Saito K, Oku T, Ata N, Miyashiro H, Hattori M, Saiki I
Department of Pathogenic Biochemistry, Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, Japan.
Biol Pharm Bull. 1997 Apr;20(4):345-8. doi: 10.1248/bpb.20.345.
In order to screen potent inhibitors of tumor invasion and metastasis, we here devised a simple and reproducible in vitro assay for tumor invasion and migration. A conventional cell-counting assay using a Transwell chamber with a microporous membrane filter is troublesome and time-consuming, involving visually counting the cells under a microscope, and the invaded or migrated cells are sometimes distributed unevenly in predetermined fields on the lower surface of the filter. Therefore, it is difficult to evaluate the invasive and migratory abilities of tumor cells easily and quantitatively by the cell counting method. In the present study, crystal violet dye was used for staining the invaded cells and colorimetrically assessing the invasive ability per filter as an absorbance. In this crystal violet assay, tumor cell invasion into a reconstituted basement membrane Matrigel was proportional to both the cell number added into the chamber and the incubation period, and inversely proportional to the amount of Matrigel barrier on the upper surface of filter. The results obtained by this dye-uptake method were highly consistent with those of a conventional cell-counting assay. Using this crystal violet assay, the anti-invasive effect of doxorubicin (DOX) was detected more easily and found to be highly proportional to that by the conventional cell-counting method. We therefore applied this convenient assay method to screen anti-invasive and anti-metastatic compounds. As a result, caffeic acid was found to be more active in the inhibition of both tumor cell invasion and migration without showing direct cytotoxicity in vitro than other related compounds.
为了筛选有效的肿瘤侵袭和转移抑制剂，我们在此设计了一种简单且可重复的肿瘤侵袭和迁移体外检测方法。使用带有微孔膜滤器的Transwell小室进行传统的细胞计数检测既麻烦又耗时，需要在显微镜下目视计数细胞，而且侵袭或迁移的细胞有时在滤器下表面的预定区域分布不均匀。因此，通过细胞计数法很难轻松且定量地评估肿瘤细胞的侵袭和迁移能力。在本研究中，结晶紫染料用于对侵袭的细胞进行染色，并通过比色法将每个滤器的侵袭能力评估为吸光度。在这种结晶紫检测中，肿瘤细胞向重组基底膜基质胶的侵袭与加入小室的细胞数量和孵育时间均成正比，与滤器上表面的基质胶屏障量成反比。通过这种染料摄取法获得的结果与传统细胞计数检测的结果高度一致。使用这种结晶紫检测，更容易检测到阿霉素（DOX）的抗侵袭作用，并且发现其与传统细胞计数法的结果高度相关。因此，我们应用这种简便的检测方法来筛选抗侵袭和抗转移化合物。结果发现，咖啡酸在抑制肿瘤细胞侵袭和迁移方面比其他相关化合物更具活性，且在体外不显示直接细胞毒性。
