of LC3-Ⅱ, Beclin1, ATG5 and ATG7 as well as the down-regulation of p62 and LC3-Ⅰ.
ABSTRACT
Objective: To observe the effect of Weichang'an on the tumor growth and lymph node metastasis, and RUN and FYVE domain-containing protein 3(RUFY3),Zinc finger protein 1(SNAI1),vascular endothelial growth factor(VEGF),epithelial-mesenchymal transition(EMT)-related proteins in nude mice with subcutaneous xenograft tumor human gastric carcinoma MKN45, so as to discuss the mechanism of Weichang'an on MKN45 human gastric metastasis. Method: The nude mice model of human gastric carcinoma MKN45 cells was established and randomly divided into normal saline group (0.5 mL/mouse), Weichang'an granule group (3.54 g·kg-1) and Weichang'an decoction group (35.49 g·kg-1). The tumor weight and volume of axillary lymph nodes in each group were observed. The morphology of lymph nodes in each group was detected by hematoxylin-eosin (HE) staining. The expressions of related proteins were detected by immunohistochemistry, including RUFY3,SNAI1,VEGF,E-cadherin,N-cadherin,Vimentin. Result: Compared with the normal saline group, the tumor weight and volume of axillary lymph node were decreased (PPPConclusion: Both Weichang'an granule and Weichang'an decoction can inhibit the tumor growth and metastasis of axillary lymph nodes, obviously down-regulate the expressions of RUFY3,SNAI1,VEGF,N-cadherin,Vimentin and up-regulate the expression of E-cadherin in human gastric MKN45 subcutaneous transplanted tumor in nude mice. This suggested that Weichang'an may inhibit the tumor metastasis through regulation expressions of RUFY3,SNAI1,VEGF and EMT-related proteins.
ABSTRACT
<p><b>OBJECTIVE</b>To establish a method for evaluating the activity of von Willebrand factor cleaving protease (vWF-cp) and evaluate its clinical application.</p><p><b>METHODS</b>Purified von Willebrand factor polymer was isolated by gel filtration from human fresh-frozen plasma as the enzyme substrate. SDS-PAGE, Western blotting, and luminographic detection were used to evaluate vWF-cp activity of 60 healthy adults, 28 patients with cerebral infarction (CI) and 7 with thrombotic thrombocytopenic purpura (TTP).</p><p><b>RESULTS</b>In the subjects involved, the method for evaluating vWF-cp activity had intra- and inter-batch coefficient of variation(CV) of 4.81% (n=8) and 8.63% (n=6), respectively. According to this method, the plasma vWF-cp activity in the 60 healthy adults was significantly higher than that in the CI patients [(86.53-/+17.49)% vs (77.15-/+16.72)%, P<0.05]. In TTP patients before plasma replacement, the vWF-cp activity was (9.06-/+7.17)% and increased significantly to (47.00-/+6.27)% 24 h after plasma replacement, respectively, but still significantly lower than that of healthy adults (P<0.01), whereas in the convalescent stage, the activity approached the normal level [(83.18-/+8.83)%, P>0.05].</p><p><b>CONCLUSIONS</b>According to the described method, which allows accurate vWF-cp activity measurement with good sensitivity, specificity and reproducibility, vWF-cp activity is lower in CI patients and even more so in TTP patients than that of healthy adults. Plasma replacement can effectively increase the vWF-cp activity in TTP patients.</p>