| Background:Juglans Mandshurica Maxim, one of traditional Chinese herb, was known to induce apoptosis in a variety of tumor cells in vitro. Juglone, one of the main active components from the roots and barks of Juglans Mandshurica Maxim. However, the molecular mechanism of apoptosis induced by Juglone in HL-60 cell remains elusive. Nowadays, it is of particular significance to use molecular biological methods to study the pharmacologic effects of traditional Chinese herbs to reiterate the underlying pharmacological mechanism and to do research in finding new anti-tumor drugs, which has also become the trend in exploiting the Chinese medicine. In such a circumference, a deeper study on the molecular mechanism of Juglone in anti-tumor effect is promising and valuable.Objects:To investigate the molecular mechanism of apoptosis induced by Juglone in HL-60 cells.Methods:1. Fluorescence microscope was used to detect the morphological changes of nuclear chromatin stained by Hoechst 33342 and AO/EB.2. Flow cytometric analysis was used to detect the percentage of apoptotic cells, mitochondrial membrane potential and ROS3. Spectrophotometric method was used to detect the activities of caspase-3, -9 and the content of GSH. 4. Western blotting was used to detect the protein level of caspase-3, -9, Bcl-2, Bax, PARP, Smac, Cyt C and AIF.5. QPCR to detect the gene changes of Bcl-2 and Bax.6. Western blotting was used to detect the effect of NAC on the apoptotic protein expression.7. Western blotting was used to detect the effect of Juglone on MAPK and PI3K/AKT pathway.Results:The main results were divided into some sections as follows:1 Juglone inhibited HL-60 cells proliferation and induced apoptosis1.1 Juglone inhibited HL-60 cells proliferationThe proliferation inhibition effect of Juglone on HL-60 cells was determined with the MTT colorimetric assay. MTT results showed that after treated with different dose Juglone for 24h, the proliferation inhibition rates of HL-60 cells were 13.56±2.87% 28.58±3.08%, 56.00±1.73%, 73.72±3.50% and 92.37±1.41%, respectively at various concentrations (0.39, 0.78, 1.56, 3.125, 6.25μg/ml) and at the dosage of 1.56μg/ml, the inhibition rate sharply rised to 56%. The IC50 was 1.5μg/ml. The results showed exposure of HL-60 cells to Juglone caused significant growth inhibition in a dose- and time-dependent manner.1.2 Juglone induce apoptosis in HL-60 cell1.2.1 Morphological changes1.2.1.1 Hoechst 33342 fluorescent staining assay was used to observe cell apoptosis. The results showed the cells of control group appeared normal appearance. Cells Juglone-treated group showed typical apoptotical morphological changes, for example condensed or fragmented nuclei.1.2.1.2 AO/EB double fluorescent staining assay was used to observe cell apoptosis. The results showed the apoptotic cells appeared in the cell Juglone-treated groups. Normal cells had a regular nuclear with green fluorimetric stain, while apoptotic cell had unregular nuclear with yellowish green stain and the capacity of cell became smaller.1.2.2 Detect apoptosis by flow cytometry 1.2.2.1 PI staining assay was used to detect apoptosis. The results showed that the percentage of apoptotic cells was increased significantly from 0.16±0.04% to 28.42±4.76%.1.2.2.2 Annexin V-FITC/PI double staining was used to detect cell apoptosis. The results showed that the percentage of apoptotic cells was increased significantly from 9.44±3.48% to 71.96±5.26%.2 Molecular mechanism of apoptosis induced by Juglone in HL-60 cell2.1 The effect of Juglone on the mitochondrial function2.1.1 The effect of Juglone on the mitochondrial transmembrane potential (ΔΨm) The results showed thatΔΨm was decreased.2.1.2 The effect of Juglone on the apoptotosis-related proteins expression2.1.2.1 The effect of Juglone on the Bcl-2 and BaxThe results showed that Bax protein and gene expression was increased and Bcl-2 protein and gene expression was decreased. The ratio of Bax/Bcl-2 was increased. Results indicated that apoptosis induced by Juglone involved the changes of Bcl-2 family.2.1.2.2 The effect of Juglone on the Cyt C, Smac and AIF proteinsThe results showed that Juglone could increase the release Cyt C, Smac and AIF proteins from mitochondrion to cytoplasm in a dose- and time-dependent manner.2.1.2.3 The effect of Juglone on the caspase-3, -9, PARP proteins expression and activity of caspase.The results showed that procaspase-3 and procaspase-9 were cut and PARP substrate of caspase was decomposed along with the elevating of concentration. The activities of caspase-3, -9 were also increased in a dose- and time-dependent manner.2.2 The effect of ROS on the apoptosis induced by Juglone in HL-60 cell2.2.1 The effect of Juglone on ROSThe results showed that the ROS increased significantly with concentration 1.0 and 1.5μg/mL compared with control group. It indicated that the ROS could be increased by Juglone in HL-60 cells.2.2.2 The effect of Juglone on GSHResults showed that the content of GSH was significantly decreased. It indicated that Juglone could induce oxidative stress in HL-60 cells. 2.2.3 The effect of NAC on the apoptosis induced by Juglone in HL-60 cellThe results showed that the percentage of apoptotic cells was 6.85±1.89%, 7.71±2.54%, 82.26±10.85% and11.67±3.26%, respectively. It indicated that NAC could inhibit the apoptosis induced by Juglone.2.2.4 The effect of NAC on the apoptotosis-related proteins expression2.2.4.1 The effect of NAC on the Bcl-2 and Bax proteins expressionThe results showed that NAC had no obviously effect on Bcl-2 and Bax proteins expression. Julgone could increase Bax protein expression and decrease Bcl-2 protein expression. The ratio of Bax/Bcl-2 was increased, While NAC could inhibit the procession.2.2.4.2 The effect of NAC on the Cyt C and Smac proteinsThe results showed that NAC had no obviously on Cyt C and Smac proteins. Juglone could increase the Cyt C and Smac protein releasing to cytoplasm, While NAC could inhibit the procession.2.2.4.3 The effect of NAC on the caspase-3, -9 proteins expressionThe results showed that NAC had no obviously effect on the procaspase-3 and procaspase-9 protein expression. Juglone could promote the procaspase-3 and procaspase-9 to cutting, while the NAC could inhibit the procession.2.3 The effect of Juglone on the MAPK pathwayThe results showed that Juglone had no obviously effect on the p38 and JNK protein levels. But Juglone promoted an increase of p-ERK in a dose dependent way. It suggested that the ERK involved in the apoptosis induced by Juglone.2.4 The effect of Juglone on the PI3K/AKT pathwayThe results showed that Juglone could induce a significant decrease p-AKT at 0.5μg/mL, p-AKT expression was inhibited by Juglone. It suggested that PI3K/AKT pathway involved in the apoptosis induced by Juglone.Conclusion:1. Juglone could inhibit the cell proliferation in a dose- and time-dependent manner.2. Juglone could induce apoptosis in HL-60 cell.3. Juglone could induce apoptosis through mitochondrial pathway in HL-60 cell. 4. Juglone could increase the ROS in HL-60 cells and activate the mitochondrial pathway to induce apoptosis.5. Juglone could activate ERK pathway and inhibit PI3K/AKT pathway. |
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