Effects Of N-[4-(4, 6-Dimethyl-2-pyrimidinyloxy)-3–methylphenyl]-N’-[2-(dimethylamino)] Benzoylurea On Human Cancer Cells And The Underlying Mechanisms

N- [4-(4, 6- dimethyl- 2- pyrimidinyloxy)- 3- methylphenyl]- N’- [2-(dimethylamino)] benzoylurea(SUD) is a novel synthesized benzoylurea derivative. The antitumor effect of SUD and the underlying mechanisms remain unknown. Thus, we investigate the effects and underliying mechanisms of SUD on hunman carcinomas. Part 1 The effects of SUD on the growth of cellsObjective: To investigate whether SUD can inhibit the growth of normol cells, cancer cells and multidrug-resistant cancer cells.Methods: MTT assay was used to investigate the effects of SUD on the growth of human normal liver cell line L-02, human normal gastric cell line GES-1, human lung cancer cell lines A549 and H1299, human breast cancer cell line MCF-7, human gastric cancer cell lines BGC-803 and BGC-823, ovarian cancer cell line SKOV3, human liver cancer cell line SMMC-7721 and human multidrug-resistant breast cancer cell line MCF-7-ADR.Results:1 The inhibitory effects of SUD on growth of the human normal gastric cell GES-1 cells and the human normal liver cell L-02 cells were very weak. Only 100 ?M of SUD could inhibit the growth of the human normal gastric cell GES-1 cells and the human normal liver cell L-02 cells after treatment for 72 hours, and the growth-inhibitory rate was(14.8 ± 1.1) % and(21.1 ± 1.2) %, respectively. The growth-inhibitory rate of 100 ?M of TAXOL on GES-1 cells was(93.8 ± 1.0) % after treatment for 72 hours, which was much higher than that of SUD(P<0.01).2 SUD inhibited the growth of human gastric cancer cell lines BGC-803 and BGC-823, human breast cancer cell lines MCF-7 and MDAMB-231, ovarian cancer cell line SKOV-3, in time- and concentration-dependent manners, and inhibited the human lung cancer cell lines A549 and H1299 and human liver cancer cell line SMMC-7721 in a concentration-dependent manner. The IC50 values(?M) of SUD against BGC-803, BGC-823, MCF-7, MDAMB-231 and SKOV-3 cells after SUD treatment for 72 hours were(1.45 ± 0.96),(1.17 ± 0.73),(0.42 ± 0.03),(1.29 ± 0.91) and(8.39 ± 0.90), respectively, which were all more than 100 ?M against A549, H1299 and SMMC-7721 cells. The growth-inhibitory effects of SUD on MCF-7, MDAMB-231, BGC-803 and BGC-823 cells were stronger than that on A549, H1299 and SMMC-7721 cells.3 The growth of the human multidrug-resistant breast cancer cell line MCF-7-ADR was significantly inhibited by SUD treatment in time- and concentration-dependent manners. The IC50(?M) of SUD after treatment for 48 and 72 hours was(0.95 ± 0.46) and(0.57 ± 0.19), respectively.Conclusion: The growth of human cancer cell lines was significantly inhibited by SUD treatment in time- and concentration-dependent manners, and the human multidrug-resistant cancer cells also were significantly inhibited by SUD treatment in time- and concentration-dependent manners, however, the growth of human normal cells was weakly inhibited by SUD treatment. Part 2 Effects of SUD on tubulinObjective:To study the effects of SUD on tubulin.Methods:The tubulin turbidity assay was used to investigate the effect of SUD on the tubulin polymerization and depolymerization. The immunocytochemistry and confocal microscopy technique were used to investigate the effect of SUD on ?-tubulin.Results:1 According to the results of the tubulin turbidity assay, the degree of tubulin polymerization and depolymerization was decreased by SUD.2 According to the results of the immunocytochemistry and confocal microscopy techniques, SUD decreased the fluorescence intensity of ?-tubulin.Conlusion:The antitumor effect of SUD was related to inhibition of tubulin polymerization and depolymerization. Part 3 Effects of SUD on the proliferation of breast cancer cells andgastric cancer cells and the underlying mechanismsObjective: To investigate the effects of SUD on the proliferation of breast cancer cells and gastric cancer cells and the underlying mechanismsMethods: The colony formation assay was used to investigate the effects of SUD on the proliferation of breast cancer cells and gastric cancer cells. Flow cytometric analysis was used to detect the effects of SUD on cell cycle progression of breast cancer cells and gastric cancer cells. Western blot and the real time fluorescence quantitative PCR were used to analyze the expression of proteins and m RNAs related to cell cycle.Results:1 The results of colony formation assay indicated that the colony formation rate of MCF-7 cells was(59.54 ± 1.07) % in control group, which was decreased to(16.83 ± 1.48) %,(14.67 ± 0.05) % and(0.81 ± 0.03) % by 0.2 ?mol/L, 1 ?mol/L and 5 ?mol/L of SUD, respectively. The colony formation rate of BGC-823 cells was(69.89 ± 2.76) % in control group, which was decreased to(26.25 ± 1.13) %,(23.08 ± 1.07)% and(2.25 ± 0.92) % by 0.2 ?mol /L, 1 ?mol/L and 5 ?mol/L SUD, respectively.2 SUD induced cell cycle arrest at G2 phase in MCF-7 cells, and G1 phase in BGC-823 cells.3 The results of Western blot and the real time fluorescence quantitative PCR indicated that SUD increased the expression of p53 and Chk1 and decreased the expression of CDK4, Cdc25 a, Cyclin B1 and CDK1 genes or proteins in a concentration-dependent manner.Conclusion: SUD could inhibit the proliferation of the MCF-7 cells and BGC-823 cells. The inhibitory effects of SUD on the the proliferation of the MCF-7 cells and BGC-823 cells were related to the cell cycle arrest induced by SUD, which was perhaps associated with up-regulation of p53 and Chk1, and down-regulation of CDK4, Cdc25 a, Cyclin B1 and CDK1. Part 4 Effects and mechanisms of SUD on the apoptosis of breast cancercells and gastric cancer cellsObjective: To study the effects and mechanisms of SUD on the apoptosis of breast cancer cells and gastric cancer cellsMethods: The whole-cell patch-clamp technique was used to investigate the effect of SUD on the resting membrane potential of breast cancer cells and gastric cancer cells; Hoechst 33258 staining was used to evaluate the apoptosis induced by SUD in the breast cancer cells and gastric cancer cells; the reactive oxygen species assay was used to detect the effect of SUD on the production of ROS in the breast cancer cells and gastric cancer cells; the SOD and MDA assay kits were used to investigate the effects of SUD on the SOD and MDA in the breast cancer cells and gastric cancer cells; Western blot was used to investigate effects of SUD on the expression of apoptosis-relevant proteins.Results:1 SUD could change the resting membrane potential significantly, and the resting membrane potential was gradually shifted in the depolarizing direction.The resting membrane potential of BGC-823 cells was shifted from(-78 ± 5) m V to(-26 ± 4) m V after 5?M SUD perfusion and to(-60 ± 6) m V,(-40 ± 5) m V and(-15 ± 4) m V after incubation with 0.2 ?M, 1 ?M and 5 ?M of SUD for 48 hours. The resting membrane potential of MCF-7 was shifted from(-78 ± 5) m V to(-21 ± 4) m V after 5?M SUD perfusion and to(-65 ± 7) m V,(-48 ± 5) m V and(-10 ± 3) m V after incubation with 0.2 ?M, 1 ?M and 5 ?M of SUD for 48 hours.2 According to the results of Hoechst 33258 stain assay, the cells treated with SUD exhibited nuclear shrinkage, chromatin condensation and nuclear fragmentation.3 According to the results of ROS assay, the production of ROS was increased by SUD treatment.4 SUD decreased the activity of SOD in a concentration-dependent manner. The activity of SOD in MCF-7 cells was decreased from(118 ± 6) U/m L to(106 ± 5),(103 ± 5) and(90 ± 4) U/m L, respectively, by 0.2??mol/ L, 1??mol/ L and 5 ?mol/ L of SUD treatment. The activity of SOD in BGC-823 cells was decreased from(188 ± 7) U/m L to(146 ± 6),(133 ± 4) and(110 ± 3) U/m L, respectively, by 0.2??mol/ L, 1??mol/ L and 5 ?mol/ L of SUD treatment.5 SUD increased the production of MDA in a concentration-dependent manner.The concentration of MDA in MCF-7 cells was increased from(1.11 ± 0.17) nmol/m L to(5.19 ± 0.32),(6.38 ± 0.43) and(14.12 ± 0.92) nmol/m L by 0.2??mol/ L, 1??mol/ L and 5 ?mol/ L of SUD treatment. The concentration of MDA in the BGC-823 cells was increased from(1.19 ± 0.29) nmol/m L to(3.82 ± 0.76),(6.18 ± 0.62) and(7.89 ± 0.98) nmol/m L by 0.2??mol/ L, 1??mol/ L and 5 ?mol/ L of SUD treatment.6 The results of Western blot analysis showed that SUD increased the level of proapoptotic protein Bax, decreased the expression of antiapoptotic protein Bcl-2, increased the Bax/ Bcl-2 ratio(P<0.05), reduced the expression of caspase-3 and-9 proteins, and increased the expression of the cleaved caspase-3 and cleaved caspase-9 proteins in a concentration-dependent manner.Conclusion: SUD could induce apoptosis in breast cancer cells and gastric cancer cells, the change of the resting membrane potential was induced by SUD, and the mechanisms of SUD-induced apoptosis were perhaps up-regulation of the expression of p53, the ratio of Bax/Bcl-2, the production of ROS and MDA and reducing the activity of SOD to activate caspases through the mitochondrial pathway, eventually inducing the apoptosis. Part 5 Effects of SUD on migration of breast cancer cells and gastriccancer cells and the underlying mechanismsObjective: To study the effects and mechanisms of SUD on the migration of breast cancer cells and gastric cancer cellsMethods: Wound-healing assay was used to evaluate the effects of SUD on the migration of cancer cells; F-actin staining was used to evaluate the effects of SUD on the F-actin of cancer cells; Western blot was used to evaluate the effect of SUD on the expression of TRPM7 protein; the Whole-cell patch-clamp technique was used to monitor the TRPM7-like current; the real-time q-PCR was used to investigate the m RNA expression of PI3 K, AKT and TRPM7.Results:1 The results of Wound-healing assay showed that SUD inhibited the migration of cancer cells and decreased the migration-enhancing effect of EGF on cancer cells.2 According to the results of F-actin staining, SUD decreased the expression of F-actin and inhibited the function of EGF enhancing the expression of F-actin in a concentration-dependent manner(P<0.05).3 The results of Western blot analysis showed that SUD reduced the expression of TRPM7 protein in a concentration-dependent manner(P<0.05).4 According to the results of whole-cell patch-clamp, the TRPM7 current in the MCF-7 and BGC-823 cells showed characteristics of strong outward rectification, and with little inward current seen at negative membrane potentials. SUD decreased the amplitude of TRPM7 current in a concentration-dependent manner.5 The results of the real time fluorescence quantitative PCR indicated SUD decreased the expression of PI3 K, AKT and TRPM7 genes in a concentration-dependent manner.Conclusion:SUD could inhibit the migration of breast cancer cells and gastric cancer cells. The inhibitory effect of SUD on migration of cancer cells was related to decreasing expression of F-action, inhibiting the effect of EGF on cell migration, and inhibiting PI3K-Akt signaling pathway by down-regulating the expression of TRPM7 gene.