Contributed equally
Radiation therapy can result in severe side-effects, including the development of radiation resistance. The aim of this study was to validate the use of oxygen nanobubble water to overcome resistance to radiation in cancer cell lines via the suppression of the hypoxia-inducible factor 1-α (HIF-1α) subunit. Oxygen nanobubble water was created using a newly developed method to produce nanobubbles in the single-nanometer range with the ΣPM-5 device. The size and concentration of the oxygen nanobubbles in the water was examined using a cryo-transmission electron microscope. The nanobubble size was ranged from 2 to 3 nm, and the concentration of the nanobubbles was calculated at 2×1018 particles/ml. Cell viability and HIF-1α levels were evaluated in EBC-1 lung cancer and MDA-MB-231 breast cancer cells treated with or without the nanobubble water and radiation under normoxic and hypoxic conditions
Progress in peri-operative management and adjuvant therapy has led to the improved survival of patients with lung cancer (
Rapidly growing tumors located at a distance from the supporting vasculature results in the characteristic tumor microenvironment of low oxygen and nutrients (
High expression levels of HIF-1α have been reported to be associated not only with radiation resistance, but also with a poor prognosis of patients with lung cancer (
Strategies for the treatment of hypoxia to overcome resistance to radiation have included the development of several radiosensitizers, and methods for directly increasing blood oxygenation, such as pure oxygen or carbogen breathing, ozone therapy, hyperbaric oxygen therapy, hydrogen peroxide injections and the administration of suspensions of oxygen carrier liquids, including ultrafine oxygen nanobubble water (
In this study, we sought to validate a newly developed method to create oxygen nanobubble water in the single nanometer range, and to examine its effect on HIF-1α expression and hypoxia-induced resistance to radiation across multiple cancer cell lines.
Oxygen nanobubble water was prepared by a nanobubble water preparation device ΣPM-5 (bellows pump type) (
The nanobubble water was diluted 100-fold for measurement. Pure water with or without diluted nanobubble was rapidly frozen using Vitrobot Mark IV (FEI Co., Ltd., Hillsboro, OR, USA). The samples were embedded in amorphous ice for observation. The sample thickness was 200 nm. Nanobubbles embedded in amorphous ice at a sample temperature of about −193°C were directly observed using a cryo-transmission electron microscope Titan Krios (FEI Co., Ltd.). The electron beam used for observation is approximately 20 electrons/Å2 by the low-dose technique, and there is almost no increase in sample temperature during photography.
The EBC-1 human lung cancer cell line was purchased from the RIKEN Cell Resource Center of Biomedical Research (Tsukuba, Japan), and the MDA-MB-231 human breast cancer cell line and BEAS-2S non-cancerous human bronchial cell line were from the American Type Culture Collection (Manassas, VA, USA). Baseline culture medium was prepared using RPMI-1640 medium (Wako, Osaka, Japan), which was dissolved in water with or without the oxygen nanobubble. The cells were cultured in filtered (0.22
Cell viability was analyzed using the Cell Counting kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). The cells were seeded 4×103/100
Protein extraction was performed using lysis buffer [10% glycerol, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 400 mM NaCl, 0.5% NP40, 4
Following a 24-h pre-incubation, the EBC-1 and MDA-MB231 cells in 96-well plates were exposed to hypoxic (1% O2) and normoxic conditions with normal or oxygen nanobubble medium for 6 and 24 h, and then treated with radiation. The O2 concentration was continuously evaluated using O2 concentration measuring devices (Oxy-M O2 monitor, Jikco) in the bags. Cell viability was evaluated by CCK-8 assay after 72 h of incubation under normoxic conditions. An X-ray machine (Faxitron RX-650; Faxitron X-Ray LLC) with 100 kV, Al 0.3 mm filter was used as the radiation source for treatment.
The cells plated into 6-well plates with RPMI-1640 normal medium and incubated at 37°C in humidified 5% CO2 for 24 h. Follwing a medium change, using the medium with or without oxygen nanobubble, the hypoxic plates were directly incubated under hypoxic conditions (1% O2). After 24 h, the cells were irradiated at various doses (0, 2, 6, 10, and 14 Gy). After 72 h, the medium was changed to normal RPMI-1640 medium and the cells were monitored every 3 days until colonies were visible. The plates were rinsed with phosphate-buffered saline (PBS), and the colonies were fixed with 99.5% ethanol and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The colonies counted up to at least 50 cells after staining. The surviva fraction (SF) was calculated as the mean (number of colonies counted/number of cells plated)/plating efficiency.
For continuous variables, the data are expressed as the means ± standard deviation. Cell viability between the treatment groups was analyzed using JMP software (SAS Institute, Cary, NC, USA). A Student’s t-test was used to compare the oxygen nanobubble group with the control group. A probability P-value <0.05 was considered to indicate a statistically significant difference.
The schematic representation of ΣPM-5 is shown in
The high velocity water through the small hole will collide with the water from the other small hole placed horizontally. The energy of water collision was calculated as follows: 1/2 mV2 + 1/2 mV2 = mV2 (m, mass). The impact force was then calculated as follows: F = mV2/½ D (D, distance between the small hole). The collision energy force with the distance adjusted at 2 mm is shown in
The oxygen nanobubble-containing water was created by ΣPM-5 from pure water and oxygen. The nanobubble water was then diluted to 1:100 and embedded in amorphous ice. The samples were then observed using a cryo-transmission electron microscope. Pure water was used as the control. As shown in
The overexpression of HIF-1α correlates with the resistance of cells to radiation (
We treated the EBC-1 and MDA-MB-231 cells with radiation at doses of 0, 2, 6, 10, and 14 Gy to examine their sensitivities to radiation under both normoxic and hypoxic conditions (
The viability of the EBC-1 lung cancer, MDA-MB-231 breast cancer and non-cancerous BEAS-2B bronchial cells was not affected by treatment with oxygen nanobubble medium under normoxic conditions (
In this study, we produced oxygen nanobubble water at a single nanometer size using the nanobubble water preparation device, ΣPM-5. The characterization of the oxygen nanobubbles using a cryo-transmission electron microscope verified that the nanobubbles were at the single-nanometer range. Moreover, the results of
Nanobubble water refers to a liquid containing small bubbles typically with <200 nm in diameter (
Resistance to radiation causes serious complications for patients with lung cancer. This resistance has been reported to be induced by several pathways, including those associated with hypoxia, tyrosine kinase receptors, AKT serine/threonine kinases, DNA damage repair, developmental pathways, adhesion pathways and inflammation (
This study utilized our oxygen nanobubble water as a modulator of radiation sensitivity under hypoxic conditions. This nanobubble water included only water and single nanometer-range oxygen bubbles, with an average size of 2–3 nm, without any chemical compounds. Small size bubbles have some advantages, including high stability and high oxygen occupancy compared to larger ones. On the other hand, the continuous administration of a high oxygen concentration is known to induce oxygen toxicity due to the production of reactive oxygen species (
Neo-adjuvant radiation and chemoradiotherapy (CRT) have been considered as effective therapeutic tools to accomplish radical resection and down-staging in patients with lung cancer (
In conclusion, in this study, we developed and characterized pure oxygen nanobubble water, in the single nanometer range, without any additives other than water and oxygen. In our human cancer cell-based experiments, oxygen nanobubble water demonstrated the ability to protect against hypoxia-induced radiation resistance through the suppression of HIF-1α. Our additive-free single nanometer-range oxygen nanobubble water may prove to be a promising modulator against hypoxia/HIF-1α-mediated radiation refractory cancers, although further studies are required in order to test its safety and effectiveness. Future studies are warranted to examine the preventative and therapeutic potential of our nanobubble water in mouse tumor models of radiation resistance. Additionally, an important challenge for future experiments will be to validate the stability of nanobubbles
This study was supported in part by the Japan Society for Promotion of Science (JSPS) Grant-in Aid for Scientific Research (Grant nos. 15K10085 and 22591450). A part of this study was supported by Osaka University Microstructural Characterization Platform as a program of ‘Nanotechnology Platform’ of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. The authors are grateful to Mr. M. Nagata and Mr. K. Yoshiura in P.D.C.A. Inc. Japan for opening up new markets.
The authors M.I., N.G., T.Y., T.A., T.N., T.A., R.K., K.A., H.Y., H.K. and M.K., declare that they have no competing interests. Y.T., K.T., and K.H. are co-inventors of a patent (Tachibana Y, Tachibana K, Harada K, Sasajima S, Tamahashi K, Honma K, Matsumoto Y: METHOD, A BUBBLE GENERATING NOZZLE, AND AN APPARATUS FOR GENERATING MICRO-NANO BUBBLES JP.5555892, JP/PCT/2013/066902, Filed August 29, 2013; issued July 23, 2014), which has been filed in relation to the formulation of nanobubble described in the study. Y.T., K.T. and D.Y. are co-inventors of a patent (Yamannouchi D, Tachibana Y, Tachibana K: AQUEOUS SOLUTION CAPABLE OF BEING ADMINISTERED TO LIVING BODY, AND METHOD FOR PRODUCING SAME. WO 2017195852. JP/PCT2017/017779, Filed November 05, 2017) (Yamanouchi D, Tachibana Y, Tchibana K: AQUEOUS SOLUTION CONTAINING OZONE NANOBUBBLES, METHOD FOR PRODUCING SAME AND UTILIZATION OF AQUEOUS SOLUTION CONTAINING OZONE NANOBUBBLES. WO 2017199827. JP/PCT/2017/017781. Filed November 05, 2017.), which has been filed in relation to the formulation and application of nanobubble described in the study.
ΣPM-5 nanobubble generator. (A) Schematic representation of ΣPM-5. Water was pumped into the pressure tank where water and oxygen were mixed at 0.4 MPa. The oxygenated water was then pushed out through the small holes in the nozzle. (B) The schematic representation of the nozzle. Two small holes were placed horizontally in the nozzle. The pressurized and oxygenated water pushed out from these holes will collide to create the nanobubbles.
Speed and energy of water in the ΣPM-5 nozzle. (A) The calculated speed of water collision based on the diameter of the small holes in the nozzle. Flow velocity through the small holes (blue) and the collision speed of water in the nozzle (red) are shown. (B) The calculated collision energies of water in the nozzle are shown.
Characterization of nanobubbles produced by the ΣPM-5 device. (A) Representative image of amorphous ice prepared from pure water by cryo-transmission electron microscopy. No contrast from nanobubbles appears in the image. (B) Representative image of oxygen nanobubble water. Nanobubbles are visible as darker spots in the image. The area encircled in red highlights isolated nanobubbles, and the area encircled in yellow highlights linear arrangement of nanobubbles. Scale bar, 50 nm.
Oxygen nanobubble water suppresses HIF-1α accumulation in hypoxic cancer cells. (A) HIF-1α and HSC70 protein expression in the EBC-1 lung cancer cell line and MDA-MB-231 breast cancer cell line was evaluated by western blot analysis after 6 and 24 h of exposure ot hypoxia. Oxygen nanobubble medium clearly suppressed HIF-1α induction under hypoxic conditions. (B) Hypoxia-induced radiation resistance was validated in both the EBC-1 and MDA-MB-231 cells. DW, normal medium; O2, oxygen nanobubble medium.
Oxygen nanobubble medium reverses hypoxia-induced radiation resistance in EBC-1 lung cancer and MDA-MB-231 breast cancer cells. (A) Cell viability assay showed that oxygen nanobubble medium suppressed hypoxia-induced radiation resistance in EBC-1 cells. A similar effect was observed with the MDA-MB-231 cells, although this was not significant; however, a similar tendency was validated. (B) Clonogenic assay revealed that oxygen nanobubble medium suppressed the hypoxia-induced resistance of EBC-1 and MDA-MB-231 cells to radiation. DW, normal medium; O2, oxygen nanobubble medium.
Oxygen nanobubble medium treatment under normoxic conditions is non-toxic to cancer and non-cancer cell lines. (A) CCK8 assay revealed that oxygen nanobubble medium did not reduce the viability of EBC-1 lung cancer, MDA-MB-231 breast cancer, or non-cancerous BEAS-2B bronchial cells compared to normal medium. (B) Clonogenic assay revealed that oxygen nanobubble medium did not affect the radiation sensitivity of EBC-1, MDA-MB-231, or BEAS-2B cells compared to normal medium.