Mild thermotherapy and hyperbaric oxygen enhance sensitivity of TMZ/PSi nanoparticles via decreasing the stemness in glioma
Glioma is a common brain tumor with a high mortality rate. A small population of cells expressing stem-like cell markers in glioma contributes to drug resistance and tumor recurrence.
Porous silicon nanoparticles (PSi NPs) as photothermal therapy (PTT) agents loaded with TMZ (TMZ/PSi NPs), was combined with hyperbaric oxygen (HBO) therapy in vitro and in vivo. To further investigate underlying mechanism, we detected the expression of stem-like cell markers and hypoxia related molecules in vitro and in vivo after treatment of TMZ/PSi NPs in combination with PTT and HBO.
NCH-421K and C6 cells were more sensitive to the combination treatment. Moreover, the expression of stem-like cell markers and hypoxia related molecules were decreased after combination treatment. The in vivo results were in line with in vitro. The combination treatment presents significant antitumor effects in mice bearing C6 tumor compared with the treatment of TMZ, PTT or TMZ/PSi NPs only.
These results suggested the TMZ/PSi NPs combined with HBO and PTT could be a potential therapeutic strategy for glioma.
KeywordsGlioma Photothermal therapy Hyperbaric oxygen Porous silicon nanoparticles Stemness
cancer stem cell
adipose-derived mesenchymal stem cell
transmission electron microscope
basic fibroblast growth factor
epidermal growth factor
cell counting kit 8
quantitative real time polymerase chain reaction
glioma stem cells
vascular endothelial growth factor
hypoxia inducible factor-1
hematoxylin & eosin staining
Gliomas are the most common brain tumors with a high mortality rate found in humans in Europe and the US [1, 2]. Surgery followed by chemotherapy or radiotherapy is the standard therapy strategy for glioma . However, patients still exhibit a poor prognosis, with a mean survival time lower than 15 months [4, 5]. Increasing evidences have indicated the existence of a small population of glioma cells with stem cell properties, referred to as glioma stem-like cells, which contribute to therapy resistance, poor prognosis, and tumor recurrence [6, 7].
Hypoxia is an important characteristic of solid tumors and plays a significant role in stem-like cell development . Hypoxia can lead to breast cancer stem cell (CSC) expansion . Hypoxia significantly favored ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and SOX2 . Hypoxia is also a distinct feature in glioma. In the absence of serum, hypoxia induced C6 cells to dedifferentiate to a CSCs phenotype . Clinically used anti-tumor drug TMZ against glioma increases the medial survival of the patient for only several months, which may happen due to chemoresistance under the hypoxia related environment [12, 13]. HBO could overcome the hypoxia microenvironment in solid tumor and increase the sensitivity of tumor cell to chemotherapy [14, 15].
Thermotherapy has long been used as a treatment method for cancer, but it is difficult to treat patients without damaging healthy cells. Among different thermotherapies, mild thermotherapy (40–44 °C) can enhance the drug effects and is more acceptable by patients [16, 17]. Heating rodent tumors at 40–42 °C was found to increase the blood flow and partial pressure of oxygen in the tumors. The increased blood flow caused by mild heat may improve the delivery of chemotherapy drugs to tumor cells . Combining photothermal therapy (PTT) with chemotherapy is an interesting research direction in nano-medicine . Nanodrug-mediated thermotherapy can eliminate CSCs . Porous silicon (PSi) can be utilized as a therapeutic agent that generates mild heat upon exposure to NIR light . Thermotherapy based on PSi under NIR light irradiation in combination with chemotherapy is an efficient technique to reduce cancer cells resistance [22, 23, 24]. Here, we hypothesized that the mild thermotherapy caused by PSi combined with HBO could increase the oxygen supply in the tumors and enhance chemosensitivity in tumor stem cells.
The boron-doped p-type silicon wafers were obtained from Virginia Semiconductor, Inc. (VA, USA) . Drug TMZ was purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). Other chemicals were of analytic grade. The experimental hyperbaric oxygen (HBO) animal chamber was purchased from Weifang Huaxin Oxygen Industry Co., Ltd (Weifang, China).
Rat glioma C6 cell line was preserved in our lab. Glioma stem cell line NCH-421K used in this study was kindly provided by the Neurosurgery Laboratory of Tongji Medical College, Huazhong University of Science and Technology. BALB/c-nude mice (male, 16–18 g) were purchased from Beijing Wei Tong Li Hua experimental animal Co., Ltd (Beijing, China). The animal protocol was approved by the Animal Experimentation Ethics Committee of College of Life Science and Technology, Huazhong University of Science and Technology.
Preparation of PSi and TMZ/PSi NPs
Preparation of PSi NPs was performed as described previously . TMZ/PSi NPs were prepared according our report with a little modification . Briefly, TMZ dissolved in 5% phosphoric acid/methanol solution was added into the NPs under stirring overnight. TMZ-loaded PSi NPs (TMZ/PSi NPs) were obtained by ultrafiltration centrifugation and washed three times. Ultraviolet spectrophotometer was used to measure the amount of TMZ/PSi NPs.
Characterization of PSi and TMZ/PSi NPs
The size and morphology were characterized by TEM (JEM-2010; JEOL, Tokyo, Japan). An 808 nm NIR laser (Changchun radium Photoelectric Technology Co., Ltd., China) was used to investigate the photothermal conversion capability of PSi and TMZ/PSi NPs.
Drug release test
HBO therapy was performed at a pressure of 2.5 ATM according to our previous work .
Photothermal therapy (PTT) treatment
Photothermal therapy (PTT) treatment was conducted with 808 nm NIR laser at 0.6 W/cm2 for 20 min. During irradiation, the temperature was monitored using thermography (E50, FLIR Systems Inc, USA). After treatment with TMZ/PSi, PTT was carried out for 20 min followed by HBO treatment in the combination treatment (PTT + HBO) groups.
NCH-421K cells were cultured in DMEM/F12 medium (Hyclone) supplemented with 20% BIT (Stemcell Technologies), 10 ng/mL basic fibroblast growth factor (bFGF, Peprotech), 10 ng/mL epidermal growth factor (EGF, Peprotech). C6 cells were cultured with DMEM medium (Hyclone) supplemented with 10% FBS (Gibco).
Cell viability assay of TMZ
Different dose of TMZ (50, 100, 200, 400, 800, 1600 μM) were used to test cell viability. NCH-421K or C6 Cells (8 × 103 cells/well) were plated into 96-well plates and incubated at 37 °C with 5% CO2 overnight before adding TMZ. Cell viability was detected at 24, 48 and 72 h using the CCK-8 after different treatments respectively.
Evaluation of TMZ/PSi on cell viability under different treatments
One critical toxicity dose of TMZ (400 μM) was used to assess the TMZ/PSi NPs effects on cancer stem-like cells. Cells were divided into PSi, TMZ or TMZ/PSi NPs (with or without PTT) groups at the different oxygen concentrations, respectively. NCH-421K cells and C6 cells were cultured in normoxia and then treated under 100% O2 for 90 min/day after adding drugs. After incubation for 72 h, SRB method was used to evaluate the cell viability .
Assay of colony formation
NCH-421K cells at a density of 2.5 × 105 cells/well were plated into 12-well plates and cultured overnight. Cells were treated with 100 μM TMZ or TMZ/PSi NPs for 24 h. Then the cells were harvested and countered, plated at a density of 2 × 104 cells/well into 24-well plates for shape observation and the surviving fraction in 24-well plates was taken a picture on day 1, 4 and 7. 1 × 102 cells/well was plated into 96-well plates for countering colony number and the number of tumorspheres in each well was recorded on day 7.
mRNA and Western-blot analysis
For qRT-PCR assays, cell and tumor tissue RNA was extracted according the manual of PrimeScript RT reagent Kit (Takara Biotechnology Co., Ltd., China) and Primers were present in Additional file 1: Table S1. The StepOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA, USA) was used. The resulting data were analyzed with the comparative cycle threshold (CT) value for relative gene expression quantification relative to GAPDH.
Cell proteins were extracted using normal method . After blocking, antibody against HIF-1α (Abcam, UK), Nestin, SOX2, VEGF and GAPDH (Proteintech, China), were added, respectively.
In vivo antitumor effects of combination treatment
To establish tumor bearing model, 3 × 106 C6 cells were subcutaneously inoculated into a nude mouse. When tumor volume was above 75 mm3, the mice were randomly divided into six groups: the control group, PSi group, TMZ group, PTT group, TMZ/PSi group, and TMZ/PSi combined with PTT and HBO group respectively. According to our previous report, HBO alone has no effects on C6 glioma . Therefore, HBO alone group was not included in this study. Then the mice bearing tumors were intratumor injected with TMZ or TMZ/PSi NPs (5 mg/kg TMZ) followed with PTT and HBO treatment on day 1, 4, 7, and 10 in the related groups, respectively. The sizes of the tumors were recorded every 2 days. The formula: tumor volume (mm3) = 0.5 × length (mm) × [width (mm)]2, was used to calculate the tumor volume. At the end of animal experiment, the mice were sacrificed, and tumor tissues and other organs were collected.
Immunohistochemistry and mRNA analysis of tumor tissue
The immunohistochemistry analysis was performed. Rabbit polyclonal Nestin and SOX2 antibody (Servicebio, China) were used to stain stemness marker of the tumor tissue, respectively. Rabbit polyclonal HIF-1α, and VEGF antibody (Servicebio, China) were used to stain hypoxia marker and vascularization of the tumor tissue, respectively. Rabbit monoclonal Ki-67 antibody (Servicebio, China) was used for analyzing cell proliferation.
Tumor tissue RNA was extracted according the manual of PrimeScript RT reagent Kit (Takara Biotechnology Co., Ltd., China) for qRT-PCR assays.
The data were expressed as mean ± SEM. Statistical significance in all experiment was determined using one-way ANOVA followed by a Student’s test for multiple comparison tests. Statistical analysis was analyzed using statistical software (IBM SPSS Statistics 20, USA).
Results and discussion
Characterization, photothermal effect and in vitro drug release
Effects of TMZ and TMZ/PSi NPs in combination with PTT and HBO on stem-like cell viability
Furthermore, the effect of TMZ/PSi NPs containing 400 μM of TMZ with or without HBO and PTT treatments on GSCs and C6 cells was studied (Additional file 1: Figure S3). The decrease of cell viability treated with TMZ/PSi NPs was observed after combination treatment with PTT and HBO compared to that without PTT or HBO treatment (Fig. 2b). Similarly, after treatment with same dose of TMZ/PSi, cell viability decreased significantly compared to free TMZ alone. Compared with TMZ/PSi, the cell viability was decreased both in TMZ/PSi + HBO group (P = 0.318) and in TMZ/PSi + PTT group (P = 0.003), whereas the cell viability was more significantly decreased in TMZ/PSi + PTT + HBO (P = 0.000063) group than that in other groups. Consistent with results in NCH-421K cells, PTT and HBO increased the sensitivity of TMZ/PSi against glioma stem-like C6 cells (Additional file 1: Figure S4).
In our study, although the amount of TMZ loaded into PSi NPs is the same with free TMZ in the treatment, nanoparticle-delivered TMZ exerted much higher cytotoxicity. Thermotherapy was thought to increase drug uptake into cells . Hyperoxia can re-sensitize chemoresistant of human glioblastoma cells to TMZ . Moreover, toxicity of combination treatment on morphology of NCH-421K was significant too. Tumorspheres of NCH-421K were fragmented significantly after treating with TMZ/PSi + PTT + HBO compared to other groups for 72 h (Fig. 2c, d). Therefore, PTT and HBO-adjuvanted with TMZ/PSi NPs enhance the effects on viability of glioma stem-like cells.
Colony formation of NCH-421K cells after different treatments
mRNA and protein analysis in NCH-421K cells after different treatments
Effects of TMZ/PSi combined with PTT and HBO on C6 cells
Antitumor effects of TMZ/PSi NPs in combination with PTT and HBO
Glioma is a solid tumor characterized with hypoxia environment . Tumor stem cells exhibit in hypoxic niches are known to be a key cause of the progression, metastasis and relapse . Efficacy of hyperbaric oxygen therapy in combination with mild heat improved the anti-tumour effects of carboplatin . In light of this, mild PTT and external nanodrug in treating glioma in vivo combined with HBO was performed. In this study, we choose C6 stem-like cells to establish tumor bearing mouse model to investigate the effects of combination treatment.
As shown in Fig. 6f, the proliferation of tumor cell (brown color) was relatively lower in the combination treatment than that in the other groups (Additional file 1: Figure S8). After H&E staining of tissues, no evident pathological changes were found (Additional file 1: Figure S9). Therefore, combination treatment of TMZ/PSi with PTT and HBO is a potential strategy for glioma therapy.
Expression of tumor stem-like markers in vivo
How to reduce stemness and increase the drug sensitive is important for glioma therapy. In this study, HBO and mild thermotherapy can perfectly adjuvant the TMZ/PSi to amend the hypoxia environment in tumor and enhance TMZ/PSi therapy on stem-like cells in glioma. The combination treatment would be a potential treatment for glioma therapy.
XZ designed and carried out experiments, analyzed data and wrote the manuscript. XT and LJ conducted in vivo treatment and imaging analysis. QW, YZ and XY supervised entire project and involved in the designing of all experiments and revised the manuscript. ZZ and MH performed the PSi preparation. YL and XB performed cell culture. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.
Consent for publication
All authors agree to be published.
Ethics approval and consent to participate
The animal studies were approved by the Animal Experimentation Ethics Committee of College of Life Science and Technology, Huazhong University of Science and Technology. All procedures were carried out strictly with the animal care guidelines of the Science and Technology Department of Hubei Province.
This work was supported by The National Natural Science Foundation of China (No. 81573013), National Basic Research Program of China (2015CB931802) and National Natural Science Foundation of China (81627901).
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