| BACKGROUND AND OBJECTIVELung cancer is one of the most deadly malignant tumors all over the world, which have been recognized as the major diseases to the human health. Therefore it is a touch challenge to clarify the pathogenesis of cancer and search for methods for curing it. Chemotherapy, which has beenwidely used in the treatment of lung cancer, has a lot of side effects. Thus it is very important to find the new agents which have less side effects to the human health.With the development of tumor biology, more and more tumor biological characteristics are gradually understood. In cancer cells, many biological processes are abnormal including cell proliferation, cell differentiation, apoptosis and autophagy in cancer cells. So it attracted people’s attention to develop new compounds playing critical roles on regulating these processes.Autophagy, an evolutionarily conserved process that regulates cell fate, is important in the cellular response to nutritional deficiencies and damages of some organelles or proteins and plays an important role in cell differentiation and growth. In cancer cells autophagy also plays important roles. It is essential for cancer cells to survive in the condition of nutritional deficiencies and anti-cancer therapy. Additionally excessive autophagy could cause autophagic cell death. Thus it is useful to develop drugs to induce autophagic cell death in cancer cells.Chemical genetics, an emerging cross-disciplinary, played an evolving role in the research of this field. Chemical genetics refers to the processes, by small molecules, interfering and modulating the cell events, exploring the molecular mechanisms of intracellular biological events, especially key factors involved. Chemical genetics. which has been generating a relatively mature technology platform, has played an increasingly important role in tumor mechanisms of research and the development of new anticancer drugs.In light of chemical genetics, we synthesized and screened two series of small molecules which could effectly inhibit the growth of A549cells. These results provided theoretical bases for developing new anti-lung adenocarcinoma agents and undersanding the structure-activity relationship of small molecules in biology.METHODS:1. A549cells were cultured in normal conditions.2. The morphological changes were observed under a phase contrast microscope.3. The cell viability was measured by SRB assay.4. Hoechst33258staining was used to detect the nuclear fragmentation and the ratio of apoptosis.5. The intracellular accumulation of acidic vesicle were analyzed by acridine orange (AO) staining under a fluorescent microscope.6. The level of LC3-Ⅱ was analyzed by western blot assay.7. LDH assay was performed to determine cells necrosis.8. The cell cycle was analyzed by the Flow CytometryRESULTS:1. Sixteen novel ethyl3-ferrocenyl-1-(2-hydroxy-3-(phenylamino)propyl)-1H-pyrazole-5-carboxylate derivatives significantly inhibited A549cell growth.1.1SRB assay showed that A549cells were significantly inhibited in a dose-dependent manner after treated with the sixteen3-aryl-1-(4-tert-butylbenzyl)-1H-pyrazole-5-carbohydrazide hydrazone derivatives in40μM at48h. The IC50of compounds (R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d were all about30μM.1.2After A549cells were treated with compounds (R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d in5μM,10μM,20μM and40μM respectively for48h, there were no significant morphological changes in treatement cells compared with cells in control groups.1.3The results from LDH assay showed that compounds (R)-4a、(S)-4a、(R)-4b、(R)-4d、(S)-4d did not induce necrosis in A549cells.1.4Heochst33258staining showed that after treated with compounds (R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d in5μM,10μM,20μM and40μM respectively for48h, A549cells did not present chromatin condensation and DNA fragmentation. The data showed that compound (R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d did not induce A549cell apoptosis.1.5AO staining showed that after A549cells were treated with compounds (R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d in5μM,10μM,20μM and40μM respectively for48h, the compounds did not increase the formation of acidic organelles. The data indicated that (R)-4a、(S)-4a、(R)-4b、(R)-4d、(S)-4d might not inhibit A549cell growth by inducing autophagy.1.6After A549cells were treated with compounds (R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d in40μM respectively for48h, the cycle distributions analyzed by the flow cytometry showed that all of the five compounds could induce A549cell cycle to arrest in G1phase.2. Two of nine2-(5-(hydroxymethyl)-3-phenyl-lH-pyrazol-1-yl)-1-phenylethanol derivatives significantly inhibited A549cell growth.2.1SRB assay showed that A549cell viability was significantly inhibited to less than50%at48h after treated with compounds4d,4e in60μM. However,4f in60μM did not display the inhibitory activity at48h.2.2After A549cells were treated with compounds4d,4e and4f in60μM respectively for48h, there were no significant morphological changes in treatement cells compared with cells in control groups.2.3The results from LDH assay showed that compounds4d,4e and4f in60μM at48h did not induce necrosis in A549cells.2.4After treated with compounds4d,4e and4f in60μM respectively for48h, A549cells did not present chromatin condensation and DNA fragmentation by Hoechst33258staining. The data showed that compounds4d,4e and4f did not induce cell apoptosis.2.5AO staining showed that after A549cells were treated with compounds4d,4e and4f in60μM respectively for48h,4d and4e increased the formation of acidic organelles, but not4f.2.6Western blot assay showed that the level of LC3-Ⅱ was obviously elevated in A549cells after treatment with4e in60μM for48h. However, the level was not increased by4d or4f in60μM for48h.2.7After A549cells were treated with compounds4d,4e and4f in60μM respectively for48h, the cell cycle distributions analyzed by the flow cytometry showed that both of4d and4e could induce A549cell cycle to arrest in Gl phase, but not4f.CONCLUSION:1.(R)-4a,(S)-4a,(R)-4b,(R)-4d,(S)-4d, five of Sixteen novel ethyl3-ferrocenyl-l-(2-hydroxy-3-(phenylamino)propyl)-1H-pyrazole-5-carboxylate derivatives could induce cell cycle to arrest in G1phase and significantly inhibite the viability of A549cells in a dose-dependent manner.2.4d and4e, two of2-(5-(hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1-phenylethanol derivatives could induce cell cycle to arrest in G1phase and cause autophagical cell death. At the same time, the two molecules could significantly inhibit the growth of A549cells in a dose-dependent manner. |
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