KLT SMMC Anti - tumor effect in vitro of coix seed oil injection on the human liver cancer cell SMMC and the mechanism study

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1 MODERN ONCOLOGY Apr VOI. 20 NO SMMC 珮 Anti - tumor effect in vitro of coix seed oil injection on the human liver cancer cell SMMC 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 China. Abstract Objective To evaluate the anti - tumor effect in vitro of coix seed oil injection on the human liver cancer cell SMMC 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 in vitro anti - tumor mechanism Modern Oncology KLT SMMC SMMC CCK - 8 Transwell Matrigel KLT KLT Western blot KLT KLT KLT G 2 /M KLT cyclin B1 cyclin D1 cyclin E KLT SMMC R A DOI /j. issn KLT E - mail yinpeipei24@ 163. com μm 4 E - mail libs@ sipi. com. cn

2 694 SMMC KLT C - 2 Matrigel DMEM MDA - MB μl KLT 20μl /ml KLT GIBCO SMMC % tritonx DMEM 10ml % RNA 1ml KLT nm CCK - 8 Dojindo SMMC % Matrigel basement membrane matrix BD Annexin V - FITC /PI Invitrogen 24h PBS DMEM 50μl /ml KLT Tris tritonx Sigma Annexin - binding buffer 1 GAPDH cyclin B1 cyclin D1 cyclin E CST Invitrogen Transwell Corning SMMC % FBS DMEM 37 5% CO % DMEM KLT PBS 0. 5% 37 5min 900r /min 3min 12% PAGE - SDS 20μg 2-3 ml / Bio - Rad 0. 65mA /cm h CCK - 8 2h cyclin D1 GAP /ml 100μl 96 DH cyclin E cyclin B1 KLT μl /ml Odessey 24h 48h μl 0. 1% DMEM CCK - 8 SPSS % 37 1h One - way ANVOA 450nm OD 2 = OD KLT SMMC OD / OD - OD 100% h 10 5 /ml 6 10μl /ml KLT SMMC μl /ml 20μl /ml KLT 40μl /ml KLT 48h 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 μl /ml KLT Transwell 40μl /ml KLT 0. 1% DMEM /ml 400μl Transwell 2. 2 CCK - 8 KLT SMMC μl 10% DMEM 37 22h CCK μl /ml KLT 24h 4% 15min 48h % % 20μl /ml 5% 10min KLT 24h 48h % % 40μl /ml KLT 20μl /ml KLT Matrigel 96 SMMC

3 MODERN ONCOLOGY Apr VOI. 20 NO Fig. 1 1 KLT SMMC h Morphological change of SMMC cells treated by KLT for 48 hours Fig. 2 2 KLT SMMC KLT inhibits cell proliferation in SMMC cells 2. 3 KLT G 2 /M KLT SMMC SMMC h G 2 /M 20μl /ml KLT 40μl /ml KLT 3 Transwell 37 40μl /ml KLT P < Matrigel Matrigel 20μl /ml KLT SMMC Matrigel 40μl /ml KLT KLT SMMC G 2 /M SMMC KLT 48h G 1 S 7 3 KLT SMMC Fig. 3 KLT inhibits SMMC migration

4 696 SMMC D1 cyclin E 8 4 Fig. 4 KLT SMMC KLT inhibits SMMC invasion 2. 5 Cyclin KLT G 2 /M KLT G 2 /M Fig. 5 Western blot KLT KLT SMMC h cyclin B1 cyclin 5 KLT SMMC Comparison of SMMC cells that migrated onto the membrane treated by KLT Fig. 6 6 KLT SMMC KLT impairs the anchor - independent growth of SMMC cells 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 % 2. 03% 6 9 KLT Annexin - V PI PI Annexin - V 50μl /ml KLT SMMC

5 MODERN ONCOLOGY Apr VOI. 20 NO Fig. 9 9 Annexin - V /PI Apoptosis assay by dual staining of AnnexinⅤand PI DNA S SMMC cyclin E CDK2 KLT G 1 /S 7 G 1 /S 15 KLT cyclin D1 8 KLT BGC - cyclin E KLT SMMC μl /ml KLT CCK - 8 KLT 16 Transwell Matrigel KLT KLT PI G 2 /M G 2 /M DNA Annexin - V /PI KLT SMMC KLT G 2 /M 18 - S KLT 20 KLT DNA S SMMC KLT HL60 KLT HT 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 KLT cyclin B1 2. J. KLT G 2 /M cyclin B1 cyclin D1 3. J G 1 /S cyclin 4. E 2 CDK2 J CDK2 5. J.

6 698 P - gp GST - π TopoII 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 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 Reuning U Magdolen V Wilhelm O et al. Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis J. Int J Oncol BGC detection methods - from morphology to gene J. J Histochem J Cytochem HT HeLa J J Yu H Yao X. Cyclin B1 conductor of mitotic symphony orchestra J. Cell Res 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 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 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 Hunter T Pines J. Cyclins and cancer Ⅱ cyclin D and CDK inhibitors come of age J. Cell J Yoshinori Otsuki Zhonglian Li Masa - Aki Shibata. Apoptosis 19. J J J ?? 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 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 / % 32 / % 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 E - mail hwdjk@ 21cn. com E - mail @ qq. com