Use of fetal bovine serum and antioxidants as delivery vehicle to improve stability and cellular uptake of lycopene in cell culture studies

Abstract

許多研究指出攝取富含茄紅素(lycopene)的食物可以降低多種癌症的發生率，如前列腺癌、肺癌等。在茄紅素體外試驗中，最常使用四氫呋喃 (tetrahydrofuran; THF) 作為溶劑，但THF不穩定，極易氧化，且具有細胞毒性，因此易造成茄紅素體外試驗之干擾。 本實驗擬使用胎牛血清(fetal bovine serum; FBS)稀釋以THF配置好的茄紅素stock solution，我們認為FBS中的脂蛋白 (lipoprotein) 可以增加茄紅素穩定性並促進細胞的吸收效果。此外，也比較多種不同載體對茄紅素的穩定性、細胞吸收效果，並分析細胞毒性以及利用剔除脂蛋白之血清(lipoprotein-deficient serum; LD-S)，驗證FBS增加細胞吸收茄紅素的作用機制。結果顯示：將茄紅素以不同載體配置後培養2、6、12和24小時時，Micelles及THF/FBS為茄紅素載體在培養基中的降解速度較其它載體慢，但是micelles對於細胞具有強烈的毒性，因此並未進一步去觀察細胞吸收micelle中茄紅素的效果。以THF/FBS為茄紅素載體時，細胞吸效果較好且較無細胞毒性；其中以培養12小時細胞吸收效果達最高。另外，比較FBS及剔除脂蛋白的血清為載體，當脂蛋白剔除時，細胞吸收茄紅素的效果與THF或THF (含BHT) 無顯著差異。 本研究試著進一步地改良THF/FBS的方法，由於使用THF/FBS為載體時，茄紅素的穩定性仍不夠好(在24小時培養後，茄紅素的剩餘量僅剩約40%)，且細胞吸收茄紅素的效率與茄紅素在培養基中的穩定度有很大的相關性(r2 = 0.71)，因此，我們嚐試藉由添加不同的抗氧化劑，避免茄紅素快速的氧化，以提高其穩定性並利於細胞吸收茄紅素的效果。結果顯示：茄紅素以FBS載體配置後，分別加入10µM的槲皮素(quercetin)、雲香苷(rutin)、生育醇(RRR-α-tocopherol)及丁基化羥基甲苯(Butylated hydroxytoluene; BHT)，培養2、6、12和24小時，發現以加入槲皮素時，茄紅素在培養基中的穩定度與細胞吸收效果皆較添加其他抗氧化劑的組別來得佳。因此進一步比較不同劑量的槲皮素對細胞吸收茄紅素的效果與穩定性的影響，發現50μM 的槲皮素減緩培養基中茄紅素的降解速率效果最好，且細胞吸收茄紅素的效果也比未添加槲皮素、添加10, 30µM的槲皮素組別分別增加了83,88和113% (P< 0.05)。 綜合以上結果，本研究證實了以FBS為茄紅素載體時，對茄紅素的穩定性、細胞吸收效果皆較其它載體好，且對細胞毒性也較低，而FBS當中的脂蛋白則是提高茄紅素穩定性和吸收率的最主要成份。再者，藉由添加槲皮素於THF/FBS載體中，更進一步能避免茄紅素快速地氧化，以增加茄紅素的穩定性而提高細胞的吸收率。因此，本研究結果對於茄紅素將來在體外的試驗中能具有一定的貢獻。

Studies have suggested that higher intakes of lycopene are associated with a reduced risk of several types of cancer, such as prostate cancer, hepatoma and coronary heart disease. Cell culture studies are useful for elucidating the mechanisms of action of lycopene, and tetrahydrofuran (THF) has commonly been used to deliver lycopene to cells. However, the use of THF as the solvent for lycopene has repeatedly been questioned because of its disadvantages in cell culture studies such as THF oxidizes readily in culture media and low cellular association. Therefore, attempts have been made by using vehicles other than THF for delivering lycopene to cells but it also suffers limitations such as cytotoxicity, poor solubility and crystallization in the medium. Here, we first compared the FBS dilution method of lycopene with other solubilization vehicles for lycopene including THF, THF containing 0.0025% butylated hydroxytoluene (THF/BHT), liposome, methyl-beta-cyclodextrin (M-β-CD) and micelles in cultured cells. We investigated that lycopene in FBS led to significantly higher stability and cellular uptake of lycopene than that in THF, THF/BHT, liposome or M-β-CD. Furthermore, when FBS was replaced with lipoprotein-deficient serum, the uptake of lycopene by DU145 cells was markedly decreased and was not significantly different from that of THF or THF/BHT. The results strongly indicate that the lipoprotein fraction of FBS plays an important role in delivering lycopene to cells. We then went on to study the improvement the methods of use THF/FBS as delivery vehicle because the stability of lycopene in this method is not satisfactory. Furthermore, the cellular uptake of lycopene was positively correlated with the degradation rate of lycopene in different delivery vehicles (r2 = 0.71) during incubation times (data not shown). We therefore used various antioxidants that may protect lycopene to decrease the degradation rate of lycopene in cultured studies. We found that use THF/FBS as delivery vehicle combined with quercetin (10µM) can better stabilize lycopene than can other antioxidants combining in this study, and similar result also suggested in cellular uptake efficiency. We further adopted the method of THF/FBS combining with quercetin in dose-dependent manners. When combining with 50 µM quercetin, the lycopene remained and cellular uptake was higher than that with 10 and 30 µM. In summary, this thesis demonstrated that the use of FBS for delivering lycopene into the two prostate cancer cell lines is superior to the use of THF, THF/BHT, liposome, M-β-CD and micelle and that the lipoprotein of FBS is likely responsible for the improved stability and cellular uptake of lycopene. Furthermore, antioxidants especially quercetin improves the use of THF/FBS as vehicle for delivering lycopene by increasing cellular uptake and increasing stability of lycopene by preventing lycopene oxidation. We conclude that using THF/FBS as vehicle for delivering lycopene combined with quercetin may contribute relatively to the in vitro studies of lycopene in the future.