Kanno Takeshi, Nishimoto Takaaki, Fujita Yumiko, Gotoh Akinobu, Nakano Takashi, Nishizaki Tomoyuki
Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya Department of Thoracic Oncology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Japan.
Cell Physiol Biochem. 2012;30(4):987-94. doi: 10.1159/000341475. Epub 2012 Sep 20.
BACKGROUND/AIMS: Evidence has pointed to the role of sphingosine in cellular differentiation, cell growth, and apoptosis. The present study investigated sphingosine-induced apoptosis in human gastric cancer cells.
Well differentiated MKN-28 and poorly differentiated MKN-45 human gastric cancer cells were cultured. MTT assay, TUNEL staining, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out in cells transfected with and without the siRNA to silence the protein kinase C (PKC)-δ-targeted gene.
Sphingosine induced apoptosis in MKN-28 cells, with the potential much greater than for MKN-45 cells. Transfection with the siRNA to silence the PKC-δ-targeted gene (PKC-δ siRNA) into MKN-28 cells significantly reduced presence of sphingosine-dependent protein kinase (SDK) in association with reduced PKC-δ expression. Sphingosine-induced apoptosis in MKN-28 cells was prevented by transfecting with the PKC-δ siRNA. Sphingosine promoted SDK production from PKC-δ and increased phosphorylated 14-3-3 protein for MKN-28 cells, but such effects were not found with MKN-45 cells. Moreover, sphingosine perturbed mitochondrial membrane potentials and activated caspase-3 and caspase-9 in MKN-28 cells, which were also inhibited by transfecting with the PKC-δ siRNA.
The results of the present study indicate that sphingosine induces apoptosis in well differentiated MKN-28 human gastric cancer cells by increasing SDK production from PKC-δ, to phosphorylate 14-3- 3 protein, thereby causing disruption of mitochondrial membrane potentials and activating caspase-9 followed by the effector caspase-3.
背景/目的：有证据表明鞘氨醇在细胞分化、细胞生长和凋亡过程中发挥作用。本研究旨在探究鞘氨醇诱导人胃癌细胞凋亡的情况。
培养高分化的MKN-28和低分化的MKN-45人胃癌细胞。对转染和未转染靶向蛋白激酶C（PKC）-δ基因的小干扰RNA（siRNA）的细胞进行MTT检测、TUNEL染色、蛋白质印迹法以及半胱天冬酶-3、-8和-9活性检测。
鞘氨醇可诱导MKN-28细胞凋亡，其诱导潜力远大于MKN-45细胞。将靶向PKC-δ基因的siRNA（PKC-δ siRNA）转染至MKN-28细胞后，鞘氨醇依赖性蛋白激酶（SDK）的表达显著降低，同时PKC-δ的表达也减少。转染PKC-δ siRNA可抑制鞘氨醇诱导的MKN-28细胞凋亡。鞘氨醇可促进MKN-28细胞中PKC-δ产生SDK，并增加磷酸化的14-3-3蛋白，但在MKN-45细胞中未发现此效应。此外，鞘氨醇可扰乱MKN-28细胞的线粒体膜电位并激活半胱天冬酶-3和半胱天冬酶-9，转染PKC-δ siRNA也可抑制此效应。
本研究结果表明，鞘氨醇通过增加PKC-δ产生SDK，使14-3-3蛋白磷酸化，从而破坏线粒体膜电位并激活半胱天冬酶-9，随后激活效应半胱天冬酶-3，进而诱导高分化的MKN-28人胃癌细胞凋亡。
