| Background and ObjectiveLung cancer remains the most common cancer in mortality,among which non small cell lung cancer(NSCLC)is the main subtype.Due to atypical symptoms of NSCLC at early stage,patients are often diagnosed at an advanced stage.Moreover,the 5-year overall survival of patients with NSCLC remains low although a number of therapeutic regimens have been approved in the past few years.Therefore,exploring novel drug therapeutics and mechanism of NSCLC provides theoretical evidence for clinical treatment,which has strong practical significance and clinical application value.Cucurbitacin B has been a hot spot in cancer research due to its anti-tumor effect in a variety of tumors,but its specific mechanism of action in NSCLC remains unknown.Therefore,the present study aimed to observe the effect of cucurbitacin B on proliferation,cell cycle,migration and apoptosis in NSCLC A549 cells,as well as the expression of CIP2A/Akt signal,through which clarifies the role and mechanism of cucurbitacin B in NSCLC and provides a theoretical basis for NSCLC treatment by cucurbitacin BMaterials and MethodsIn this study,human lung adenocarcinoma A549 cell was used as the experi-mental models,which were treated with Cucurbitacin B at the final concentrations of 0.20,40,80 nmol/l for 24 and 48 hours,the following experiments were conducted to evaluate the effect of cucurbitacin B on A549 cells in vitro:1.MTS assay to detect cell viability and proliferation,2.scratch test to detect the migration ability of cells,3.flow cytometry to detect cell cycle,4.flow cytometry to detect apoptosis;The final concentration of 40nmol/L cucurbitacin B was used for 24 and 48 hours.The final concentration of 40nmol/L cucurbitacin B was used to treat A549 cells for 24 and 48 hours.Western blot method was used to determine the protein expression levels of CIP2A,p-AKT and AKT in A549 cells,and the possible mechanism of cucurbitacin B on A549 cells was evaluatedResults1.MTS assay showed that Cucurbitacin B could significantly inhibit the proliferation of A549 cells.The survival rate of tumor cells decreased with the elevation of Cucurbitacin B concentration and application time,showing a negative correlation.Compared with control group,different concentrations and application time of Cucurbitacin B have significant differences(P<0.05).The survival rate of tumor cell was lowest at 80 nmol/L and 48 h group(54.8±2.8)%,(P<0,0001).2.Wound healing assay showed that Cucurbitacin B significantly inhibited the migration of A549 cells.The cell relative mobility of tumor cell was lowest at 80 nmol/L and 48 h group(0.075±0.006)%,(P<0,0001).3.Flow cytometry showed that the number of cells at G2/M stage was significantly increased after the application of different concentrations of Cucurbitacin B for different time(P<0.05).Moreover,with the elevation of Cucurbitacin B concentration and application time,the ratio of cells at G2/M stage was notably promoted,showing a negative correlation.The number of cells at G2/M stage was highest at 80 nmol/L and 48 h group(23.04±0.689)%,(P<0,0001)4.Flow cytometry showed that the apoptosis of A549 cell was elevated after applying different concentrations of Cucurbitacin B for different times.When the concentration or the application time of Cucurbitacin B was increased,the apoptosis ratio of A549 cells was elevated,showing a positive correlation.The most significant difference was detected at 80 nmol/L and 48 h group with the apoptosis ratio of(31.74±0.533)%,(p<0.0001).5.Western blot showed that after 40nmol/L cucurbitacin B treated A549 cells for 48 hours,cucurbitacin B significantly inhibited the expression of CIP2A and p-AKT,while the expression of total AKT had no significant effect.Conclusions1.Cucurbitacin B inhibited the proliferation,migration,retarded cell cycle at G2/M phase and induced apoptosis of A549 cells in a concentration-and time-dependent way in vitro.2.The antitumor activity of Cucurbitacin B was associated with the inhibition of CIP2A/AKT pathway. |
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