The Effect Of Lappaconitine And Its Synergistic Effect With Docetaxol And Oxaliplatin On Lung Cancer

Objective:To study the effect of proliferation inhibition, cell cycle changes, apoptosis induction and MRP1,VEGF-A,CyclinEl gene expression on the Human lung cancer A549 cell line by Lappaconitine alone or by its synergistic treatment with Oxaliplatin or Docetaxol correspondingly. To investigate the cell kinetic changes of the A549 cell line pre-treated by LA at concentration of 0.24mg/ml for 30 days and 2 days. To clarify the probable mechanism by which Lappaconitine alone or combined with Oxaliplatin or Docetaxol induces cell death in cultured human lung cancer A549 cell line.Method:The cytotoxic effect was determined by MTT assay. The cell apoptosis and cell cycle change was demonstrated by flow-cytometric analysis. The clone formation rate was determined by flat plate clone formation method. The gene expression was detected by RT-PCR method. The morphological changes of A549 cell was investigated by atomic force microscope (AFM).Results:(1) Lappaconitine at concentration of 0.48-3.6 mg/ml inhibited proliferation of A549 cells in vitro and was in a Dose dependent manner.2μg/ml Oxaliplatin and 0.8μg/ml Docetaxol had significant proliferation inhibitory effect on A549. Lappaconitine at concentration of 0.48-0.96 mg/ml had synergistic proliferation inhibitory effect on A549 cells with either Oxaliplatin or Docetaxol correspondingly; (2)The graphics of apoptosis showed that Lappaconitine had an apoptotic effect on A549 cell, and also had synergistic apoptotic effect on A549 cells with Oxaliplatin and Docetaxol; (3) Lappaconitine acted in G0/G1 arrest and downregulating CyclinE1 gene expression in A549 cells. Lappaconitine and Oxaliplatin acted synergistically in G0/G1 arrest and downregulating CyclinEl gene expression in A549 cells; (4)Detected by AFM, compared with the control group, Lappaconitine had apoptotic effect on A549 cell. Lappaconitine had synergistic apoptotic effect on A549 cell with oxaliplatin; (5) Group of A549 cell, which was pre-cultured with 0.24 mg/ml of Lappaconitine for 30 days, showed tumoristatic effect, G0/G1 arrest, diminished Clone formation rate. After investigation of cell growth curve, the group, which was pre-cultured with lappaconitine for 30 days, showed prolonged cell doubling time (Dt), and increased cell loss; (6) Lappaconitine inhibited VEGF-A expression in A549 cell in mRNA level. Lappaconitine acted synergistically with Oxaliplatin or Docetaxol in VEGF-A expression inhibition in A549 cell in mRNA level. Lappaconitine alone and combined with Oxaliplatin or Docetaxol showed no obvious change in MRP1 gene expression in A549 cell in mRNA level.Conclusion:1. Lappaconitine had a proliferation inhibition effect and a synergistic inhibitory effect on A549 cell, when combined with Oxaliplatin or Docetaxol.2. Lappaconitine exhibited apoptotic effect on A549 cell, especially in late apoptotic effect. Lappaconitine also had a synergistic apoptotic effect on A549 cell, when combined with Oxaliplatin or Docetaxol. Morphologically, LA showed apoptotic effect on A549 cell. LA combined with OXAL had a synergistic effect on apoptotic effect. Lappaconitine blocked A549 cells in G0/G1 stage and inhibited CyclinE1 gene expression in A549 cell. Lappaconitine combined with Oxaliplatin synergistically blocked A549 cell in G0/G1 stage, and synergistically inhibited CyclinEl gene expression in A549 cell. There was no obvious synergistic effect with Docetaxol on CyclinEl gene expression.3. Group, which was preincubated in 0.24 mg/ml Lappaconitine for 30 days, was a tumoristatic effect towards A549 cell. It promoted A549 cells to escape from the cell cycle into the Quiecent Stage, which lead to a cell loss in cell cycle and at last induced proliferation inhibition effect on A549 cells. LA inhibited VEGF-A gene expression. LA combined with OXAL or Doc showed synergistical inhibitory effect on VEGF-A expression, but no obvious change in MRP1 expression in each group.