Combined use of irinotecan and p53 activator enhances growth inhibition of mesothelioma cells

Abstract

Malignant mesothelioma (MM) is an aggressive malignant neoplasm which rapidly invades pleural tissues and has a poor prognosis. Here, we explore enhancement of the effect of irinotecan [camptothecin-11 (CPT-11)] by the p53-dependent induction of carboxylesterase 2 (CES2), a CPT-11-activating enzyme, in MM. The level of CES2 mRNA was greatly increased on treatment with nutlin-3a. A combination of CPT-11 and nutlin-3a inhibited the growth of MM cells more effectively than either drug alone. Knocking down CES2 in MM cells reduced the effect of the drug combination, and its forced expression in MESO4 cells enhanced the growth inhibitory activity of CPT-11 in the absence of nutlin-3a. Enhancement of the growth inhibitory activity of CPT-11 by nutlin-3a suggests a possible new combinatorial MM chemotherapy regimen.

Keywords: carboxylesterase 2; irinotecan; mesothelioma; p53.

Fig. 1

MM cell growth after treatment with nutlin‐3a or Dox. (A) Cell growth analysis. After treatment with vehicle (V), 10 µm of nutlin‐3a (N), or 1 µm of Dox (D) for 24 h, cell growth of four MM cell lines, MESO1, H28, 211H, and MESO4, was analyzed by the XTT assay. The figure shows the mean percent cell growth with SD (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Dunnett's test. The * and *** represent P < 0.05 and P < 0.001, respectively, relative to the vehicle‐treated control cells. (B) Induction of CES2 and p53 target genes in MM cell lines by nutlin‐3a and Dox. MESO1, H28, 211H, and MESO4 cells were treated with DMSO (vehicle; V), 10 µm of nutlin‐3a (N), or 1 µm of Dox (D) for 24 h. The levels of CES2, CDKN1A, and NOXA mRNA were examined by qRT‐PCR analysis. The figure shows the mean expression with SD (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Dunnett's test. The *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively, relative to the vehicle‐treated control cells. (C) Expression of p53, CES2, and p21 proteins. Protein samples from MESO1, H28, 211H, and MESO4 cells treated with DMSO (vehicle; V), 10 µm of nutlin‐3a (N), or 1 µm of Dox (D) for 24 h were analyzed by western blotting with anti‐p53, anti‐CES2, anti‐p21, and anti‐GAPDH antibodies.

Fig. 2

Enhancement of the growth suppressive activity of CPT‐11 by nutlin‐3a in MESO4 cells. (A) Increased expression of CES2 and p53 target genes by CPT‐11 treatment. The levels of CES2, CDKN1A, and NOXA mRNA in CPT‐11‐treated cells with indicated concentrations were examined by qRT‐PCR. The figure shows the mean expression with SD (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Williams' test. The ** and *** represent P < 0.01 and P < 0.001, respectively, relative to the vehicle‐treated control cells. (B) Cell growth analysis. After treatment with drugs for 24 or 48 h, cell growth was analyzed by the XTT assay. The figure shows the mean percent cell growth with SD (n = 3), relative to the vehicle‐treated control cells. C = vehicle control, N = nutlin‐3a (10 µm), 5C = 5 µg·mL⁻¹ of CPT‐11, 20C = 20 µg·mL⁻¹ of CPT‐11. Statistical significance was determined by Dunnett's test. The * and *** represent P < 0.05 and P < 0.001, respectively, relative to the vehicle‐treated control cells. (C) Expression of CES2 and p53 target genes. The levels of CES2, CDKN1A, and NOXA mRNA in nutlin‐3a or/and CPT‐11 treated cells were examined by qRT‐PCR. The figure shows the mean with SEM relative to the vehicle‐treated control cells (n = 3). C = vehicle control, N = nutlin‐3a, 5C = 5 µg·mL⁻¹ of CPT‐11, 20C = 20 µg·mL⁻¹ of CPT‐11. The figure shows the mean expression with SD (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Dunnett's test. The *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively, relative to the vehicle‐treated control cells. (D) Expression of p53, CES2, and p21 proteins. Cells were treated with drugs as indicated for 48 h. Protein samples were analyzed by western blotting with anti‐p53, anti‐CES2, anti‐p21, and anti‐GAPDH antibodies. (E) Dose response curve of the combined treatment. Cells were treated with various concentrations of CPT‐11 in the presence (N+) or absence (N−) of 10 µm nutlin‐3a for 48 h. The cell growth was analyzed by XTT assay. The figure shows the mean percent cell growth with SEM (n = 3), relative to the vehicle‐treated control cells. Statistical significance at each CPT‐11 concentration was determined by Student's t‐test. The **, and *** represent P < 0.01, and P < 0.001, respectively, between N+ and N− cells. (F) MESO4 cells were treated with various concentrations of SN‐38 in the presence (N+) or absence (N−) of 10 µm nutlin‐3a for 48 h. The cell growth was analyzed by XTT assay. The figure shows the mean percent cell growth with SEM (n = 3), relative to the vehicle‐treated control cells. Statistical significance at each SN‐38 concentration was determined by Student's t‐test. The * and ** represent P < 0.05, and P < 0.01, respectively, between N+ and N− cells. (G) Expression of p53 and p21 proteins. Cells were treated with SN‐38 at indicated concentrations for 48 h. V; vehicle (DMSO) control. Protein samples were analyzed by western blotting with anti‐p53, anti‐p21, and anti‐GAPDH antibodies.

Fig. 3

Combined efficacy of nutlin‐3a and CPT‐11 on MM cell lines. (A) Cell growth analysis. After treatment with drugs for 48 h, the growth of MESO1, H28, and 211H was analyzed by the XTT assay. The graphs show the mean percent cell growth with SD (n = 3), relative to the vehicle‐treated control cells. C = vehicle control, N = 10 µm of nutlin‐3a, 5C = 5 µg·mL⁻¹ of CPT‐11, 20C = 20 µg·mL⁻¹ of CPT‐11. Statistical significance was determined by Dunnett's test. The * and *** represent P < 0.05 and P < 0.001, respectively, relative to the vehicle‐treated control cells. (B) Expression of p53, CES2, and p21 proteins. Protein samples from H28 and 211H treated with indicated drugs for 48 h were analyzed by western blotting with anti‐p53, anti‐CES2, anti‐p21, and anti‐GAPDH antibodies. The sample from 211H cells treated with both nutlin‐3a and 20 µg·mL⁻¹ CPT‐11 was not included because massive cell death was occurred (see below) making the protein sample not suitable for analysis. (C) Cell death analysis. After treatment with vehicle (V), 10 µm nutlin‐3a (N), 20 µg·mL⁻¹ CPT‐11 (C), or both 10 µm nutlin‐3a and 20 µg·mL⁻¹ CPT‐11 (N + C), cells death was assessed by trypan blue exclusion assay. The graphs show mean percent cell death with SD (n = 3). Statistical significance was determined by Tukey's test. The * and *** represent P < 0.05 and P < 0.001, respectively, relative to the vehicle‐treated control cells. The ^# and ^### represent P < 0.05 and P < 0.001, respectively, relative to the nutlin‐3a‐treated cells. The ^† and ^††† represent P < 0.05 and P < 0.001, respectively, relative to the CPT‐11‐treated cells.

Fig. 4

Suppression of the combined effect of nutlin‐3a and CPT‐11 by RNAi for CES2 or pharmacological inhibition of p53. (A) Validation of CES2 siRNAs using CES2‐overexpressing cells. CES2‐overexpressing ACC‐MESO4 cells (see Fig. 5) were treated with a control double‐stranded RNA (N.C.) or four different siRNAs for CES2 (CES2siRNA#1, CES2siRNA#2, CES2siRNA#3, and CES2siRNA#4) for 48 h. The level of CES2 mRNA was examined by qRT‐PCR. The figure shows the mean relative expression level with SEM (n = 3). (B) Western blot analysis. MESO4 cells were treated with nutlin‐3a (10 µm) and then with CPT‐11 (20 µg·mL⁻¹) and a control siRNA or CES2 siRNA (CES2siRNA#1 or ‐#3; experimental condition as in C). Protein samples were analyzed by western blotting with anti‐CES2 and anti‐GAPDH antibodies. (C) Suppression of combined efficacy of nutlin‐3a and CPT‐11 by CES2 siRNA in MESO4 cells. MESO4 cells were treated with vehicle (V) or 10 µm of nutlin‐3a (N) and then treated with a control (N.C.) or CES2 siRNAs (CES2siRNA#1 and CES2siRNA#3) with or without 20 µg·mL⁻¹ of CPT‐11 (C). Cell growth was assessed by the XTT assay. The figure shows the mean percent cell growth with SEM (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Student's t‐test. The * represents P < 0.05 between indicated cells. (D) Suppression of combined efficacy of nutlin‐3a and CPT‐11 by CES2 siRNA in H28 cells. H28 cells were treated and analyzed as in B. The figure shows the mean percent cell growth with SEM (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Student's t‐test. The * represents P < 0.05 between indicated cells. (E) Effect of pifithrin‐α (p53 inhibitor) on CES2 expression. MESO4 cells were treated with 10 µm of nutlin‐3a in the presence (+) or absence (−) of 30 µm pifithrin‐α for 48 h. Cell extracts were analyzed by western blotting with anti‐CES2 and anti‐GAPDH antibodies. (F) Suppression of combined efficacy of nutlin‐3a and CPT‐11 by pifithrin‐α (p53 inhibitor) treatment. MESO4 cells were treated with vehicle (V), 10 µm nutlin‐3a (N), 10 µm nutlin‐3a and 20 µg·mL⁻¹ CPT‐11 (N + C), 10 µm nutlin‐3a, 20 µg·mL⁻¹ CPT‐11, and 30 µm pifithrin‐α (N + C + p53 inhibitor) for 48 h. Cell growth was assessed by the XTT assay. The figure shows the mean percent cell growth with SEM (n = 3), relative to vehicle‐treated control cells. Statistical significance was determined by Student's t‐test. The ** represents P < 0.01 between indicated cells.

Fig. 5

Enhanced efficacy of CPT‐11 in CES2‐overexpressing cells. (A) Expression of CES2 in established cell lines. The level of CES mRNA in two empty vector‐carrying (C1 and C2) and two CES2‐overexpressing MESO4 cells (CES2‐1 and CES2‐2) was examined by qRT‐PCR. The figure shows the mean relative expression with SEM (n = 3). (B) Detection of CES2 enzyme activity. Protein lysates from control (C1 and C2) and CES2‐overexpressing cells (CES2‐1 and CES2‐2) were analyzed by gel‐based ABPP. The arrow indicates the CES2 band. Then, the gel was subjected to western blotting with anti‐GAPDH antibody. The size markers for ABPP are indicated on the left (kD). (C) Cell growth analysis. Control cells (C1 and C2) and CES2‐overexpressing cells were treated for 24 h with vehicle or CPT‐11 (20 µg·mL⁻¹) and cell growth was assessed by the XTT assay. The figure shows the mean percent cell growth with SEM (n = 3), relative to the vehicle‐treated control cells. Statistical significance was determined by Student's t‐test. The ** represents P < 0.01. n.s., not significant.