Study On The Anti-tumor Activities And Molecular Mechanism Of Thalidomide And Its Enantiomer In Vitro

Background and ObjectiveThe chiral drugs exert them pharmacological action by chiral matching and molecular recognition and show a marked stereoselectivity, the different enantiomeres of a chiral drug can have distinct effects on the pharmacodynamic action, drug metabolism and toxic reactions. Thalidomide(TD) is an immunomodulatory drug, have a single chiral center in its molecular structure, consisting of S(-) and R(+) enantiomers. Once it was widely used to treat vomiting of pregnancy in europen countries, but then was withdrawn from the markets for its severe side effects of teratogenesis. When inquiring into the reason of its teratogenic, researchers find that thalidomide might prevent the growth of blood vessels, its severe malformations were the result of the drug's interference with vasculogenesis. Tumor's growth and metaptosis depend on its new blood vessel formation, so TD has became the anti-tumor research focus due to its potency of antitumor. Researchs in vivo and vitro have shown that the drug has antitumoral properties, and promising results have been reported after thalidomide treatment in patients with myeloma, myelodysplastic syndrome and melanoma, renal carcinoma and a variety of other solid tumors. But its mechanism of antitumor action are also unclear. As report, the S(-) form potently inhibits release of tumour necrosis factor a (TNF-a) and result in newborn teratogenicity, whereas the R(+) form seems to act as a sedative.As a result, to research the antineoplasmic activity of single enantiomer have theorial and practical significance.The aim of this research is to investigate the effect and difference of racemic thalidomide (TD) and its laevoisomer (S-TD) on the proliferation of three tumor cells line (NCI-H460 cells, A549cells, HepG2) and to research the anti-tumor molecular mechanism of TD and S-TD.Methods1. NCI-H460 cells, A549cells, HepG2 cells were cultured in vitro respectively, the effects of TD and S-TD on cell growth activity was determined by MTT assay.2. Flow cytometry analysis were used to examine the distribution of NCI-H460 cell cycles.3. DAPI staining were used to analyze apoptosis-induced effect on NCI-H460 cells. 4. To collective the supernatants of cultured cells(A549cells, HepG2 cells), the VEGF and MMP-9 protein level in supernatants were determined by enzyme-linked immunosorbent assay (ELISA).Results1. NCI-H460 cells were treated with TD, S-TD at 10.0～200.0μg-mL-1 for 24h could inhibited the cell proliferation (p< 0.05) and for 48h inhibited the cell proliferation significantly (p < 0.01), the inhibitory effect of TD was more potent than that of S-TD at the same concentration. A549 cells were treated with TD,S-TD at 2.0～200.0μg·mL-1 for 48h have no inhibitory effect on cell proliferation (p> 0.05). HepG2 cells were treated with TD,S-TD at 200.0μg·mL-1 for 48h have weak inhibitory effect on cell proliferation (p< 0.05).2. With the increase of drug concentration, G2/M phase cells increased and S phase cells decreased after the treatment of 200.0μg·mL-1TD, S-TD on NCI-H460 cells for 48h; Morphological changes such as chromatin condensation, chromatin margination were not observed in NCI-H460 cells.3. For A549 cells, the VEGF protein level in supernatant decreased after the treatment of 200.0μg·mL-1 TD,S-TD for 48h, the MMP-9 protein level decreased after the treatment of 100～200.0μg·mL-1 TD, S-TD for 48h. For HepG2 cells, the VEGF protein level in supernatants had no apparent decrease after the treatment of 2.0～200.0μg·mL-1 TD,S-TD for 48h, the MMP-9 protein level decreased after the treatment of 100.0～200.0μg·mL-1 TD and 100μg·mL-1 S-TD for 48h.ConclusionTD have selectivity on proliferation of tumor cells. Both TD and S-TD have no inhibition on cell proliferation of A549 cells and HepG2 cells in vitro, at certain concentration have inhibition effects on cell proliferation in cultured NCI-H460 cell, the inhibition on cell proliferation of TD was slightly greater than that of S-TD. The regulation of cell cycle may be involved in the inhibition effect on NCI-H460 cells, to inhibit the cell proliferation through delaying cell G2/M phase. TD and S-TD exert antiangiogenesis effects through decreasing the MMP-9 level of protein, and the activitie have no notable difference.