Abstract Objective To observe the simvastatin-induced Caspase 3 apoptosis of K562 cells, changes in the activity of Caspase . Different concentrations of simvastatin in K562 cells, cell morphology, flow cytometry apoptosis, colorimetric determination of Caspase 3, Caspase 9 activity. Results of 5, 10, 20, μmol / L simvastatin in K562 cells after 48h nuclear condensation, nuclear fragmentation and apoptotic bodies morphological changes, apoptosis rates were (4.00 ± 0.13)%, (6.24 ± 0.18)%, (9.41 ± 0.22)%, compared with the control group difference was statistically significant (P <0.01). For 72h, the apoptosis rates were (7.62 ± 0.21)%, (12.41 ± 0.32)%, (19.08 ± 0.26)%, compared with the control group, the difference was statistically significant (P <0.01). And Caspase 3, Caspase 9 activity was significantly higher, 10 μmol / L and 20μmol / L group Caspase 3, Caspase 9 activity with the control group, the difference was statistically significant (P <0.01). Conclusion Simvastatin may by caspase 9, and activation of Caspase 3 induced apoptosis of K562 cells.
[Keyword] simvastatin Caspase apoptosis
Apoptosis blocked significant characteristics of malignant cells to find effective ways to induce tumor cell apoptosis is one of the hotspots of the medical profession today. Statins (statins), the drug is the endogenous cholesterol synthesis limiting enzyme hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase competitive inhibitors, can inhibit the mevalonate pathway, affecting intracellular signal transduction. Recent studies have shown that statins can induce mesothelioma , glioma , acute myeloid leukemia  and melanoma  and other tumor cell apoptosis and normal cells obvious side effects, but the mechanism of apoptosis is not yet completely clear. Cysteine aspartate-specific proteases (Cysteinyl aspartate specific protease, Caspase) is closely related to apoptosis, studies have shown that statins induced melanoma and sarcoma cells during apoptosis in both human rhabdomyosarcoma Caspase activation [4 , 5]. Simvastatin induced human chronic myelogenous leukemia K562 cells during apoptosis whether Caspase activation has not been reported, this study uses the spectrophotometric determination of Caspase 3, Caspase 9 activity observed in the simvastatin-induced K562 changes in apoptosis.
1 Materials and methods
1.1 cell lines of human chronic myelogenous leukemia K562 cells were purchased from Sichuan University Huaxi campus of Preclinical and Forensic Medicine Immunology.
The 1.2 main reagent simvastatin provided by the China Pharmaceutical and Biological Products. Ethanol dissolved with amount of simvastatin substances. Concentration NaOH, 50 ° C water bath for 2h, pH adjusted to 7.20, filter-sterilized, the concentration of 10 mmol / L, packing after stored at -20 ℃ before use The RPMI1640 dilution culture system. RPMI1640 were purchased from Gibco, fetal calf serum was purchased from the U.S. GM companies, Annexin Ⅴ FITC kit was purchased from Biological Engineering Co., Ltd. of Shenzhen Jingmei Caspase 3 of Caspase 9 spectrophotometric kit purchased from Nanjing KGI biotechnology Development Limited.
1.3 Cell culture K562 containing 10% fetal calf serum, penicillin 100U/ml, streptomycin 100U/ml RPMI 1640 medium at 37 ℃, saturated humidity routinely cultured in 5% CO 2 incubator, each 2 to 3 days The medium was changed subculture.
1.4 cell morphology
Take the logarithmic growth phase cells, different concentrations of simvastatin culture system, cultured cell morphology after 48h the cells were collected smear Wright stain light microscope.
1.5Annexin Ⅴ FITC detection of apoptosis
To collect cells in the logarithmic growth phase, adjusting the cell concentration of 1 × 105 cells / ml, the experimental group Member simvastatin, the final concentration of 5,10,20 μmol / L, each concentration were performed three holes, while set negative control group, and cultured under the same conditions 48,72 h. The collection of the cells in each group, the 4 ° C cold PBS the cells were washed twice, the binding buffer, re-suspended cells, adjusting the cell concentration of 1 × 106 cells / ml. Take 100μl cell suspension in the a 5ml flow tube, adding 5μl Annexin Ⅴ FITC and 10μl propidium iodide solution, mix dark at room temperature and incubated 15min, the reaction tube was added 400 μl PBS, flow cytometry.
1.6Caspase 3, Caspase-9 activity was measured
To collect cells in the logarithmic growth phase, adjusting the cell concentration of 1 × 105 cells / ml, the experimental group Member simvastatin, the final concentration of 5,10,20 μmol / L, each concentration were performed three holes, while set The negative control, cultured under the same conditions, 48h, 72h. Collection of cells in each group, PBS washed 2 times, 2 000r / min centrifugation 5min, collecting 3 ~ 5 × 106 cells with 60μl ice of Lysis Buffer suspension cells, -20 ℃ placed 20min, 4 ° C 10 the 000r/min centrifugation 3min measured cell Lysates supernatant protein concentration and adjust the protein concentration of 1.8μg/μl. Each the bore draw 50μl cell lysate supernatant containing 90μg protein and, at the same time take 50μl Lysis Buffer blank control. Each 50μl 2 × Reaction Buffer by adding 0.5μl DTT, every hole draw 50μl already prepared 2 × Reaction Buffer, then add 5μl Caspase 3 Substrate Substrate Caspase 9, at 37 ° C for Incubate 4h microplate determination A405.
All data ± s, using SPSS11.0 statistical software analysis, and comparison groups using one-way ANOVA, P <0.05 was considered statistically significant.
2.1 changes in cell morphology
5,10,20 μmol / L simvastatin for after K562 48h, light microscopy showed typical apoptotic morphological changes, cell shrinkage, cell membrane integrity, cytoplasmic vacuoles, nuclear chromatin condensation, fragmentation into massive dispersed in the cytoplasm, and apoptotic bodies.
2.2 simvastatin-induced apoptosis of K562 cells
AnnexinV / PI double staining method is sensitive apoptosis detection, early apoptotic cells AnnexinV FITC staining, late apoptotic cells AnnexinV FITC / PI double staining. Simvastatin in K562 showed that apoptosis was significantly increased, as shown in Table 1, Figure 1. Table 1 simvastatin K562 cell apoptosis induced effects (apoptosis rate%, ± s)
group 48h 72hcontrol 1.88 ± 0.14 4.20 ± 0.195μmol / L 4.00 ± 0.13 * 7.62 ± 0.21 * 10μmol / L 6.24 ± 0.18 * 12.41 ± 0.32 * 20μmol / L 9.41 ± 0.22 * 19.08 ± 0.26 *
Note: n = 3, the control group * P <0.012.3 simvastatin activated Caspase 3, Caspase 9
Caspase-3, of Caspase reactive test results as shown in Table 2, 10 μmol / L, 20 μmol / L simvastatin for K562 cells 48h, 72h after Caspase 3, Caspase 9 activity was significantly increased compared with the control group differences statistically significant (P <0.01), indicating that simvastatin can be activated Caspase 3, Caspase 9.
With the in-depth study of apoptosis induced apoptosis drugs are constantly being discovered. With the surface of apoptotic cells phosphatidylserine-specific binding by flow cytometry AnnexinV / PI double staining method using AnnexinV, PI could be specifically through the principle of dead cell membrane so that staining apoptosis in early and late cells and dead cells distinguish , is commonly used method of detection of apoptosis. In this study, K562 cells were treated with simvastatin, simvastatin can induce apoptosis of K562 cells by flow cytometry AnnexinV / PI double staining confirmed.
In the study of the mechanism of apoptosis, the proposed two major signaling pathways that mitochondrial pathway and death receptor pathway . Caspase 9 key protease of the mitochondrial apoptotic pathway in Caspase “waterfall" Activate the top, the activation of the mitochondrial the withered Table 2 simvastatin K562 cells were treated Caspase 3, Caspase 9 activity assay results Note: n = 3, compared with the control group, * P <0.01 apoptosis pathway activation is particularly important. Caspase 9 zymogens by proteolysis size subunit, cytochrome C, caspase-activating factor 1 (Apaf 1 form active dimer) and dATP participation of further activation of Caspase 3. Activated Caspase 3 can cleave DNA repair-related molecule inhibitor of apoptosis proteins, extracellular matrix proteins and cytoskeletal proteins, etc., to promote apoptosis. Shellman et al  found that lovastatin-induced apoptosis in human melanoma Caspase 3 activation. Wang et al  showed that lovastatin HL60 cells during apoptosis induced activation of Caspase 3, Caspase-3 inhibitors can inhibit Caspase activity and DNA fragmentation. Cafforio et al  in the study of statin-induced human bone marrow tumor the MCC 2 apoptosis found that statins can be activated Caspase 3, Caspase 8 and Caspase 9 expression of Caspase 9 inhibitor significantly reduce cell withered mortality Caspase 8 inhibitor can not show that statins primarily through Caspase 9 activation and further activation of Caspase 3 induced apoptosis in myeloma. In this study, we have different concentrations of simvastatin-induced apoptosis of K562 cells by spectrophotometric detection of Caspase-3, Caspase 9 activity, found that simvastatin induced apoptosis of K562 cells in Caspase 3, Caspase 9 activity was significantly increased, suggesting that Caspase 3, Caspase 9 involved in the regulation of simvastatin-induced apoptosis of K562 cells. Simvastatin may by caspase 9 and further activation of Caspase 3 induced apoptosis of K562 cells.