LTX-401

Cell experiment:

The MTT assay is employed to investigate the in vitro cytotoxicity of LTX-401 against a selection of both cancer and non-malignant cell lines. Pre-cultured cells are seeded at a density between 1x104-1.5x104 cells/well. The cytotoxic activity of LTX-401 against human red blood cells (RBCs) is determined by a hemolytic assay using freshly isolated blood from healthy individuals who gave their signed informed consent. RBCs are resuspended to a 10% hematocrit solution before being incubated for 1 h at 37°C with LTX-401 dissolved in PBS at concentrations ranging from 136-1358 μM (50-500 μg/mL). RBCs with PBS and 1% Triton solution alone serves as a negative and positive control, respectively. After centrifuging the samples at 4,000 rpm for 5 minutes, the absorbance of the supernatant is measured at 405 nm on a spectrophotometric microliter plate reader[1].

Animal experiment:

Mice[1]Female C57BL/6 wild-type mice, 5-6 weeks old, are used. All animals are housed in the same room and in cages (containing 2-5 mice) specially designed for mice, with a 12 h/12 h day-night cycle, and allowed ad libitum access to high quality food and water. Animals are weighed weekly and monitored several times per week, and no unexpected deaths are observed. B16F1 cells are harvested, washed in serum-free RPMI and injected intradermally (i.d) into the right side of the abdomen in C57BL/6 mice (7.5x104 B16F1 cells per mouse/50 μL RPMI-1640). Palpable tumors (20-30 mm2) are injected intratumorally (i.t) with either LTX-401 (5 mg/mL) or a vehicle (saline) once per day for three consecutive days. Animals are euthanized according to humane endpoints such as a weight loss of >10%, general physical discomfort or reduced activity, severe tumor necrosis or ulceration, or if the tumor size exceeds 130 mm2. All animals are euthanized using cervical dislocation[1].

References:

[1]. Liv-Marie Eike, et al. The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma. PLoS One. 2016; 11(2): e0148980.
[2]. Heng Zhou, et al. The oncolytic compound LTX-401 targets the Golgi apparatus. Cell Death Differ. 2018 Jan; 25(1): 227–228.