Lind D S, Kontaridis M I, Edwards P D, Josephs M D, Moldawer L L, Copeland E M
Department of Surgery, University of Florida, Gainesville 32610, USA.
J Surg Res. 1997 May;69(2):283-7. doi: 10.1006/jsre.1997.5015.
Recently, several antitumor drugs have been shown to stimulate nitric oxide (NO) production.
To determine if adriamycin induces NO production in breast cancer cells in vitro and whether NO contributes to adriamycin's antitumor effect in vivo.
Murine breast cancer cells (EMT-6) were incubated with adriamycin (ADRIA, 0, 10, 100, 1000 microM) in the presence or absence of the NO synthase inhibitor aminoguanidine (AG, 1 mM). Twenty-four hours later nitrite accumulation (Greiss reagent) and cell viability (MTT assay) were assessed. Supernatants from adriamycin-stimulated cells were also analyzed at 6, 8, and 24 hr for TNF, IL-1, and IFN gamma (ELISA). For in vivo experiments, 10(5) EMT-6 cells were injected into the flank of BALB/c mice (n = 20) and 1 hr later mice received one of four treatments: (1) saline, (2) ADRIA (10 mg/kg ip), (3) AG (100 mg/kg sc BID), or (4) ADRIA (10 mg/kg ip) and AG (100 mg/kg sc BID). Two weeks later tumor size was measured and in situ tumor cell apoptosis was determined by fluorescent microscopy and flow cytometry.
Adriamycin was cytotoxic to EMT-6 cells with 100 microM resulting in nearly 100% killing (P < 0.01). Adriamycin also stimulated nitrite accumulation with 100 microM producing 6.5 +/- 0.26 microM nitrite (P < 0.001). AG blocked adriamycin-stimulated nitrite accumulation (P < 0.05), but did not inhibit cytotoxicity in vitro. In vivo, adriamycin inhibited tumor size by nearly 400% (P < 0.001), while AG attenuated adriamycin's effect on tumor growth (P < 0.05). There was no difference in the detection of apoptotic tumor cells between the adriamycin and adriamycin and AG groups as determined by immunohistochemistry and flow cytometry.
These findings suggest that adriamycin stimulated NO production in EMT-6 cells, but adriamycin's cytotoxicity in vitro was NO-independent. In vivo, adriamycin inhibited tumorigenesis partially via an NO-dependent, nonapoptotic mechanism.
最近，几种抗肿瘤药物已被证明可刺激一氧化氮（NO）的产生。
确定阿霉素在体外是否能诱导乳腺癌细胞产生NO，以及NO是否有助于阿霉素在体内的抗肿瘤作用。
将小鼠乳腺癌细胞（EMT-6）与阿霉素（ADRIA，0、10、100、1000微摩尔）在有或无NO合酶抑制剂氨基胍（AG，1毫摩尔）的情况下孵育。24小时后，评估亚硝酸盐积累（格里斯试剂）和细胞活力（MTT法）。还在6、8和24小时对阿霉素刺激的细胞的上清液进行TNF、IL-1和IFNγ分析（ELISA）。对于体内实验，将10⁵个EMT-6细胞注射到BALB/c小鼠的胁腹（n = 20），1小时后小鼠接受四种处理之一：（1）生理盐水，（2）阿霉素（10毫克/千克腹腔注射），（3）AG（100毫克/千克皮下注射，每日两次），或（4）阿霉素（10毫克/千克腹腔注射）和AG（100毫克/千克皮下注射，每日两次）。两周后测量肿瘤大小，并通过荧光显微镜和流式细胞术确定原位肿瘤细胞凋亡情况。
阿霉素对EMT-6细胞具有细胞毒性，100微摩尔时导致近100%的细胞死亡（P < 0.01）。阿霉素还刺激亚硝酸盐积累，100微摩尔时产生6.5±0.26微摩尔亚硝酸盐（P < 0.001）。AG阻断了阿霉素刺激的亚硝酸盐积累（P < 0.05），但在体外未抑制细胞毒性。在体内，阿霉素使肿瘤大小抑制了近400%（P < 0.001），而AG减弱了阿霉素对肿瘤生长的作用（P < 0.05）。通过免疫组织化学和流式细胞术测定，阿霉素组与阿霉素加AG组在凋亡肿瘤细胞的检测上没有差异。
这些发现表明阿霉素刺激EMT-6细胞产生NO，但阿霉素在体外的细胞毒性不依赖于NO。在体内，阿霉素部分通过一种依赖于NO的非凋亡机制抑制肿瘤发生。
