Asakura Tadashi, Maeda Kazuhiro, Omi Hiroko, Matsudaira Hiroshi, Ohkawa Kiyoshi
Department of Biochemistry, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Int J Oncol. 2008 Aug;33(2):389-95.
We investigated the induction of apoptosis via deamidation of Bcl-xL and translocation of Bax to the mitochondria by treatment with GSH-DXR. GSH-DXR treatment of HepG2 cells, which did not express GST P1-1, exhibited deamidation of Bcl-xL, and the degree of deamidation was related to the activation of caspase-3. Overexpression of GST P1-1 in HepG2 cells decreased both the Bcl-xL deamidation and caspase-3 activation induced by treatment with GSH-DXR. Bcl-xL deamidation and caspase-3 activation were also suppressed by co-treatment with SP600125, a specific inhibitor of JNK activity. Overexpression of wild-type Bcl-xL in HepG2 decreased GSH-DXR-induced apoptosis although deamidation was observed. However, expression of the deamidated mutant of Bcl-xL, in which aspartic acid was substituted for both arginine 52 and 66 (N52,66D-Bcl-xL), exhibited high sensitivity for the induction of apoptosis. Expression of the Bcl-xL mutant, in which alanine was substituted for both arginine 52 and 66 (N52,66A-Bcl-xL), suppressed deamidation and showed resistance to the induction of apoptosis by treatment with GSH-DXR. On the other hand, endogenous Bax and overexpressed Flag-Bax were localized in the cytosolic fraction of HepG2 cells. Treatment of the cells with GSH-DXR caused translocation of Flag-Bax to the mitochondrial fraction following the induction of apoptosis. The induced apoptosis was enhanced by the expression of Flag-Bax. Moreover, Flag-Bax was partly located in the mitochondrial fraction in N52,66D-Bcl-xL-expressed cells without the induction of apoptosis. Therefore, the induction of apoptosis by treatment of HepG2 with GSH-DXR was enhanced, thereby facilitating the release of cytochrome c by both deamidated inactivation of Bcl-xL and functional translocation of Bax to the mitochondria via JNK activation. Deamidation of Bcl-xL might be induced in order to translocate Bax to the mitochondria.
我们研究了通过谷胱甘肽-柔红霉素（GSH-DXR）处理使Bcl-xL脱酰胺以及Bax转位至线粒体从而诱导细胞凋亡的过程。对不表达谷胱甘肽S-转移酶P1-1（GST P1-1）的HepG2细胞进行GSH-DXR处理后，出现了Bcl-xL的脱酰胺现象，且脱酰胺程度与半胱天冬酶-3（caspase-3）的激活有关。在HepG2细胞中过表达GST P1-1可降低GSH-DXR处理诱导的Bcl-xL脱酰胺和caspase-3激活。用JNK活性的特异性抑制剂SP600125共同处理也可抑制Bcl-xL脱酰胺和caspase-3激活。在HepG2细胞中过表达野生型Bcl-xL可降低GSH-DXR诱导的细胞凋亡，尽管观察到了脱酰胺现象。然而，Bcl-xL的脱酰胺突变体（其中天冬氨酸取代了精氨酸52和66，即N52,66D-Bcl-xL）的表达对细胞凋亡诱导表现出高敏感性。用丙氨酸取代精氨酸52和66的Bcl-xL突变体（N52,66A-Bcl-xL）的表达可抑制脱酰胺，并对GSH-DXR处理诱导的细胞凋亡表现出抗性。另一方面，内源性Bax和过表达的Flag-Bax定位于HepG2细胞的胞质部分。用GSH-DXR处理细胞会在诱导细胞凋亡后使Flag-Bax转位至线粒体部分。Flag-Bax的表达增强了诱导的细胞凋亡。此外，在未诱导细胞凋亡的情况下，Flag-Bax部分位于表达N52,66D-Bcl-xL的细胞的线粒体部分。因此，用GSH-DXR处理HepG2细胞诱导的细胞凋亡增强，从而通过Bcl-xL的脱酰胺失活和通过JNK激活使Bax功能性转位至线粒体来促进细胞色素c的释放。Bcl-xL的脱酰胺可能是为了使Bax转位至线粒体而被诱导的。
