ABSTRACT
ObjectiveTo study the effect and underlying mechanism of Stemona tuberosa alkaloids on the proliferation and apoptosis of human non-small cell lung cancer NCI-H460 cells. MethodNon-small cell lung cancer NCI-H460 cells were divided into a blank group and S. tuberosa alkaloids groups （50， 100， 150， 200， and 250 mg·L-1）. The effect of S. tuberosa alkaloids on the proliferation of human NCI-H460 cells was observed by thiazolyl blue tetrazolium bromide （MTT） assay and colony formation assay. Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry. Real-time fluorescence-based polymerase chain reaction （Real-time PCR） was used to detect the effect of S. tuberosa alkaloids on the mRNA expression of cysteinyl aspartate-specific protease 3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and epidermal growth factor receptor （EGFR）. The protein expression levels of Caspase-3， Bax， Bcl-2， protein kinase B （Akt）， phosphorylated （p-）Akt， EGFR， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （p38 MAPK）， and p-p38 MAPK were measured by Western blot. ResultCompared with the blank group， the S. tuberosa alkaloids groups showed increased inhibition rate on cell proliferation （P<0.01）， reduced number of cell clones formed and the rate of cell clonal formation （P<0.05， P<0.01）， and increased karyopyknosis， cytoplasmic aggregation， and cell apoptosis rate （P<0.01）. The S. tuberosa alkaloids groups at 100， 150， 200， and 250 mg·L-1 showed increased Caspase-3 mRNA expression （P<0.05）， decreased EGFR mRNA expression （P<0.05， P<0.01）， up-regulated protein expression of Caspase-3 and p-JNK （P<0.01）， and down-regulated protein expression of EGFR and p-Akt （P<0.05， P<0.01）. Additionally， compared with the blank group， the S. tuberosa alkaloids groups showed increased expression of Bax mRNA （P<0.01）， decreased expression of Bcl-2 mRNA （P<0.01）， up-regulated protein expression of Bax and p-p38 MAPK （P<0.01）， and down-regulated protein expression of Bcl-2 （P<0.01）. ConclusionsS. tuberosa alkaloids can inhibit proliferation and induce apoptosis of human non-small cell lung cancer NCI-H460 cells， and the mechanism may be related to the inhibition of EGFR protein expression and phosphorylation of Akt protein， as well as the activation of the JNK/p38 MAPK signaling pathway.
ABSTRACT
AIM To optimize the aqueous extraction for polysaccharides from Astragali Radix and to evaluate the in vitro antitumor activity.METHODS With extraction temperature,extraction time and solid-liquid ratio as influencing factors,extraction rate of polysaccharides as an evaluation index,the extraction was optimized by uniform design.The effect of polysaccharides on the proliferation of non-small cell lung cancer NCI-H460 cells,the apoptosis rate and cell cycle of NCI-H460 cells,and the expressions of Caspase-3,Bax and Bcl-2 were detected by MTT assay,flow cytometry and Western blot,respectively.RESULTS The optimal conditions were determined to be 100 ℃ for extraction temperature,1 h for extraction time,and 1 ∶ 35 for solid-liquid ratio,the extraction rate of polysaccharides was 3.62％.Compared with the control group,the proliferation of NCI-H460 cells was significandy inhibited in a dose-dependent manner (P ＜ 0.01),the S phase ratio,early apoptosis rate,late apoptosis rate and total apoptosis rate were markedly increased (P ＜ 0.01),and the Caspase-3 expression and Bax/Bcl-2 ratio were also obviously increased (P ＜ 0.01) in the polysaccharides group.CONCLUSION This fast,stable and reliable method can be used for the aqueous extraction for polysaccharides from Astragali Radix,which can significantly inhibit the proliferation of NCI-H460 cells and induce apoptosis of NCI-H460 cells.
ABSTRACT
AIM To optimize the aqueous extraction for polysaccharides from Astragali Radix and to evaluate the in vitro antitumor activity.METHODS With extraction temperature,extraction time and solid-liquid ratio as influencing factors,extraction rate of polysaccharides as an evaluation index,the extraction was optimized by uniform design.The effect of polysaccharides on the proliferation of non-small cell lung cancer NCI-H460 cells,the apoptosis rate and cell cycle of NCI-H460 cells,and the expressions of Caspase-3,Bax and Bcl-2 were detected by MTT assay,flow cytometry and Western blot,respectively.RESULTS The optimal conditions were determined to be 100 ℃ for extraction temperature,1 h for extraction time,and 1 ∶ 35 for solid-liquid ratio,the extraction rate of polysaccharides was 3.62％.Compared with the control group,the proliferation of NCI-H460 cells was significandy inhibited in a dose-dependent manner (P ＜ 0.01),the S phase ratio,early apoptosis rate,late apoptosis rate and total apoptosis rate were markedly increased (P ＜ 0.01),and the Caspase-3 expression and Bax/Bcl-2 ratio were also obviously increased (P ＜ 0.01) in the polysaccharides group.CONCLUSION This fast,stable and reliable method can be used for the aqueous extraction for polysaccharides from Astragali Radix,which can significantly inhibit the proliferation of NCI-H460 cells and induce apoptosis of NCI-H460 cells.
ABSTRACT
Objective:To determine whether the bcl-2 antisense oligonucleotide (ASODN) can increase the sensitivity of NCI-H460 cell lines to docetaxel or radiation. Methods: Docetaxel in combination with bcl-2 ASODN or non-sense oligonucleotide (NSODN) was used to treat NCI-H460 cells, and then IC_~50 of docetaxel against NCI-H460 cells was determined with MTT method. In radiation group, NCI-H460 cells were divided into pure radiation, radiation plus bcl-2 ASODN, and radiation plus NSODN groups. The inhibition rates of cell growth were assayed by MTT, and the expression levels of Bcl-2 protein were assayed by immunofluorescence. Results: IC_~50 in bcl-2 ASODN plus docetaxel, pure docetaxel and NSODN plus docetaxel groups was (0.101?0.009) ?mol/L,(0.183?0.018)?mol/L, and (0.179?0.016)?mol/L, respectively. The differences between the first group and the latter 2 groups were significant (P