Induction of apoptosis by anesthetics in the human cancer cell lines.

与吗啡相关化合物相比，可待酮相关化合物对人类癌细胞株的抗增殖作用的研究相对较少。作者先前发现，在10种阿片类药物中，可待因的氧化代谢产物可待酮对人早幼粒白血病细胞株(HL-60)具有最高的细胞毒活性(DNA片段化诱导活性)。这被抗氧化剂N-乙酰-L-半胱氨酸(NAC)所抵消。这些发现促使我们对可待酮治疗后的细胞凋亡诱导进行了更详细的研究。用可待因或可待酮处理HL60细胞1~6h，诱导细胞凋亡。二氨基苯基吲哚(DAPI)染色后的DNA。Hoechst(H)-33342染色后用显微镜观察凋亡细胞的出现，Annexin染色后用荧光激活细胞分类器观察细胞的凋亡情况。用Western印迹法检测线粒体细胞色素C和细胞色素氧化酶的释放及caspase 3的激活。细胞内caspase3样活性用细胞通透性底物进行流式细胞仪检测。分别用聚丙烯酰胺凝胶电泳法和逆转录聚合酶链式反应(RT-PCR)法检测线粒体含锰超氧化物歧化酶(MnSOD)活性和mRNAmRNA表达。在HL-60细胞中，可待酮比可待因更有效地诱导核小体间DNA断裂和产生Annexin阳性的凋亡细胞。Codeinone可促进细胞色素C和细胞色素氧化酶的释放以及对原天冬氨酸氨基转移酶3的裂解，而对MnSOD的活性和表达均无明显影响。除了已报道的抗伤害活性外，可待酮被发现具有诱导细胞凋亡和诱导坏死的活性，进一步证实了其抗肿瘤的潜力。

There are relatively few studies about antiproliferative effects of codeinone-related compounds on human cancer cell lines, compared with those of morphine related compounds. The authors previously found that codeinone, an oxidation metabolite of codeine, among 10 opiois, showed the highest cytotoxic activity (DNA fragmentation-inducing activity) agains human promyelocytic leukemic cell lines (HL-60). This was counteracted by an antioxidant, N-acetyl-L-cysteine (NAC). These finings prompted us performed a more detailed study of apoptosis induction after codeinone treatment. Apoptosis was induced by treating HL60 cells for 1-6h with codeine or codeinone. DNA after staining with diamidinopheylindole (DAPI). The appearance of apoptotic cells was monitored bymicroscopic observation after staining with Hoechst (H)-33342,and fluorescence actiated cell sorter (FACS) after staining with Annexin. The release of cytochrome C and cytocrome oxidase from mitochondria and activation of caspase 3 were monitored by Western blot analysis. Intracellular caspase 3 like activity was confirmed by FACS, using cell permeable substate. Mitochondrial manganese-containing superoxide dismutase (MnSOD) activity and mRNAexpression were assayed by ativity staining after separation on the polyacrylamide gel electrophoresis, and reverse transcriptase-poly-merase chain reaction (RT-PCR), respectively. Codeinone induced internucleosomal DNA fragmentation and production of Annexin-positive apoptotic cells more potently than codeine in HL-60 cells. Codeinone stimulated the release of both cytochrome C and cytochrome oxidase, and cleavage of procaspase 3 without significant changes in both the activity and expression of MnSOD. Codeinone was found to possess both apoptosis and necrosis-inducing activity, in addition to the reported antinociceptive activity, further substantiated its antitumor potential.