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Survivin Mediates Paclitaxel Effect in Hela That Survivin May Play an Important Role in Hela Western Blot Cells Apoptosis17

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Survivin Mediates Paclitaxel Effect in Hela That Survivin May Play an Important Role in Hela Western Blot Cells Apoptosis17 European Review for Medical and Pharmacological Sciences 2017; 21: 3504-3509 Down-regulation of survivin enhances paclitaxel-induced Hela cell apoptosis F. GU1, L. LI2, Q.-F. YUAN3, C. LI4, Z.-H. LI4 1Department of Obstetrics, Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China 2Department of Pediatrics, Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China 3Department of Gynecology and Obstetrics, Huangdao Qingdao District Wang Tai Center Hospital, Qingdao, China 4Department of Gynecology, Qingdao Haici Hospital, Qingdao, Shandong Province, China Abstract. – OBJECTIVE: Paclitaxel is one of success rate of cervical cancer treatment is a big the common anticancer drugs in the treatment challenge in the medical field. Targeted therapy of cervical cancer, while the mechanism of re- is the first choice in clinic8,9. The curative effect straining and killing cancer cells is still unclear. This study aimed to investigate the molecular of current molecular targeting anti-apoptotic pro- mechanism of paclitaxel in regulating prolifera- teins, such as survivin and apollon on cervical tion and apoptosis of cervical cancer Hela cells. cancer, is still not satisfactory. Therefore, it is MATERIALS AND METHODS: Paclitaxel at 2 urgently required to explore more effective mole- μmol/L was used to treat Hela cells for 48 h. MTT cular targets for the treatment of cervical cancer assay and flow cytometry were applied to test He- in clinic. Paclitaxel is a kind of important first-li- la cells proliferation and apoptosis respective- ne anticancer drug for the treatment of cervical ly. Western blot was adopted to determine the ex- 10 pression of survivin. SiRNA was performed to cancer . Paclitaxel catalyzes the rapid synthesis suppress survivin protein expression in Hela cells. of excitron protein, which binds to the micro- RESULTS: Paclitaxel restrained Hela cells tubules, leading to stabilization and prevention growth and induced apoptosis. Also, paclitaxel of microtubule depolymerization. Paclitaxel can treatment significantly reduced survivin protein inhibit mitosis, promote apoptosis, as well as expression in Hela cells. Moreover, survivin siR- NA transfection further promoted Hela cells apop- inhibit tumor cell migration through directly or tosis after intervention by 2 μmol/L paclitaxel. indirectly suppressing the expression and activity CONCLUSIONS: Down-regulation of survivin of myosin and actin11. After injected to the blo- promoted paclitaxel-induced apoptosis of cervi- od, paclitaxel is quickly decomposed into smaller cal cancer Hela cells. albumin-paclitaxel polymer, and further binds to Key Words: albumin GP60 receptor on the surface of vascu- Paclitaxel, Survivin, Cervical cancer, Hela, Apoptosis. lar endothelial cells12. Then, the albumin-paclita- xel is transported out of the blood vessel through Introduction endocytosis and aggregates around the tumors to regulate tumor formation. It was showed that the Cervical cancer is a common reproductive concentration of abraxane was 1.33-fold higher in system tumor in female with a higher morbidi- the tumor tissue, while half in normal tissue com- ty rate1,2. The pathogenesis of cervical cancer is pared with general solvent-based paclitaxel. Cur- very complicated and is thought to be influenced rent guidelines recommend single use of abraxa- by viral infections, sexual behavior, and delivery ne in the treatment of metastatic breast cancer13, times3-5. Also, genetic factors affect the develop- pancreatic cancer14, and other solid tumors15. Up ment of cervical cancer6. Although the curative to now, however, the molecular mechanism of effect of combined therapy is significant, it has paclitaxel in inhibiting and eliminating cervical numerous shortcomings and side effects, such cancer cells remains poorly understood. Anti-a- as bleeding7. At present, a variety of treatment poptosis protein survivin is a kind of protein kina- strategies have been developed in clinical practi- se involving in apoptosis, necroptosis, autophagy, ce7,8. However, how to improve the accuracy and and NF-κB signaling pathway16. It was speculated 3504 Corresponding Author: Lei Li, MD; e-mail: [email protected] Survivin mediates paclitaxel effect in Hela that survivin may play an important role in Hela Western Blot cells apoptosis17. However, the exact mechanism The cells were treated with lysis buffer and sepa- by how survivin induces Hela cells apoptosis re- rated by sodium dodecyl sulphate-polyacrylamide mains unclear. The aim of the present study was gel electrophoresis (SDS-PAGE). Primary antibodies to investigate the specific regulatory mechanism for survivin and actin were applied for detection. of survivin on Hela cells apoptosis. Statistical Analysis All data analysis was performed with SPSS18.0 Materials and Methods software (SPSS Inc., Chicago, IL, USA). Data were represented as mean ± standard deviation Reagents (SD). Student’s t-test was performed for compa- Cell apoptosis detection kit was purchased ring the difference between two groups. One-way from Beyotime (Shanghai, China). MTT, lipo- ANOVA and Dunnet post hoc test were perfor- fectamine reagent escort™, and rabbit anti-hu- med to assess the statistical significance among man survivin primary antibody were purchased multiple treatment groups. A significant differen- from Sigma-Aldrich (St. Louis, MO, USA). Sur- ce was depicted as p < 0.05. vivin siRNA and control siRNA were designed and synthetized by Genepharma (Shanghai, China). The sequences were 5’GCGATGATTG- Results GTTATCGT3’, and 5’AAAATAGGTCATCA- TGGT3’, respectively. Dulbecco’s Modified Ea- Paclitaxel Inhibited Hela Cells gle Medium (DMEM) was provided by Beijing Proliferation Dingguo Changsheng Biotechnology Co. Ltd The impacts of paclitaxel on Hela cell survival (Beijing, China). and proliferation were tested by MTT assay. As shown in Figure 1, paclitaxel significantly sup- Cell Culture pressed Hela cell growth (p < 0.05), suggesting it Hela cells were purchased from ATCC. The cel- affects the proliferation of Hela cells. ls were resuscitated and routinely cultured. Paclita- xel at 2 μmol/L or dimethyl sulfoxide (DMSO) was Paclitaxel Reduced Cell Membrane applied to treat the cells for 48 h. Potential of Hela Cells Mitochondrial membrane potential of Hela cel- Lipofectamine Escort™ Transfection ls was evaluated by flow cytometry after paclita- The cells were seeded and transfected using xel treatment. It was found that paclitaxel obviou- survivin siRNA and Lipofectamine reagent sly declined mitochondrial membrane potential of Escort™ according to the manual. Hela cells (p < 0.05) (Figure 2), indicating that paclitaxel may induce the apoptosis of Hela cells. Flow Cytometry Cell membrane potential specific dye TMRE Paclitaxel-induced Hela Cell Apoptosis and cell membrane phosphatidylserine specific To confirm whether paclitaxel induces apop- dye FITC-Annexin V were applied to test the tosis of Hela cells, we measured the phosphati- apoptosis of cervical cancer cell line by flow cytometry. MTT Assay The cells were seeded into 96-well plate at a density of 10.000 cells/well for analysis of cell proliferation by methylthiazolyl tetrazolium bro- mide (MTT) assay. Caspase-3 Activity Detection The cells were cracked on ice for 10 min and added into the 96-well plate. Then, the chro- mophoric substrate was added to the plate for me- Figure 1. Paclitaxel inhibited Hela cells proliferation. * p asuring the caspase-3 activity. < 0.05 vs. control. 3505 F. Gu, L. Li, Q.-F. Yuan, C. Li, Z.-H. Li Figure 2. Paclitaxel reduced cell membrane potential in Hela cells. *p < 0.05 vs. control. dylserine exposure of Hela cells after paclitaxel Inhibition of Survivin Promoted treatment. Results showed that after paclitaxel Paclitaxel-induced Hela Cells Apoptosis intervention, Hela cells exhibited markedly in- As shown in Figure 6, survivin level in Hela creased phosphatidylserine exposure (p < 0.05) cells was suppressed after survivin siRNA tran- (Figure 3), revealing that paclitaxel induces Hela sfection, whereas, caspase-3 activity was enhan- cell apoptosis. ced in survivin siRNA transfected Hela cells treated with paclitaxel, revealing that down-re- Paclitaxel Activated Caspase-3 gulation of survivin facilitated paclitaxel-induced in Hela Cells Hela cells apoptosis. Considering paclitaxel-induced apoptosis of Hela cells, caspase-3 activity was also measured. As shown in Figure 4, caspase-3 activity in Hela Discussion cells was obviously enhanced after paclitaxel tre- atment. Paclitaxel is an important anti-cancer drug in clinic. In this study, we discussed the im- Paclitaxel Down-regulated Survivin pacts of paclitaxel on the growth and apopto- Protein Expression in Hela Cells sis of cervical cancer cell line Hela. Firstly, we Western blot was adopted to assess survivin explored the influence of paclitaxel on cervi- protein expression in Hela cells. It was revealed cal cancer Hela cells growth and survival and that survivin protein level was down-regulated showed paclitaxel significantly decreased the in Hela cells after treated with paclitaxel (Fi- proliferation of Hela cells, which is consistent gure 5). with previous study. To evaluate the effect of 3506 Survivin mediates paclitaxel effect in Hela Figure 3. Paclitaxel-induced Hela cell phosphatidylserine exposure. * p < 0.05 vs. control. Figure 5. Paclitaxel downregulated survivin protein expres- sion in Hela cells. Figure 4. Paclitaxel activated caspase-3 in Hela cells. *p < 0.05 vs. control. paclitaxel on
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