| Part I The effects of different analgesics on the biological behaviours of liver cancer QGY-7703 cellsObjective:To investigate effects of ibuprofen,celecoxib, morphine and ketamine on the biological behaviours of liver cancer QGY-7703 cells.Methods:The liver cancer QGY-7703 cells were divided, according to the method of random number, into one control group(C group) and twelve treatment groups cultured by different analgesics with different concentrations. Groups IB 1-3 were exposed to ibuprofen with the final concentrations of 250, 500 and 1000umol/L. Groups CE1-3 were exposed to celecoxib with the final concentrations of 25,50 and 100umol/L. Groups MO1-3 were exposed to morphine with the final concentrations of 0.01,10 and 100umol/L. Groups KE1-3 were exposed. to ketamine with the final concentrations of 250,500 and 1000umol/L respectively, while the control group was exposed to normovolemic RPM-1640 nutrient solution. Set 6 repeat in each group. The proliferation of cells were determined by CCK-8 assay after being incubated for 24,48 and 72h. The migration and invasion of cells with diverse incubating time from different groups were determined by Transwell, as well as the cell cycle and apoptosis of cells was examined by Flow cytometry (FCM) after being incubated for 48h.Results:Compared with C group, the rates of proliferation inhibition and apoptoticrate in the IB1-3, CE1-3, MO1-3 and KE1-3 groups were significantly increased, and the differences were statistically meaningful. This property of ibuprofen on cell proliferation inhibition and apoptoticrate was positively relevant with the concentration and incubation time. (P<0.05). Compared with C group, the ability of migration and invasion in the IB 1-3, CEi-3, MO1-3 and KE1-3 groups were significantly decreased, and the differences were statistically meaningful(P<0.05). This property of analgesics on cell migration and invasion inhibition was positively relevant with treated concentration (P< 0.05).Compared with C group,the percentages of G1 phase cells were also markedly higher in the group in the IB1-3, CE1-3, MO1-3 and KE1-3 groups after being incubated for 48h (P<0.05), as well as the percentages of M phase cells in KE1-3 groups were up-regulated in a concentration-dependent manner with statistically meaningful differences(P<0.05).Conclusions:These results suggested ibuprofen, celecoxib, morphine and ketamine could inhibit migration These results suggested ibuprofen,celecoxib, morphine and ketamine can affect the QGY-7703 cell by inhibiting the proliferation, reducing the ability of migration and invasion,which may result from the inducing apoptosis and arresting the cell cycle.Part Ⅱ The mechanism of inhibitory effect of Ibuprofen on Liver Cancer QGY-7703 CellsObjective:To investigate the inhibitory effect of ibuprofen on growth of QGY-7703 via PI3K/Akt/mTOR and IKKβ/IκBα/NF-κB signaling pathway.Methods:The liver cancer QGY-7703 cells were divided, according to the method of random number, into one control group(C group)and one teatment group. Then the treatment group was divided into three subgroups cultured by ibuprofen with different concentrations (IB1-3 groups).Groups IB1 was exposed to ibuprofen with the final concentrations of 250umol/L, Groups IB2 with the final concentrations of 500umol/L, and IB3 with the final concentrations of 1000umol/L. The control group was exposed to normovolemic RPM-1640 nutrient solution. Real-TimePCR was used to measure the expression levels of IKKβ, Bcl-2, PI3K, PTEN, MMP-9 mRNA after being incubated for 48h. Western Blot was used to evaluate the expression levels of PI3K/Akt/mTOR and IKKβ/IκBα/NF-κB signaling pathway related proteins, after being incubated for 48h.Results:1.Expression of PI3K/Akt/mTOR pathway related factorCompared with C group, the expression levels of PTEN mRNA and protein in IB1-3 groups were up-regulated in a concentration-dependent manner with statistically meaningful differences(P< 0.05). Compared with C group, the expression levels of Akt, mTORprotein and MMP-9 mRNA had no significant change(P> 0.05).Compared with C group, the expression levels of p-Akt, p-mTOR, MMP-9 protein and MMP-9 mRNA were down-regulated in B1-3 groups, which also showed a negative correlation with ibuprofen-treated concentration (P<0.05).2.Expression of IKKβ/IκBα/NF-κB pathway related factorCompared with C group, the expression levels of IKKβmRNA and Bcl-2mRNA in IB 1-3 groups were down-regulated in a concentration-dependent manner with statistically meaningful differences. Compared with C group, the expression levels of KKPmRNA protein, Bcl-2mRNA protein, p-IKKβ, p-IκBa and NF-κBp65 protein was down-regulated in IB1-3 groups, which also showed a negative correlation with ibuprofen-treated concentration (P<0.05).Conclusions:The inhibitory effect of ibuprofen on growth of QGY-7703, which may probably be related with its regulation on the activity of PI3K/Akt/mTOR and IKKβ/IκBα/NF-κB signaling pathway.PartⅢ Screening of differentially expressed genes after treatment analgesics in liver cancer cell line QGY-7703Objective:The mechanism of inhibitory effect of celecoxib, morphine and ketamine on liver cancer QGY-7703 cellsMethods:The liver cancer QGY-7703 cells were divided, according to the method of random number, into one control group(C group) and three treatment groups cultured by different analgesics with different concentrations. Group CE were exposed to celecoxib with the final concentrations of 50 umol/L. Groups MO were exposed to morphine with the final concentrations of lOumol/L. Groups KE were exposed to ketamine with the final concentrations of 500umol/L respectively, while the control group was exposed to normovolemic RPM-1640 nutrient solution. Set 3 repeat in each group. Human gene-wide expression profile chip was then employed to screen the differentially expressed genes, after being incubated for 48h, and bioinformatics analysis of the differentially expressed genes was performed.Results:Compared with group C, has 141 genes expression in CE cells increased,50 genes expression decreased; In the KE group has 1032 genes expression increased, fall 899 gene expression; In the MO group has 766 genes expression in rise, fall 311 gene expression. The KE group and CE two groups of common control gene has 34; KE group and MO group two groups of common control gene,195; MO CE group and two groups of 18 common regulation genes; KE, CE group and MO group three common regulation 17 genes.Bioinformatics analysis showed that Compared with group C, these differentially expressed genes in CE Group are involved in many cellular biological processes such as response to stress, negative regulation of cellula, cellular macromolecule metab, PPAR, MAPK and mTOR signaling pathway; Compared with group C, these differentially expressed genes in KE Group are involved in many cellular biological processes such as cell cycle process, cell cycle go, RNA metabolic process, Lysosome,and P53 signaling pathway; Compared with group C, these differentially expressed genes in MO Group are involved in many cellular biological processes such as cellular macromolecule metabolic processes, RNA metabolic process, antigen processing and presentation, natural killer cell mediated cytotoxicity and P53 signaling pathway.Conclusions:This study successfully screened analgesics (celecoxib, morphine and ketamine) processing the QGY-7703 liver cancer cells differentially expressed genes, bioinformatics and pathway analysis showed analgesic drugs inhibit the growth of QGY-7703 cells involved in different genes and pathways, to study the effect of analgesia drug resistance of liver cancer provides effective clues and has a certain guiding significance. |
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