Elevated cellular PpIX potentiates sonodynamic therapy in a mouse glioma stem cell-bearing glioma model by downregulating the Akt/NF-κB/MDR1 pathway

Glioblastoma (GBM) has high mortality rates because of extreme therapeutic resistance. During surgical resection for GBM, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is conventionally applied to distinguish GBM. However, surgical intervention is insufficient for high invasive GBM. Sonodynamic therapy (SDT) combined with low-intensity ultrasonication (US) and PpIX, as a sonosensitizer, is an emerging and promising approach, although its efficacy is limited. Based on our previous study that down-regulation of multidrug resistant protein (MDR1) in GBM augmented the anti-tumor effects of chemotherapy, we hypothesized that elevation of cellular PpIX levels by down-regulation of MDR1 enhances anti-tumor effects by SDT. In high invasive progeny cells from mouse glioma stem cells (GSCs) and a GSC-bearing mouse glioma model, we assessed the anti-tumor effects of SDT with a COX-2 inhibitor, celecoxib. Down-regulation of MDR1 by celecoxib increased cellular PpIX levels, as well as valspodar, an MDR1 inhibitor, and augmented anti-tumor effects of SDT. MDR1 down-regulation via the Akt/NF-κB pathway by celecoxib was confirmed, using an NF-κB inhibitor, CAPÉ. Thus, elevation of cellar PpIX by down-regulation of MDR1 via the Akt/NF-κB pathway may be crucial to potentiate the efficacy of SDT in a site-directed manner and provide a promising new therapeutic strategy for GBM.

Glioblastoma (GBM) is the most common primary malignant brain tumor and has high recurrence and mortality rates 1,2 . Various current therapeutic approaches, including surgery, chemotherapy, radiotherapy, and immunotherapy, are clinically applied for treating GBMs 3,4 . However, surgical intervention is insufficient for eradicating GBM because of its extremely high invasiveness 3 . Chemotherapy and radiotherapy not only damage tumor cells but also partly harm normal cells. Furthermore, tumor cells acquire chemo-resistance during treatment, thus deterring GBM treatment. Though various approaches including neoadjuvant anti-PD-1 immunotherapy have been studied 5,6 , novel therapeutic strategies are required to improve the prognosis of GBM patients.
Sonodynamic therapy (SDT) is a promising and emerging noninvasive approach for cancer treatment. SDT involves a combination of ultrasonication (US) via low-intensity ultrasound imaging and specialized chemical agents known as sonosensitizers including 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) 7,8 . After oral administration, 5-ALA can accumulate in GBM and surrounding infiltrating cancer cells such as glioma stem cells (GSCs) outside of the tumor bulk. Once formed, PpIX emits fluorescence at a peak wavelength of 635 nm after excitation with light near the Soret band peak around 410 nm 9 . Microbubbles generated during US implode and release markedly high energy and initiate the emission of sonoluminescence light, subsequently leading to the generation of reactive oxygen species (ROS). The anti-tumor effect of SDT is primarily attributed to the generated ROS, suggesting that mitochondrial ROS generation is promoted under the presence of PpIX upon SDT 10 , resulting in severe damage to tumor cells through hydrodynamic shear forces and selectively destroying tumor cells 10,11 . Currently, 5-ALA is commonly administered to visualize GBM during resection surgery. It is selectively taken up by tumor cells upon delivery and localizes to the mitochondria, being converted to PpIX 10 . Because of its specificity and effectiveness towards highly invasive GBM cells 11 , SDT might potentially be a novel strategy for glioma therapy. However, SDT monotherapy cannot completely eradicate tumors. Since the efficacy of SDT depends on cellular PpIX levels to induce intrinsic caspase-dependent apoptosis [9][10][11][12][13] , it may be necessary to elevate the cellular PpIX level to promote the effects of SDT with new methods, including highintensity focused US 14 .
P-Glycoprotein, referred to as multidrug resistance receptor (MDR1), is a transmembrane glycoprotein functioning as an efflux pump and conferring multidrug resistance in brain tumors 15,16 . We have previously reported high expression of MDR1 in GBM and demonstrated that down-regulation of MDR1 via Akt/NF-κB pathways upon transfection of the Ad-DKK3 gene augmented the anti-tumor effects of temozolomide in GBM cells and in a GBM-xenograft model 17 . We also reported that a selective cyclooxygenase-2 (COX-2) inhibitor, celecoxib exerted anti-tumor effects associated with the down-regulation of Akt/NF-κB pathways in mouse glioma stem cells (GSCs) and GSCs-bearing glioma model 18,19 . Based on these findings, we hypothesized that down-regulation of MDR1 by celecoxib via Akt/NF-κB pathways may promote the uptake of 5-ALA into GBM, thereby elevating cellular PpIX levels and enhance the anti-tumor effects of SDT.
This study shows that elevation of cellular PpIX through celecoxib-mediated MDR1 down-regulation potentiates anti-tumor effects of SDT in a mouse GSC-bearing malignant glioma model which is highly invasive and similar to GBM in many respects 20,21 .

Results
Effects of SDT on GSC cell lines. Based on clinical usage in GBM patients, we first confirmed the time course of PpIX fluorescence in a GBM cell line, U251 (Fig. 1a).and in GSCs (Fig. 1b). Since the luminescence intensity peaked at 3 h after treatment with 5-ALA and was retained for 12 h in both cell lines (Fig. 1a,b), we decided to perform US (1 MHz, 2 W/cm 2 for 2 min) at 3 h after treatment with 1 μM 5-ALA, based on previous study 10,12 . The effects of SDT in GSCs was assessed at 3, 6, 12, 24 and 48 h after SDT and compared with nontreatment control, treatment with 5-ALA or US alone (Fig. 1c,d). A significant reduction of cell viability was observed at 6-48 h after SDT.

An increase in cellular PpIX via celecoxib-mediated MDR1 down-regulation augmented the anti-tumor effect of SDT.
To enhance the anti-tumor effects of SDT, we examined whether MDR1 downregulation can increase the cellular PpIX levels after 5-ALA injection. Based on our previous findings in human glioma cells 17 , we first observed that the expression levels of COX-2 and MDR1 in human glioblastoma and the mouse GSC-bearing glioma model were higher than those in normal brain tissue (Fig. 3a). Next, using a COX-2 inhibitor, celecoxib, we assessed the effects on the MDR1 expression in GSCs. Treatment with celecoxib 60 μM (IC 50 dose) decreased MDR1 expression levels compared to diluted DMSO as vehicle control and the PpIX levels were significantly higher upon combination treatment with 1 μM 5-ALA and celecoxib than with 5-ALA monotherapy (Fig. 3b). To confirm the effects of MDR1 down-regulation on PpIX levels, we examined the effects of an MDR1 inhibitor, valspodar (Fig. 3c). MDR1 was down-regulated by 1 μM valspodar (Fig. 3c) and cellular PpIX levels increased as expected, resulting in the enhanced anti-tumor effects of SDT by the combination of valspodar and SDT in GSCs (Fig. 3d). The increase in cellular PpIX levels through MDR1 decreased by celecoxib may be at least partly attributable to the enhancement of the anti-tumor effects of SDT.

Combination therapy with SDT and celecoxib enhanced apoptosis induction, thereby enhancing anti-tumor effects in the mouse GSC-bearing glioma model. To further confirm whether SDT
with down-regulation of MDR1 by celecoxib augments anti-tumor effects, we assessed the efficacy in the mouse www.nature.com/scientificreports/ GSC-bearing glioma model (Fig. 4a). Compared to normal tissue, the expression of MDR1 was elevated in brain tumor tissue after GSCs injection, which was reduced upon treatment with 10 mg/kg celecoxib for 7 days before SDT (Fig. 4b). Concurrent with this finding, cellular PpIX levels in brain tumor after 5-ALA injection were significantly increased (Fig. 4b). The elevation of cellular PpIX levels upon combination therapy with SDT and celecoxib escalated the induction of intrinsic and extrinsic apoptosis 1 day after SDT (Fig. 4c), thus decreasing the tumor volume (Fig. 4d) and prolonging survival (Fig. 4e). The combination therapy with SDT and celecoxib may be a promising means for GBM treatment.

Enhancement of anti-tumor effects by SDT was attributed to celecoxib-mediated MDR1 down-regulation through the AKT/NF-κB pathway.
To clarify the mechanisms underlying celecoxibmediated MDR1 down-regulation, we treated (i.p.) the mouse GSC-bearing glioma model with 10 mg/kg celecoxib for 7 days (Fig. 5a). AKT2, pAkt, pNF-κB, and MDR1 were significantly up-regulated in the glioma model than in normal tissue and were attenuated through celecoxib treatment (Fig. 5a). Furthermore, to confirm the transcriptional regulation of MDR1, we used a proteasome inhibitor, caffeic acid phenethyl ester (CAPE), which inhibits the phosphorylation of inhibitor of κB (I-κB), thus facilitating the phosphorylation of NF-κB (pNF-κB) and regulating the transcription of target genes. In GSCs, up-regulation of MDR1 mRNA and protein was significantly attenuated upon treatment with 1 μM CAPE, accompanied by the reduction of pI-κB and pNF-κB (Fig. 5b), suggesting transcriptional regulation of MDR1 through NF-κB. Together, celecoxib-mediated MDR1 down-regulation through the Akt/NF-κB pathway may increase cellular PpIX levels by increasing the intratumoral uptake of 5-ALA, thus promoting apoptosis induction via SDT (Fig. 5c). MDR1 down-regulation may be attributable to the improvement of not only chemo-resistance, but also the therapeutic efficacy of SDT in GBM.

Discussion
In this study, we initially show that combination of SDT and celecoxib to down-regulate MDR1 augmented the anti-tumor effects in GSCs and the mouse GSC-bearing malignant glioma model indicating invasive and therapeutic resistance. First, we confirmed that SDT monotherapy exerted anti-tumor effects in GSCs and GSCsbearing glioma model; however, it displayed transient and limited efficacy. Since we have demonstrated the antitumor effects by a COX2 inhibitor, celecoxib 18,19 , we assessed the efficacy of SDT combined with pre-treatment by celecoxib. Expectedly, the pre-treatment by celecoxib decreased the expression of MDR-1 and elevated the cellular PpIX levels induced by 5-ALA, resulting in the enhanced anti-tumor effects of the combination therapy in the GSC-bearing glioma model. Next, to confirm the mechanisms underlying the downregulation of MDR-1 by celecoxib, we used valspodar as an MDR-1 inhibitor. It decreased the expression of MDR-1 and elevated the cellular PpIX levels induced by 5-ALA, thereby reducing cell viability. Finally, we verified the down-regulation of MDR-1 by celecoxib via Akt/NF-κB pathway, using CAPE, a NF-κB inhibitor. The combination therapy of SDT and celecoxib enhanced the induction of both intrinsic and extrinsic apoptosis pathways in the GSCs-bearing malignant glioma model. Taken together, combination therapy with SDT and celecoxib may be a promising strategy to retard GBM recurrence. SDT has been developed as a novel promising noninvasive approach derived from photodynamic therapy (PDT) 22,23 . Because PDT is tightly focused with a short penetration depth of light in soft tissue up to several tens of centimeters, PDT is not effective for the treatment of deep-seated tumors 24,25 . In contrast, SDT is anticipated to overcome the major limitation of PDT. It may be a promising treatment method for brain tumors and other tumors 26 . PpIX, converted from 5-ALA in mitochondria, is used in heme formation in a reaction catalyzed by www.nature.com/scientificreports/ the enzyme ferrochelatase. In GBM, the decrease of ferrochelatase may contribute to the accumulation of PpIX within gliomas and GSCs and lead to its preferential localization in tumor 9 . Although we did not assess the effects of ferrochelatase, we suggested that the increased cellular PpIX level may be at least partly attributable to the uptake of 5-ALA into brain or GSCs through down-regulation of MDR-1 by celecoxib, thereby enhancing anti-tumor effects. Because GBM occurs in the brain parenchyma surrounded by the skull and differs from other epithelial cancers, thus far, SDT has been performed for GBM patients only during surgical intervention 27 . Although the ultrasound instrument allows the repeated application of STD, this technique is limited to only a few institutes. Therefore, to enhance the repeatability of SDT, the development of feasible and safe equipment with new sonosensitizers 28 are urgently required. MDR1 (ABCB1) gene encodes P-glycoprotein, a drug transporter that is a critical component of the bloodbrain barrier, which prevents entry of many potentially toxic compounds into the central nervous system 29 . We previously reported that the reduction of MDR1 upon transfection of DKK3 gene in human glioma cells enhanced the anti-tumor effects of temozolomide 17 . Concurrent with a previous report indicating increased mRNA levels of MDR1 in COX-2-overexpressing cells, the present study showed that MDR1 was highly upregulated and COX-2 was overexpressed in GSC-derived glioma tissue and in human glioma tissue. COX-2, an inducible form of the enzyme catalyzing the first step in prostanoid synthesis, has been reported to be overexpressed in various tumors and possesses proangiogenic and anti-apoptotic properties. MDR1 is expressed in normal liver and kidney tissue, where it functions to actively transport lipophilic xenobiotic compounds and serves as an efflux pump for chemotherapeutic agents 30 . Thus, up-regulation of MDR1 and COX-2 may be associated with not only chemotherapeutic resistance but also with the limited uptake of 5-ALA in GSCs and the mouse GSC-bearing glioma model. To confirm the enhanced efficacy of SDT through the cellular up-take of 5-ALA upon MDR1 down-regulation in GSCs, we treated GSCs with a COX-2 inhibitor, celecoxib, or an MDR1 inhibitor, valspodar. As expected, cellular PpIX levels increased upon MDR1 down-regulation by each chemical compound in GSCs. Whereas celecoxib is applicable in a clinical setting, valspodar has not shown evidence www.nature.com/scientificreports/ of clinical efficacy. Therefore, we used celecoxib but not valspodar to reduce MDR1 in our in-vivo study and confirmed the efficacy of SDT enhanced by celecoxib.
Although glioma-associated macrophages may take up large amounts of 5-ALA, we did not directly assess the effects of celecoxib on macrophages. However, we previously verified the reduction on chemokines/its receptors (CCL2/CCR2 and CXCL10/CXCR3) by celecoxib 18 . The reduction of these molecules may inhibit the recruitment of macrophages into the tumor, resulting in anti-tumor effects in the GSC-bearing glioma model. Therefore, the ability to take up 5-ALA into macrophages may be limited in the presence of celecoxib. Taken together, treatment with celecoxib before SDT may contribute to increase the uptake of 5-ALA into the tumor, thereby enhancing the anti-tumor effects.
In addition, MDR1 expression is associated with several cellular signaling pathways and protein kinases, chaperons, ubiquitin-related enzymes, and transcription factors 31 . As the COX-2 inhibitor induced apoptosis by inhibiting the AKT pathway in low-grade glioma cells in a previous study 18 , we observed MDR1 down-regulation through the Akt/NF-κB pathway upon celecoxib treatment. Furthermore, we observed the down-regulation of MDR1 by a NF-κB inhibitor, CAPE. Thus, celecoxib may affect both apoptosis induction and MDR1 downregulation by inhibiting the Akt/NF-κB pathway, resulting in an enhanced anti-tumor effects of not only SDT but also other anti-tumor agents.
This study has some limitations. Within a short period after SDT, apoptosis induction was enhanced in combination with celecoxib, and the cell viability was decreased. Therefore, we need the assessment of anti-tumor effects through repeated SDT upon combination during a longer period treatment with celecoxib. We previously confirmed that on combination treatment with 5-ALA and US, ROS generation and anti-tumor effects were greater than those upon monotherapy 10 . Unfortunately, in the current study we did not assess ROS generation, although the elevated cellular PpIX level was confirmed. Further investigation is necessary to determine whether elevated PpIX in combination therapy with SDT and celecoxib is associated with the enhanced ROS generation, thereby leading to the augment of the anti-tumor effects. SDT activates the mitochondrial caspase pathway and down-regulates ATP-binding cassette transporters such as MDR1, thus selectively improving the uptake of chemotherapeutic drugs into tumor cells and reducing the toxic effects on normal cells and tissues 31,32 . Therefore, sequential treatment with celecoxib after SDT may improve the uptake of celecoxib by itself into glioma cells, contributing to the prolonged survival in our glioma model. Although combination therapy with SDT and celecoxib may exert various synergistic therapeutic effects on GBM, we cannot exclude the favorable effects of celecoxib after SDT beyond MDR1 down-regulation 33 . Further studies are required to develop clinically applicable, handy, repeatable, and feasible devices for SDT.
In conclusion, the combination therapy with SDT and celecoxib resulted in enhanced anti-tumor efficacy among GSCs and a mouse GSC-bearing glioma model. MDR1 down-regulation via the Akt/NF-κB pathway may be a promising mean for treatment of GBM patients. Our results warrant for further verification of the anti-tumor effects of combination therapy with SDT and other chemotherapeutic agents and the development of new US systems capable of penetrating the skull.

Method
Study approval and informed consent. This study was approved by the ethical review board of Tokushima University Hospital for human study and the ethics committee of Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan. Human tissue samples were obtained during routine clinical procedures after informed consent including the use of tissue sample from patients with brain tumors at the Department of Neurosurgery, Tokushima University Hospital. For the use of samples obtained from patients, we have obtained a statement attesting to informed consent for all patients with or without neurosurgery in our department. Each sample was fixed in 4% formalin in phosphate-buffered saline (PBS) and processed for paraffin embedding. The samples were classified by neuropathologists in accordance with the WHO classification of brain tumors. Sections from non-neoplastic regions (NNRs) were purchased from BioChain Institute (Newark, NJ, USA). The study was performed in accordance with the tenets of the Declaration of Helsinki. All animal experiments were approved and performed in accordance with the animal care guidelines of Tokushima University. Cell viability assay. GSCs (1 × 10 3 cells/well) were plated in 96-well tissue culture plates. To enumerate viable cells, the conversion of WST-8 to formazan by metabolically active cells was quantified using WST-8 reagent (Dojindo, Osaka, Japan) on a microplate reader (Infinit F200 PRO, TECAN) at 450 nm. We used PBS-treated cells as the control to represent 100% viability and the percent viability was determined in each treatment.
Establishment of the animal model and assessment of anti-tumor effects. All experimental protocol was approved by Tokushima University institutional committee (No. T27-2) and carried out in compliance with the animal care guidelines of Tokushima University and the ARRIVE guidelines (PLoS Bio 8 (6) www.nature.com/scientificreports/ Six-week-old male C57BL/6 mice were subjected to inhalation anesthesia with isoflurane and a stereotactic apparatus was placed in the right brain. With a dental drill, a small hole was bored into the skull 2.0 mm lateral to the bregma. In a malignant glioma model with mouse GSCs, established by Sampetrean and Saya 20 , and Shibao, et al. 21 , GSC progeny cells (1 × 10 3 ) in 2 μl of Hank's balanced salt solution (Sigma-Aldrich) were injected into the right cerebral hemisphere 3 mm below the brain surface, using a 10-μl Hamilton syringe. To examine the anti-tumor effects of SDT, the mice were randomized and treated with 5-ALA, US, or SDT and compared to the non-treated control (Fig. 2). For SDT, a 0.2-ml solution of 5-ALA in PBS was intraperitoneally injected at a dose of 200 mg/kg body weight. Three hours later, the mouse right brain was placed on the stereotactic apparatus and subjected to US imaging (1 MHz, 2 W/cm 2 for 2 min) under inhalation anesthesia. On day 1 after SDT, apoptosis induction by SDT was confirmed and tumor volume on days 3, 7, and 14 and the survival rate were analyzed. Mice were euthanized and their brains were sliced on a brain slicer matrix at 1.0-mm intervals and the tumor volume, represented by the GFP-positive area, was microscopically determined (Keyence BZ-X710, Osaka, Japan).
In addition, to assess the effect of combination therapy of SDT and celecoxib, another set of mice were randomized and treated with vehicle, celecoxib, SDT, or a combination of celecoxib and SDT (Fig. 4). Celecoxib, lysed with dimethyl sulfoxide (DMSO) and hydroxypropyl-β-cyclodextrin (HBC), was injected (i.p.) at a dose of 10 mg/ kg consecutively after mouse GSC implantation. Vehicle controls received equivalent doses of DMSO/HBC and normal saline at the same dosing schedule. To validate the efficacy of celecoxib during SDT, tumor volume on day 14 and the survival rate during the observation period were assessed in each group (Fig. 4) as described above.
Measurement of cellular PpIX levels and SDT. Luminescence was measured 1, 2, 3, 6, 12 or 24 h after treatment of human GBM U251 cells with 1 μM 5-ALA in human GBM U251 cells and GSCs, using the image analyzer in the BZ-X710 microscope (KEYENCE). Thereafter, the viability assessed for 3-48h of GSCs upon combination therapy with SDT and US (1 MHz, 2 W/cm 2 for 2 min) at 3 h after 5-ALA treatment was assessed after 24 h.
Celecoxib (Sigma-Aldrich, PHR1683) was dissolved in DMSO and supplemented in the culture medium at a final concentration of 60 μM. After 3-h incubation of cells in medium supplemented with 1 μM 5-ALA (SBI ALA promo, Tokyo, Japan), the medium was replaced with fresh complete medium, and the 96-well plate was exposed to LED irradiation (630 nm, 80 mW/cm 2 ) for 5 min. The LED light spot was an equally illuminated rectangular spot encompassing the entire culture plate. SDT was performed using the ultrasonic generator UST-770 (ITO Co. Ltd., Tokyo, Japan). In the mouse GSC-bearing glioma model, the tumor area was extracted 3 h after treatment with 200 mg/kg 5-ALA and PpIX levels were analyzed as previously described 20 .
Quantitative real-time PCR (qRT-PCR). Total RNA was isolated and extracted using the MagNA Pure