Onoda M, Inano H
First Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, Chiba, 263-8555, Japan.
Nitric Oxide. 2000 Oct;4(5):505-15. doi: 10.1006/niox.2000.0305.
We have hypothesized that one aspect of the antitumor activity of curcumin (diferuloylmethane) during the promotion stage of mammary gland tumorigenesis may be linked to reduction of free radicals (Inano et al., Carcinogenesis, 20: 1011-1018, 1999). Nitric oxide (NO) has been found to inflict damage on important biomolecules, and the overproduction of NO in diseases may be implicated in carcinogenesis and tumor progression. We have reported that the presence of three isoforms of nitric oxide synthases (NOS) and NO generation in the mammary gland correlate with the mammary gland development and mammary carcinogenesis. We, therefore, investigated the inhibitory activity of curcumin for the production of NO in rat mammary glands by using an organ culture system to validate the effectiveness and usefulness of curcumin in the pathophysiology of the mammary gland. A diced mammary gland (approximately 3 mm cubes) from the inguinal part of a female Wistar-MS rat treated with estradiol and progesterone was cultured with 2 ml of 5% FCS/DMEM in the presence or absence of LPS (0.5 microg/ml) for 2-3 days. Curcumin ( approximately 100 microM) was added at the same time to the LPS-treated cultures. In some experiments, curcumin was added to the culture after the LPS had been washed out. The NO production was significantly increased (by almost 20-fold compared to the control) by the addition of LPS to the culture system. This enhancement of NO production by LPS was reduced to 76 and to 56% by addition of 30 and 100 microM curcumin, respectively, to the culture. When LPS was eliminated from the culture after prestimulation for 1 day, the production of NO by the mammary gland dropped off, although some NO was still detectable. Curcumin did not further inhibit the production of NO by the prestimulated mammary gland after the elimination of LPS from the culture. The inducible nitric oxide synthase (iNOS, 122 kDa) and endothelial nitric oxide synthase (eNOS, 152 kDa) isoforms were detected in the mammary gland extracts at the end of the organ culture. The quantity of iNOS was apparently increased in the gland treated with LPS, while the eNOS expression was clearly diminished. Curcumin (100 microM) obviously suppressed the iNOS expression in the mammary glands cultured with LPS, and a recovery in the eNOS expression was observed. On the other hand, curcumin exhibited scavenging activity for the NO released from N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC 12), a NO donor compound, in the coincubation mixture. These results indicate that curcumin has the ability to inhibit iNOS induction by LPS in the mammary gland and to scavenge NO radicals, which might explain, at least partly, its therapeutic properties in inflammation of the mammary gland.
我们曾提出假说，姜黄素（二阿魏酰甲烷）在乳腺肿瘤发生促进阶段的抗肿瘤活性的一个方面可能与自由基的减少有关（稻野等人，《癌变》，20: 1011 - 1018，1999）。已发现一氧化氮（NO）会对重要生物分子造成损害，疾病中NO的过量产生可能与致癌作用和肿瘤进展有关。我们曾报道，乳腺中三种一氧化氮合酶（NOS）同工型的存在及NO的生成与乳腺发育和乳腺癌发生相关。因此，我们利用器官培养系统研究了姜黄素对大鼠乳腺中NO生成的抑制活性，以验证姜黄素在乳腺病理生理学中的有效性和实用性。将经雌二醇和孕酮处理的雌性Wistar - MS大鼠腹股沟部位切成小块的乳腺（约3毫米见方），在有或无脂多糖（LPS，0.5微克/毫升）存在的情况下，用2毫升5%胎牛血清/杜氏改良伊格尔培养基培养2 - 3天。同时向经LPS处理的培养物中加入姜黄素（约100微摩尔）。在一些实验中，在洗去LPS后向培养物中加入姜黄素。向培养系统中加入LPS后，NO生成显著增加（与对照相比几乎增加了20倍）。向培养物中分别加入30和100微摩尔姜黄素后，LPS诱导的NO生成增强分别降至76%和56%。在预刺激1天后从培养物中去除LPS，乳腺中NO的生成下降，尽管仍可检测到一些NO。从培养物中去除LPS后，姜黄素并未进一步抑制预刺激乳腺中NO的生成。在器官培养结束时，在乳腺提取物中检测到诱导型一氧化氮合酶（iNOS，122千道尔顿）和内皮型一氧化氮合酶（eNOS，152千道尔顿）同工型。在用LPS处理的腺体中，iNOS的量明显增加，而eNOS的表达明显减少。姜黄素（100微摩尔）明显抑制了与LPS共同培养的乳腺中iNOS的表达，并观察到eNOS表达的恢复。另一方面，姜黄素对共孵育混合物中N - 乙基 - 2 -（1 - 乙基 - 2 - 羟基 - 2 - 亚硝基肼基）乙胺（NOC 12，一种NO供体化合物）释放的NO具有清除活性。这些结果表明，姜黄素具有抑制LPS诱导乳腺中iNOS的能力并能清除NO自由基，这可能至少部分解释了其在乳腺炎症中的治疗特性。
