Amino acid deprivation in cancer cells with compensatory autophagy induction increases sensitivity to autophagy inhibitors.

Inhibition of autophagy is an important strategy in cancer therapy. However, prolonged inhibition of certain autophagies in established cancer cells may increase therapeutic resistance, though the underlying mechanisms of its induction and enhancement remain unclear. This study sought to elucidate the mechanisms of therapeutic resistance through repeated autophagy inhibition and amino acid deprivation (AD) in an in vitro model of in vivo chronic nutrient deprivation associated with cancer cell treatment. In the human cervical cancer cell line HeLa and human breast cancer cell line MCF-7, initial extracellular AD induced the immediate expression of endosomal microautophagy (eMI). However, repeated inhibition of eMI with U18666A and extracellular AD induced macroautophagy (MA) to compensate for reduced eMI, simultaneously decreasing cytotoxicity. Here, hyperphosphorylated JNK was transformed into a hypophosphorylated state, suggesting conversion of the cell death signal to a survival signal. In a nutrient medium, cell death could not be induced by MA inhibition. However, since LAT1 inhibitors induce intracellular AD, combining them with MA and eMI inhibitors successfully promoted cell death in resistant cells. Our study identified a novel therapeuic approach for promoting cell death and addressing therapeutic resistance in cancers under autophagy-inhibitor treatment.

Secretory production of a camelid single-domain antibody (VHH, nanobody) by the Serratia marcescens Lip system in Escherichia coli.

Escherichia coli is one of the most popularly used hosts to produce recombinant proteins. Most recombinant proteins are produced in the cytoplasm and periplasm, requiring multiple steps to extract and purify recombinant proteins. The Serratia marcescens Lip system (LipB-LipC-LipD) is a type 1 secretion system that selectively secretes LipA from the intracellular to extracellular space in a single step. This study aimed to establish a secretory production system for nanobodies, camelid-derived small molecule antibody fragments, using the S. marcescens Lip system. Surprisingly, E. coli harboring only LipC, a membrane fusion protein of the Lip system, could secrete an anti-green fluorescent protein (GFP)-Nb, a nanobody against GFP, without the addition of a long amino acid sequence. The LipC-based secretion system recognized the Val-Thr-Val sequence at the C-terminus of the nanobody. Finally, Strep-tagged anti-GFP-Nb was purified from culture supernatants of E. coli harboring LipC by Strep-affinity chromatography at a final yield of >5 mg per liter of culture supernatant. These results potently supported that the S. marcescens LipC-based secretion system has the potential to establish an efficient secretory production system for nanobodies.