Korean J Physiol Pharmacol.  2010 Dec;14(6):365-369. 10.4196/kjpp.2010.14.6.365.

Melatonin Induces Apoptotic Cell Death via p53 in LNCaP Cells

• Kim CH
• Yoo YM

• KMID: 2071707
• DOI: http://doi.org/10.4196/kjpp.2010.14.6.365

Abstract

In this study, we examined whether melatonin promotes apoptotic cell death via p53 in prostate LNCaP cells. Melatonin treatment significantly curtailed the growth of LNCaP cells in a dose- and time-dependent manner. Melatonin treatment (0 to 3 mM) induced the fragmentation of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3, caspase-8, and caspase-9. Moreover, melatonin markedly activated Bax expression and decreased Bcl-2 expression in dose increments. To investigate p53 and p21 expression, LNCaP cells were treated with 0 to 3 mM melatonin. Melatonin increased the expressions of p53, p21, and p27. Treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that the melatonin-induced apoptosis was p21-dependent, but ERK-independent. With the co-treatment of PD98059 and melatonin, the expression of p-p53, p21, and MDM2 did not decrease. These effects were opposite to the expression of p-p53, p21, and MDM2 observed with SP600125 and SB202190 treatments. Together, these results suggest that p53-dependent induction of JNK/p38 MAPK directly participates in apoptosis induced by melatonin.

Keyword

Melatonin; p53; p38; JNK; LNCaP cells

MeSH Terms

Anthracenes
Apoptosis
Caspase 3
Caspase 8
Caspase 9
Cell Death
Flavonoids
Imidazoles
Melatonin
Poly(ADP-ribose) Polymerases
Prostate
Protein Kinases
Pyridines
Anthracenes
Caspase 3
Caspase 8
Caspase 9
Flavonoids
Imidazoles
Melatonin
Poly(ADP-ribose) Polymerases
Protein Kinases
Pyridines

Figure

• 

  Fig. 1. Viability of melatonin-treated LNCaP cells. LNCaP cell viability was determined using the Cell Counting Kit-8 assay (A) 48 hr after exposure to melatonin at varying doses and (B) at varying times after exposure to 3 mM melatonin. In (A) and (B), results for cells not treated with melatonin are shown for comparison. Results are the means of 3 independent experiments (bars represent SD). ∗∗p<0.01, ∗∗∗p<0.001 vs. control.

• 

  Fig. 2. Induction of LNCaP cell apoptotic cell death by melatonin. LNCaP prostate cancer cells were cultured in DMEM containing 10% FBS and then treated with melatonin at varying doses for 48 h. (A) Cell lysates prepared at the indicated culture times were separated by 10% SDS-PAGE and immunoblotted with antibodies to PARP, caspase-3, –8, –9, and GAPDH. (B) Cell lysates prepared at the indicated culture times were separated by 12% SDS-PAGE and immunoblotted with antibodies to Bax, Bcl-2, and GAPDH. (C) The relative amounts of Bax and Bcl-2 were quantified as described in Materials and Methods. ∗∗∗p<0.001 vs. control. ††p<0.01, †††p<0.001 vs. control.

• 

  Fig. 3. Activation of p53, p21, and p27 in melatonin-treated LNCaP cells. Cells were cultured in DMEM medium containing 10% FBS and then treated with melatonin at varying doses for 48 h. (A) Cell lysates prepared at the indicated culture times were separated by 12% SDS-PAGE and immunoblotted with antibodies to p53, p21, p27, and GAPDH. The relative amounts of p53 (B), p21 (C), and p27 (D) were quantified as described in Materials and Methods. ∗∗p<0.01, ∗∗∗p<0.001 vs. control.

• 

  Fig. 4. Activation of p-p53, p21, and MDM2 by blockade with PD98059, SP600125, and SB202190 inhibitors on melatonin-induced apoptotic cell death. Inhibitor concentrations of 10 to 30 μM for PD98059, SP600125 and SB202190 were added to cells 1 hr before melatonin (3 mM) treatment for 48 hr. (A) LNCaP cells were cultured with melatonin at varying doses. (B), (C), and (D) Cells were treated with 3 mM melatonin for 48 hr in the presence of various concentrations of PD98059 (B), SP600125 (C), or SB202190 (D). Cell lysates prepared at the indicated culture times were separated by 12% SDS-PAGE and immunoblotted with antibodies to p-p53, p21, MDM2, and GAPDH. ∗∗∗p<0.001 vs. control.

Reference

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