Lactaptin Induces p53-Independent Cell Death Associated with Features of Apoptosis and Autophagy and Delays Growth of Breast Cancer Cells in Mouse Xenografts

Abstract

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.

Selection of antitumor displayed peptides for the specific delivery of the anticancer drug lactaptin

“…The cytotoxic activity of RL2 in relation to tumor cells is due to its ability to induce apoptosis in these cells (6,7). The current study assessed the ability of the fusion proteins T1_RL and T2_RL to induce apoptosis in MDA-MB-231 cells by cytometry.…”

“…As is known, RL2-induced apoptosis is accompanied by the activation of effector caspase-3 and −7, and mitochondrial transmembrane potential dissipation (7,13). Activation of caspase-3 and −7 in MDA-MB-231 cells by the recombinant proteins RL2, T1_RL and T2_RL was analyzed by cytofluorometry in the present study.…”

“…A series of recombinant analogs of the natural peptide has been created (4,5). It has been demonstrated that the lactaptin analog, RL2, induces apoptosis in mice and human tumor cells in vitro , and reduces the growth and metastasis of animal and human tumors in vivo (6,7). The preclinical trials of the RL2-based therapeutic drug lactaptin have been successful (7,8).…”

Comparative analysis of lactaptin activity when produced in bacterial or eukaryotic expression systems

“…Анализ цитотоксической активности кондиционированной среды, со держащей аналог лактаптина EL1. Ранее нами показано, что IC50 RL2 для чувствительных опухолевых клеток в среднем составляет 300 ± 100 мкг/мл (Koval et al, 2014. Полученные данные по содержанию белков EL1 и EL2 в кондиционированной среде свидетельствуют о том, что концентрация EL1 и EL2 приблизительно в 500 раз ниже значения IC50 RL2.…”

“…При исследовании механизма цитоток-сического действия RL2 показано, что гибель клеток аденокарциномы молочной железы человека MCF7 и MDAMB231, индуцируемая действием RL2, проходит по механизму апоптоза (Semenov et al, 2010). Исследова-ние активности рекомбинантного лактаптина RL2 in vivo также свидетельствовало о его противоопухолевой актив-ности в отношении опухолей мыши и человека (Koval et al, 2012(Koval et al, , 2014. Тем не менее при системном введении RL2 животным трудно достичь его высокой концентрации в опухоли, поскольку время его полувыведения из кровотока составляет 0.26 ч (Бондаренко и др., 2015).…”