Trichostatin A sensitizes cisplatin-resistant A549 cells to apoptosis by up-regulating death-associated protein kinase

Abstract

Aim: To investigate the apoptosis-inducing effect of trichostatin A (TSA) in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP) and to examine whether TSA can enhance sensitivity to cisplatin treatment and the underlying molecular mechanisms of such an enhancement.

Methods: Cell viability was evaluated using the Neutral Red assay. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry analysis. Protein expression was detected by Western blotting. To determine the role of Death-associated protein kinase (DAPK) in TSA-induced apoptosis in the A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has a higher expression level of DAPK compared to endogenous expression, and DAPK activity was inhibited by both over-expression C-terminal fragment of DAPK which may competitive binding DAPK substrates to inhibit the function of DAPK and RNA interference.

Results: TSA induced apoptosis in both A549 cells and A549/CDDP cells. TSA enhanced the sensitivity of A549/CDDP cells to cisplatin, along with concomitant DAPK up-regulation. When DAPK was over-expressed, A549/CDDP cells became sensitive to cisplatin and the cytotoxicity of TSA could be increased. Moreover, the cytotoxicity of TSA could be alleviated by inhibition of DAPK activity by the expression of a recombinant C-terminal fragment of DAPK or RNA interference.

Conclusion: TSA induced sensitivity to cisplatin treatment in cisplatin-resistant A549 cells. The up-regulation of DAPK is one of the mechanisms mediating sensitization to TSA-induced apoptosis in cisplatin-resistant cells.

Figure 1

The sensitivity of A549 cells and A549/CDDP cells to cisplatin. (A) The effect of cisplatin on A549 cells and A549/CDDP cells. The plot shows the viability of A549 cells and A549/CDDP cells treated with indicated concentrations of cisplatin. The IC₅₀ of cisplatin to A549 cells and A549/CDDP cells is 2.36±0.10 μmol/L and 30.27±1.50 μmol/L, respectively. (B) The expression level of DAPK in A549 cells and A549/CDDP cells.

Figure 2

The effect of TSA on A549 cells and A549/CDDP cells. (A) Cells were treated by TSA for 24 h and stained with Hocest 33258/PI to measure apoptosis. Apoptotic cells were stained with light-blue and pictures were taken under a 400×objective. n=4. Mean±SD. ^bP<0.05, ^cP<0.01 vs A549 control group. ^eP<0.05, ^fP<0.01 vs A549/CDDP control group. (B) Cell viability is measured by Neutral Red assay after 24 h TSA treatment at indicated concentrations.

Figure 3

Apoptosis (sub-G₁) analyzed by flow cytometry. DNA profile was analyzed by PI staining with flow cytometry. The percent apoptosis is calculated as the percentage sub-G₁ peak as determined by cell-cycle analysis (arrow indicated sub-G₁ peak).

Figure 4

The effect of TSA on the sensitivity of A549/CDDP cells to cisplatin, The IC₅₀ of cisplatin to A549/CDDP cells is at 31.34±1.23 μmol/L in absence of TSA, while the IC₅₀ values are at 20.61±1.80 μmol/L and 18.48±0.70 μmol/L in presence of 31.25 nmol/L and 62.5 nmol/L TSA, respectively. n=4. Mean±SD.

Figure 5

The effect of TSA on DAPK expression and serineS308-phosphorylation level in A549/CDDP cells. (A) Western blot analysis of DAPK expression and serineS308-phosphorylation level induced with indicated concentrations of TSA. 1: control; 2: A549/CDDP cells treated with 125 nmol/L TSA; 3: A549/CDDP cells treated with 250 nmol/L TSA; 4: A549/CDDP cells treated with 500 nmol/L TSA; 5: A549/CDDP cells treated with 1000 nmol/L TSA. (B) The relative level of DAPK serineS308-phosphorylation induced by indicated concentrations TSA was measured by gray scale calculating with AlphaEaseFC software. n=3. Mean±SD. ^bP<0.05,^cP<0.01 vs control group.(C) The changes of DAPK expression and serineS308-phosphorylation level induced with 500 nmol/L TSA for different times were analyzed with Western blot method. 1: control; 2: A549/CDDP cells treated with 500 nmol/L TSA for 6 h; 3: A549/CDDP cells treated with 500 nmol/L TSA for 12 h; 4: A549/CDDP cells treated with 500 nmol/L TSA for 18 h; 5: A549/CDDP cells treated with 500 nmol/L TSA for 24 h. (D) The relative of DAPK serineS308-phosphorylation level induced with 500 nmol/L TSA for indicated time periods was measured by gray scale calculating with AlphaEaseFC software. n=3. Mean±SD. ^cP<0.01 vs control group

Figure 6

The effect of cisplatin on DAPK expression in A549/CDDP cells. 1: control; 2: A549/CDDP cells treated with 6.66 μmol/L cisplatin; 3: A549/CDDP cells treated with 13.32 μmol/L cisplatin; 4: A549/CDDP cells treated with 26.64 μmol/L cisplatin; 5: A549/CDDP cells treated with 53.28 μmol/L cisplatin.

Figure 7

The sensitivity of A549/CDDP cells to TSA and cisplatin is affected by DAPK expression and activity. (A) Western blot analysis of DAPK expression in A549/CDDP cells transfected by various expression vectors. n=3. Mean±SD. ^cP<0.01 vs control group.1: non-transfected group; 2: pcDNA3.1(+)-tranfected group; 3: pcDNA3.1(+)-DCTP-tranfected group; 4: pcDNA3.1(+)-DAPK-transfected group. (B) Western blot analysis of DAPK expression in TSA-treated A549/CDDP cells transfected by various expression vectors. 1: non-transfected group in absence of TSA; 2: non-transfected group induced by 500 nmol/L TSA; 3: pcDNA3.1(+)-tranfected group in absence of TSA; 4: pcDNA3.1(+)-tranfected group induced by 500 nmol/L TSA; 5: pcDNA3.1(+)-DCTP-tranfected group in absence of TSA; 6: pcDNA3.1(+)-DCTP-tranfected group induced by 500 nmol/L TSA; 7: pcDNA3.1(+)-DAPK-transfected group in absence of TSA; 8: pcDNA3.1(+)-DAPK-transfected group induced by 500 nmol/L TSA. (C) The sensitivity of A549/CDDP cells to TSA affected by DAPK expression. n=4. Mean±SD. (D) The sensitivity of A549/CDDP cells to cisplatin affected by DAPK expression. n=4. Mean±SD.

Figure 8

TSA changes the sensitivity of A549/CDDP cells to the cisplatin treatment by affecting DAPK expression. (A) Western blot analysis of DAPK expression in A549/CDDP cells transfected by interference vectors. 1: Non-transfected group 2: control vector group; 3: Endogenous DAPK expression silence group. (B) Western blot analysis of DAPK expression in TSA-treated A549/CDDP cells transfected by interference vectors. 1: non-transfected group in absence of TSA; 2: non-transfected group induced by 500 nmol/L TSA; 3: Control vector group in absence of TSA; 4: Control vector group induced by 500 nmol/L TSA; 5: Endogenous DAPK expression silence group in absence of TSA; 6: Endogenous DAPK expression silence group induced by 500 nmol/L TSA. (C) Effect of endogenous DAPK expression silence on TSA-induced A549/CDDP cells death. n=4. Mean±SD. (D) Effect of endogenous DAPK expression silence on cisplatin-induced A549/CDDP cells death. n=4. Mean±SD.