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Objective: joint research and honokiol artemisinin role CNE-2, SPC-A-1, MGC-803 cells with synergistic anti-tumor effect; combination of two drugs to further explore the role of the CNE-2 cells produce synergy mechanism for Both clinical application of combination chemotherapy provides an experimental basis. : 1, will be in the logarithmic phase of CNE-2, SPC-A-1, MGC-803 LO-2 cells with different concentrations of honokiol or artemisinin processing and analysis of different concentrations of the test material The inhibition rate, to select the optimal drug concentration for further combination. Will be in the logarithmic growth phase of CNE-2, SPC-A-1, MGC-803 cells respectively to Magnolol to fixed optimum concentration, select a range of concentrations of artemisinin-treated cells 48 hours after MTT assay to detect inhibition rate of different ratio of the concentration of the test; obtained by analyzing the experimental data, clear and magnolol with artemisinin whether synergistic anti-tumor effect. 2, collected in the logarithmic growth phase CNE-2 cells, thymidine double block method cell synchronization, and honokiol artemisinin monotherapy and honokiol, single-drug artemisinin treated cells 24h after collection of cells in the control group and the test treatments, and the impact of each sample on the cell cycle distribution by flow cytometry. 3, collected in the logarithmic growth phase CNE-2 cells, and observed under a fluorescence microscope for cells 48h after DAPI staining and the honokiol artemisinin monotherapy and Magnolol single-drug artemisinin treatment The trial of the CNE-2 cell morphology. Flow cytometry was used drug-treated cells apoptosis after 48h. Double thymidine block method cell synchronization, and Artemisinin Magnolol, single drug honokiol, single-drug artemisinin treatment cells 24h or 48h, in accordance with the RT-PCR process Preparation of RNA samples, analysis of the test material impact on of cyclinD1 mRNA, cyclinE1 mRNA and P27mRNA expression. 5, respectively and honokiol, artemisinin alone and combination role 0h, 6h, 12h, cells were collected by Western Blot detect the cytoplasm ctyochrome c expression. 6, respectively honokiol, artemisinin alone and combination role 0h, 24h, 48h after the cells were collected by Western Blot detection of bcl-2, bax expression. 7, respectively and honokiol, artemisinin alone and combination after 48h the cells were collected with caspase-3 protein expression by Western Blot. 8, respectively and honokiol, artemisinin alone and combination after 5h cells were collected using flow cytometry mitochondrial membrane potential, respectively honokiol, artemisinin alone and The combined effects of 0h, 24h the cells were collected 48 hours after its death receptor using flow cytometry. 10 and honokiol, artemisinin and combination of the role of the COX-2 gene and protein expression using RT-PCR and Western Blot method detection CNE-2 cells after 48h. 11 Statistical analysis: Statistical analysis was performed using the statistical software SPSS13.0 version statistically using one-way ANOVA, t-test, p-lt; 0.05 difference significantly significance synergy effect according to the Guinness Book of formula and obtained. Results: 1 (1) and Magnolia phenol, artemisinin CNE-2, SPC-A-1, MGC-803 LO2 cell lines, growth inhibition and honokiol CNE-2, SPC-A-1 MGC-803, L-O2 cells with IC50 8.58μg/ml, 8.20μg/ml, 3.95μg/ml, 8.04μg/ml. Artemisinin of CNE-2, SPC-A-1, MGC-803 LO2 cells ICso were 62.24μg/ml, 53.52μg/ml, 64.85μg/ml, 93.92μg/ml. ② honokiol with artemisinin combination application of CNE-2, SPC-A-1, MGC-803 cell line growth inhibition select honokiol final concentration: 7.5,5.0 μg / ml (CNE- 2, SPC-A-1) 3.5,2.0 μg / ml (MGC-803); selected artemisinin final concentration: 7.5,10.0,12.5,15.0,20.0,30.0 μg / ml. each concentration and the thick Park phenols with different concentrations of artemisinin combination, acting on the above cells for 48 hours. The results show, and the honokiol and artemisinin inhibition rate of the combined treatment group was significantly higher than the single drug group. Honokiol with artemisinin combined effects of CNE-2, SPC-A-1 cells Q value gt; 1.15, indicating that honokiol with artemisinin combination with synergistic anti CNE-2, SPC-A-1 Cells have a synergistic or additive combination MGC-803 cells. 2, the and honokiol artemisinin on the results of the CNE-2 cell cycle that synchronization after the release of Oh sampling, G1, S, G2 phase cells ratio were (72.64 ± 5.005)%, (24.60 ± ± 4.35)%, (0.00%). Blank control group G1, S, G2 phase cells were (66.59 ± 3.34)%, (32.80 ± ± 3.28)% (0.00%); honokiol combined effects of artemisinin and both CNE-2 cells 24h After honokiol (7.5μg/ml) G1, S, G2 phase cell proportion were (67.86 ± ± 1.59)%, (30.39 ± ± 1.37)%, (0.00%), artemisinin (7.5μg / ml) G1, S, G2 phase cells ratio (66.77 ± 2.25)%, (33.23 ± 2.26)% (0.00%), the combined treatment group G1, S, G2 phase cells, respectively (90.78 ± 1.49) %, (2.38 ± 0.24)%, (6.70 ± ± 9.46)%, and honokiol or artemisinin monotherapy group than in the G1 phase cells increased the proportion of the decline in the proportion of S-phase cells (P lt; 0.05). Tips and honokiol with artemisinin combination synergistically induced cell cycle arrest in G1 phase. 3, fluorescence microscopy after DAPI staining clearly visible under the edge of the negative group, even dyeing and honokiol artemisinin on apoptosis of CNE-2 (1). Issued honokiol treatment group than normal group of strong fluorescence cytoplasmic concentrated nuclear chromosome condensation, artemisinin treatment were observed in the nucleus complete, no significant apoptosis, but with the combination of honokiol after significant enhancements honokiol ability to induce apoptosis in CNE-2 cells, a significant increase in the microscope, the number of apoptotic cells, most of the cells shrink emit strong blue fluorescence concentrated cytoplasmic nuclear chromosome is a high degree of cohesion, marginalization , nuclear fragmentation was fragmented, crescent typical apoptotic bodies. ② AnnexinV / PI results show after 7.5μml and honokiol 2.5,5.0,7.5 μg / ml of artemisinin combination, CNE-2 the apoptosis rate increase, significantly higher than the concentration of honokiol the artemisinin group (P lt; 0.05), the tips and honokiol artemisinin combination may be synergistic mode induced apoptosis in CNE-2 cells. 4 honokiol Joint artemisinin application of cyclinD1 mRNA, cyclinE1 mRNA, and P27mRNA expression by RT-PCR results showed that honokiol and / or artemisinin role CNE-2 cells 24h able cyclinD1 mRNA suppression Wei , cyclinE1 mRNA expression to raise P27mRNA (P lt; 0.05). With the normal control group, and honokiol group, artemisinin group, and honokiol artemisinin combination therapy group, the more significant to inhibit cyclinD1mRNA, cyclinE1mRNA expression (P lt; 0.05). CNE-2 cells for 24 h and honokiol group, artemisinin group, and honokiol with artemisinin combination therapy treatment P27mRNA expression increased (P lt; 0.05), compared with the control group, no significant difference ( P gt; 0.05). 5, and the of honokiol of artemisinin role of cytochrome c expression affect Western blot showed that: and honokiol (7.5μg/ml), artemisinin (7.5μg/ml) and both combined effects of CNE-2 cells 6h, 12h, released by the mitochondrial cytochrome c in the cytoplasm are increased. Released more than drugs alone, artemisinin monotherapy group and the normal control group, and honokiol monotherapy group compared to the combined treatment group (P lt; 0.05), indicating that honokiol (7.5μg/ml ) (7.5μg/ml) the combined effects of artemisinin synergistic increase in the release of cytochrome c in the mitochondria. 6 and honokiol artemisinin role of Bax, Bcl-2 protein expression and honokiol (7.5μg/ml) and artemisinin (7.5μg/ml) combined effects of CNE-2 cells 24h, 48h , Bax, Bcl-2 expression was significantly enhanced, over and honokiol (7.5μg/ml), artemisinin (7.5μg/ml) expression alone, indicating that honokiol (7.5μg/ml) the combined effects of artemisinin (7.5μg/ml) can significantly increase Bax, Bcl-2 expression, but Bax expression than Bcl-2 increased trend. Honokiol with artemisinin combination CNE-2 cells by upregulating the expression of Bax-induced apoptosis. 7, the impact and of honokiol artemisinin combination role of caspase-3 protein expression Western Blot results and the honokiol artemisinin and both combined effects of CNE-2 cells after 48h Procaspase-3 occurs gradually degraded the shearing get caspase-3, caspase-3 and honokiol with artemisinin combination therapy group, the expression level is slightly stronger than the honokiol stronger than the artemisinin group. 8, and the honokiol artemisinin role of mitochondrial membrane potential flow cytometry results show that the combined effects of honokiol, artemisinin and both CNE-2 cells after 5h, mitochondrial membrane potential, respectively. (1.51 ± 0.65)%, (4.68 ± 0.79)%, (4.43 ± 1.12)%, (21.56 ± 1.28)%, and honokiol group, artemisinin group, the combined treatment group of mitochondrial membrane potential showed depolarization (P lt; 0.05). 9, and the of honokiol of artemisinin role of the Fas / FasL protein expression results showed that the drug action CNE-2 cells 24h, 48h, FasL protein expression did not increase in Fas protein expression is essentially the same. CNE-2 cell apoptosis induced by honokiol with artemisinin not dependent on the interaction of the Fas / FasL. 10, and the of honokiol of artemisinin role of COX-2 expression RT-PCR, Western Blot two detection methods Results show that 0 honokiol (7.5μg/ml), artemisinin (7.5μg / ml) and honokiol (7.5μg/ml) artemisinin (7.5μg/ml) the role of CNE-2 cells 48h, the combination group COX-2 gene or protein expression were reduced, there is a significant difference (p lt; 0.05). Conclusion: 1 honokiol inhibit human nasopharyngeal carcinoma CNE-2, the growth of lung adenocarcinoma SPC-A-1 cells, gastric cancer MGC-803 cells and normal liver cell line LO2 was concentration-dependent. , Artemisinin human nasopharyngeal carcinoma CNE-2, lung adenocarcinoma SPC-A-1 cells, did not inhibit the growth of human gastric cancer MGC-803 cells, little toxicity to normal liver cell line L02. Honokiol with artemisinin combination of human nasopharyngeal carcinoma CNE-2, lung adenocarcinoma SPC-A-1 cells have a synergistic effect, a synergistic or additive effect on human gastric cancer MGC-803 cells. Honokiol with artemisinin combination can induce human nasopharyngeal carcinoma CNE-2 cell apoptosis and cell cycle arrest. Honokiol with artemisinin combination induced CNE-2 cell cycle arrest process involved of cyclinD1 mRNA involved cyclinE1 mRNA in gene expression changes during apoptosis, Bcl-2, Bax, cytochromec caspase-3 , COX-2 changes. Apoptosis process is not dependent on the interaction of the Fas / FasL but CNE-2 cells induced apoptosis through the mitochondrial pathway. 6, our experimental study new ideas and experimental basis for the choice of chemotherapy for nasopharyngeal carcinoma.
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