Combination of sonodynamic with temozolomide inhibits C6 glioma migration and promotes mitochondrial pathway apoptosis via suppressing NHE-1 expression

doi:10.1016/j.ultsonch.2017.05.013

Ultrasonics Sonochemistry

Volume 39, November 2017, Pages 654-661

https://doi.org/10.1016/j.ultsonch.2017.05.013 Get rights and content

Abstract

Temozolomide (TMZ) was used for clinical postoperative or non-surgical chemotherapy patients. However, its effect remains unsatisfactory and gradually discovered that the presence of chemoresistance. To explore more effective therapy using TMZ, we investigate the effects of combination of application of TMZ together with Sonodynamic therapy (SDT), which is based on the ultrasonic activation of a sonosensitizer, with low toxicity, noninvasive, deeper penetrability and a promising approach for treating malignant glioma by inducing apoptosis on glioma cells in vitro. Sodium–hydrogen exchanger isoform 1 (NHE1), which enable glioblastoma cells to escape TMZ-mediated toxicity via increased H⁺ extrusion and affect the apoptosis effect on C6 glioma cells in vitro. The C6 cells survival rate and time point of TMZ resistance were tested by the Cell Counting Kit-8 (CCK8) viability assay. Western blot analysis results showed that the expression of NHE1 and matrix metalloproteinase-2 (MMP-2) protein obviously decreased by TMZ + SDT. Meanwhile, combined treatments enhanced the expression of mitochondrial pathway apoptosis proteins, as well as suppressed MMP-2 to weaken the migration ability in TMZ-resistant C6 cell line. These results provided the first evidence that the sensitivity of TMZ chemotherapy in resistant malignant glioma may be improved by SDT.

Introduction

Glioblastoma is the most common malignant primary brain tumor with median survival of 14 months and survival rate of less than 10% at 5 years after diagnosis [1], [2]. Current treatment of glioma included surgical resection, radiation and temozolomide (TMZ) chemotherapy which is the standard chemotherapeutic plan and prolonged survival rate which showed benefit for this malignant disease [3]. However, application of TMZ alone in the treatment was ineffective, intrinsic and acquired resistance to TMZ is still a major obstacle in the treatment of Glioblastoma for this agent. There are some well-recognized DNA repair enzyme and systems, including the highly expression of O6-methylguanine–DNA methyltransferase (MGMT) [4], base excision repair (BER) [5], mismatch repair (MMR) systems [6], and different pathways (eg, Akt, Notch3, PI3K, mTOR, STAT3, GD3) [7], [8], [9], [10], [11], [12]. Therefore, to understand the molecular mechanisms about TMZ resistance and therapeutic strategies which can overcome the chemotherapy resistance with less side effect are urgently needed.

Sodium–hydrogen exchanger isoform 1 (NHE1) protein is the important one of membrane transporter proteins family which is in charge of exchanging of one extracellular Na⁺ for one intracellular H⁺ [13], and maintain intracellular homeostasis of many tumor cells by extruding H⁺ [14]. Through our previous study, we have found that the resistance of glioma cells to TMZ is associated with high level expression of NHE-1 protein which maintain an alkaline tumor extracellular microenvironment and enhance their invasion by extruding H⁺, and obtain ability of invading surrounding normal brain tissues [15]. Sonodynamic therapy (SDT) has become an promising non-invasive method for anti-tumor treatment because of its high selectivity, low toxicity, deep penetration and effectivity to a certain degree. Up to now, no studies have demonstrated that the effect of SDT to the expression of NHE-1 of TMZ-resistant gliomas and the combination of TMZ with SDT could effectively inhibit C6 cell proliferation and migration in resistant gliomas by affecting NHE1 protein expression, and the apoptosis mechanisms among them are needed to be further understood.

In this study, we detected that SDT inhibited the expression of NHE1 and then enhanced C6 glioma cell sensitive to TMZ-mediated apoptosis by decreasing NHE1 protein expression. TMZ chemotherapy combined SDT was not only reduced NHE1 expression which may be associated with the inhibition of proliferation and migration, but also regulated the function of mitochondrial pathway apoptosis proteins to kill C6 cells. Taken together, these new findings demonstrate that SDT exposure enhanced apoptosis in C6 cells which were treated by TMZ chemotherapy at the same time through downregulation of MMP-2 and NHE1 protein expression, suggesting a potential therapeutic strategy for the anti TMZ-resistant effect and enhancing treatment of gliomas effectively.

Section snippets

Cell cultures

The C6 glioma cell line was obtained from the science experiment center of Harbin Medical University, Harbin, P. R. China, and cultured in RPMI 1640 medium (Hyclone, thermo) supplemented with 10% fetal bovine serum (PAN Biotech), 100 U/ml penicillin and 100 μg/ml streptomycin incubated in a water-jacketed incubator (3111, Thermo scientific) at 37 °C with 5% CO2 + 95% air. When the C6 cells in the exponential phase of growth at the bottom of the 75 cm² culture bottle (Corning), trypsinized and

SDT or TMZ inhibits proliferation of C6 glioma cells

To assess the apoptosis potency of SDT in vitro, its inhibitory rate on proliferation of C6 glioma cells was determined by CCK8 assay at different time points post SDT treatment (0–24 h). It shows that survival rates are 96.8 ± 7.9%, 94.5 ± 6.6%, 61.1 ± 13.3%, 30.1 ± 4.4%. 28.5 ± 2.9%, 26.3 ± 4.1%, 25.4 ± 4.2%, 19.3 ± 3.8% after 1, 2, 4, 6, 8, 12, 18, 24 h insonation respectively. The inhibition rate was sharp decreased occurred only when the exposure time was from 3 h extended to 6 h, which was the median

Discussion

Although Temozolomide has been considered as an effective chemotherapy for glioma patients, unsatisfactory killing effects exhibited in the previous work [22]. From we known, during chemotherapy there exists TMZ resistance which is a complex process involving interactions among related cellular ions proteins or pathways, tumor cells, and their microenvironment [23]. There is a significant challenge to put fundamentals into clinical trails. Our previous findings that NHE1 has been proved to

Conclusions

We demonstrated that SDT suppress NHE1 expression to regulate intracellular and extracellular microenvironment and in response to TMZ-mediated apoptosis via the mitochondrial pathway. Inhibition of MMP2 activity and affection of mitochondrial related apoptosis proteins increased the sensitivity of C6 cells to the cytotoxic effects of TMZ in vitro. Considering the remarkable effectivity of HMME-SDT in chemoresistant C6 cells, it will be widely used to improve the sensitivity of resistant gliomas

Conflict of interest

The authors declare no conflict of interest.

Acknowledgement

This work was financially supported by the National Natural Science Foundation of China (Grant No. 61575058); Institute of science and technology of Heilongjiang province, China (Grant No. 201609); The Science and Technology cooperation Project Foundation of Heilongjiang Medical Science Institute (Grant No. ys14c11); Self-made topic Foundation of Heilongjiang Scientific Research Institute (Grant No. 201609 and 201405).

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Combination of sonodynamic with temozolomide inhibits C6 glioma migration and promotes mitochondrial pathway apoptosis via suppressing NHE-1 expression

Abstract

Introduction

Section snippets

Cell cultures

SDT or TMZ inhibits proliferation of C6 glioma cells

Discussion

Conclusions

Conflict of interest

Acknowledgement

Cancer Lett.

Tumor Biol.

Ultrason. Sonochem.

Ultrasonics

Cancer Lett.

Ultrason. Sonochem.

Cytotherapy

Curr. Opin. Cell Biol.

Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma

CA Cancer J. Clin.

CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2006–2010

Neuro-Oncology