Influence of estrogen and polyamines on mifepristone-induced apoptosis in prostate cancer cells

Abstract

Purpose: Although androgens are the main steroids controlling the growth of prostate glands, estrogens are also important in the regulation of its growth. Prostate cancer cells, like other cancer cells, maintain high levels of polyamines. In LNCaP cells, apoptosis is induced by mifepristone. During the process of cell death, the regulation of ROS production, caspase-3 activation and poly (ADP-ribose) polymerase cleavage were investigated in the presence of estrogen and polyamines to identify their possible roles.

Materials and methods: The cell growth was assessed using the MTT assay, and the intracellular ROS production by the DCFH-DA assay. The p53 protein expression, activation of caspase-3 and PARP cleavage were checked by Western blotting, with specific antibodies to each.

Results: The growth and viability of the cells were significantly inhibited, in a dose- and time-dependent manners, by mifepristone (MIF) treatment. The production of ROS were dependent on the MIF dosage. The activation of caspase-3 and cleavage of PARPalso increased with the duration of MIF treatment. The expression of p53 protein also increased with increases in the MIF incubation time. E(2) severely inhibited the ROS production, caspase-3 activation and PARP cleavage. However, polyamines only inhibited the ROS production, without influencing the caspase-3 activation or PARP cleavage.

Conclusion: In LNCaP cells, MIF induces apoptosis through ROS production. The expression of p53 protein, caspase-3 activation and PARP cleavage accompanied the process of apoptosis. The apoptotic processes were inhibited by E(2), but polyamines only inhibited the ROS production, implying the multifunctional role of E(2), in addition to its role as a free radical scavenger.

Keywords: Apoptosis; Estrogen; Mifepristone; Polyamine; Prostate cancer cell.

Fig. 1

Influence of MIF on the proliferation of LNCaP cells. Cells seeded in 48-wells, at 1×10³ cells per well, were treated with MIF (5~20 µM) for 4 days. The viability was measured by the MTT assay. Statistical analysis was performed by a one-way analysis of variance followed by the student t-tests. ^*p<0.05 vs. control for the 3rd day; ^†p<0.05 vs. control for the 4th day.

Fig. 2

Effects of E₂ and polyamines on MIF-induced cell death. Cells were treated with 10 nM of the E₂ and each of the PAs (Put 5 mM; Spd 10 µM; Spm 10 µM) for 24 h prior to the addition of 17 µM MIF. The viability was measured by the MTT assay.

Fig. 3

MIF-induced ROS generation in LNCaP cells. MIF treated-cells were incubated for 2 h, in 25 µM/ml DCFH-DA, in a final volume of 200 µl/well, at 37℃. After incubation, the fluorescence was measured at excitation and emission wavelengths of 485 and 530 nm, respectively. H₂O₂ was used as a vehicle for the ROS measurement. ^*p<0.01 vs. control.

Fig. 4

The effects of the E₂ and polyamines on the MIF-induced ROS generation in the LNCaP cells. Cells were treated with 10 nM of the E₂ and each of the PAs (Put 5 mM; Spd 10 µM; Spm 10 µM) for 24 h prior to the addition of 17 µM MIF. ^*p<0.01 vs. control; ^†p<0.05 vs. MIF alone; ^‡p<0.01 vs. MIF alone.

Fig. 5

Influence of MIF on the p53 expression in the LNCaP cells. A: Western blotting of the p53 in the LNCaP cell treated with MIF (17 µM, 2~4 days). B: The relative amount of p53 expression was determined by scanning densitometry.

Fig. 6

The effects of the E₂ and polyamines on the MIF-induced caspase-3 activation. Cells were treated with 10 nM of the E₂ and each of the PAs (Put 5 mM; Spd 10 µM; Spm 10 µM) for 24 h prior to the addition of MIF 17 µM. Activation of caspase-3 is indicated by the disappearance of the precursor band. A: Western blotting of caspase-3 in LNCaP cells treated with MIF for 4 days. B: The relative amount caspase-3 activation was determined by scanning densitometry

Fig. 7

The effects of the E₂ and polyamines on the MIF-induced PARP cleavage. The culture conditions were the same as in Fig. 6. A: Western blotting of PARP in LNCaP cells treated with MIF for 4 days. B: The relative amount PARP cleavage was determined by scanning densitometry.