RESUMO
ObjectiveTo study the mechanism of Shenbai Jiedu prescription inhibiting the proliferation of HCT116 colorectal cancer （CRC） cells by regulating the phosphatase and tensin homolog deleted on chromosome ten （PTEN）/phosphatidylinositol 3-kinase （PI3K）/ protein kinase B （Akt） signaling pathway. MethodShenbai Jiedu prescription was extracted by water extraction and alcohol precipitation to prepare freeze-dried powder. HCT116 cells were cultured in vitro， and treated with different concentrations of Shenbai Jiedu prescription （2， 4， 8， 16 g·L-1）. The inhibitory effect of Shenbai Jiedu prescription on the proliferation of HCT116 cells was tested by methyl thiazolyl tetrazolium （MTT）. Real-time quantitative PCR was used to detect the mRNA expression levels of PTEN， PI3K， Akt， glycogen synthase kinase-3β （GSK-3β）， c-Myc， survivin and Cyclin D1. Western blot was employed to measure the protein expression levels of PTEN， phosphorylated PTEN （p-PTEN）， PI3K， Akt， phosphorylated Akt （p-Akt）， GSK-3β， phosphorylated GSK-3β （p-GSK-3β）， c-Myc， survivin and Cyclin D1， β-catenin nuclear import was explored by immunofluorescence assay. ResultCompared with the control group， Shenbai Jiedu prescription inhibited the proliferation of HCT116 cells in a dose-dependent manner （P<0.01）. Compared with the control group， the mRNA expression levels of PTEN and GSK-3β were up-regulated whereas those of PI3K， Akt， c-Myc， survivin and CyclinD1 were down-regulated after treatment with Shenbai Jiedu prescription （P<0.01）. The protein expression levels of PTEN， p-PTEN and GSK-3β were up-regulated whereas those of PI3K， Akt， p-Akt， GSK-3β， p-GSK-3β， c-Myc， survivin and CyclinD1 were down-regulated （P<0.05， P<0.01）. Immunofluorescence assay showed that Shenbai Jiedu prescription suppressed β-catenin nuclear import in HCT116 cells. ConclusionShenbai Jiedu prescription inhibited the proliferation of HCT116 cells via the mechanism of regulating the PTEN/PI3K/Akt signaling pathway.
RESUMO
ObjectiveTo investigate the mechanism by which Shenbai Jiedu prescription （SBJDF） inhibits the proliferation of colorectal cancer （CRC） HCT116 cells. MethodAfter 48 h treatment of HCT116 cells with SBJDF （0， 0.25， 0.5， 1， 2， 4 g·L-1）， the viability of HCT116 cells were determined by methyl thiazolyl tetrazolium （MTT） colorimetry. Following the classification of cells into blank control group and SBJDF （1， 2， 4 g·L-1） groups， the effect of SBJDF on HCT116 cell morphology was observed under an inverted microscope. The effects of SBJDF on the proliferation of HCT116 cells and mitochondrial membrane potential （Δψm） were detected by colony formation assay and JC-1 probe， respectively. The flow cytometry was then performed for determining cell cycle distribution and apoptosis. The effects of SBJDF on cell cycle-， apoptosis-， and nuclear factor kappa-B （NF-κB） signaling pathway-related proteins were determined by Western blot. ResultSBJDF effectively inhibited the vitality of HCT116 cells and changed their morphology in a concentration-dependent manner. Compared with the blank control group， SBJDF at 1， 2， 4 g·L-1 significantly reduced cell colony formation （P<0.05， P<0.01），and SBJDF at 2 and 4 g·L-1 arrested the HCT116 cell cycle at G0/G1 phase （P<0.05， P<0.01）. Compared with the blank control group， SBJDF at 1， 2， 4 g·L-1 remarkably down-regulated the protein expression of CyclinD1 （P<0.05， P<0.01）. SBJDF at 2 and 4 g·L-1 lowered the CyclinA2 and cyclin-dependent kinase 4 （CDK4） （P<0.05， P<0.01）. SBJDF at 4 g·L-1 reduced the cyclin-dependent kinase 1 （CDK1） （P<0.01）. Compared with the blank control group， SBJDF at 2 and 4 g·L-1 induced HCT116 cell apoptosis， down-regulated the protein expression of anti-apoptosis-related proteins Bcl-2 and Bcl-xl as well as the NF-κB signaling pathway-related proteins IκB kinase α （IKKα），inhibitor α of NF-κB （IκBα），and phospho-NF-κB p65 （p-p65） （P<0.05， P<0.01）， and diminished the mitochondrial membrane potential of HCT116 cells. ConclusionSBJDF inhibits the proliferation of HCT116 cells， which may be related to its inhibition of the activation of NF-κB signaling pathway and the induction of cell cycle arrest and apoptosis.