Hypoxia Enhanced Glioblastoma Resistance to Erastin-Induced Ferroptosis by Up-Regulating GPX4 via PI3K/AKT/HIF-1α Axis

Background Glioblastoma is the deadliest type of primary brain tumor with a high rate of recurrence and treatment resistance. Hypoxia contributed much to radiotherapy resistance and chemoresistance of cancer. Ferroptosis is a nonapoptotic, oxidative cell death and identified as a potential anticancer mechanism in recent years. Erastin acts as a ferroptosis activator and shows a potential role in tumor treatment but the relationship between hypoxia and erastin resistance in glioblastoma has not been explained. This study aimed to investigate the role and underlying mechanism of hypoxia in erastin-induced ferroptosis in glioblastoma. Methods Cell proliferation and viability were determined by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, TUNEL assays, and clone formation assay. Lipid peroxides level was analyzed by Malondialdehyde (MDA) assay and flow cytometry using C11-BODIPY dye. The correlation between HIF-1α and GPX4 expression was detected in data from the TCGA database and was determined by ChIP-qPCR assay and luciferase reporter assay. Subcutaneous xenograft and orthotopic xenograft models were established to test our findings in vivo. Results Hypoxia for at least 16 hours significantly suppressed erastin-induced ferroptosis by up-regulating glutathione peroxidase 4 (GPX4) expression in U87 and U251 cells. Hypoxia promotes GPX4 expression via enhancing the PI3K/AKT/HIF-1α pathway. Mechanistically, HIF-1α directly bound to the GPX4 gene promoter region and promoted GPX4 transcription. AKT inhibitor MK-2206 and HIF-1α inhibitor PX-478 could significantly reverse the effect. Besides, under normoxia, PX-478 could induce a higher lipid peroxidation level by decreasing GPX4 expression in U87 and U251 cells but cannot induced cell death directly, and it could significantly enhance the tumor cell killing effect of erastin. In vivo, combination of PX-478 and erastin had a coordinated intensification effect on anticancer activity uncovered by subcutaneous xenograft and orthotopic xenograft mouse models. Conclusions Hypoxia enhanced glioblastoma resistance to erastin-induced ferroptosis by activating PI3K/AKT/HIF-1α pathway and promoting GPX4 expression in a transcriptional regulation way. Combination therapy of PX-478 and erastin may be a potential strategy against glioblastoma.

A lignan from Alnus japonica inhibits glioblastoma tumorspheres by suppression of FOXM1

Introduction Glioblastoma (GBM) is the most common and aggressive malignant brain tumor. Forkhead Box M1 (FOXM1) has been shown to regulate cell proliferation, apoptosis, angiogenesis, DNA damage repair and tumorigenesis. The lignin, (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS), from Alnus japonica (Betulaceae) has shown anti-cancer effects against colon cancer cells by suppressing FOXM1. However, the efficacy of DFS in GBM has not yet been determined. The present study hypothesized that DFS can have anti-cancer effects against GBM tumorspheres (TSs). Methods Immunoprecipitation and luciferase reporter assays were performed to evaluate the ability of DFS to suppress nuclear translocation of β-catenin through β-catenin/FOXM1 binding. GBM TSs were treated with DFS to assess the ability of DFS to inhibit GBM TSs and to evaluate their transcriptional profiles. The in vivo efficacy of DFS was examined in orthotopic xenograft models of GBM. Results Expression of FOXM1 was higher in GBM than in normal tissues. DFS-induced FOXM1 protein degradation blocked β-catenin translocation into the nucleus and consequently suppressed downstream target genes of FOXM1 pathways. DFS considerably inhibited cell viability and ATP levels in GBM TSs, while increasing the proportion of apoptotic cells. Treatment with DFS also reduced neurosphere formation and the invasive properties of GBM TSs. Transcriptome analyses showed that DFS reduced the activities of transcription factors related to tumorigenesis, stemness, and invasiveness. In addition, DFS significantly inhibited tumor growth and prolonged the survival rate of mice in orthotopic xenograft models of GBM. Conclusions DFS inhibits the proliferation of GBM TSs by suppressing FOXM1. These findings suggest that DFS may be a potential therapeutic agent to treat patients with GBM.

Histopathological Tumor and Normal Tissue Responses after 3D-Planned Arc Radiotherapy in an Orthotopic Xenograft Mouse Model of Human Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. Innovative treatment concepts may enhance oncological outcome. Clinically relevant tumor models are essential in developing new therapeutic strategies. In the present study, we used two human PDAC cell lines for an orthotopic xenograft mouse model and compared treatment characteristics between this in vivo tumor model and PDAC patients. Tumor-bearing mice received stereotactic high-precision irradiation using arc technique after 3D-treatment planning. Induction of DNA damage in tumors and organs at risk (OARs) was histopathologically analyzed by the DNA damage marker γH2AX and compared with results after unprecise whole-abdomen irradiation. Our mouse model and preclinical setup reflect the characteristics of PDAC patients and clinical RT. It was feasible to perform stereotactic high-precision RT after defining tumor and OARs by CT imaging. After stereotactic RT, a high rate of DNA damage was mainly observed in the tumor but not in OARs. The calculated dose distributions and the extent of the irradiation field correlate with histopathological staining and the clinical example. We established and validated 3D-planned stereotactic RT in an orthotopic PDAC mouse model, which reflects the human RT. The efficacy of the whole workflow of imaging, treatment planning, and high-precision RT was proven by longitudinal analysis showing a significant improved survival. Importantly, this model can be used to analyze tumor regression and therapy-related toxicity in one model and will allow drawing clinically relevant conclusions.