Article Figures & Data
Figures
- Figure 1.
Growth inhibition and morphological changes of CAOV-3 and SKOV-3 ovarian cancer cells by HDACi and calpeptin. A. CAOV-3 ovarian cancer cells were treated with 0.5 mM sodium butyrate (SB), 2 μM suberanilohydroxamic acid (SAHA), 5 μg/ml calpeptin (Calp) or combination of SB and Calp (p-value=0.0002) and SAHA and Calp (p-value=0.0001), at the same concentrations. B. SKOV-3 ovarian cancer cells were treated with 1 mM SB, 10 μM SAHA, 10 μg/ml Calp or combination of SB and Calp (p-value=0.0001) and SAHA and Calp (p-value=0.0001), at the same concentrations. Viable cells were washed, trypsinized and counted after four days. All treated cells are expressed as a percent relative to the arbitrarily set 100% of the dark shaded column of control cells. C. CAOV-3 ovarian cancer cells were treated with 1 mM SB, 10 μM SAHA, 20 μg/ml Calp or a combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. D. SKOV-3 ovarian cancer cells were treated with 1 mM SB, 10 μM SAHA, 20 μg/ml Calp or a combination of SB and Calp and, also, SAHA and Calp, at the same concentrations. Morphology changes and number of cells are different in the cells with inhibitors when compared to control cells. Photographs were taken after four days of treatment.
- Figure 2.
Transwell inhibition of CAOV-3 and SKOV-3 ovarian cancer cell motility. A. CAOV-3 ovarian cancer cells were treated with 0.5 mM sodium butyrate (SB), 2 μM suberanilohydroxamic acid (SAHA), 5 μg/ml calpeptin (Calp) or combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. B. SKOV-3 ovarian cancer cells were treated with 1 mM SB, 10 μM SAHA, 10 μg/ml Calp or combination of SB and Calp and, also, SAHA and Calp, at the same concentrations. After 48 h, ovarian cancer cells were harvested, suspended in serum-free media and added to the upper chamber. The lower chamber contained conditioned media from untreated control cells. After 12 h, cells were removed from the upper chamber and the membrane was stained and viewed under the microscope. Cell counts were taken from triplicates. Untreated control cell counts were normalized to 100% motility. HDACi, Calp and combination treatment results are shown in percentage motility compared to the untreated control.
- Figure 3.
Re-expression of tumor suppressor genes: ARHI, p21, RARβ2. A. CAOV-3 ovarian cancer cells were treated with 10 μM suberanilohydroxamic acid (SAHA) for 48 h. B. SKOV-3 ovarian cancer cells were treated with 10 μM SAHA for 48 h. Total RNA was isolated, cDNA was prepared. qPCR for ARHI, p21 and RARβ2 transcripts were performed. The results were normalized for each sample to the level of β-actin transcripts for each gene in each sample. Average of triplicate data was expressed as a fold re-expression compared to the untreated control cells.
- Figure 4.
Cell-cycle inhibition, apoptosis and autophagy. A. CAOV-3 ovarian cancer cells were treated for 48 h with 1 mM sodium butyrate (SB), 10 μM suberanilohydroxamic acid (SAHA) and 10 μg/ml calpeptin (Calp) or combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. B. SKOV-3 ovarian cancer cells were treated for 48 h with 1 mM SB, 10 μM SAHA and 10 μg/ml Calp or combination of SB and Calp and, also, SAHA and Calp, at the same concentrations. The cells were stained with PI and FACS analysis was performed to determine the stages of cell-cycle inhibition in each sample. C. CAOV-3 ovarian cancer cells were treated with 1 mM SB, 10 μM SAHA, 10 μg/ml Calp or combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. D. SKOV-3 ovarian cancer cells were treated with 1 mM SB, 10 μM SAHA, and 10 μg/ml Calp or combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. After a 48-h treatment, the ovarian cancer cells were stained with annexin conjugated to FITC and PI. Apoptosis quantities were measured with flow cytometry and plotted. E. Graph representation of CAOV-3 cancer cell apoptotic percentages from data of C. F. Graph representation of SKOV-3 cancer cell apoptotic percentages from data of D. G. CAOV-3 ovarian cancer cells were treated for 96 h with 0.5 mM SB, 2 μM SAHA, 5 μg/ml Calp or combination of SB and Calp, as well as SAHA and Calp at the same concentrations. The arrow indicates one of the several LC3b-associated phagolysosomes in cells. H. SKOV-3 ovarian cancer cells were treated for 96 h with 1 mM SB, 10 μM SAHA, 10 μg/ml Calp or combination of SB and Calp and, also, SAHA and Calp, at the same concentrations. Next, cells were treated with antibodies followed by DAPI addition and incubated. Cells were viewed and photographed with a fluorescent microscope. The arrow indicates one of the several LC3b-associated phagolysosomes in cells.
- Figure 5.
Effects of HDACi and calpeptin on Akt and ERK phosphorylation. A. CAOV-3 ovarian cancer cells (upper left panel) and SKOV-3 ovarian cancer cells (upper right panel) were treated with 1 mM sodium butyrate (SB), 1 μM suberanilohydroxamic acid (SAHA), 20 μg/ml calpeptin (Calp), or combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. Western analysis was performed for Akt phosphorylation (pAkt) in CAOV-3 and SKOV-3 ovarian cancer cells. B. CAOV-3 ovarian cancer cells (lower left panel) and SKOV-3 ovarian cancer cells (lower right panel) were treated with 1 mM SB, 10 μM SAHA, 20 μg/ml calpeptin (Calp) or combination of SB and Calp, as well as SAHA and Calp, at the same concentrations. Western analysis was performed for ERK phosphorylation (pERK) in CAOV-3 and SKOV-3 ovarian cancer cells. Blots were stripped and a western blot followed for Akt and ERK, respectively.
- Figure 6.
Model of combination therapy using HDACi and calpeptin.
