Effects Of Wangzaozin A, An Ent-kaurane Diterpenoid On Vimentin Intermediate Filaments Of Human Pulmonary Adenocarcinoma A549 Cell

So far, few of ideal drugs specifically for intermediate filaments(IFs) have been found. Thus, to some extent, it hinders development of the study of function and assembly dynamics of IFs. As anti-cancer drugs from traditional cytotoxic drugs, develop into new antitumor drugs for specific targets or in specific mechanisms. Cytoskeleton has been became new target for tumor chemotherapy. Ent-kaurane diterpenoid compounds, a class of drugs which have good antitumor activity, have not reported related research of targets for cytoskeleton yet. non-small cell lung adenocarcinoma A549 cells are tested cell line to study effects of diterpenoids kaurane Wangzaozin A on cell growth and vimentin intermediate filaments. Wangzaozin A was isolated from Isodon excisoides in Gansu Province, which is one of ent-kaurane diterpenoids with strong cytotoxicity. Trypan blue exclude staining, MTT assay, flow cytometry and indirect immunofluorescence techniques and rhodamine-labeled phalloidin were used to explore the impact of Wangzaozin A on A549 cells. The main results as follows:1. The results of trypan blue staining showed that 1 ?M, 2 ?M, 4 ?M, 6 ?M and 8 ?M Wangzaozin A significantly inhibited A549 cells proliferation in a time and dose dependent manners, the cell growth was completely inhibited by 6 ?M Wangzaozin A, lethal effect was induced by 8 ?M Wangzaozin A on A549 cells.2. The results of MTT assay showed that Wangzaozin A had a low cytotoxicity at 1 ?M, 2 ?M, 4 ?M on A549 cells for 24 h, but 6 ?M and 8 ?M Wangzaozin A had a higher cytotoxicity.3. The results of flow cytometry showed that 2, 4 and 6 ?M Wangzaozin A treated A549 cells had low cytotoxicity,more than 90 % were normal cells indicating that such range of Wangzaozin A concentration did not lead to A549 cells apoptosis.4. The results of cytoskeleton change of A549 cells was observed after the treatment of with microfilaments and microtubule antagonist by indirect immunefluorescence. 4 ?g·m L-1 cytochalasin B could specialty destroy microfilaments both in well spread and spreading A549 cells, but did not influence assembly of vimentin IFs; A549 cells were exposed to 0.15 ?g·mL-1 or 0.02 ?g·m L-1 colchicine, and then microtubule were specialty disturbed, hardly affecting vimentin IFs; A549 cells treated with combination of 4 ?g·mL-1 cytochalasin B and 0.15 ?g·mL-1 or 0.02 ?g·m L-1 colchicine demonstrated that both microfilaments and microtubule were destroyed, but vimentin IFs remained exist in cytoplasm. The phenomenon above showed that vimentin Ifs assemble independent of microfilaments and microtubule assemble in A549 cells.5. Indirect immunofluorescence was used to investigate the effect of Wangzaozin A on well spreaded and spreading A549 cells. A549 cells were exposed to 2, 4 and 6 ?M or high concentration(15 ?M and 20 ?M)Wangzaozin A for 24 h, with the increasing of concentration, cytoplasmic vimentin intermediate filaments were significantly decreased and gradually aggregates to the one side of nuclear,however the content of vimentin had no change after the detection of flow cytometer. In the spreading cells, assemble of vimentin IFs were inhibited by Wangzaozin A leading to the inhibition of spreading A549 cells. These results indicated that assembly of vimentin IFs in A549 cells could be inhibited dramatically by Wangzaozin A in a direct way. 4 ?g·m L-1 cytochalasin B and 0.15 ?g·mL-1 colchicine respectively acted on A549 cells combining with 2, 4 and 6 ?M Wangzaozin.Further confirmed that effects of Wangzaozin A on vimentin IFs of A549 cells were remarkable.6. A549 cells were exposed to 2, 4 and 6 ?M Wangzaozin A for 24 h, filopodia were disappeared, however, slightly influence on stress fibers and lamellipodia. The results indicated that the Wangzaozin A inhibited assembly of vimentin intermediate fiber caused the depolymerization of filopodia. However, A549 cells exposed to 2 and 4 ?M Wangzaozin A for 24 h, there was no significant change of microtubule. Nevertheless, Indicating the resemble of vimentin Ifs may affect the rearrangement of microtubules leading to the some kind of link between microtubules and vimentin intermediate filaments7. Adding 15 ?M and 20 ?M Wangzaozin A after the vimentin fibrous of extracellular system was developed according to cell extraction,we observed that the medicine had not cutting effect.In conclusion,Wangzaozin A had direct inhibiting effect on vimentin of A549 cells in 2~6 ?M concentration range. However the medicine had not cutting effect on vimentin in vitro.