| Objective:1. To investigate the effects of emodin on the cell proliferation and apoptosis of K562 cells and its molecular mechanisms.2. To synthesize emodin derivatives and evaluate its anti-tumor activities in vitro.Methods:1.1 MTT assay was employed to evaluate the effects of emodin on the proliferative inhibition of K562 cells after they had been treated with various concentrations of emodin for different time.1.2 The morphologic changes of K562 cells were observed in microscopy by Wight-Giemsa. AnnexinⅤ-FITC/PI staining, Electronmicroscope, TUNEL were performed to confirm the apoptosis of K562 cells incubated with emodin for different time.1.3 Cell cycle of K562 cells treated with emodin for 24h was assayed by flow cytometry.1.4 Colorimetric method was used to measure the activities of caspase-3,-8,-9 of K562 cells incubated with emodin for 48h.1.5 RT-PCR was employed to analyze the mRNA expression levels of caspase-3 in K562 cells after having been treated with emodin for 48h.2.1 A new emodin trimethoxy derivatives were synthesized from emodin with (Me)2SO4 and (Me)2CO3. The physicochemical property of the new derivatives was identified with routine methods. And its structure was characterized by HPLC and ESI-MS spectra.2.2 The effect of the new derivatives on K562 cell proliferation was detected by MTT, as well as the cell cycle by flow cytometry.Results:1. Emodin and emodin trimethoxy derivatives could inhibit proliferation of K562 cells in a time dependent manner. The anti-tumor activitve of emodin derivatives is more than emodin at low concentration.2. Typical morphological changes of K562 cells were observed in microscopy and electronmicroscope. A typical apoptotic morphological change and TUNEL cells were observed after K562 cells had been treated with 40μmol/L emodin for 48h. K562 cells could be induced to undergo apoptosis after 40, 80μmol/L emodin treatment for 12h,the apoptotic rate was (8.89±0.21)%,(26.06±0.13)%,Compared with the control group, it was significantly higher(P<0.01).3. The result of flow cytometry indicated that the cell cycle was blocked in G0/G1 phase, After treated with emodin. Emodin derivatives could arrest K562 cell cycle from G0/G1 phase to S phase.4. The caspase-3,-8,-9 activities of K562 cells which had been treated with emodin for 72h elevated remarkably. Compared with the control group, the caspase-3,-8,-9 activities of 40μmol/L emodin treated groups were markedly higher (P<0.01).5. RT-PCR results showed that emodin could increase the expression of Caspase-3 mRNA.Conclusions:1. Emodin can inhibit proliferation of K562 cells in a time dependent manner by arresting cell cycle in G0/G1 phase. Emodin can induce K562 cells to undergo apoptosis, and the underlying mechanism might be related to upregulation of caspase-9 activity which subsequently transforms caspase-3 into its active form.2. Emodin derivatives could arrest cell cycle from G0/G1 phase to S phase and inhibit proliferation. The anti-tumor activitve of emodin derivatives is more than emodin at low concentration. |
