Chemosensitize And Radiosensitizing Effect Of SAHA On Cervical Cancer SiHa Cells

ObjectiveTo observe the sensitizing effects of the HDAC inhibitor suberoylanilide ydroxamic acid (SAHA) combined with DDP or X ray to cervical cancer SiHa cells and the mechanism of sensitization as well through studying the growth inhibition and apoptosis, cell colons formation rate and the result of the radiation sensitive parameters of cerical cancer cells.Methods1. SiHa cells were treated with0.25,0.5,1,2,4and6μmol/L of SAHA, and1.2,4,6,8and10μg/mL of DDP, respectivly. SiHa cells were treated with the vary combination dose of the two drugs. The inhibitory rate of SiHa cells were analyzed with MTT; Cell cycle distribution was detected by flow cytometry; Apoptosis of cells was measured by Annexin V-FITC.2. SiHa cells were incubated with SAHA at the concentration of20%IC50for24h and then were exposed to X-ray irradiation (IR)(0,2,4,6,8Gy). Colonies and surviving fraction (SF) of cells were detected by Clonogenic assay, the SF、Do and sensitizing enhancement ratio (SER) of each groups and the cell survival curve was gained by single-hit multi-target model.3. P21, Bax and Ku70’s mRNA and protein expression levels were investigated by RT-PCR and Western bolt in SiHa cell. Results1. SAHA was capable of inhibiting SiHa cells growth comparing with control group (P<0.05); SAHA appears more inhibiting SiHa cells growth effect attending by the increasing dose and time.2. The combined groups inhibited the cell growth obversely than single drug groups after24hours (P<0.05); Inhibition rate increased gradually with SAHA increasing dose combined with DDP; The effect of SAHA combined With DDP showed that low doses of DDP combination is synergy, while higher doses of DDP combination has simple addition effect.3. When1,2,4umol/L of SAHA (S group) combined with the DDP1,2,4ug/ml (D group) respectively, all the groups appear arresting cell at G1/G0phase, while the G2/M+S period cells proportion markedly reduced. PI reduced compared with control group significantly (P<0.05). GO/G1phase ratio increased significantly in SD group, while the G2/M+S period cells proportion markedly reduced. PI reduced significantly compared with the S group and D group (P<0.05).4. SAHA combing with DDP induced more severe apoptosis than separate groups (P<0.05). Apoptosis in2ug/mL DDP group was not obviously, but after pretreatment by2umol/L SAHA, apoptosis rate increased significantly.5. Compared the expression of Bax mRNA with the control group, there was no statistical significance of D, S group(P>0.05), while the expressions of combination groups were higher then other groups(P<0.05); P21expression in each group is raised significantly, especially in combination groups comparing with D group and control group (P<0.05).6. Bax protein expression between SAHA groups and the control group was not statistically significant (P>0.05). The combination of two drugs increased Bax protein level (P<0.05). P21protein in each group are raised higher than D group and control group, especially in combination groups, significantly (P<0.05).7. Cell survival fraction was obviously lower after treatment with the combination of SAHA and radiation than that of radiation alone (P<0.05). The cell survival curve of combination groups were to the left compared with radiotherapy groups. The front one was relatively flat and the shoulder area is not obvious. Do of the X-ray groups and the combination groups were2.329and1.213respectivly. The Dq were1.721and0.823respectively. The SFR was1.92.8. Expression of Bax mRNA of radiotherapy groups were higher then the control groups(P<0.05). The expressions of combination groups of Ku70mRNA were were lower then than the radiotherapy groups (P<0.05).Conclusions1. SAHA has the synergistic effect with DDP on SiHa cells.2. SAHA can arrest cell cycle and induce apoptosis of SiHa cells with DDP.3. The mechanism might be associated with up-regulating expression of P21and Bax mRNA and protein.4. Low concentrations of SAHA enhanced the radiosensitivity of SiHa cells.6. Up-regulating the expression of Bax and down-regulating the expression of Ku70response to the sensitization enhancement of SAHA to Siha cells.