Effect of Modified Huangqi Gancaotang on Proliferation， Apoptosis， Invasion， Migration， and Epithelial-mesenchymal Transition of Non-small Cell Lung Cancer Cells Based on Wnt/β-catenin Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate effect of lyophilized powder of modified Huangqi Gancaotang on proliferation， apoptosis， invasion， migration， and epithelial-mesenchymal transition of human non-small cell lung cancer cells （A549， PC9） and possible mechanism. MethodEffect of 1.0， 2.0， 4.0， 8.0， 12.0 g·L-1 modified Huangqi Gancaotang on the proliferation of non-small cell lung cancer cells was detected by cell counting kit-8 （CCK-8） assay. A549 and PC9 cells were classified into the blank group and the low-， medium-， and high-dose Huangqi Gancaotang groups （2.0， 4.0， 8.0 g·L-1）. Plate cloning assay was used to examine the effect of modified Huangqi Gancaotang on cell cloning ability. Hoechst 33342 staining and flow cytometry were employed to detect the apoptosis， and scratch assay and Transwell migration assay were applied to examine cell migration and invasion abilities， respectively. Mammosphere assay was used to examine the sphere-forming ability of tumor cells， and real-time polymerase chain reaction （Real-time PCR） to detect the mRNA expression of stemness-related molecules octamer-binding transcription factor 4 （Oct-4）， human sex-determining region Y-box 2 （Sox2）， and homeobox transcription factor （Nanog） to assess cancer stem cell activity. The protein expression of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated death promoter （Bad）， Bcl-2-associated X protein （Bax）， cleaved Caspase-3， Caspase-3， E-cadherin， N-cadherin， vimentin， matrix metalloproteinase-2 （MMP-2）， β-catenin， c-Myc， Cyclin D1， and zinc-finger transcription factor （Slug） was determined by Western blot. ResultThe proliferation ability of A549 and PC9 cells was significantly inhibited after 24 h and 48 h treatment with 1.0， 2.0， 4.0， 8.0， and 12.0 g·L-1 lyophilized powder of modified Huangqi Gancaotang compared with that in the blank group and the inhibition was dose- and time-dependent （P<0.05， P<0.01）. Compared with the blank group， the low-， medium-， and high-dose modified Huangqi Gancaotang suppressed the cloning ability of A549 and PC9 cells （P< 0.05， P<0.01）， and high-dose modified Huangqi Gancaotang induced apoptosis of A549 and PC9 cells （P<0.01）. In comparison with the blank group， the low-， medium-， and high-dose modified Huangqi Gancaotang inhibited the invasion and migration of A549 and PC9 cells （P<0.05， P<0.01）. Compared with the blank group， the low-， medium-， and high-dose modified Huangqi Gancaotang significantly decreased volume of the microspheres of A549 cells and the mRNA expression of Oct-4， Sox2， and Nanog in A549 and PC9 cells （P<0.05， P<0.01）. Compared with the blank group， the medium- and high-dose modified Huangqi Gancaotang down-regulated the expression of the anti-apoptotic protein Bcl-2 （P<0.05， P<0.01）， up-regulated the expression of pro-apoptotic proteins Bad， Bax， and cleaved Caspase-3/Caspase-3 （P<0.05， P<0.01） in A549 and PC9 cells， decreased the expression of MMP-2， N-cadherin， and vimentin （P<0.05， P< 0.01）， and raised the E-cadherin expression （P<0.05， P<0.01）. Moreover， the medium-dose and high-dose modified Huangqi Gancaotang all reduced the expression of β-catenin， c-Myc， Cyclin D1， and Slug in A549 and PC9 cells （P<0.01）. ConclusionModified Huangqi Gancaotang can inhibit the proliferation， invasion， migration， activity of cancer stem cells， and epithelial-mesenchymal transition of human non-small cell lung cancer （A549， PC9） cells and induce apoptosis， and the mechanism is the likelihood that it regulates Wnt/β-catenin signaling pathway.