Figure 1.
HDAC expression and the effect of panobinostat on high-risk neuroblastoma cell lines.
Panel A: Protein extracts from log phase SK-N-AS, SK-N-DZ, SK-N-SH and SK-N-BE(2) cells were subjected to Western blots probed by anti-HDAC or -β-actin antibody. Panels B and C: SK-N-AS, SK-N-DZ, SK-N-SH or SK-N-BE(2) cells treated with a range of concentrations of panobinostat (0–80 nM) for 48 h were harvested and lysed. Soluble proteins were analyzed on Western blots probed by anti-acetylated (ac)-H4, -H4, -ac-tubulin, -α-tubulin or –β-actin antibody. Panel D: SK-N-AS, SK-N-DZ, SK-N-SH or SK-N-BE(2) cells were cultured with a range of concentrations of panobinostat in complete medium in 96-well plates at 37°C for 48 h, cell viabilities were determined using the MTT reagent and a visible light microplate reader. The IC50 values were calculated as the concentrations of drug necessary to inhibit 50% growth compared to control cells treated with vehicle control. The data are presented as mean values ± standard errors from at least 3 independent experiments.
Figure 2.
Panobinostat treatments potently induced apoptosis in high-risk neuroblastoma cell lines.
Panels A and B: SK-N-AS, SK-N-DZ, SK-N-SH or SK-N-BE(2) cell lines were treated with variable concentrations of panobinostat for 48 h. Apoptosis was measured by detecting sub-G1 population with propidium iodide (PI) staining and flow cytometry analyses. The experiment was repeated twice and the results are presented as means ± standard errors of triplicates from one representative experiment. Panels C and D: SK-N-AS, SK-N-DZ or SK-N-SH cells were treated with variable concentrations of panobinostat for 48 h. Apoptosis was measured by Annexin V-FITC and PI double staining and flow cytometry analyses. The results are presented as means ± standard errors of triplicates from one representative experiment. Panels E and F: SK-N-AS, SK-N-DZ, SK-N-SH or SK-N-BE(2) cells treated with variable concentrations of panobinostat for 48 h were harvested and lysed. Soluble proteins were analyzed on Western blots probed by anti-cleaved form caspase3 (-CF-Casp3), -PARP, or –β-actin antibody.
Table 1.
Cell cycle distribution of non-sub-G1 cells after panobinostat treatment of neuroblastoma cell lines.
Figure 3.
Synergistic antitumor interactions between panobinostat and doxorubicin in SK-N-BE(2) cells.
Panels A, C, and E: The SK-N-BE(2) cells were treated with variable concentrations of panobinostat and doxorubicin with three different administration schedules including simultaneous treatment with the two drugs for 48 h (designated PAN+DOX, panel A), pretreatment with panobinostat for 24 h followed by simultaneous treatment with the two drugs for 24 h (designated PAN→DOX, panel C), and pretreatment with doxorubicin for 24 h followed simultaneous treatment with the two drugs for 24 h (designated DOX→PAN, panel E). Viable cells were measured by MTT assays and the data are presented as means ± standard errors from at least three independent experiments. Panels B, D, and F: Standard isobologram analyses of antitumor interactions between panobinostat and doxorubicin were performed in the SK-N-BE(2) cells treated under the three different administration schedules described above. The IC50 values of each drug are plotted on the axes; the solid line represents additive effect, while the points represent the concentrations of each drug resulting in 50% inhibition of growth. Points falling below the line indicate synergism, whereas those above the line indicate antagonism. PAN, panobinostat; DOX, doxorubicin. The same abbreviations were used throughout the study unless otherwise stated.
Table 2.
Effects of panobinostat on the cytotoxicities of etoposide, doxorubicin, or cisplatin against high-risk neuroblastoma cell lines.
Figure 4.
Panobinostat potently enhanced apoptosis induced by etoposide, doxorubicin or cisplatin in SK-N-SH and SK-N-BE(2) cells.
SK-N-SH and SK-N-BE(2) cells were treated with etoposide, doxorubicin or cisplatin for 48 h in the absence or presence of 20 nM panobinostat administered simultaneously. Cell death was determined by trypan blue exclusion (panels A and B), while apoptosis was measured by PI staining and flow cytometry analyses and Western blotting measuring cleavage of caspase3 and PARP (panels C-F). Cell cycle progression was determined by PI staining and flow cytometry analyses (panels C and D). Changes in the CHK1-CDK1 signaling pathway were measured by probing Western blots with phosphorylation-specific antibodies. Results from trypan blue exclusion are presented as mean percentages ± standard errors (relative to control cells treated with vehicle control) from three independent experiments. ** indicates p<0.005, while *** indicates p<0.0005. The PI staining and flow cytometry analysis experiment was repeated two times and the data are presented as means of triplicates from one representative experiment. ETO, etoposide; CIS, cisplatin. The same abbreviations were used throughout the study unless otherwise stated.
Figure 5.
Effects of LY2603618 on the cytotoxicities of cisplatin, doxorubicin, or etoposide in SK-N-BE(2) cells.
Panel A: SK-N-BE(2) cells were treated with variable concentrations of LY2603618 for 48 h and cell viabilities were determined by MTT assays. The data are presented as means ± standard errors from three independent experiments. Panel B: SK-N-BE(2) cells treated with variable concentrations of LY2603618 for 48 h were harvested and subjected to Western blots probed by anti-CF-Casp3, -PARP, -CHK1, -p-CDC25CS216, -CDK1, -p-CDK1Y15, or –β-actin antibody. Panels C-F: The SK-N-BE(2) cells were treated with etoposide, doxorubicin, or cisplatin for 48 h in the absence or presence of 8 µM LY2603618 administered simultaneously. Cell death was determined by trypan blue exclusion (panel C), while apoptosis and cell cycle progression were determined by PI staining and flow cytometry analyses (panels D and E). Soluble proteins were subjected to Western blots probed by anti-CF-Casp3, -PARP, -CHK1, -p-CDC25CS216, -CDK1, -p-CDK1Y15, or -β-actin antibody (panel F). The trypan blue exclusion data are presented as means ± standard errors from three independent experiments, while the PI staining and flow cytometry analysis experiment was repeated two times and the data are presented as means of triplicates from one representative experiment. * indicate p<0.05. LY, LY2603618.
Table 3.
Effects of LY2603618 on the cytotoxicities of etoposide, doxorubicin, or cisplatin against high-risk neuroblastoma cell lines.