assessed and modified the manuscript

assessed and modified the manuscript. in glioblastoma. Our results showed that nitazoxanide suppressed cell growth and induced cell cycle arrest in glioblastoma by upregulating ING1 expression with a favorable toxicity profile. Nitazoxanide inhibited autophagy through blockage of late-stage lysosome acidification, resulting in decreased cleavage of ING1. A combination with chloroquine or Torin1 enhanced or Adriamycin impaired the chemotherapeutic effect of nitazoxanide in glioblastoma cells. Taken Adriamycin together, these findings indicate that nitazoxanide as an autophagy inhibitor induces cell cycle arrest in glioblastoma via upregulated ING1 due to increased transcription and decreased post-translational degradation by late-stage autophagic inhibition. Introduction Glioma is the most common type of malignant brain tumor in adults, accounting for 27% of all primary central nervous system (CNS) tumors. Among these, glioblastoma multiforme (GBM, WHO grade IV) is the most lethal CNS tumor and is characterized by excessive proliferation, aggressive invasion and high resistance to conventional therapies1,2. Chemotherapy is widely used in adjuvant approaches for Adriamycin the treatment of brain tumors, especially glioma. Currently, numerous antineoplastic drugs, such as temozolomide, carmustine wafer and bevacizumab, have been approved for treatment of glioma; these drugs alter MGMT promoter methylation, DNA and RNA crosslinking, cell cycle arrest, VEGF, and autophagy2,3. Despite these current advances in the clinical treatment of glioma, little improvement has been made in the median survival time of initially diagnosed GBM patients, which is 15C18 months on average2. Therefore, identification and development of new therapeutics for glioma patients is urgently needed. Drug repurposing, also known as drug repositioning is a novel therapeutic switching strategy that has gained popularity in the development of new agents4,5. The repurposing of existing treatments, such as sildenafil and metformin, for alternative disorders can save time and money in drug design and development6. Nitazoxanide (NTZ), an antiprotozoal drug used against protozoan, bacterial or viral infections such as Cryptosporidia, Helicobacter or Hepatitis C, has shown a wide spectrum of pharmacological functions in infectious and neoplastic diseases7C9. However, the chemotherapeutic role of NTZ in glioma remains unclear. To date, the pharmacological effects of NTZ include mediating the unfolded protein response (UPR), reversing chemotherapy detoxification, targeting the c-Myc signaling pathway, stimulating the immune response, and especially regulating autophagy9C13. Autophagy is an intracellular lysosomal degradation process regulated by a variety of highly conserved autophagy-related genes (ATGs) through different mechanisms14. This homeostatic process could affect or be induced by multiple cellular stressors and signaling pathways involved in nutrient and growth factor status, energy sensing, hypoxia, oxidative and endoplasmic reticulum (ER) stress, pathogen infection, or chemotherapy resistance15,16. Interestingly, inhibition or activation of autophagy may produce synergistic or contradictory effects on cancer therapy depending on the cellular context17,18. Thus, whether autophagy is involved in the chemotherapeutic effects of NTZ and whether NTZ combined with inhibition or activation of autophagy enhances or impairs the chemotherapeutic efficacy still need to be confirmed. In the present study, we demonstrated the therapeutic efficacy of NTZ either alone or Adriamycin combined with an autophagy inducer or inhibitor on glioma growth in vitro and in vivo. We FLJ14936 further screened target genes of NTZ and investigated the underlying molecular mechanism of NTZ-associated autophagic suppression in glioma treatment. Results NTZ decreases glioma cell viability and proliferation To investigate the effect of NTZ on glioma cell viability, we exposed LN229, U87, A172, and HUVECs to different NTZ concentrations ranging from 100 to 1600?M for 48?h and 72?h. As shown in Fig.?1a, NTZ inhibited cell proliferation in the 4 cell lines in a dose-dependent and time-dependent manner, which significantly reduced Adriamycin cell viability in the 48?h and 72?h groups. The 48?h IC50 values of NTZ were 383.39?M for LN229, 398.66?M for A172, 411.72?M for U87 and 659.93?M for HUVECs. Inhibition of cell proliferation was augmented after 48?h of NTZ treatment as shown by light microscopy (Fig.?1b). The fluorescence results further indicated that expression of the proliferative marker Ki67 was decreased in the LN229 cell line (Fig.?1c). Similarly, colony formation assays showed that colony formation was significantly decreased after NTZ exposure (Fig.?1d). These results indicate that NTZ exhibits cytotoxicity and inhibits cell growth in glioma cells. Open in a separate window Fig. 1 NTZ inhibits glioma cell growth in vitro.a Cell viability of LN229, A172, U87, and HUVECs determined by MTT assays after 48?h and 72?h of NTZ treatment. b Phase contrast microscopy of LN229 cells inhibited by NTZ. Scale bar represents 100 or 250?m. c Fluorescence microscopy of Ki67 expression after treatment of the LN229 cell line with NTZ at concentrations of 0, 200, and 400?M for 48?h. Scale bar represents 100?M. d.