Resveratrol induced apoptosis and cell cycle arrest and its mechanism _1307

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Resveratrol induced apoptosis and cell cycle arrest and its mechanism
 
 
Resveratrol induced apoptosis and cell arrest in cancer cells   【Abstract】 AIM: To study the mechanism of resveratrol induced HepG2 and K562 cells apoptosis and cell arrest. METHODS: Cell morphological method and Annexin V technology were used to detect HepG2 and K562 cells apoptosis, flow cytometry (FCM) was used to detect the cell cycle and MTT was used to detect the medicine sensitivity of HepG2 and K562 cells. RESULTS: Apoptosis was seen in human liver cancer HepG2 cell treated with resveratrol and nuclear chromatine condensation and fragmentation were observed in HepG2 cells. Typical ladder patterns of DNA fragmentation were also observed. The highest rate of HepG2 cell apoptosis was 33.7% by Annexin V. Cell cycle stopped at S phase and cell apoptosis rate was 9.97% by FCM. Time and dose dependent effects of resveratrol to HepG2 cell were detected by MTT. Resveratrol had no effect on K562 cells. CONCLUSION: Resveratrol induces HepG2 cell apoptosis and inhibits the development of human liver cancer with a time and dose dependent effect. The resveratrolinduced cell apoptosis is specific to cancer cells.   【Keywords】 resveratrol; apoptosis; cell cycle; hepG2; K562   Abstract Objective: To investigate the resveratrol (RES) induced hepatocellular carcinoma HepG2, human CML K562 tumor cell apoptosis and cell cycle arrest and its mechanism. Methods: Morphological study, Annexin V fluorescence staining HepG2, K562 cell apoptosis, using flow cytometry (FCM ) to detect cell cycle, the application MTT assay (MTT) test HepG2, K562 tumor cells, RES drug sensitivity. Results: RES in human hepatoma HepG2 cells significantly inhibited the growth and induce tumor cell apoptosis. apoptotic cells showed cell shrinkage, nuclear chromatin fragmentation. Annexin V fluorescence staining was 33.7% in HepG2 cell apoptosis, cell cycle was detected by FCM was to stop at G1 phase, while the apoptosis rate of K562 cells is 9.97%. MTT assay showed that human hepatoma HepG2 cells RES in a dose-response relationship. But the RES on the human K562 cells significantly inhibited the growth of no. Conclusion: RES induced HepG2 cell apoptosis by inhibiting tumor growth, and a dose of effect relationship. Meanwhile RES inhibition of tumor cells and induction of apoptosis are cell selective.   Key words resveratrol; apoptosis; cell cycle; HepG2; K562   0 Introduction   Resveratrol (Resveratrol, RES) is an important active compounds, widely present in plants. has both cis and trans structures with biological trans- activity. The chemical name of anti-3,4 ', 5-trihydroxy stilbene, it has the on cardiovascular disease and cancer prevention and health effects has caused widespread international concern and in-depth study. RES have broad anti-cancer and anti-cancer role in the carcinogenic effect of three main stages [1] with the chemical anti-cancer activity. Therefore, we are induced hepatoma HepG2 RES and the CML K562 tumor cell apoptosis and to explore the role of RES induced tumor cell apoptosis and cell cycle arrest mechanisms.   1 Materials and methods   1.1 Materials   human hepatoma HepG2 and K562 CML cells by the first Fourth Military Medical University, Department of Biochemistry and Molecular Biology Laboratory IV frozen. RES was purchased from Sigma, dissolved in dimethyl sulfoxide (DMSO, purchased from Sigma Company), diluted to the concentration of 5 × 106 mol / L, - 20 ℃ spare. RPMI 1640 and newborn calf serum were purchased from Invitrogen Corporation. MTT was purchased from Sino-American Biotechnology Company, dissolved in 1 × PBS (10 mmol / L, pH 7.4) to a concentration of 5 g / L, 0.22 μm micropore 4 ℃ after the backup filter sterilization. DNA fluorescent dye (PI and Annexin V) was purchased from Coulter Corporation,You are not allowed to view links. Register or Login, 4 ℃ spare.   1.2 Methods   1.2.1 cultured human liver cancer cells 1 × 105 HepG2 and K562 CML cell cultures containing 100 mL / L newborn calf serum in RPMI 1640 medium, add 1 × 105 u / L penicillin, 100 mg / L streptomycin, placed in incubator at 37 ℃, 50 mL / L CO2 under the conditions of saturated humidity culture experiments logarithmic growth phase cells.   1.2.2 morphology to 5,10,20,50 and 100 μmol / L RES the role of cells, 24 ~ 72 h after immunization was observed under inverted microscope difference HepG2 and K562 cell morphology changes.   1.2.3Annexin V fluorescence staining cells collected 1 × 106 cells (HepG2 cells collected before Used Annexin V 5 μL 37 ℃ incubation 10 min), PBS washed 2 times, 800 ~ 1000 r / min centrifugation 5 min, discard supernatant, add ice-cold binding buffer resuspended cells, add 5 μL PI and 5 μL Annexin V in the cell suspension (the establishment of apoptotic cells per sample were negative control and positive control to adjust the best work sample testing voltage and two-color fluorescence compensation), mix gently 4 ℃ after 10 min dark stain , flow cytometry (FCM) analysis.   1.2.4FCM collect cell cycle 1 × 106 cells, PBS washed 2 times, 800 ~ 1000 r / min centrifugation 5 min, discard supernatant, add 1 mL saline into single cell suspension. adding 2 mL pre-cooled fast mixing ethanol, fixed cells. abandoned fixative, PBS washed 2 times, 1000 r / min centrifugation 5 min, adding 1 mL DNA fluorescent dye PI , 4 ℃ dark stained 15 min, FCM analysis.   1.2.5 MTT assay (MTT) test the impact of RES on cell growth cells were collected, cells were re-inoculated to 96 well plates so that cell number per well 1 × 103 ~ 1 × 104, namely to 20,50 and 100 μmol / L RES effector cells, adding 100 mL / L calf serum RPMI 1640 culture medium. in the post-dosing 24, 48 and 72 h were detected. each hole by adding MTT solution of 20 μL, 37 ℃ cultured for 4 h, supernatant was removed, each well add 150 μL DMSO dissolved samples were measured by enzyme-linked immunosorbent assay absorbance of 490 nm of the hole values, calculate the average growth curve. 

  2 results   2.1RES role in morphological changes of the tumor cells by the RES 5 ~ 100 μmol / L effect, the difference between immune HepG2 cells seen under inverted microscope typical morphological features of apoptosis: cell shrinkage, cytoplasmic condensation, nuclear fixed, chromosome condensation, and some formation of apoptotic bodies. With the extended role of necrotic cells increased visibility , expressed as cell swelling, broken (Fig 1,2). but no significant change in K562 cells.   2.2RES RES on the cell cycle of K562 cells and act on 48 h, HepG2 cells, FCM detection of cells Although there were no cell cycle period of the previous subG1 G1 peak, but the K562 cells in G1 phase cells accounted for the percentage of cells were detected by 50.72%, G2 period is 22.81%, apoptosis rate was 0.59%; HepG2 cells in G1 phase was 67.75%, G2 period is 13.69%, apoptosis rate was 9.97%. which RES function was detected after the HepG2 cell cycle was blocked at the G1 phase.   2.3RES role in apoptosis of the tumor cells of different concentrations of RES no effect on K562 cells, while more significant role in HepG2 cell apoptosis, apoptosis and necrosis coexist. necrosis with time gradually increased. Annexin V fluorescence staining HepG2 cell apoptosis rate was the highest 13.7% (Fig 3 ).   2.4RES MTT cell growth of human hepatocellular carcinoma HepG2 show RES cells in a dose-response relationship, with time, RES concentration, the less cell survival, A490 nm and cell survival proportional. 48 h after the results started more pronounced effect (Fig 4). but the RES on the human K562 cells significantly inhibited the growth of no.   3 Discussion   With RES in-depth research and found [1] RES in the carcinogenic effect of three main stages with a chemical anti-cancer activity, anti-initiation activity, promote the role of anti-and anti-development role. More important is a broad anti-cancer effect, that is comparable with paclitaxel anticancer one of the most promising. The topics to RES, respectively, HepG2 and K562 tumor cells induced apoptosis and to explore the role of RES on the tumor cell apoptosis. apoptosis and tumor and is closely related to inhibition. We found that RES induces apoptosis by inhibiting tumor growth. apoptotic morphological change is the most direct indicators. immunological difference between HepG2 cells seen under inverted microscope typical apoptotic morphological features. Annexin V staining and FCM were detected in HepG2 cell apoptosis induced by RES. RES is by inducing apoptosis to its anti-tumor activity. of [2-4] RES effect on the tumor that RES and p53, bcl2, bax oncogenes such as the interaction of multiple signal transduction pathways by inducing apoptosis, thereby inducing apoptosis.   RES [5] can inhibit cancer cells from S phase to G2 / M phase, the cell resistance Delay in S phase by inhibiting DNA synthesis, thereby inhibiting the growth of cancer cells, and dose dependent. Yu Leung et al [6] reported that RES by inhibiting the expression of cell cycle proteins affect cell cycle progression
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