The Cytotoxicity And Molecular Mechanism Of Paris Polyphylla Monomer PP-26 In Human Gastric Cancer Cells

Objective:To study the proliferative inhibition effect of PP-26 on human gastric cancer cells and explore the underlying molecular mechanism of PP-26 induced cytotoxicity, so that provide experimental support for the dedevelopment and utilization of Paris polyphylla active monomer PP-26.Methods:1. The MTT assay and clone formation inhibition test were used to detect the proliferative inhibition effect of PP-26 on human gastric cancer cells.2. The levels of cell apoptosis of human gastric cancer cells by PP-26 were evaluated by Annexin V-FITC/PI staining and flow cytometry; and the cytomorphological changes of human gastric cancer cells induced by PP-26 were tested with Hoechst 33258 staining.3. To explore the corelation between proliferation inhibition and mitochondrial apoptotic pathway,the changes of mitochondrial transmembrane potential induced by PP-26 on human gastric cancer cells were examined by JC-1 staining, the expression of Bcl-2 family proteins and apoptosis-associated proteins were analyzed by western blotting, the expression of apoptosis-associated proteins treated with Caspase inhibitor VI(Z-VAD- fmk) was analyzed by western blotting.4. To explore the relation between proliferation inhibition and Akt signal pathway,the Akt proteins expression of human gastric cancer cells induced by PP-26 were evaluated by western blotting.Result:1. MTT assay results show the cytotoxicity of PP-26 in human gastric cancer MGC-803 and BGC-823 cells were higher than normal human liver L02 cells and human embryonic kidney HEK293 cells. IC50 of PP-26 in MGC-803 cells for 48 h and 72 h were 2.45 ±0.11、1.57 ±0.07 μmol/L, IC50 of PP-26 in BGC-823 cells for 72 h were 2.41± 0.08 μ mol/L; IC50 of PP-26 on L02 and HEK293 cells were not tested. Cell colony formation inhibition assay demonstrated that cell clones were decreased and cell coloning ability were inhibited at 1~1.25 μmol/L PP-26 in MGC-803 and BGC-823 cells.2. The flow cytometry assays with Annexin V-FITC/PI staining showed that the levels of cell apoptosis increased after treatment with different concentrations of PP-26 in MGC-803 cells for 24 h and BGC-823 cells for 48 h. Hoechst 33258 staining results showed typical apoptotic morphology changes like nuclear pyknosis, nuclear cracking and apoptotic bodies.3. High green and low red fluorescences were detected by JC-1 staining and the membrane potential of mitochondria reduced post treatment with PP-26 at 2.5 μmol/L in MGC-803 cells for 24 h and BGC-823 cells for 48 h. Western blot results showed that the Bcl-2 family proteins were changed with increasing concentrations of PP-26: the decreased expressions of Bcl- xl、Mcl-1, the increased expression of Bax were observed in both cells; Bak was upregulated in MGC-803 cells, Bcl-2 was downregulated in MGC-803 cells. Moreover, Caspase-9、Caspase-3 and PARP were downregulated on both cells, cleaved Caspase-3 and cleaved PARP were upregulated.4. Phospho-Akt expression increased after treatment with different concentrations of PP-26 at times and points in MGC-803 cells for 24 h and BGC-823 cells for 48 h. The phosphorylation of downstream protein of Akt, p-GSK-3β were also downregulated. The results demonstrated that Akt signal pathway was inhibited by PP-26.Conclusion:1. PP-26 inhibited the proliferative of human gastric cancer MGC-803 and BGC-823 cells, but had weaker cytotoxicity to normal human liver L02 cells and human embryonic kidney HEK293 cells.2. The proliferative inhibition effect of PP-26 is achived by inhibiting Akt signal pathway and futher activating mitochondrial apoptotic pathway.