Kok Stanton Hon Lung, Gambari Roberto, Chui Chung Hin, Lau Fung Yi, Cheng Gregory Yin Ming, Lai Paul Bo San, Lam Wing Sze, Chan Albert Sun Chi, Cheng Chor Hing, Teo Ivy Tuang Ngo, Yu Michael Wing Yiu, Tang Johnny Cheuk On, Cheung Filly, Wong Raymond Siu Ming
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, P.R. China.
Int J Mol Med. 2007 Jun;19(6):971-5.
There are several scientific approaches for the determination of cellular growth influences of known or novel substances under in vitro conditions, among which colourimetric absorption measurement is considered to be one of the convenient methods. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay is one of the commonly used colourimetric absorption assays based on the ability of dehydrogenase from viable cells to produce the brown soluble formazan detectable at 490 nm. Here we have tested the possible growth influence of iron (II) sulphate on two human cancer cell lines, the K562 chronic myelogenous leukaemia and T47D breast carcinoma cells, based on the MTS assay. We found that iron (II) sulphate possessed an inhibitory effect when added at 16- to 125-microM concentrations, but iron (II) sulphate became growth stimulatory when its concentration was further increased to 1000 microM. In addition, a dose-dependent increase in absorbance at the same wavelength was observed when we repeated the experiments without the addition of MTS and phenazine methosulfate. When we further repeated the cell growth determinations using adenosine triphosphate content assay for K562 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for T47D, iron (II) sulphate showed a consistent dose-dependent growth inhibitory effect. Morphological investigation after methylene blue staining clearly demonstrated that iron (II) sulphate, at a concentration of 1000 microM, is cytotoxic to T47D cells. Interestingly, a consistent increment for the absorbance at 490 nm was further observed with increased iron (II) sulphate concentration either in the presence or absence of MTS even in a cell-free environment. Thus we conclude that iron (II) sulphate is actually growth inhibitory and even cytotoxic at high concentrations towards the K562 and T47D cancer cells and the paradoxical proliferative activity of iron (II) sulphate on these two cancer cell lines using the MTS assay was solely due to the oxidation of initial pale green iron (II) to brownish iron (III) during incubation in the aqueous condition.
在体外条件下，有几种科学方法可用于测定已知或新型物质对细胞生长的影响，其中比色吸收测量被认为是方便的方法之一。3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑鎓测定法是常用的比色吸收测定法之一，其基于活细胞中的脱氢酶产生可在490nm处检测到的棕色可溶性甲臜的能力。在此，我们基于MTS测定法测试了硫酸亚铁对两种人类癌细胞系，即K562慢性粒细胞白血病细胞和T47D乳腺癌细胞的可能生长影响。我们发现，当以16至125微摩尔浓度添加硫酸亚铁时，其具有抑制作用，但当硫酸亚铁浓度进一步增加至1000微摩尔时，其变为生长刺激剂。此外，当我们在不添加MTS和吩嗪硫酸甲酯的情况下重复实验时，在相同波长下观察到吸光度呈剂量依赖性增加。当我们进一步使用K562的三磷酸腺苷含量测定法和T47D的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐(MTT)测定法重复细胞生长测定时，硫酸亚铁显示出一致的剂量依赖性生长抑制作用。亚甲蓝染色后的形态学研究清楚地表明，浓度为1000微摩尔的硫酸亚铁对T47D细胞具有细胞毒性。有趣的是，即使在无细胞环境中，无论是否存在MTS，随着硫酸亚铁浓度的增加，在490nm处的吸光度进一步观察到一致的增加。因此，我们得出结论，硫酸亚铁实际上对K562和T47D癌细胞具有生长抑制作用，甚至在高浓度下具有细胞毒性，并且使用MTS测定法时硫酸亚铁对这两种癌细胞系的矛盾增殖活性完全是由于在水性条件下孵育期间初始浅绿色的亚铁(II)氧化为棕色的铁(III)。
