Chonnam Med J.  2008 Dec;44(3):162-168. 10.4068/cmj.2008.44.3.162.

Protective Effects of K6PC-5, A Sphingosine Kinase Activator, Against 1-methyl-4-phenylpyridinium-induced Dopaminergic Neuronal Cell Death

• Jang S
• Choi SS
• Kim SH
• Park JS
• Jeong HS

• KMID: 2172296
• DOI: http://doi.org/10.4068/cmj.2008.44.3.162

Abstract

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metaolite, regulates multiple cellular responses such as Ca2+ signaling, cellular growth and survival, and differentiation. Sphingosine kinase (SphK) is a key enzyme in modulating the levels of S1P and is emerging as an important regulating enzyme. Here we have investigated whether K6PC-5, a newly synthesized SphK activator, plays a neuroprotective role by activating cell survival systems such as protein kinase C, phosphatidylinositol-3-kinase (PI3K), and acting on the anti-apoptotic and the pro-apoptotic genes in SN4741 dopaminergic cells. 1-methyl-4-phenylpyridinium is a neurotoxin that selectively inhibits the mitochondrial functions of dopaminergic neurons in the substantia nigra pars compacta. In the present study, we found that MPP+ induced a decrease in SN4741 mouse dopaminergic cell viability. K6PC-5 restored the reduced phospho-PKC and phospho-PI3K activities caused by MPP+ toxicity. In addition, gene expression analysis revealed that K6PC-5 prevented both the MPP+ -induced expression of the pro-apoptotic gene mRNA, Bax, and the decrease of the anti-apoptotic gene mRNA, bcl-w. These results suggest that the neuroprotective mechanism of K6PC-5 against MPP+ -induced apoptotic cell death includes stimulation of PKC and PI3K, and modulation of cell survival and death genes.

Keyword

K6PC-5 compound; Sphingosine kinase-1; 1-methyl-4-phenylpyridinium; Parkinson disease; Dopaminergic agents

MeSH Terms

1-Methyl-4-phenylpyridinium
Amides
Animals
Cell Death
Cell Survival
Dopamine Agents
Dopaminergic Neurons
Gene Expression
Lysophospholipids
Mice
Parkinson Disease
Phosphotransferases
Phosphotransferases (Alcohol Group Acceptor)
Protein Kinase C
RNA, Messenger
Sphingosine
Substantia Nigra
1-Methyl-4-phenylpyridinium
Amides
Dopamine Agents
Lysophospholipids
Phosphotransferases
Phosphotransferases (Alcohol Group Acceptor)
Protein Kinase C
RNA, Messenger
Sphingosine

Figure

• 

  Fig. 1 MTT+ neurotoxicity and protection by K6PC-5 in SN4741 mouse dopaminergic neuronal cell line as measured by MTT reduction assay (A, B, and C). (A) Compare to media treated cells, treatment of cells with DMSO showed no significant decrease in the cell viability. (B) The viability of SN4741 cells was increased with increasing concentration of K6PC-5. (C) Protective effect of K6PC-5 against dopaminergic neuronal cell death in SN4741 cells. SN4741 cells were pretreated with varying concentrations of K6PC-5 (0.1, 1, 5 uM) for 1 hr, followed by incubation with 1 mM MPP+ for 24 hr. The viability was markedly reduced by a 24 hr treatment with 1 mM MPP+, whereas 1 hr pretreatment with K6PC-5 (0.1, 1, 5 uM) conferred significant protection against MPP+-induced neurotoxicity. These data represent the mean±SE (standard error) of three independent experiments, with each point done in quadruplicate.

• 

  Fig. 2 Modulation of anti- and pro-apoptotic gene expressions by K6PC-5 and MPP+. SN4741 cells were treated with K6PC-5 (0.1, 1, 5 uM), MPP+ (1 mM), or the combination of the two compounds for 6 hr. Cellular RNA from each treatment was extracted and the mRNA expression for Bcl-2, Bcl-w, Bcl-xl, Bad, Bax, Caspase-6, p21, and GAPDH was analyzed by RT-PCR.

• 

  Fig. 3 Effects of K6PC-5 and MPP+ on p-PKC, p-PI3K, p-JNK, p-GSK, p-p38, and p-ERK immunoreactivities in SN4741 cells. Cells were treated with K6PC-5 (0.1, 1, 5 uM), MPP+ (1 mM), or the combination of the two drugs for 3 hr. Cell lysis buffer extracted cellular proteins from each treatment.Toxicity test of MPP+ (A) and EGCG (B) compounds in SN4741 mouse dopaminergic neuronal cell line. In vitro cytotoxic cell death for MPP+ and EGCG were assessed by MTT reduction assay in SN4741 cells at 24h.

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