2012 4 20 4 MODERN ONCOLOGY Apr. 2012 VOI. 20 NO. 04 693 SMMC - 7721 珮 Anti - tumor effect in vitro of coix seed oil injection on the human liver cancer cell SMMC - 7721 and the mechanism study YIN Beipei YAN Pingping LIU Chang LU Hongqi LI Bingsheng State Key Laboratory of Innovative Medicines and Pharmaceutical Technology Shanghai Institute of Pharmaceutical Industry Shanghai 200437 China. Abstract Objective To evaluate the anti - tumor effect in vitro of coix seed oil injection on the human liver cancer cell SMMC - 7721 and study the mechanism of action. Methods Human HCC cells were treated with KLT. CCK - 8 cell viability assay scratch assay transwell assay and matrigel sprout assay were performed to investigate the effect of KLT on cell proliferation migration and invasion. Impact of KLT on apoptosis and cell cycle of cancer cells were measured by flowcytometry. The expression level of target genes of KLT in cancer cells were studied by Western blot. Results KLT suppressed the cell proliferation migration and invasion of human HCC cells. Cell cycle of HCC cells was arrested in G 2 /M phase when treated with KLT and late stage of apoptosis was observed. KLT was capable of inducing the expression of cyclin B1 while down - regulating the expression of cyclin D1 cyclin E. Conclusion KLT possesses well acknowledged anti - tumor activity in vitro which may result from cell cycle arrest apoptosis down - regulation of oncogenes. Key words coix seed extract KLT SMMC - 7721 in vitro anti - tumor mechanism Modern Oncology 2012 20 04 0693-0698 KLT SMMC - 7721 SMMC - 7721 CCK - 8 Transwell Matrigel KLT KLT Western blot KLT KLT KLT G 2 /M KLT cyclin B1 cyclin D1 cyclin E KLT SMMC - 7721 R735. 7 A DOI 10. 3969 /j. issn. 1672-4992. 2012. 04. 11 1672-4992 - 2012 04-0693 - 06 6 3 2 1 2011-11 - 09 2011-12 - 07 200437 1984 - KLT E - mail yinpeipei24@ 163. com 0. 1-1 μm 4 E - mail libs@ sipi. com. cn 5 1948-3
694 SMMC - 7721 KLT - 20 4 C - 2 Matrigel DMEM 1 3 96-9 MDA - MB - 231 6 100μl 96 37 KLT 20μl /ml KLT 1000 1. 2. 6 6 1 1. 1 1. 1. 1 GIBCO SMMC - 7721 1% tritonx - 100 DMEM 10ml 10 0. 01% RNA 1ml 10 1. 1. 2 KLT 0809051-1 488nm CCK - 8 Dojindo 1. 2. 7 SMMC - 7721 10% Matrigel basement membrane matrix BD Annexin V - FITC /PI Invitrogen 24h PBS DMEM 50μl /ml KLT Tris tritonx - 100 Sigma Annexin - binding buffer 1 GAPDH cyclin B1 cyclin D1 cyclin E CST Invitrogen Transwell Corning 1. 2 1. 2. 1 SMMC - 7721 10% 1. 2. 8 3 10 5 FBS DMEM 37 5% CO 2 6 10% DMEM KLT PBS 0. 5% 37 5min 900r /min 3min 12% PAGE - SDS 20μg 2-3 ml / Bio - Rad 0. 65mA /cm 2 2. 5h 1. 2. 2 CCK - 8 2h cyclin D1 GAP- 5 10 4 /ml 100μl 96 DH cyclin E cyclin B1 KLT 10 20 40μl /ml Odessey 24h 48h 96 1. 3 100μl 0. 1% DMEM CCK - 8 SPSS 11. 5 10% 37 1h One - way ANVOA 450nm OD 2 = OD - 2. 1 KLT SMMC - 7721 OD / OD - OD 100% 1. 2. 3 3 48h 10 5 /ml 6 10μl /ml KLT SMMC - 7721 20μl /ml 20μl /ml KLT 40μl /ml KLT 48h 3 10 5 KLT 48h 2 70% 5μl 1mg /ml 30min 10 6 /ml 100μl 4μl Alexa Fluor 488 Annexin - V 1μl 100μg /ml PI 100μl 15min 400μl Annexin - binding buffer SMMC - 7721 40μl /ml KLT 1. 2. 4 Transwell 40μl /ml KLT 0. 1% DMEM 1 1 10 5 /ml 400μl Transwell 2. 2 CCK - 8 KLT SMMC - 7721 300μl 10% DMEM 37 22h CCK - 8 10μl /ml KLT 24h 4% 15min 48h 80. 54% 60. 99% 20μl /ml 5% 10min KLT 24h 48h 64. 85% 4 200 50. 22% 40μl /ml KLT 20μl /ml KLT 1. 2. 5 Matrigel 96 SMMC - 7721
2012 4 20 4 MODERN ONCOLOGY Apr. 2012 VOI. 20 NO. 04 695 2 Fig. 1 1 KLT SMMC - 7721 48h Morphological change of SMMC - 7721 cells treated by KLT for 48 hours Fig. 2 2 KLT SMMC - 7721 KLT inhibits cell proliferation in SMMC - 7721 cells 2. 3 KLT G 2 /M KLT SMMC SMMC - 7721 48h - 7721 G 2 /M 20μl /ml KLT 40μl /ml KLT 3 Transwell 37 40μl /ml KLT P < 0. 01 4-5 Matrigel Matrigel 20μl /ml KLT SMMC - 7721 Matrigel 40μl /ml KLT 6 2. 4 KLT SMMC - 7721 G 2 /M SMMC - 7721 KLT 48h G 1 S 7 3 KLT SMMC - 7721 Fig. 3 KLT inhibits SMMC - 7721 migration
696 SMMC - 7721 D1 cyclin E 8 4 Fig. 4 KLT SMMC - 7721 KLT inhibits SMMC - 7721 invasion 2. 5 Cyclin KLT G 2 /M KLT G 2 /M Fig. 5 Western blot KLT KLT SMMC - 7721 48h cyclin B1 cyclin 5 KLT SMMC - 7721 Comparison of SMMC - 7721cells that migrated onto the membrane treated by KLT Fig. 6 6 KLT SMMC - 7721 KLT impairs the anchor - independent growth of SMMC - 7721cells Fig. 7 7 Cell cycle distribution was determined by Flow Cytometry Fig. 8 8 KLT Cyclin KLT impairs the expression of Cyclins 2. 6 KLT Annexin - V /PI Annexin - V PI Annexin - V PI Annexin - V PI 50μl /ml KLT 24h SMMC - 7721 12. 38% 2. 03% 6 9 KLT Annexin - V PI PI Annexin - V 50μl /ml KLT SMMC - 7721
2012 4 20 4 MODERN ONCOLOGY Apr. 2012 VOI. 20 NO. 04 697 3 Fig. 9 9 Annexin - V /PI Apoptosis assay by dual staining of AnnexinⅤand PI DNA S SMMC - 7721 cyclin E CDK2 KLT G 1 /S 7 G 1 /S 15 KLT cyclin D1 8 KLT BGC - cyclin E KLT 823 9 SMMC - 7721 20μl /ml KLT CCK - 8 KLT 16 Transwell Matrigel KLT KLT PI G 2 /M G 2 /M DNA Annexin - V /PI KLT SMMC - 7721 17 KLT G 2 /M 18 - S KLT 20 KLT DNA S SMMC - 7721 KLT HL60 KLT HT - 29 21 KLT G 2 /M KLT 10 cyclin B1 G 2 /M 1 CDK1 cyclin D1 CDK1 cyclin E KLT cyclin B1 M G 2 /M cyclin B1 M M 1. J. 2008 11 28 10 908-910. KLT cyclin B1 2. J. KLT G 2 /M 2001 39 10 742-744. cyclin B1 cyclin D1 3. J. 2009 44 1 11-18. 12-14 G 1 /S cyclin 4. E 2 CDK2 J. 2007 38 6 946-948. CDK2 5. J.
698 P - gp GST - π TopoII 2008 10 3 135-138. 6 Woo JH Li DP. Coix seed extract a commonly used treatment for cancer in china inhibits NFκB and protein kinase C signaling J. Cancer Biol Ther 2007 6 12 2005-2010. 7 Pang XF Yi TF Yi ZF et al. Morelloflavone a biflavonoid inhibits tumor angiogenesis by targeting Rho GTPases and extracellular signal - regulated kinase signaling pathways J. Cancer Res 2009 69 2 518-521. 8 Reuning U Magdolen V Wilhelm O et al. Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis J. Int J Oncol 1998 13 5 893-960. 9. BGC - 823 detection methods - from morphology to gene J. J Histochem J. 2010 294 3 283-286. Cytochem 2003 38 3 275-339. 10. HT - 29 18. HeLa J. 2006 21 6 531 J. 2002 22 6 481-482. - 533. 11 Yu H Yao X. Cyclin B1 conductor of mitotic symphony orchestra J. Cell Res 2008 18 2 218-220. 12 Weinstat - Saslow D Merino MJ Manrow RE et al. Overexpression of cyclin D mrna distinguishes invasive and in situ breast carcinomas from non - malignant lesions J. Nat Med 1995 1 1257-1260. 13 Gautschi O Ratschiller D Gugger M et al. Cyclin D1 in non - small cell lung cancer a key driver of malignant transformation J. Lung Cancer 2007 55 1 1-14. 14 Udhayakumar G Jayanthi V Devaraj N et al. Interaction of MUC1 with beta - catenin modulates the Wnt target gene cyclind1 in Hpylori - induced gastric cancer J. Mol Carcinog 2007 46 9 807-817. 15 Hunter T Pines J. Cyclins and cancer Ⅱ cyclin D and CDK inhibitors come of age J. Cell 1994 79 6 573-582. 16. J. 2003 20 4 48-51. 17 Yoshinori Otsuki Zhonglian Li Masa - Aki Shibata. Apoptosis 19. J. 2003 23 8 629. 20. J. 2006 23 5 307-310. 21. J. 2002 29 12 869-871.?? P - gp GST - π TopoII Expression and it's clinical significance of P - glycoprotein GST - π and TopoⅡ in salivary gland mucoepidermoid carcinoma HE Wei CHEN Yongji LIU Caiyun NI Xiaobing CHEN Rui Department of Stomatology Taihe Hospital Hubei Medical University Hubei Shiyan 442000 China. Abstract Objective To investigate the clinical significance of P - glycoprotein GST - π and TopoII expression in mucoepidermoid carcinoma of the salivary glands. Methods The expression of P - glycoprotein GST - π and TopoII in 37 cases of epidermoid carcinoma and 12 cases of normal salivary glands was detected immuohistochemically by SP method. Results P - glycoprotein GST - π and TopoII expression was found in both mucoepidermoid carcinoma and normal salivary glands. The positive rates of P - glycoprotein GST - π and TopoII in mucoepidermoid carcinoma of salivary glands were 91. 9% 34 /37 86. 5% 32 /37 21. 6% 8 /37. There was a negative correlation between the expression of TopoII and degree of differentiation. Conclusion P - glycoprotein GST - π and TopoII play an important role in the multidrug resistance in epidermoid carcinoma of the salivary glands. P - glycoprotein GST - π and TopoII detection may be helpful to chemotherapy and should also be considered as a significant indicator of prognosis in epidermoid carcinoma of the salivary glands. 2011-06 - 29 2011-07 - 22 442000 1975 - E - mail hwdjk@ 21cn. com 1968 - E - mail 75137390@ qq. com