Lys05 induces lysosomal membrane permeabilization and increases radiosensitivity in glioblastoma.

Glioblastoma (GBM) is one of the most malignant primary brain tumors and its prognosis is very poor. Lysosome-dependent cell death is mainly caused by lysosomal membrane permeabilization (LMP), a process in which the lysosome loses its membrane integrity and lysosomal contents are released into the cytosol. Lysosomotropic agent, a kind of compound that selectively accumulates in the lysosomes, is one of the most important inducers of LMP. As a newly-synthetic lysosomotropic agent, Lys05 showed efficient autophagy inhibiting and antitumor effect. But its mechanisms are not well illustrated. Here, we studied whether Lys05 has antiglioma activity. We found that Lys05 decreased cell viability and reduced cell growth of glioma U251 and LN229 cells. After Lys05 treatment, autophagic flux is inhibited and lysosome function is impaired. We also found that Lys05 caused LMP and mitochondrial depolarization. Finally, Lys05 increased radiosensitivity in an LMP-dependent manner. For the first time, our findings indicate that LMP contributes to radiosensitivity in GBM cells. Therefore, LMP inducer, Lys05 might be a promising compound in the treatment of GBM cells.

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 µM and 2.1-5.1 µM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.

Lys05 - A Promising Autophagy Inhibitor in the Radiosensitization Battle: Phosphoproteomic Perspective.

BACKGROUND: Autophagy is a crucial factor contributing to radioresistance during radiotherapy. Although Lys05 has proven its ability to improve the results of radiotherapy through the inhibition of autophagy, molecular mechanisms of this inhibition remain elusive. We aimed to describe the molecular mechanisms involved in Lys05-induced inhibition of autophagy. MATERIALS AND METHODS: Radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative) and methods of quantitative phosphoproteomics were employed to define the molecular mechanisms involved in Lys05-induced inhibition of autophagy. RESULTS: We confirmed that at an early stage after irradiation, autophagy was induced, whereas at a later stage after irradiation, it was inhibited. The early-stage induction of autophagy was characterized mainly by the activation of biosynthetic and metabolic processes through up- or down-regulation of the critical autophagic regulatory proteins Sequestosome-1 (SQSTM1) and proline-rich AKT1 substrate 1 (AKT1S1). The late-stage inhibition of autophagy was attributed mainly to down-regulation of Unc-51 like autophagy-activating kinase 1 (ULK1) through phosphorylation at Ser638. CONCLUSION: This work contributes to emerging phosphoproteomic insights into autophagy-mediated global signaling in lung cancer cells, which might consequently facilitate the development of precision medicine therapeutics.