Koval Olga A, Tkachenko Anastasiya V, Fomin Alexandr S, Semenov Dmitry V, Nushtaeva Anna A, Kuligina Elena V, Zavjalov Eugeny L, Richter Vladimir A
Institute of Chemical Biology and Fundamental Medicine SB RUS, Novosibirsk, Russia; Novosibirsk State University, Novosibirsk, Russia.
Institute of Chemical Biology and Fundamental Medicine SB RUS, Novosibirsk, Russia.
PLoS One. 2014 Apr 7;9(4):e93921. doi: 10.1371/journal.pone.0093921. eCollection 2014.
Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.
乳凝素是人类母乳κ-酪蛋白的蛋白水解片段，可诱导多种培养的癌细胞死亡。乳凝素处理后导致细胞死亡的机制尚未完全明确。在本研究中，检测了重组乳凝素类似物（RL2）的体内和体外效应。用重组乳凝素类似物处理后，检测到强烈的半胱天冬酶-3、-7激活。由于半胱天冬酶激活，我们观察到出现了具有亚二倍体DNA含量的亚G1期细胞群。评估了凋亡相关基因的mRNA和蛋白质水平的动态变化。在对照细胞和经RL2处理的细胞之间，未发现p53 mRNA水平或蛋白质水平有统计学上可靠的差异。我们观察到RL2在MDA-MB-231细胞中持续抑制bcl-2 mRNA表达并下调Bcl-2蛋白表达。我们证明RL2可穿透癌细胞和未转化细胞。对乳凝素类似物的细胞靶点鉴定显示，α/β-微管蛋白和α-辅肌动蛋白-1是与RL2结合的蛋白。由于细胞对蛋白质刺激的活力改变不仅可以通过凋亡实现，还可以通过自噬实现，因此我们研究了自噬在RL2依赖性细胞死亡中的作用。我们还发现RL2处理可诱导LC3加工，这是自噬的一个标志。自噬抑制剂氯喹增强了RL2对MDA-MB-231细胞的细胞毒性，表明RL2依赖性自噬的促生存作用。在携带MDA-MB-231细胞的小鼠异种移植瘤中，对RL2的抗肿瘤潜力进行了体内研究。我们证明重组乳凝素类似物显著抑制实体瘤的生长。我们的结果表明，乳凝素可能是一种用于开发抗癌药物的新分子。
