PDF(Pubmed)
Abstract：
Tryptophan is an essential amino acid required for tumor cell growth and is also the precursor to kynurenine, an immunosuppressive molecule that plays a role in limiting anticancer immunity. Tryptophanase (TNase) is an enzyme expressed by different bacterial species that converts tryptophan into indole, pyruvate and ammonia, but is absent in the Salmonella strain VNP20009 that has been used as a therapeutic delivery vector. We cloned the Escherichia coli TNase operon tnaCAB into the VNP20009 (VNP20009-tnaCAB), and were able to detect linear production of indole over time, using Kovács reagent. In order to conduct further experiments using the whole bacteria, we added the antibiotic gentamicin to stop bacterial replication. Using a fixed number of bacteria, we found that there was no significant effect of gentamicin on stationary phase VNP20009-tnaCAB upon their ability to convert tryptophan to indole over time. We developed a procedure to extract indole from media while retaining tryptophan, and were able to measure tryptophan spectrophotometrically after exposure to gentamicin-inactivated whole bacterial cells. Using the tryptophan concentration equivalent to that present in DMEM cell culture media, a fixed number of bacteria were able to deplete 93.9% of the tryptophan in the culture media in 4 h. In VNP20009-tnaCAB depleted tissue culture media, MDA-MB-468 triple negative breast cancer cells were unable to divide, while those treated with media exposed only to VNP20009 continued cell division. Re-addition of tryptophan to conditioned culture media restored tumor cell growth. Treatment of tumor cells with molar equivalents of the TNase products indole, pyruvate and ammonia only caused a slight increase in tumor cell growth. Using an ELISA assay, we confirmed that TNase depletion of tryptophan also limits the production of immunosuppressive kynurenine in IFNγ-stimulated MDA-MB-468 cancer cells. Our results demonstrate that Salmonella VNP20009 expressing TNase has improved potential to stop tumor cell growth and reverse immunosuppression.
摘要：
色氨酸是肿瘤细胞生长所需的必需氨基酸，也是犬尿氨酸的前体，在限制抗癌免疫中起作用的免疫抑制分子。色氨酸酶（TNase）是由不同细菌物种表达的酶，可将色氨酸转化为吲哚，丙酮酸和氨，但在已用作治疗性递送载体的沙门氏菌VNP20009中不存在。我们将大肠杆菌TNase操纵子tnaCAB克隆到VNP20009(VNP20009-tnaCAB)中,并且能够随着时间的推移检测吲哚的线性产生，使用Kovács试剂。为了使用整个细菌进行进一步的实验，我们添加了抗生素庆大霉素来阻止细菌复制。使用固定数量的细菌，我们发现庆大霉素对稳定期VNP20009-tnaCAB对其随着时间的推移将色氨酸转化为吲哚的能力没有显著影响。我们开发了一种从培养基中提取吲哚同时保留色氨酸的程序，并且能够在暴露于庆大霉素灭活的全细菌细胞后通过分光光度法测量色氨酸。使用相当于DMEM细胞培养基中存在的色氨酸浓度，固定数量的细菌能够在4小时内耗尽培养基中93.9%的色氨酸。在VNP20009-tnaCAB耗尽的组织培养基中，MDA-MB-468三阴性乳腺癌细胞无法分裂，而用仅暴露于VNP20009的培养基处理的细胞继续分裂。将色氨酸重新添加到条件培养基中恢复了肿瘤细胞生长。用摩尔当量的TNase产物吲哚治疗肿瘤细胞，丙酮酸盐和氨仅引起肿瘤细胞生长的轻微增加。使用ELISA测定法，我们证实,在IFNγ刺激的MDA-MB-468癌细胞中,色氨酸的TNase耗竭也限制了免疫抑制性犬尿氨酸的产生.我们的结果表明，表达TNase的沙门氏菌VNP20009具有阻止肿瘤细胞生长和逆转免疫抑制的潜力。
