Oncogenic Ras is downregulated by ARHI and induces autophagy by Ras/AKT/mTOR pathway in glioblastoma
Glioblastoma is a disease with high heterogeneity that has long been difficult for doctors to identify and treat. ARHI is a remarkable tumor suppressor gene in human ovarian cancer and many other cancers. We found over-expression of ARHI can also inhibit cancer cell proliferation, decrease tumorigenicity, and induce autophagic cell death in human glioma and inhibition of the late stage of autophagy can further enhance the antitumor effect of ARHI through inducing apoptosis in vitro or vivo.
Using MTT assay to detect cell viability. The colony formation assay was used to measure single cell clonogenicity. Autophagy associated morphological changes were tested by transmission electron microscopy. Flow cytometry and TUNEL staining were used to measure the apoptosis rate. Autophagy inhibitor chloroquine (CQ) was used to study the effects of inhibition at late stage of autophagy on ARHI-induced autophagy and apoptosis. Protein expression were detected by Western blot, immunofluorescence and immunohistochemical analyses. LN229-derived xenografts were established to observe the effect of ARHI in vivo.
ARHI induced autophagic death in glioma cells, and blocking late-stage autophagy markedly enhanced the antiproliferative activites of ARHI. In our research, we observed the inhibition of RAS-AKT-mTOR signaling in ARHI-glioma cells and blockade of autophagy flux at late stage by CQ enhanced the cytotoxicity of ARHI, caused accumulation of autophagic vacuoles and robust apoptosis. As a result, the inhibition of RAS augmented autophagy of glioma cells.
ARHI may also be a functional tumor suppressor in glioma. And chloroquine (CQ) used as an auxiliary medicine in glioma chemotherapy can enhance the antitumor effect of ARHI, and this study provides a novel mechanistic basis and strategy for glioma therapy.
KeywordsARHI Autophagy Apoptosis Glioblastoma Ras
adjacent normal tissue
Aplasia Ras homolog member I
late or degradative autophagic vacuoles
initial autophagic vacuoles
central nervous system
transmission electron microscopy
Although great progress has been made in current medical science, cancer remains a principal enemy threatening human health and life. Glioma, which is associated with low median overall survival and a high rate of occurrence in brain tumors, is a disease that needs to be urgently addressed . New and effective therapeutic targets should be promptly identified. Autophagy is a highly conserved catabolic process by which cells can recycle organelles and long-lived intracellular proteins . Depending upon the cellular microenvironment, the induction of autophagy can either protect or kill metabolically active cancer cells . In the short term, autophagy can sustain cancer cells with multiple cellular stressors . However, dysregulated or excessive autophagy could cause autophagic cell death, the type II programmed cell death. Aplasia Ras homolog member I (ARHI) is a powerful tumor suppressor gene belonging to the Ras superfamily located on human chromosome 1p31.3 and includes one promoter, two exons and one intron with a 687 bp protein-coding region that encodes a 26 kDa protein with 50–60% homology to Ras and Rap [5, 6]. However, the features of ARHI are totally different from those of the oncogene Ras. ARHI is downregulated in multiple malignant tumors, including ovarian cancer, breast cancer, lung cancer, prostate cancer, thyroid cancer, pancreatic cancer and glioma , and over-expression of ARHI at physiological levels can retard proliferation, reduce motility and enhance cancer cell dormancy . ARHI has been recently reported to induce autophagy in ovarian and breast cancer cells and ARHI over-expression can decrease tumor growth, migration, and invasion in human glioma [9, 10]. But whether over-expression of ARHI can induce autophagy in human glioma cells and promote autophagic death are still unknown. Inhibition of autophagic function with chloroquine (CQ) can induce cancer death through necroptosis . However, whether ARHI regulates autophagy in glioma and the relationship between this autophagy and glioblastoma has not been reported. Factors regulating the formation of ARHI induced autophaghy in glioma cells are still not completely understood.
Human cancer cells grown as subcutaneous xenografts in immunodeficient mice represent one of the most frequently used in vivo models for drug target validation and preclinical drug testing in translational cancer research. These cells are often engineered for inducible expression/suppression of a given gene of interest to enable a more precise assessment of its functions. So, we built subcutaneous xenografts to test the result in vivo. In the present study, we found that ARHI can induce autophagy-mediated cell death in glioma cells, the therapeutic efficacy of ARHI on glioma growth was preliminary verified in vitro and in vivo. We also found that using CQ after over-expression of ARHI enhanced apoptosis in glioma cells. ARHI-associated autophagy in glioma was further studied at molecular mechanism level. We found that ARHI can negatively regulate oncogenic Ras and inhibit RAS-AKT-mTOR signaling in glioma cell. Thus, ARHI may serve as a tumor suppressor gene and can regulate autophagy in glioma.
Cell lines and reagents
Human glioblastoma cell lines (LN229, T98G, U87, U251) and human brain astrocyte cell line NHA were obtained and authenticated from the China Infrastructure of Cell Line Resource at July 2017. All cell lines were recently authenticated by STR analysis and tested for mycoplasma contamination. LN229 (CRL-2611), T98G (CRL-1690), and U87 (HTB-14) were distributed by ATCC. CQ was purchased from Sigma Aldrich (St. Louis, MO, USA). 3-MA were purchased from MedChem Express (MCE, USA).
Patient tissue preparation
A total of 9 glioma and 3 normal brain samples were obtained from the First Affiliated Hospital of Harbin Medical University between 2012 and 2014. The patients’ clinical characteristics, such as age, gender, and WHO grade, were collected. For qRT-PCR and western blot analysis, tissues were immediately frozen in liquid nitrogen.
The ARHI plasmids and lentiviruses used for ARHI overexpression and the negative control plasmids and lentiviruses were purchased from Genechem Co., Ltd. (Shanghai, China). Before transfection, glioma cells were cultured in six-well plates at 5 × 104 cells per well. Then, ARHI lentiviruses were introduced into glioma cells treated with 8 μg/ml polybrene (Genechem). Transfection effects were observed by a fluorescence microscope after 48 h. Puromycin was used to purify the infected cells.
Total RNA in cells and tissues was extracted using Trizol reagent, and the concentration of these RNA samples was measured using a spectrophotometer (Thermo Scientific™ NanoDrop 2000c). The RNA specimens were reverse transcribed into cDNA using a PrimeScript RT reagent kit (ToYoBo). The primer sequences for ARHI were forward 5′-CATAAGTTCCCCATCGTGC-3′; and reverse 5′-GAACAGCTCCTGCACATTCA-3′, and those for GAPDH used as a standard were forward 5′-ACCACAGTCCATGCCATCAC-3′; and reverse 5′-TCCACCACCCTGTTGCTGTA-3′.
The tissues and cells were lysed by RIPA buffer (Beyotime Institute of Biotechnology, Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor. The equal amounts of lysates were separated by 8–12.5% SDS-PAGE gels and transferred onto PVDF membranes. Antibody SQSTM1 (#23214), LC3B(#3868), Ras (#8955), Cleaved Caspase3 (#9662), mTOR (#2972), AKT (#9272) and Beta actin (#3700) were purchased from Cell Signaling Technology, ARHI antibody (ab107051) was purchased from abcam.
Cell proliferation and clone formation assay
A density of 5000 cells were planted into 96-well plates for each well and mixed 10 μl MTT at 72 h. After 24 h of transfection with the ARHI and control plasmid, cells (500 cells/well) were seeded into a six-well plates and cultured for two weeks. Then, 0.1% crystal violet was used to stain clones, and cells were photographed using a ChemiDocTM MP system (Bio-Rad, USA).
Cells were plated on coverslips, and after treatment, cells were washed twice with phosphate-buffered saline (PBS). They were then fixed with 4% paraformaldehyde for 20 min, blocked with 5% goat serum at 37 °C for 30 min and then treated with 0.3% Triton X-100 for 10 min. Next, 50 μl TUNEL reaction mix (Wanleibio, WLA030a) was added to each sample, and cells were incubated for 60 min at 37 °C in the dark. They were then incubated with DAPI (Beyotime, C1005), and photographs were captured by using a FSX100 Bio Imaging Navigator system (Olympus, Japan).
Transmission electron microscopy
GBM cells were seeded onto 6-well plates at a density of 1 × 105 cells per well. After treatment, cells were harvested and fixed using 2.5% glutaraldehyde at 4 °C overnight. Then, the harvested cells were dehydrated with ethanol and acetone and fixed with 1% osmium tetroxide. Samples were observed and captured by using transmission electron microscope (Hitachi H-7650, Japan).
A total of 5 × 104 cells were seeded onto 35-mm glass-bottomed dishes. After treatment, cells were harvested and fixed with 4% paraformaldehyde. Then, cells were treated with 0.1% Triton X-100 and blocked with 5% bovine serum albumin (BSA, BOSTER, AR0004). Cells were incubated with primary antibody at 4 °C overnight and then incubated with Alexa Flour 594 AffiniPure goat anti-rabbit IgG (ZSGB-BIO, ZF-0516) and DAPI (Beyotime, C1005).
Flow cytometry detected cell apoptosis
After transfecting ARHI for 24 h, cells were seeded onto a six-well plate and fixed with 3-MA or CQ. After 48 h, cells were harvested and detected using an Annexin V-PE Apoptosis Detection Kit (BD Bioscience, 556,422). Then, samples were measured by an Accuri C5 flow cytometer (BD Bioscience).
GFP-RFP-LC3 lentivirus transfection and fluorescence imaging
Cells were transfected with GFP-RFP-LC3 lentivirus according to the manufacturer’s protocol. After treatment, the autophagosomes (yellow dots) and autolysosomes (red dots) were determined by augmented microscopy (BioTek Instruments, USA).
Tumor xenograft model
All 28-day-old (15-20 g) BALB/C nude mice were purchased from the Vital River Animal Center (Beijing, China) randomly divided into four groups and each group have 6 mice. The required sample sizes were calculated and tabulated with different levels of accuracies and marginal errors with 95% confidence level for estimating and for various effect sizes with 80% power for purpose of testing as well. LN229 cells (5 × 106 ARHI-LN229 cells was subcutaneously injected into the right hips of each mouse. In drug treatment group, the mice were injected with CQ (30 mg/kg/d) for 4 weeks. Tumor volume was assessed and calculated (volume = (width)2 × (length)/2) every 2 days. Mice were sacrificed on day 34, all mice had no panic, struggling, shouting. The procedure is as follows: After using Avertin (2, 2, 2-Tribromoethanol) anesthesia (the injection dose should reach lethal dose:> 0.5 mg/20 g, intraperitoneal injection), the heartbeat、breath and various reflexes of mice disappeared. No painful reaction was observed in the whole course of anesthesia. Then, mice were sacrificed by breaking neck for next step. After photography, tumor weight was measured. All experimental ethics and animal experiments were conformed to the European Parliament Directive (2010/63/EU) and were approved by the Institutional Animal Care and Use Committee at Harbin Medical University (No. HMUIRB-2008-06).
Formalin-fixed and using paraffin embedded samples were sliced into 5-μm-thick sections. Then, sample sections were immunostained using primary antibodies Ki67, Ras, cleaved caspase-3 and ARHI at 4 °C overnight. Then, applying secondary antibodies at 37 °C for 30 min. Next, samples were visualized according to manufacturer’s protocol and using a diaminobenzidine (DAB) substrate kit for 10 min. After intensive washing, samples were counterstained with hematoxylin, dehydrated and coverslipped. Obtaining pictures by using FSX100 Bio Imaging Navigator (Olympus, Japan).
Three independents experiments data are shown as the means ± standard deviations (SD). Two groups statistical analysis was performed using Student’s t test (two-tailed), *indicated a statistical significance of P-value< 0.05; **indicated a strong statistical significance of P-value< 0.01; *** indicated an even strong statistical significance of P-value< 0.001.
ARHI has a lower expression level in glioma than in normal brain tissue and over-expression of ARHI can induce LN229 and T98G cells proliferation arrest
Over-expression of ARHI can induce dysregulated autophagy in glioma cells
Over-expression of ARHI can cause autophagic cell death and can reduce the levels of p-AKT and p-mTOR by negatively regulating Ras
CQ can cause excessive autophagy in ARHI-glioma cells
Inhibition of autophagy at late stage can promote apoptosis in ARHI-LN229 cells through the excessive autophagy and reduction of anti-apoptotic protein Bcl-2
CQ enhances the cancer suppression of ARHI in vivo
In our study, we found that ARHI was frequently downregulated in glioma tissues compared with normal brain tissues by qRT-PCR and western blot. Moreover, the expression level of ARHI was inversely associated with tumor grade. Furthermore, over-expressing ARHI can induce autophagy-mediated cell death in glioma, which further supports the hypothesis that ARHI represents a tumor suppressor gene in glioma. Previous studies have shown that ARHI gene expression products can inhibit the expression of Ras and ERK which is the downstream of Ras. Further studies found that the expression of ARHI decreased both PI3K and Ras/MAP signaling by downregulating EGFR through enhanced internalization and degradation leading to a shortened half-life, and decreased mTOR activity initiates autophagy. Mammalian target of rapamycin (mTOR) signaling pathway play a direct role in regulating autophagy . Decreased p-AKT can inhibit mTOR activity through the AKT/mTOR pathway. According to our study, over-expression of ARHI can decrease the expression of Ras in glioma cells, thus indirectly suppressing mTOR activity and ultimately initiating autophagy. Moreover, ARHI can inhibit GBM cells proliferation and decrease tumorigenicity through inducing autophagic death. CQ can promote this effect through increasing AVd, enhancing the accumulation of cytotoxic protein and reducing anti-apoptotic protein Bcl-2, suggesting that ARHI may be a potential therapeutic target for glioma. Autophagosome formation has been implicated in the process of apoptosis [38, 39], and inhibition of the early stagy of autophagy reduced the activation of caspase-3, while the effect of the late stage of autophagy inhibition was opposite [40, 41, 42].
In our study, we found that both autophagy and apoptosis were induced after over-expressing ARHI in glioma cells. Inhibiting autophagy by 3-MA led to decreased apoptosis and lower levels of the apoptosis marker cleaved caspased-3, indicating that inhibition of the early stage of autophagy reduced caspase-dependent apoptosis. CQ, as an inhibitor of late autophagy, combined with ARHI can enhance the accumulation of autophagic vacuoles and indirectly enhance the accumulation of cytotoxic protein and promoting apoptosis in glioma cells (Fig. 7). Thus, to sum up, ARHI may be a functional tumor suppressor in glioma. And CQ used as an auxiliary medicine in glioma chemotherapy can enhance the antitumor effect of ARHI, and this study provides a novel mechanistic basis and strategy for glioma therapy.
The Natural Science Foundations of Heilongjiang Province of China (D9619), the fund of the health and family planning commission of Heilongjiang Province (grant numbers 2016–020 to XC), the Project of Science and Technology of Education Department of Heilongjiang Province (12511z016) and the Special Fund for Translational Research of Sino-Russia Medical Research Center in Harbin Medical University (grant number CR201410). The funding bodies had no role in the design of the study, collection, analysis, and interpretation of data or in writing the manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
SGZ, CZ conceived, designed, coordinated and directed this experiment.CZ, MTS, JYY, XXW, CX, ZDL performed the statistical analysis in this study. CZ, XC, BXZ, WYZ, MTS, ZXZ, HQZ, MG, KKW and ZQY performed all experiments. CZ wrote this paper. XC modified the manuscript. All authors reviewed and approved the final manuscript.
Ethics approval and consent to participate
All experimental ethics and animal experiments were conformed to the European Parliament Directive (2010/63/EU) and were approved by the Institutional Animal Care and Use Committee at Harbin Medical University (No. HMUIRB-2008-06).
Consent for publication
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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