Yamamoto-Yamaguchi Yuri, Okabe-Kado Junko, Kasukabe Takashi, Honma Yoshio
Saitama Cancer Center Research Institute, 818 Komuro, Ina, Saitama 362-0806, Japan.
Anticancer Res. 2003 May-Jun;23(3B):2537-47.
Differentiation-inducing agents for myeloid leukemic cells, such as dimethyl sulfoxide (DMSO), Na-butyrate and hexamethylene-bis-acetamide (HMBA), barely induce irreversible differentiation or growth inhibition in solid tumors when used alone. However, we previously reported that combined treatment with differentiation-inducing agents and interferon (IFN)-alpha effectively suppressed the growth of human lung cancer cell lines in vitro and in vivo. We show here that combined treatment-induced cell death is caused by apoptosis.
Induction of apoptosis was examined by expression of several apoptotic markers.
Combined treatment-induced cell death is caused by apoptosis, which is characterized by the induction of apoptotic morphological changes and the activation of caspase-3-like protease. The expression of Apo2.7, an early apoptotic change, is also induced by this combined treatment, and is suppressed by the addition of Z-Asp-CH2-DCB, a pan caspase inhibitor. The signal transduction pathway of IFN-alpha is rapidly observed in lung cancer cells, although it does not induce significant growth inhibition when used alone. We analyzed the effect of IFN-alpha on the apoptotic pathway and found that IFN-alpha but not DMSO induces the expression of caspase-4. The combination of IFN-alpha and DMSO did not cause further increase in the expression of caspase-4. In many cases of the induction of apoptosis, cytochrome c is released from mitochondria, which induces the formation of large caspase-activating complexes. Caspase-3-like-activities induced by the combination of DMSO or Na-butyrate with IFN-alpha were analyzed by gel filtration and detected equally in fractions with molecular weights of 200-300 kDa, suggesting that caspase-3 is activated in large complexes in lung cancer cells. However, Na-butyrate and HMBA, when used alone, induced the release of cytochrome c and enhanced the loss of mitochondrial membrane potential, whereas DMSO had no effect.
These results suggest that the combination of differentiation-inducing agents with IFN-alpha effectively induces apoptosis in lung cancer cells whereas the mechanism of such induction varies depending on the differentiation-inducing agent used.
用于髓系白血病细胞的分化诱导剂，如二甲基亚砜（DMSO）、丁酸钠和己二甲双乙酰胺（HMBA），单独使用时几乎不会在实体瘤中诱导不可逆的分化或生长抑制。然而，我们之前报道，分化诱导剂与干扰素（IFN）-α联合治疗可在体外和体内有效抑制人肺癌细胞系的生长。我们在此表明，联合治疗诱导的细胞死亡是由凋亡引起的。
通过几种凋亡标志物的表达来检测凋亡的诱导情况。
联合治疗诱导的细胞死亡是由凋亡引起的，其特征是诱导凋亡形态学变化和激活类半胱天冬酶-3蛋白酶。联合治疗还诱导了早期凋亡变化Apo2.7的表达，而添加泛半胱天冬酶抑制剂Z-Asp-CH2-DCB可抑制这种表达。尽管单独使用IFN-α不会诱导明显的生长抑制，但在肺癌细胞中可快速观察到其信号转导途径。我们分析了IFN-α对凋亡途径的影响，发现IFN-α而非DMSO可诱导半胱天冬酶-4的表达。IFN-α与DMSO的联合使用并未导致半胱天冬酶-4表达的进一步增加。在许多凋亡诱导情况下，细胞色素c从线粒体释放，从而诱导形成大型半胱天冬酶激活复合物。通过凝胶过滤分析了DMSO或丁酸钠与IFN-α联合使用诱导的类半胱天冬酶-3活性，在分子量为200 - 300 kDa的组分中均能检测到，这表明在肺癌细胞中半胱天冬酶-3在大型复合物中被激活。然而，单独使用丁酸钠和HMBA时会诱导细胞色素c的释放并增强线粒体膜电位的丧失，而DMSO则无此作用。
这些结果表明，分化诱导剂与IFN-α联合使用可有效诱导肺癌细胞凋亡，而这种诱导机制因所使用的分化诱导剂而异。
