Study Of Anti-cancer Effect And Mechanism Of Flavonoids On Leukemia HL-60 Cells

Leukemia is a malignant disease of the hematopoietic system. The risk of onset in children and young people is more than the other cancers. It is a significant research topic to seek natural and potent anti-cancer compounds to prevent and cure leukemia. Flavonoids are a large class of polyphenolic compounds, which are ubiquitously present in the plant world and our common diet, such as vegetables, fruits and plant-derived beverages. Several beneficial biological activities have ascribed to flavonoids, including anti-oxidant, anti-inflammatory, anti-cancer and anti-estrogenic properties. Among these pharmacological properties, there has been an increasing scientific interest in the anti-cancer activity of flavonoids due to their potent inhibition effect on the carcinogenesis, proliferation, migration, invasion, angiogenesis and drug resistance of cancer. Our previous study evaluated cytotoxicities of 23 different flavonoids in human cancer cells, and found that 3,6-dihydroxyflavone and 2′-hydroxyflavanone exhibited the most potent cytotoxic effect. This study was based on these researches.Repaid proliferation is the noticeable characteristic of cancer cells; it is the basic ability for anti-cancer compounds to inhibit proliferation of cancer cells. Cells survival, that is the rate of living cells, reflects the viability of cells. Cells apoptosis appears morphological changes including cell shrinkage and cytoplasmic condensation. The abolition of mitochondria membrane potential and the release of cytochrome-c from mitochondria was the key step for apoptosis originated. Cyto C could activate caspases family in the presence of dATP, and start the apoptosis signal transduction.The function of antioxidase of cells was important for the regulation of reactive oxygen species (ROS) and the proliferation, apoptosis, inflammatory reaction of cells life activities. The activity of antioxidase in cancer cells was lower than normal cells; ROS could selectively kill the cancer cells. Mitogen-activated protein kinases (MAPK) is an important signal pathway in cells. It has been reported that ROS and oxidative stress could change the MAPK signal pathway and induced apoptosis. Previous study has shown that 3,6-dihydroxyflavone could change the ROS level of HL-60 cells, whether it influence MAPK pathway need further study.Based on the analysis mentioned above, we selectively study the anti-cancer effect of 3,6-dihydroxyflavone and 2′-hydroxyflavanone on leukemia HL-60 cells. We observed the effect of these two flavonoids on the proliferation and apoptosis of HL-60 cells. By flow cytometric analysis, laser confocal scanning microscopy and western blot, we investigated the changes of mitochondria membrane potential, the release of cytochrome-c, the activation of caspases and MAPK signal pathway under the 3,6-dihydroxyflavone treatment.The main results and conclusions were summarized as follows:1. Cells growth curve showed that the proliferation of normal HL-60 cells was rapid; different dose of 3,6-dihydroxyflavone and 2′-hydroxyflavanone treatment could reduce the proliferation of HL-60 cells differently. The proliferation inhibition effect showed dose-dependent relationship. The treatment of 10μM 3,6-dihydroxyflavone or 20μM 2′-hydroxyflavanone could inhibit proliferation of HL-60 cells, but could not prevent the growth of living cells; 20μM 3,6-dihydroxyflavone could reduce the amount of living cells, showed potent anti-cancer activity. Cells viability assay indicated the similar results that 3,6-dihydroxyflavone and 2′-hydroxyflavanone treatment could dose, time-dependently decrease the cells viability of HL-60 cells, the living cells rate was significantly reduced after the flavonoids treatment.2. We examined the morphological changes of HL-60 cells after the treatment of 3,6-dihydroxyflavone and 2′-hydroxyflavanone. The normal HL-60 cells showed good growth condition, no cell fraction was found. Treated with 3,6-dihydroxyflavone and 2′-hydroxyflavanone for 24h, dramatic pro-apoptotic morphological changes in comparison with the control were observed, including cell shrinkage and cytoplasmic condensation. 3. The apoptosis analysis by flow cytometric assay showed that 20μM 3,6-dihydroxyflavone or 2′-hydroxyflavanone treatment could significantly induce apoptosis in HL-60 cells. The apoptosis rate was markedly increased after the treatment. The further study showed that after the treatment of 20μM 3,6-dihydroxyflavone, the mitochondria membrane potential was gradually decreased and was significantly lower than the control after 8 h. The level of cytochrome-c in cytosol was significantly increased after treatment. These results indicated that 3,6-dihydroxyflavone treatment could decrease the mitochondria membrane potential and increase the release of cyto C, induce apoptosis.4. Results of western blot indicated that the caspases in normal HL-60 cells was mainly in inactive form, the level of cleaved-caspases was low. After the treatment of 3,6-dihydroxyflavone, the level of caspases decreased while the level of cleaved-caspases increased, the rate of cleaved-caspase 3/caspase 3, cleaved-caspase 9/caspase 9 and cleaved-PARP/PARP were significantly increased. It indicated that 3,6-dihydroxyflavone actived caspases apoptosis signal transduction.5. Results of antioxidase activities detection indicated that 3,6-dihydroxy- flavone could significantly decrease the activity of SOD, CAT and GSH-Px. The MDA level was markedly increased. It showed that 3,6-dihydroxyflavone could decrease the antioxidases of HL-60 cells and induce oxidative damage.6. Our study showed that 3,6-dihydroxyflavone could significantly change the MAPKs signal pathway. The protein level of phosphorylation of ERK in HL-60 cells which were treated for 2h, 8h, 24h by 3,6-dihydroxyflavone was decreased significantly compared with untreated group, the expression of phosphorylation of JNK and p38MAPK increased significantly. It indicated 3,6-dihydroxyflavone influenced more on JNK and p38MAPK signal pathway.In all, our study indicated potent proliferation inhibition and pro-apoptosis effect of 3,6-dihydroxyflavone or 2′-hydroxyflavanone on leukemia HL-60 cells. Further study showed 3,6-dihydroxyflavone could significantly decrease the antioxidases activities, induce the oxidative damage, cause the abolition of mitochondria membrane potential and the release of cytochrome-c, active caspases apoptosis signal transduction. The anti-cancer mechanism may be associated with the influence on MAPKs signal pathway.