| Objective:To study anti-tumor effect and mechanism of Patchouli alcohol and Pogotone in acute myloid leukemia cell lines MV4-11.Methods:1. MTT assay was used to detect the inhibitory effect of Patchouli alcohol on 15 kinds of cancer cell lines and 2 kinds of normal cell lines and calculate the IC50, filtrating the most sensitive one.2. After treated with 25,50,100 ? g/mL Patchouli alcohol or Pogostone, using hoechst 33258 staining method to observed the morphology changes of apoptosis; cell cycle was measured by flow cytometry using PI staining; Apoptosis was measured by flow cytometry using Annexin-V-FITC/PI staining.3. Western blot was used to detect the protein expressions of NF-?B, P-PKM2 and Caspase-3 inducing by Patchouli Alcohol; to detect the protein expressions of ERK and Cystine aspartic acid protease-7 (Caspase-7) inducing by pogostone.Results:1. The influence on kinds of tumor cell lines:Patchouli alcohol and pogostone could efficiently inhibit the proliferation on 15 kinds of cell lines. The IC50 between 19.6 and 100 ?g/mL, the acute myloid leukemia cell lines MV4-11 was the most sensitive one, and the IC50 was 23.2±4.9 ?g/mL,22.6±3.7?g/mL, respectively.2. The influence of cell cycle and apoptosis on MV4-11:After treated with 25,50,100 ?g/mL Patchouli alcohol or Pogostone, we observed that the cell nucleus of MV4-11 which was treated by staining Hoechst become shrinkage, turn round, fine and close, even spliting into multiple apoptotic body under the fluorescence microscope; By using flow cytometry to detect the DNA content in tumor cells, we confirmed that patchouli alcohol could block MV4-11 cells in G2/M phase, comparied with the control group (41.6%), the percentages of cell accumulation in G2/M phase when treated with patchouli alcohol increased from 48.7%(25 ?g/mL),55.1%(50?g/mL) and 62.8%(100 ?g/mL); pogostone could block MV4-11 cells in G0/G1 phase, comparied with the control group (33.2%), while treated with pogostone increased from 38%(25 ?g/mL),44.4%(50 ?g/mL) and 57.1%(100 ?g/mL), with statistically significant difference compared with control group (P<0.05); After treated with 25,50 or 100?g/mL patchouli alcohol and pogostone could induce apoptosis of MV4-11 cells, with the apoptosis rates of 8.9%,13.6%,22.0% when treated with patchouli alcohol; with the apoptosis rates of 1.6%,10.1%,14.1% when treated with pogostone, with statistically significant difference compared with control group (P<0.05);3. The influence of protein expression on MV4-11:Western blot result indicate that patchouli dose-dependent down-regulated the expression of phosphorylate PKM2, with no change of its protein expressions, while the exopression of NF-?B and Caspase-3 were changed(P<0.05); pogostone dose-dependent down-regulated the expression of phosphorylate ERK signaling pathways, with no change of its protein expression, while activated the caspase-7(P<0.05).Conclusion:(1) Patchouli alcohole and Pogostone could inhibit the proliferation of kinds of cancer cell lines. Human acute myeloid leukemia cell lines MV4-11 was the most sensitive one; (2) Patchouli alcohole and pogostone were both able to induce apoptosis on MV4-11 cell line. Patchouli alcohole caused cell cycle arrest of the MV4-llcells in the G2/M phase, while Pogostone arrested cell cycle in G0/G1 phase. (3) The mechanism of apoptosis on MV4-11 cells may be associated with the changes of NF-?B and p-PKM2 protein expressions by patchouli, and then induced the dependent endogenous apoptosis of caspase-3; Pogostone induced MV4-11 cells death is due to induce apoptosis involving ERK Signaling Pathway, and then induced the dependent endogenous apoptosis of caspase-7. |
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