Higher levels of dynamin-related protein 1 are associated with reduced radiation sensitivity of glioblastoma cells

Background : Dynamin-related protein 1 (DRP1) is a GTPase involved in mitochondrial fission, mitochondrial protein import, and drug sensitivity, suggesting an association with cancer progression. This study was conducted to evaluate the prognostic significance of DRP1 in glioblastoma multiforme (GBM). Methods : DRP1 expression was measured by immunohistochemistry and Western blotting. Correlations between DRP1 expression and clinicopathological parameters were determined by statistical analysis. Differences in survival were compared using the log-rank test. Results : DRP1 expression was detected in 87.2% (41/47) of the investigated patients with GBM. The patients with higher DRP1 levels had worse survival ( p = 0.0398). In vitro , the silencing of DRP1 reduced cell proliferation, invasive potential, and radiation resistance. The addition of shikonin inhibited DRP1 expression and increased drug uptake. Moreover, shikonin reduced the nuclear entry of DNA repair-associated enzymes and increased radiation sensitivity, suggesting that reducing DRP1 expression could inhibit DNA repair and increase the radiation sensitivity of GBM cells. Conclusions : Our results indicate that DRP1 overexpression is a prospective radio-resistant phenotype in GBM. Therefore, DRP1 could be a potential target for improving the effectiveness of radiation therapy. calf intestinal phosphatase the 85-kDa band gradually but of 80-kDa protein band increased, suggesting that 85-kDa protein could be a phosphorylated form of 80-kDa DRP1. an internal to ensure that equal of were added each respective well. To validate the supposition that hypoxia could DRP1, cytoplasm results corresponded well with our previous study of lung

DRP1 is an 80-kDa GTPase, that mediates the budding and scission of a variety of transport vesicles and organelles [24,28,29], including mitochondria [30]. A number of anticancer drugs, e.g., epipodophyllotoxins and cisplatin, induce mitochondrial fragmentation, a phenomenon that is closely associated with apoptosis and chemotherapeutic cytotoxicity [31]. A reduction in the levels of DRP1 protein/ transcript causes ATM-dependent G2/M arrest and aneuploidy [32]. Disturbance in mitochondrial dynamics is due to an increased expression of Drp1 fission protein in vitro and in vivo [33]. A better understanding of DRP1 on drug activity could, therefore, provide more valuable information to improve disease management. In addition, the aforementioned chemotherapeutic agents might become vital probes for studying the essential function as well as the regulation mechanisms of DRP1 and other fusion/fission-related proteins in intracellular material transport and organelle damage [24,25,28,29]. However, the role of DRP1 in GBM has not previously been studied.
In this study, therefore, we used immunohistochemistry and Western blotting to determine DRP1 expression in GBM. We then evaluated the prognostic significance of DRP1 expression in GBM patients. Moreover, we investigated the effect of shikonin and suberoylanilide hydroxamic acid (SAHA, vorinostat), a histone deacetylase (HDAC) inhibitor, on DRP1 expression as well as radiation sensitivity in vitro.

Methods
The protocols of this study were approved by the Medical Ethics Committee of Taichung Veterans General Hospital (Approval number: CF12026B#2).

1.
Tissue specimens and immunohistochemical detection of DRP1 expression.
The validation cohort consisted of 47 cases selected from the primary cohort based on the following

2.
Cell culture and alteration of DRP1 expression using lentivirus-carrying shRNA or ectopic plasmid.
Briefly, 30 μg of total cell lysate was separated on a 10% polyacrylamide gel with a 4.5% stacking gel.
After electrophoresis, proteins were transferred to a nitrocellulose membrane. The membrane was probed with specific antibodies. The proteins were visualized by exposing the membrane to an X-Omat film with enhanced chemiluminescence reagent (Merck, Darmstadt, Germany). The respective primary antibodies were mouse anti-DRP1 and mouse anti-b-actin. These mouse monoclonal antibodies to DRP1 were home-made and have previously been characterized [24]. The digital images on X-Omat film were processed in Adobe Photoshop 7.0 (http://www.adobe.com/). The results were analyzed and quantified by the Image J software program (NIH, Bethesda, MD).

Confocal immunofluorescence microscopy.
Purified shikonin (>98%, HPLC) was purchased from Sigma-Aldrich (Saint Louis, Mo). The method for immunofluorescence confocal microscopy has been described previously [24,28,29]. Briefly, the cells on slides were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 prior to staining with mouse anti-DRP1. After washing off of the primary antibodies, the slides were incubated with Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen, Grand Island, NY). The nuclei were stained with 4', 6-Diamidino-2-phenylindole (DAPI), and the slides were examined under a laser confocal microscope (Olympus FV-1000, Tokyo, Japan). Images of the cells were analyzed using the FV10-ASW 3.0 software (Tokyo, Japan).

5.
Colony formation assay and the culture of GBM stem cells.
T98G-shLuc and T98G-DRP1 KD cells, U87-shLuc and U87-DRP1 KD cells, and GSC-shLuc and GSC-  was confirmed by Western blotting (Fig 1C). In addition, we speculated that the protein of DRP1 in the DRP1-positive nuclei was likely to consist of the phosphorylated state of DRP1 ( Supplementary Fig.   S2C). Interestingly, the molecular weights of the DRP1 in 7 of 12 surgical specimens were higher than the anticipated 80-kDa and three samples clearly had two protein bands, indicating that the DRP1 in biopsies could be post-translationally modified [24]. It is worth mentioning that the anti-DRP1 monoclonal antibody has been proved and characterized, and is a highly specific antibody [24].

The impact of DRP1 overexpression on GBM patient prognosis
The survival of patients with low DRP1 levels was significantly better than that of patients with high

Silencing of DRP1 expression in GBM cells decreases cell growth and mobility, but increases radiation sensitivity
In vitro, the protein levels of DRP1 were examined by Western blotting analysis in three human glioma cell lines (H4, U87MG, and T98G). All three cell lines expressed both 80-and 85-kDa proteins fold. Interestingly, CD-133 + GBM stem cells (GSC) also highly expressed DRP1, in particular the 85-kDa protein (Fig 3C). The silencing of DRP1 expression inhibited the cell growth of GSCs, including the number of cells and the spheroid formation ( Fig 3D). These results confirmed our previous findings that DRP1, which is essential for mitochondrial protein import, is involved in cell growth and genotoxic resistance [24, 28, 29], suggesting that reducing the total intracellular DRP1 expression or nuclear DRP1 levels could enhance the anticancer efficacy of radiation and anticancer drug therapies.

The respective effects of shikonin and SAHA on DRP1 expression and cell survival
Our previous studies showed that DRP1 is involved in an alternative mitochondrial import route, and the disruption of this route induces autophagy [28,29]. Using DRP1 as a target, we found that several we analyzed the related protein expression.
As shown in Fig. 4A, shikonin decreased the levels of both 80-and 85-kDa DRP1 and increased that of an autophagic marker, LC3B-II. Using fluorescence microscopy, we found that shikonin clearly induced the formation of autophagosomes (Fig. 4B). Although SAHA did not affect DRP1 expression (Fig. 4C) and daunorubicin levels, in T98G cells, we found that shikonin not only markedly increased nuclear DAPI and daunorubicin, but also significantly increased cell sensitivity to daunorubicin (Figs. 5A-5C).
We further found that glioblastoma cells are sensitive to daunorubicin even through it is not used for 13 brain tumors. Moreover, shikonin treatment reduced the nuclear accumulation of ATM (Fig. 5D, left panel), supporting our previous results indicating that the inhibition of DRP1 expression restricted the nuclear import of DNA repair-related enzymes and induced bulging of the MAM (Fig. 5D, right panel).
Using a transmission electron microscope, we further showed that shikonin treatment increased nuclear envelope damage (Figs. 5E1 & 5E2). Shikonin causes nuclear envelope breaks (Fig. 5e2, arrow), and leads to the disruption of the inner and outer nuclear membrane in T98G cells, which implies that membrane disintegration starts after the addition of shikonin.

Discussion
Our results showed that DRP1 was highly expressed in the investigated newly diagnosed GBM Both TMZ and radiation induce nuclear and mitochondrial genome DNA breakage. TMZ affects both mitochondrial electron transport and oxidative phosphorylation [53]. Radiation, on the other hand, induces the translocation of ATM, which is important for the repair of DNA breaks, to the nucleus and mitochondria [54]. An ATM deficiency, whether induced by a genetic or a biochemical method, reduces genomic DNA repair functions as well as mitochondrial biogenesis and oxidative respiratory functions [55]. By demonstrating that extranuclear ATM is bound to ER-associated peroxisome targeting signal type 1 (PTS1) receptor (also named peroxisomal biogenesis factor 5, Pex5), Watters et al suggested that besides the nucleus, ATM could be targeted to the MAM [56]. In a gene knockout study, Baumgart et al further showed that a defect in the Pex5 gene reduced peroxisomal metabolism, as well as the expression and activities of the mitochondrial respiration system [57].
Their results strongly suggested that MAM and its associated enzymes, in particular, DRP1, a GTPase, play a pivotal role in allocating materials, that are essential for maintaining organelle morphology, as to an increase in the apoptosis response of glioblastoma cells (Fig. 4D). Obviously, the lack of DRP1 similar to the inhibition of autophagy, contributed to the sensitivity of cells to both chemo-and radiotherapy [58]. In spite of the fact that this study could not clearly explain the role played by DRP1 in the autophagy process, it revealed evidence indicating that DRP1 is likely to play a role in mitochondrial DNA stability (Fig. 4E) [58].

Conclusions
In conclusion, our results showed that DRP1 is overexpressed in GBM, while the inhibition of DRP1 expression induces autophagy and enhances radiation sensitivity. This effect is specific to cancer cells, which overexpress not only DRP1 but also ATAD3A, AKR1C1, eukaryotic elongation factor

Funding
The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgement
Not Applicable.