Fig 1.
Panobinostat inhibits colony formation and induces cell death of SKOV-3 cells assessed by flow cytometry after 72 hours of treatment.
A: Representative images of the colony formation after exposure to various drug concentrations. The blue dots represent vital cell clusters. B: Percentages of viable colonies relative to control (untreated) cells. C: Apoptosis for SKOV-3 measured by annexin V/PI staining. Viable cells (lower left quadrant) were negative for both annexin V and PI, apoptotic cells were positive for annexin V staining and negative for PI, and the dead/late apoptosis cells were positive for both annexin V and PI (upper right quadrant). D: Percentages of the different cell death stages for SKOV-3. All results were presented as mean ± SD (n = 3), * P < 0.05, ** P < 0.005 and *** P < 0.001 compared to controls, calculated by unpaired t-test. E, F: Apoptosis by annexin V/PI staining and percentage of the different cell death stages for CaOv3.
Fig 2.
Apoptosis on SKOV-3 cells after treatment with panobinostat, carboplatin or a combination of these drugs for 72 hours.
The combination of panobinostat and carboplatin resulted in synergism. A: Nuclear morphology was evaluated by Hoechst 33342 staining. Abnormal nuclei with condensed chromatin were consistent with cell apoptosis/death (highlighted in white). B: The percentages of apoptotic nuclei are shown as mean ± SD (n = 3). C: Cell viability for SKOV-3 is measured with the use of the WST-1 assay. D: The effect of combining panobinostat and carboplatin on SKOV-3 was calculated from the response to each of the drugs alone, compared to the expected response by combining similar concentrations (calculated by Bliss independence analysis). A positive difference between actual response and expected was then ascribed to synergy. E,F: Cell viability for CaOv3, and Bliss independence analysis of expected and actual responses for combinational therapy of panobinostat and carboplatin for CaOv3.
Fig 3.
Exposure of SKOV-3 cells to panobinostat for 24 hours results in cell cycle arrest and upregulation of proteins regulating cell cycle arrest, histone acetylation and cell death.
A, B: Cell cycle arrest revealed by cell cycle analysis. C: Upregulation of p21 and H2B. PARP1 and caspase-3 cleavage and activation of cdc2 detected by immunoblotting techniques. D, E: Increased phosphorylation of H2AX examined by phosphoflow cytometry.
Fig 4.
Chemotherapeutic in vivo cohorts.
A: Illustration of weekly bioluminescent image analysis of representative groups of xenografted mice: a) control, b) panobinostat, c) carboplatin and d) combination of panobinostat and carboplatin. B: Relative tumour growth measured by BLI. C: Kaplan-Meyer cumulative survival curves of control, panobinostat-, carboplatin- and a combination of panobinostat- and carboplatin-treated mice. D: Median survival time and increase in survival time (%) for the variously treated groups.
Fig 5.
A: Illustration of weekly bioluminescent image analysis of representative group of xenografted mice: a) control, b) surgery, c) surgical and panobinostat/carboplatin-treated and d) combination of panobinostat and carboplatin. B: Relative tumour growth measured by BLI. C: Kaplan-Meyer cumulative survival curves of control, panobinostat-, carboplatin- and a combination of panobinostat- and carboplatin-treated mice. D: Median survival time and increase in survival time (%) for the variously treated groups.