Liu Jian-Wen, Kayasuga Atsushi, Nagao Norio, Masatsuji-Kato Eiko, Tuzuki Toshi, Miwa Nobuhiko
Department of Cell Biochemistry, Hiroshima Prefectural University School of BioSciences, Shobara, Hiroshima 727-0023, Japan.
Int J Oncol. 2003 Dec;23(6):1561-7.
Our previous study showed that tumor invasion of human fibrosarcoma cells HT-1080 is hardly inhibited by ascorbic acid itself (Asc), but inhibited by 2-O-phosphorylated Asc-6-O-palmitylester (Asc2P6Plm) more markedly than 2-O-phosphorylated Asc or Asc-6-O-palmitylester, and that the inhibitory effect may be attributed to an increase in intracellular Asc derived from Asc2P6Plm. In the present study, the mechanism underlying the inhibitory effect of Asc2P6Plm on tumor invasion was analyzed. Migratory ability of the tumor cells was shown to be inhibited in a dose-dependent manner by either treatment with Asc2P6Plm at 50-300 micro M for 1 h or at 10-50 micro M for 18 h as assessed by cell sheet scratching assay. Hydroxyl radicals in homogenates of Asc2P6Plm-treated HT-1080 cells were markedly diminished relative to those of non-treated cells as evaluated by electron spin resonance method using the spin trapping agent DMPO. This may be closely related to attenuation of intracellular gross reactive oxygen species by Asc2P6Plm as was shown with the redox indicator CDCFH-DA. Actin was localized in the vicinity of the cell membrane abundantly in non-treated cells, but was diminished in a time-dependent manner in Asc2P6Plm-treated cells together with disappearance of pseudopods as shown with the actin-directed agent NBD-phallacidin and by immunocytochemical stain. The cell adhesion-controling molecule RhoA was increased time-dependently in the cytoplasm of Asc2P6Plm-treated cells as shown by Western blots. Thus the inhibition of tumor invasion by Asc2P6Plm was shown to be attributed to decrease in both the cell migratory ability and the actin localization near the cell membrane, which may result from an increase in cytoplasmic RhoA and reduction of intracellular ROS that is achieved by enrichment of intracellular Asc derived from Asc2P6Plm.
我们之前的研究表明，抗坏血酸本身（Asc）几乎不能抑制人纤维肉瘤细胞HT-1080的肿瘤侵袭，但2-O-磷酸化抗坏血酸-6-O-棕榈酸酯（Asc2P6Plm）比2-O-磷酸化抗坏血酸或抗坏血酸-6-O-棕榈酸酯更能显著抑制肿瘤侵袭，且这种抑制作用可能归因于源自Asc2P6Plm的细胞内抗坏血酸增加。在本研究中，分析了Asc2P6Plm对肿瘤侵袭抑制作用的潜在机制。通过细胞单层划痕试验评估，用50-300微摩尔/升的Asc2P6Plm处理1小时或10-50微摩尔/升处理18小时，肿瘤细胞的迁移能力均呈剂量依赖性受到抑制。用自旋捕获剂DMPO通过电子自旋共振法评估，Asc2P6Plm处理的HT-1080细胞匀浆中的羟自由基相对于未处理细胞明显减少。这可能与Asc2P6Plm使细胞内总活性氧减少密切相关，这已通过氧化还原指示剂CDCFH-DA得到证实。在未处理细胞中，肌动蛋白大量定位于细胞膜附近，但在Asc2P6Plm处理的细胞中，肌动蛋白随时间减少，同时伪足消失，这通过肌动蛋白导向剂NBD-鬼笔环肽和免疫细胞化学染色得以显示。如蛋白质印迹所示，细胞黏附控制分子RhoA在Asc2P6Plm处理的细胞胞质中随时间增加。因此，Asc2P6Plm对肿瘤侵袭的抑制作用归因于细胞迁移能力和细胞膜附近肌动蛋白定位的降低，这可能是由于胞质RhoA增加以及细胞内活性氧减少所致，而细胞内活性氧减少是由源自Asc2P6Plm的细胞内抗坏血酸富集实现的。
