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Objective@#To observe the effect of cinobufacin on the expression of phosphorylated PTEN protein in human breast cancer MCF-7 and MDA-MB-231 cells.@*Methods@#The MCF-7 and MDA-MB-231 cells were divided into control group, high dose group and low dose group according to random number table method. Low and high dose groups were added with 0.5, 5.0 mg/ml cinobufacin, 100 μl/well for intervention, respectively, while the control group was added with equal volume of RPMI-1640 medium. Cell Counting Kit-8 was used to detect cell proliferation assay at 24, 48, and 72 h following intervention. Immunoblotting was used to detect the expression of AKT/mTOR pathway protein expression and p-PTEN.@*Results@#At 24, 48, and 72 h after intervention, the proliferation of MCF-7 and MDA-MB-231 cells in high dose group were significantly decreased (P<0.05). Compared with the control group, the expression of p-AKT (MCF-7: 0.357 ± 0.064, 0.215 ± 0.056 vs. 0.924 ± 0.085; MDA-MB-231: 0.310 ± 0.022, 0.194 ± 0.019 vs. 0.811 ± 0.089), p-mTOR (MCF-7: 0.476 ± 0.039, 0.217 ± 0.038 vs. 0.838 ± 0.058; MDA-MB-231: 0.300 ± 0.031, 0.223 ± 0.025 vs. 0.896 ± 0.096), p-S6 (MCF-7: 0.551 ± 0.068, 0.428 ± 0.041 vs. 1.254 ± 0.264; MDA-MB-231: 0.281 ± 0.014, 0.197 ± 0.012 vs. 0.748 ± 0.022), p-PTEN (MCF-7: 0.487 ± 0.170, 0.184 ± 0.135 vs. 1.003 ± 0.284, P<0.05; MDA-MB-231: 0.261 ± 0.184, 0.170 ± 0.105 vs. 1.014 ± 0.206) in the low and high dose group significantly decreased (P<0.05).@*Conclusions@#Cinobufacin inhibited the proliferation of MCF-7 and MDA-MB-231 cells, which may be mediated by down-regulation of the expression of the upstream of the AKT/mTOR signaling pathway p-PTEN.
ABSTRACT
Objective To observe the effect of cinobufacin on the expression of phosphorylated PTEN protein in human breast cancer MCF-7 and MDA-MB-231 cells. Methods The MCF-7 and MDA-MB-231 cells were divided into control group, high dose group and low dose group according to random number table method. Low and high dose groups were added with 0.5, 5.0 mg/ml cinobufacin, 100 μl/well for intervention, respectively, while the control group was added with equal volume of RPMI-1640 medium. Cell Counting Kit-8 was used to detect cell proliferation assay at 24, 48, and 72 h following intervention. Immunoblotting was used to detect the expression of AKT/mTOR pathway protein expression and p-PTEN. Results At 24, 48, and 72 h after intervention, the proliferation of MCF-7 and MDA-MB-231 cells in high dose group were significantly decreased (P<0.05). Compared with the control group, the expression of p-AKT (MCF-7: 0.357 ± 0.064, 0.215 ± 0.056 vs. 0.924 ± 0.085; MDA-MB-231: 0.310 ± 0.022, 0.194 ± 0.019 vs. 0.811 ± 0.089), p-mTOR (MCF-7:0.476 ±0.039, 0.217 ±0.038 vs. 0.838 ±0.058; MDA-MB-231: 0.300 ±0.031, 0.223 ±0.025 vs. 0.896 ±0.096), p-S6 (MCF-7: 0.551 ±0.068, 0.428 ±0.041 vs. 1.254 ±0.264; MDA-MB-231: 0.281 ±0.014, 0.197 ±0.012 vs. 0.748 ±0.022), p-PTEN (MCF-7: 0.487 ±0.170, 0.184 ±0.135 vs. 1.003 ±0.284, P<0.05; MDA-MB-231: 0.261 ± 0.184, 0.170 ± 0.105 vs. 1.014 ± 0.206) in the low and high dose group significantly decreased (P<0.05). Conclusions Cinobufacin inhibited the proliferation of MCF-7 and MDA-MB-231 cells, which may be mediated by down-regulation of the expression of the upstream of the AKT/mTOR signaling pathway p-PTEN.
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Objective To investigate the effect of salidroside on xenograft tumor of human pancreatic cancer cells BxPC-3 in nude mice.Methods BxPC-3 subcutaneous xenograft tumor nude mice model was established,and the mice were randomly divided into control group,salidroside 25 mg group and salidroside 50 mg group using random number method (6 mice in each group).25 mg/kg and 50 mg/kg salidroside by intragastric administration was performed per day for 3 weeks and the tumor was resected.The tumor was weighed and the diameter was measured.Cell apoptosis was detected by Tunel staining.Results As result,tumor weight was (1.561 ± 0.416)g in the control group,(0.742 ± 0.272)g in the 25 mg group and (0.276 ± 0.064) g in the 50 mg group respectively;tumor volume was (2354.35 ± 523.11) mm3,(991.43 ± 348.91) mm3 and (403.60 ± 130.98) mm3,respectively;and the apoptosis rate of the transplanted tumor cells was (23.74 ± 5.88) ％,(49.30 ± 6.75) ％ and (64.97 ± 6.99) ％,respectively,with significant difference among three groups (all P ＜ 0.01).The effect of salidroside on inhibiting proliferation and promoting apoptosis was dose dependent.Conclusions Salidroside can significantly inhibit the proliferation and growth of pancreatic cancer BxPC-3 cells via promoting cell apoptosis and inhibiting cell proliferation.
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Objective To construct the eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1?(HIF-1?) and get ready for the coloning and expressing of HIF-1? gene. Methods The total RNA was isolated from blood cells,and cDNA library was constructed by reverse transcription PCR method.PIREGFP containing EGFP fragment and cDNA were used as templet,the three designed primers (EGFP-linker,HIF-1? upstream and HIF-? downstream)were put into,after amplification the target gene fragment was inserted into the shuttle vector T,and transfected with E.coli DH5?,the bacterial colonies containing recombinant plasmids were identified by LB/KANA-agar plate,and the recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.The correct sequences were cloned into the pVAX1 vector.Results The amplified fragments were about 870,1 199,672 bp by PCR,they were inserted into the shuttle vector T and sequenced. The result of sequencing was identical with that provided by GenBank.The final plasmid about 1 800 bp was obtained by the identification of enzyme digestion and it had same molecular size as expected.Conclusion The eukaryotic expression plasmid pVAX1-EGFP-linker-HIF-1? of recombinant human HIF-1? is successfully constructed.