Fig 1.
The sensitivity of ZL55 cells to Ptac2S and cisplatin.
(A) Cells were treated with or without increasing concentration of Ptac2S or CDDP for 12, 24 and 48 h; (B) or were continuously exposed to 100 μM CDDP or Ptac2S. Cell viability was monitored by SRB assay and data are presented as means ± S.D. of six-independent experiments with eight replicates in each, and are presented as percent of control. (C) Clonogenic survival assay in cells treated with the indicated amounts of Ptac2S or CDDP for 2 h, and after 15 days of growth, colonies consisting of more than 50 cells were scored. The percentage of number colonies represents the means ± S.D. of six-independent experiments. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (D) After the treatment with 5 μM Ptac2S or 50 μM CDDP for 24 hours, cell cycle profiling was performed by FACSCanto flow cytometer as described under Materials and methods. Representative FACS histogram from six separate experiments is shown. (E) Comparison of sub-G1 DNA content in Ptac2S or CDDP treated cells. *P < 0.01, significantly different from saline control; #P < 0.01, significantly different between Ptac2S and CDDP. Inset: The IC50 values to CDDP and Ptac2S calculated after 24 and 48 h.
Fig 2.
Ptac2S and CDDP provoked apoptosis.
(A, B, C) ZL55 cells treated or not with 5 μM Ptac2S or 50 μM CDDP for the indicated times were stained with DAPI. (A) The representative fields of one of five independent experiments after 24 hours of incubation are shown. Scale bar = 50 μm.The quantification of the percentage of apoptotic nuclei obtained from cells stained with DAPI (mean ± S.D.; n = 5) and incubated with Ptac2S (B) or CDDP (C). Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (D) Cytosolic and nuclear proteins were obtained from ZL55 cells, treated or not with 5 μM Ptac2S (right) or 50 μM CDDP (left). Samples were dissolved in SDS buffer and separated on SDS gel. Immunoblotting was performed using monoclonal antibodies specific to PARP, caspases-3, -7 and -9, Bax and Bcl2. Sequential incubation with anti-actin confirmed the equal protein loading. These results are representative of six independent experiments. (E) Densitometic analysis of Bcl-2 and BAX or active caspase-3 (f-Casp 3), active caspase-7 (f-Casp 7), active caspase-9 (f-Casp 9) and fragmented PARP-1 (f-PARP) normalized to β-actin and fragmented PARP (f-PARP) normalized to H3/4 of experiments shown in (D). The data are means ± S.D. of fIve different experiments. P < 0.0001 by one-way ANOVA for Bcl-2, f-caspases and f-PARP (n = 5); P < 0.001 by one-way ANOVA for BAX (n = 5); values with shared capital and lower case letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Fig 3.
Effect of Ptac2S on ΔΨm and apoptotic proteins in ZL55 cells.
(A) Fluorescent spectra of JC-1 in ZL55 cells treated or not with 5 μM Ptac2S for the indicated time. The data are means ± S.D. of six different experiments and are presented as red J-aggregates/green monomer JC-1 fluorescence ratio. Asterisks indicate values that are significantly different (p < 0.05) from control at the same time point. (B) Mitochondrial and cytosolic fractions were prepared at the indicated times of Ptac2S treatment (5 μM), and the kinetics of Bax and Bcl-2 cytosol-to-mitochondria translocations and the release of mitochondrial cytochrome c were examined by western blotting. Porin and β-actin served as a mitochondrial and cytosolic indicators, respectively. The Fig is representative of six independent experiments. (C) Densitometic analysis of cytochrome c, Bcl-2 and Bax, normalized to β-actin (cytosolic fractions) or to porin (mitochondrial fractions), of experiments shown in (B). The data are means ± S.D. of six different experiments and are presented as the ratio of the densitometric values between Bcl-2 or BAX and β-actin/porin. P < 0.0001 by one-way ANOVA for all (n = 6); values with shared capital and lower case letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Fig 4.
PKC-δ induces apoptosis in ZL55 cells.
ZL55 cells were treated without or with 5 μM Ptac2S for the indicated times. For PKCs translocation studies, cytosol (cyt), membrane (mem), nuclei (nuc) (A) and mitochondrial (B) fractions were analysed by Western blotting with specific antibodies. The Figs are representative of six independent experiments. (C and D) The data of densitometic analysis are means ± S.D. of six different experiments and are presented as the ratio of the densitometric values between PKCs and β-actin (cytosolic fractions) or α-subunit of Na+/K+ATPase (membrane fractions), or H3/4 (nuclear fractions) or porin (mitochondrial fractions). P < 0.0001 by one-way ANOVA for all (n = 6); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (E) Cells were transfected with siRNA–PKC-δ and then were incubated or not with Ptac2S for the indicated times. The data are means ± S.D. of six different fluorescent spectra of JC-1 presented as red J-aggregates/green monomer JC-1 fluorescence ratio. Asterisks indicate values that are significantly different (p < 0.05) from control at the same concentration and time point. (F) Cells were transfected with siRNA–PKC-δ or control siRNA (NS) and then were incubated with Ptac2S. Viable cell number was determined 24 h later by SRB assay. The data are means ± S.D. of six different experiments run in eight replicates and are presented as percent of control; values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. Western blotting of total lysates was performed with specific anti-PKC-δ or with anti-caspase-7 or anti-PARP-1 antibodies. The Fig is representative of six independent experiments. (G) Densitometic analysis of f-caspase-9 (f-Casp-9) and f-PARP-1, normalized to β-actin are shown. The data are means ± S.D. of five different experiments. P < 0.0001 by one-way ANOVA for all (n = 5); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Fig 5.
Role of PKC-ε in Ptac2S-induced apoptosis in ZL55 cells.
(A) Cells were treated or not with 5 μM Ptac2S or with 50 μM CDDP for indicated time. Cell lysates were analysed by Western blotting with anti-unphosphorylated and phosphorylated p38MAPK antibodies. (B) Densitometic analysis of p-p38MAPK normalized to p38MAPK. The data are means ± S.D. of five different experiments. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (C) Cells were transfected with siRNA–PKC-δ or siRNA–PKC-ε or pretreated with SB203580 (15, 30 μM) and then incubated with Ptac2S; viable cell number was determined 24 h later by SRB assay. The data are means ± S.D. of five different experiments run in eight replicates and are presented as percent of control; values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (D) Cells were transfected with siRNA–PKC-ε or control siRNA (NS) and then were incubated with Ptac2S. Cytosolic or nuclear (for PARP-1) fractions were analysed by Western blotting with antibodies against PKC-ε, phosphorylated p38MAPK, cytochrome c (Cyt. c), caspase-9 (Casp-9) and PARP-1; β-actin was used as a control for protein loading. Representative immunoblots of five experiments are depicted. (E) Densitometic analysis of f-caspase-9 (f-Casp-9), f-PARP-1 and cytochrome c (Cyt. c), normalized to β-actin are shown. The data are means ± S.D. of five different experiments. P < 0.0001 by one-way ANOVA for all (n = 5); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Fig 6.
Growth inhibitory effect of Ptac2S and CDDP in vitro and in a xenograft model of Mesothelioma.
Balb/c nude mice carrying mesothelioma developed by injection of ZL55 cells (around 50 mm3) received intravenous Ptac2S (10 mg/kg) or CDDP (10 mg/kg); tumour volume (A) and body weight of mice (B) were measured every 3 days for 35 days in total. The results are presented as mean ± S.D. (animals per group n = 8). *P < 0.05, significantly different from saline control; #P < 0.05, significantly different between Ptac2S and CDDP. (C) After killing, the tumors were collected and measured.