| ObjectiveTo discuss the effect of bufalin on celluar proliferation and apoptosis in lung cancer cells in vitro, which is an effective compound extracted from Chan Chu skin, based on the therapeutic method of counteracting toxin with toxin in Chinese Medicine. Furthermore, to detect the change of mitochondrial membrane potential and proteins related to mitochondrial pathway mediated apoptosis, for a deep insight into the molecular mechanism of bufalin against lung cancer. Thus, in the age of rapid development in anti-cancer drugs, to provide necessary theoretical and experimental evidences for futher research on anti-tumor herbs from natural sources, and clinical application of the theory that counteracting toxin with toxin.Methods1. The effect of bufalin on A549 cell proliferation was examined by measuring the CCK-8 dye absorbance of living cells, to screen out the effective dose range and determine the dose-response relationship.2. The extent of apoptosis caused by bufalin with low, medium, high doses for 48h were analyzed by Annexin V-FITC/PI kit through flow cytometer. Also within FCM the cell cycle of A549 cells was detected under bufalin of 4ng/mL.3. A549 cells incubated adherently were treated with various groups of bufalin for 48h, and then examined and photographed by inverted phase contrast microscope. Ultrastructure and apoptotic morphology of cells with bufalin of 4ng/mL can be observed directly using transmission electron microscopy(TEM).4. JC-1 mitochondrial membrane potential detection assay kit was for detection of mitochondrial depolarization under a fluorescent microscope during the early stages of apoptosis, when treated with bufalin of 4ng/mL.5. The proteins related to mitochondrial pathway such as Cytochrome C, Caspase-3 were analyzed by western blot, when cells were treated with low, medium, high doses of bufalin for 48h, a Caspase inhibitor(Z-VAD-FMK) also was used to confirm the molecular mechanism of bufalin in triggering apoptosis via mitochondrial pathway.Results1. Compared to the control group, each treated group exhibited excellent effect of growth inhibition in A549 cells(P<0.01). Also the effect was enhanced along with the increasing concentration from 1 to 16ng/mL of bufalin respectively(P<0.05). The exhibition of dose-response relationship could be proved well. The difference of OD value between bufalin(4ng/mL) and DDP(4μg/mL) showed no significance(P>0.05), suggested bufalin a powerful activity against cancer in vitro, and decided 4ng/mL as the optimal dose in subsequent experiments.2. Results assessed by FCM showed bufalin a clear potency of promoting apoptosis on A549 cells, the differences were statistically significant when compared to the control group(P<0.05). The total and early-stage apoptotic rate were both growed with the increasing concentration. As to the apoptotic rate of early-stage, the compare between each group brought a significant difference(P<0.05), except for the groups between 4ng/mL and 8ng/mL, indicated that the apoptosis caused by bufalin exhibited a positively correlationship with its concentration. Also under the analysis of FCM, the proportion in each phase of cell cycle changed to some extent when cells treated with bufalin of 4ng/mL for 48h. The percentage of S phase changed significantly(P<0.01), while there were no statistically difference in other phases, suggested the inhibition of bufalin probably be related to S phase arrest.3. Compared to the control group incubated in a good condition, A549 cells exposed to various concentration of bufalin for 48h presented typical apoptotic changes in morphology by inverted phase contrast microscope, such as cell shrinkage, nuclear condensation, reduction of cell density, increase in cytoplasmic granules. TEM also revealed various morphological alterations in A549 cells after treated with bufalin(4ng/mL) for 48h. Chromatin condensation and marginalization could be observed in treated cells, as well as mitochondrial reduction in the amount and damage in the cristae structure, but no classical apoptotic bodies, while in the control cells there were smooth and complete celluar surface, some microvilli, uniform cytoplasm density with no vacuoles, uniform distribution of nuclear chromatin, and complete nucleoli, as same as the ultrastructure of organelle.4. For the fluorimetric analysis of △ψm in the intact cells, the two groups were exposured to the same intensity. The result showed that green fluorescence intensity in treated group were equivalent, while red fluorescence intensity weaker than the control one. Compared with the control group, a Aym loss was observed as a significant decrease in the red/green fluorescence intensity ratio when cells treated with bufalin(4ng/mL) for 48h(.P<0.05). The data indicated that apoptosis triggered by bufalin could be related to disruption of △ψm.5. The expression levels of Cyt C in the cytosolic and total protein were detected by western blot analysis. The results revealed an up-regulation in cytosolic Cyt C after bufalin treatment in A549 cells, meanwhile, the relative density of Cyt C in the cytosol fraction increased compared to the control. Accordingly it could be considered that a release of Cyt C was involved in bufalin-mediated apoptosis. Furthermore, the activation of apoptotic effector Caspase-3 was detected through the cleaved form of Caspase-3. With the increasing dose of bufalin treatment, the density of cleaved Caspase-3 enhanced. To clear that bufalin activated Caspase-3, Z-VAD-FMK, a specific inhibitor of caspases was used, and found that it could inhibit expression of cleaved Caspase-3 induced by bufalin in A549 cells. Taken together, these results suggested that bufalin caused the release of Cyt C from the mitochondria and subsequently increased the activity of Caspase-3, which finally induced apoptosis in A549 cells.Conclusions1. In vitro bufalin inhibited the proliferation of A549 cells in a dose-dependent manner. The cytotoxic effect of bufalin-induced cell death was probably due to the induction of apoptosis and arrest of cell cycle.2. Bufalin induced early-stage apoptosis to inhibit cell growth, caused the typical changes of apoptosis in cell morphology. The dose-response relationship were proved positive within a certain range. In addition, interference in S phase of A549 cells may be one of the mechanisms.3. Apoptosis caused by bufalin depended on mitochondria in cells. Bufalin exposure led to decrease of membrane potential and loss of function in mitochondria.4. On molecular level, bufalin triggered apoptosis through activation of intrinsic signaling pathway, which caused the up-regulation of Cytochrome C in the cytosol, as well as the activation of Caspase-3, which exerted apoptosis finally. |
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