Radiation therapy is often used for destroying cancer cells and as palliative therapy [1
], but often induces radiation resistance; indeed, high-dose radiation may cause acute and chronic severe side effects, including lethal radiation-induced pneumonitis and fibrosis [3
]. Therefore, the development of radiation sensitizers without side effects is urgently needed to overcome radiation resistance and decrease radiation-related lethal severe side effects in patients with refractory cancer.
Growing tumors result in complex tumor microenvironments with low-oxygen areas because of the severe structural abnormalities of tumor microvessels [5
]. Hypoxia restricts the physiological functions of organs, tissues, and cells, including cancer cells. To survive in hostile hypoxic environments, hypoxia-inducible factors (HIFs) can function as essential regulators of cellular oxygen homeostasis and hypoxia adaptation in tumor tissues [7
]. HIFs are heterodimers, including one of three HIF-α subunits, HIF-1α, HIF-2α, or HIF-3α (and one constitutive HIF-1β subunit known as ARNT), which form HIF-1, HIF-2, and HIF-3 transcriptional complexes, respectively [10
]. Among their subunits, HIF-1α has been well studied and is known to activate several downstream genes transcriptionally related to angiogenesis, anaerobic metabolism, and resistance to radiation in cancer tissues [11
]. Many researchers have reported the importance of an accumulation of HIF-1α in clinical cancer tissues, causing radiation resistance, cancer aggressiveness, and poor prognosis [10
]. However, an HIF-1α-targeting strategy may induce deleterious side effects in non-cancerous tissues [12
]. Therefore, the development of therapeutic tools targeting HIF-1α without side effects is urgently needed for refractory cancers with radiation resistance.
Previously, we reported the development of oxygen nanobubble (NBO2
) water in the single-nanometer range, which was produced by Sigma Technology Inc. (Hitachinaka, Japan) [15
]. Our in vitro analysis clearly showed that NBO2
water suppressed hypoxia-induced radiation resistance in cancer cell lines via the downregulation of HIF-1α accumulation under hypoxic conditions. Notably, NBO2
water did not affect the in vitro cell viability and radiation reactivity of either cancer or non-cancerous cell lines under normoxic conditions, suggesting that NBO2
water might have the potential to overcome hypoxia-induced radiation resistance without side effects, even in an animal model. Therefore, we designed this study to examine the effects of NBO2
water in a tumor-bearing mouse model.
The purpose of this study was to elucidate whether NBO2 water could act as a radiosensitizer via regulation of HIF-1α in a tumor-bearing mouse model and whether it has any side effects consistent with previous in vitro experiments.
Radiation treatments can cause acute tissue effects (mucositis, radiodermatitis, pneumonia, and others) based on DNA damage, inflammation, and vascular depletion, and late adverse tissue effects (hardening of irradiated organs such as lung or breast tissues; malabsorption, small bowel narrowing, radiation-induced secondary carcinogenesis, and others) due to fibrosis and vascular changes associated with ischemia and hypoxia. Among these various changes in the radiation treatment process, hypoxia/ HIF-1α levels have been involved in fibrosis and vascular changes [4
]. HIF-1α activates TGF-β signaling and integrin expression in stromal cells, causing fibrosis in vital organs. Furthermore, hypoxia/HIF-1α induces the secretion of CXCL12, VEGF, and ANG1/2, leading to vascular changes, such as fragile tumor angiogenesis, through the recruitment of vascular endothelial cells. Therefore, the therapeutic strategy of suppressing hypoxia/HIF-1α in tumor tissues using NBO2
water may affect both radiation sensitivity and radiation-related side effects.
In this study, we found that NBO2
water sensitized radiation reactivity via suppression of radioresistance-related HIF-1α and CA IX expression in a tumor-bearing mouse model, consistent with our previous in vitro data [15
]. Notably, NBO2
water administration for 28 days downregulated the accumulation of HIF-1α in the xenografted tumors and did not affect the vital organs or biochemical examinations in healthy wild-type mice. We did not observe obvious side effects (appearance, body weight alteration, or treatment-related deaths as acute side effects) between radiation with or without NBO2
water treatment. This may be because our radiation settings had already been adjusted to complete the animal experiments. As our study was conducted to evaluate the combined effects of NBO2
water and radiation in a tumor-bearing mouse model, we could not obtain data on the relationship between high-dose radiation-related late complications and NBO2
administration under our experimental conditions. Future studies are needed to elucidate the influence of NBO2
administration on radiation-related side effects. On the other hand, amifostine has been approved as a clinical radiation protector to reduce the radiation-related side effect in normal tissues [19
]. Therefore, we expect that amifostine combined with NBO2
water could allow for higher radiation doses, thereby enhancing the therapeutic effect without increasing radiation-related side effects.
Surgical operation, radiation, and anticancer drugs are recognized as essential treatment methods in current cancer care [20
]. However, some patients with poor general condition cannot undergo highly invasive treatments. In clinical practice, despite the existence of promising therapeutic options such as surgery or chemotherapy, patients with advanced or recurrent cancer and patients without tolerance to invasive treatments are often not provided with any other alternatives and rather offered the best supportive care [23
]. This study focused on NBO2
water, which suppressed intratumor hypoxia/HIF-1α and showed radio-sensitizing effects without side effects. Of particular interest to us was that the NBO2
water group, even the group with NBO2
water alone, showed a certain degree of antitumor effects compared to the control water group. As mentioned above, NBO2
water targets intratumoral hypoxia/HIF-1α, which is known to cause therapeutic resistance to radiation and various anticancer drugs [25
]. Thus, overcoming hypoxia/HIF-1α is a promising therapeutic strategy for hypoxia-related refractory cancer patients. If targeting intratumoral hypoxia/HIF-1α using NBO2
water without side effects is made possible, treatment of high-risk cancer patients who cannot tolerate surgery or systemic chemotherapy would be feasible. We hope that treatment with NBO2
alone or in combination with NBO2
water with dose-reduced chemotherapy or radiation will improve the quality of life and prolong the prognosis in cancer patients who cannot tolerate highly invasive treatments.
Free drinking water was selected as a general and straightforward administration route of NBO2 water in the animal model. However, oral intake is often impossible for patients with advanced cancer with poor conditions. Therefore, in the future, we would like to investigate the relationship between the hypoxia/HIF-1α suppression effect and the route of administration of NBO2 water, such as intravenous or intraperitoneal injections.
4. Materials and Methods
4.1. Production of NBO2 Water
water with ultrafine oxygen bubbles of single-nanometer size was provided by Sigma Technology Inc. and produced by their original device based on patented technology [15
]. Briefly, oxygen and pure water were mixed at 0.4 MPa and pushed out of a nozzle. Oxygen-containing water collided at a high speed to produce the NBO2
water with single nanometer-sized oxygen bubbles.
4.2. Cell Lines
Human lung cancer cell line EBC-1 and human colon cancer cell line HCT116 were purchased from the RIKEN Cell Resource Center of Biomedical Research (Tsukuba, Japan). Culture media were prepared using RPMI1640 powder (Wako, Osaka, Japan) and dissolved in control water (water) or water with ultrafine oxygen bubbles (NBO2). Cells were cultured in filtered (0.22 μm) RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin antibiotics and incubated at 37 °C and 5% CO2. For hypoxia induction, the cells were incubated under hypoxic conditions (1% O2) using a BIONIX-1 hypoxic culture kit (Sugiyamagen, Tokyo, Japan).
4.3. Hypoxia Detection by Fluorescent Hypoxic Probe
Cells were seeded at a density of 4 × 103/100 μL per well in 6-well plates. After 48 h, the culture medium was changed to media with or without NBO2 and the plates were incubated with 1 μM hypoxic probe MAR (Goryo Chemical, Inc., Sapporo, Japan) under normoxic or hypoxic conditions. After 24 h of incubation, the fluorescence signal was detected using an All-in-One BZ-X710 fluorescence microscope (KEYENCE Corporation).
4.4. Protein Extraction and Western Blotting
Protein extraction was performed using lysis buffer (10% glycerol, 10 mM Tris-HCl (pH7.5), 1 mM EDTA, 400 mM NaCl, 0.5% NP40, 4 μg/mL aprotinin, PMSF, proteasome inhibitor MG-132, and 1mM DTT). Total protein (10 µg) was electrophoresed on a 10% polyacrylamide gel and then electroblotted at 300 mA for 90 min on a nitrocellulose membrane (Invitrogen, Waltham, MA, USA). Western blotting was used to confirm the expression of HIF-1α and β-actin proteins, which were detected using anti-HIF-1α rabbit polyclonal antibody (1:1000) (Cell Signaling Technology, #3716, Danvers, MA, USA) and β-actin mouse monoclonal antibodies (1:1000) (A5316; 1:1000; Sigma, St. Louis, MO, USA), respectively. β-actin was used as a loading control. The signals were detected using the ECL Select Western Blotting Detection System (GE Healthcare Life Sciences, Chicago, IL, USA) and Image Quant LAS 4000 (GE Healthcare Life Sciences).
4.5. Safety Assessment of the NBO2 Water in Mice
Six-week-old female BALB/c mice were used in this experiment. Control water or NBO2 water was administered via free oral drinking for 28 days. The evaluation of the general condition and body weight in each group was continued until day 28 and the vital organs were harvested for macroscopic and histological evaluation on day 28. Biochemical examinations were performed by Oriental Yeast Co., Ltd., Tokyo, Japan.
4.6. Nude Mouse Xenograft Model
Six-week-old female BALB/c nu/nu nude mice were subcutaneously injected with 5 × 106 EBC-1 and 5 × 106 HCT116 cells. Tumor volume was calculated using the following formula: volume = S2 × L/2, where S is the shortest length of the tumor (mm) and L is the longest length of the tumor (mm). Tumor volumes and body weights were determined every 4 days. After the tumor volume reached 100 mm3, the mice were randomly divided into four groups: water group as control, NBO2 water alone, radiation alone, and radiation plus NBO2 water. Each group consisted of six mice. Water or NBO2 was administered via free oral drinking for 28 days. Radiation of 3 Gy (HCT116) or 4 Gy (EBC-1) was administered on days 4 and 8. Tumor volume and mouse body weight were evaluated until day 28, and xenograft tumors were harvested for further analysis on day 28. The animal experiments were approved by the Review Committee on Animal Use of Gunma University (approval number 15-046, 7 September 2015).
4.7. Radiation Treatment against Xenograft Tumors
We performed local radiation treatment of xenograft tumors with 6 mm lead shielding using a subcutaneous xenograft mouse model. Radiation was performed at a dose of 3 or 4 Gy for HCT116 and EBC-1 cell lines, respectively (on days 4 and 8) using a TITAN-225S (Shimadzu Mectem, Inc., Tokyo, Japan) at 200 kV and 14.6 mA with Al 0.5 mm and Cu 0.5 mm filtration at a distance of 47.3 cm from the target.
4.8. Detection of Hypoxic Conditions in Xenograft Tumors
Pimonidazole was used as a marker of tumor hypoxia. Following the manufacturer’s instructions, pimonidazole (Hypoxyprobe-1 Omni Kit, #HP3-100) at a dose of 60 mg/kg body weight was administered to the tumor-bearing mice intraperitoneally 30 min before tumor excision. Hypoxic tumor regions were detected immunohistochemically. Hematoxylin was used as a counterstain. Area fractions showing pimonidazole staining were identified as hypoxic areas.
Four-micron sections were cut from paraffin blocks of resected xenograft tumors and each section was mounted on a silane-coated glass slide, deparaffinized, and soaked in 0.3% H2O2/methanol for 30 min at room temperature to block endogenous peroxidases. The sections were then heated in boiling water using an Immunosaver (Nishin EM, Tokyo, Japan) for 45 min at 98 °C. Non-specific binding sites were blocked by incubating the sections with Protein Block Serum-Free (DAKO, Santa Clara, CA, USA) for 30 min. Anti- HIF-1α antibody (Novusbio, #NB100-479, Littleton, Co, USA), anti-pimonidazole antibody (Hypoxyprob-1 Omni Kit, #HP3-100), and CA IX antibody (Abcam, ab15086, Cambridge, UK) were each applied in dilutions of 1:100 (HIF-1α and pimonidazole) and 1:500 (CA IX) for 24 h at 4 °C. The primary antibody was visualized using the Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. The chromogen 3,30-diaminobenzidine tetrahydrochloride was used as a 0.02% solution containing 0.005% H2O2 in 50 mM ammonium acetate–citrate acid buffer. The sections were lightly counterstained with Mayer’s hematoxylin and mounted. Negative control specimens were incubated without primary antibodies and no detectable staining was evident.
4.10. Statistical Analysis
Data are expressed as the mean ± standard deviation for continuous variables. The differences between groups were compared using JMP Pro 15 software (SAS Institute, Cary, NC, USA). The Mann-Whitney U-test and Steel–Dwass test were used to compare the groups. A probability (p) value of less than 0.05 was considered statistically significant.