Calcium salicylate-mediated apoptosis in human HT-1080 fibrosarcoma cells

Abstract

Salicylates are novel biologically active compounds that exhibit multiple therapeutic activities. The anti-cancer effectiveness of calcium salicylate has been investigated on human HT-1080 fibrosarcoma cell lines at relatively low concentrations (predominantly 0.4 mM) compared to those previously reported. Although low calcium salicylate concentrations did not retard tumour growth progression significantly, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and time-lapse assays, its cytotoxic characteristics were proven to be prominent by various morphological and immunocytological techniques. The results here demonstrate evidence for approximately 25% apoptosis after treatment with calcium salicylate, which up-regulatd the expression of p53, p21 and Bax, and down-regulated Bcl-2 in HT-1080 cells.

Figures

Figure 1

Chemical structures of salicylic acid and aspirin (acetylsalicylic acid).

Figure 2

Concentration‐ and time‐dependent effects of CaSa on the viability of HT‐1080 cells. (a) 0–0.4 mm CaSa (b) 0.4–8 mm CaSa. Cells were grown in a 96‐well plate for different periods and drug concentrations (cell seeding density in = 2 × 104 cells/well, 100 µl of optimal DMEM, growth under standard growth conditions). The data represent the mean ± SEM of three replicates. Cell viability was measured by MTT assay.

Figure 3

Human HT‐1080 fibrosarcoma cell tracking image for first*, second** mitotic cycle and cell cycle*** durations when cells were cultured for 22 h in the presence of different salicylates. (a) The series of images illustrates a sample of the mitotic process during the designated time events for different stages. (b) Concentration‐ and time‐dependent effects of CaSa on the growth rate of HT‐1080 cells. (c) A representation for the effect of CaSa (0.4, 0.8, 1.2 mm) on the duration of first, second mitotic cycles and cell cycle duration.
*First mitotic duration refers to the observed HT‐1080 cells that underwent mitosis for the first time during the 22 h. The number of observed cells that completed the first mitotic cycle was: 44, 18, 44, 29, for the treatments in the graph, respectively, from left to right. **Second mitosis duration refers to the observed HT‐1080 cells that completed mitosis after the first mitosis during the 22 h of culturing. The number of observed cells was: 8, 6, 9, 7 for the treatment in this graph, respectively, from left to right. ***Cell cycle duration refers to the time from the end of first mitosis to the beginning of the second mitosis. The number of observed cells was: 16, 5, 12, respectively, from left to right.
Comparison of different treatments using one way anova for:
1 – The first mitotic cycle duration (P value); CaSa 0.4 CaSa 1.2 (0.014).
2 – The cell cycle duration (P value): control/CaSa 1.2 (0.002); CaSa 0.4/CaSa 1.2 (0.014).

Figure 4

FACS results of the cell cycle for untreated HT‐1080 cells (control). Cells were cultured under standard conditions, and arrested with serum‐free DMEM. Cells were then harvested at the indicated time points and were labelled with propidium iodide. Results are an average of two independent experiments.

Figure 5

FACS results of the cell cycle for HT‐1080 cells treated with CaSa for 2, 6, 12 and 24 h. Cells were cultured under standard conditions, arrested with serum‐free DMEM treated with CaSa at the indicated time points and labelled with propidium iodide.

Figure 6

Differential staining of CaSa‐treated HT‐1080 cells. White arrows refer to filopodial attachment. The cells were grown at standard conditions and were treated for 24 h with the CaSa compound.

Figure 7

Immunohistochemical illustration for treated HT‐1080 cells labelled with annexin‐V and treated with 0.4 mm CaSa. White arrows refer to apoptotic cells.

Figure 8

The effect 0.05 mm and 0.4 mm concentration of CaSa on the expression of p21, p53, Bax and Bcl‐2 in HT‐1080 cancer cells. Cells were cultured under standard conditions, arrested with 10% serum‐free DMEM and treated for 24 h. The data represent the mean ± SD of two independent gels for CaSa treatments and four control gels. One‐way anova (P value): p21‐control versus CaSa 0.4 mm (0.017); p53‐control versus CaSa 0.05 mm (0.004); Bax‐ 0.05 versus 0.4 mm CaSa (0.029), control versus 0.05 mm CaSa. (0.028), control versus 0.4 mm CaSa (0.005); BCl‐2‐control versus 0.05 mm CaSa (006), control versus 0.4 mm CaSa (0.07).