Oxygen nanobubbles revert hypoxia by methylation programming
Abstract
| Targeting the hypoxic tumor microenvironment has a broad impact in cancer epigenetics and therapeutics. Oxygen encapsulated nanosize carboxymethyl cellulosic nanobubbles were developed for mitigating the hypoxic regions of tumors to weaken the hypoxia-driven pathways and inhibit tumor growth. We show that 5-methylcytosine (5mC) hypomethylation in hypoxic regions of a tumor can be reverted to enhance cancer treatment by epigenetic regulation, using oxygen nanobubbles in the sub-100 nm size range, both, in vitro and in vivo. Oxygen nanobubbles were effective in significantly delaying tumor progression and improving survival rates in mice models. Further, significant hypermethylation was observed in promoter DNA region of BRCA1 due to oxygen nanobubble (ONB) treatment. The nanobubbles can also reprogram several hypoxia associated and tumor suppressor genes such as MAT2A and PDK-1, in addition to serving as an ultrasound contrast agent. Our approach to develop nanosized oxygen encapsulated bubbles as an ultrasound contrast agent for methylation reversal is expected to have a significant impact in epigenetic programming and to serve as an adjuvant to cancer treatment.
Introduction
Epigenetics plays an important role in regulating the expression of genes and corresponding cellular and molecular pathways1. DNA methylation (i.e. covalent addition of a methyl group to the C-5 carbon of the cytosine group in DNA) constitutes an important step in epigenetic programming and has been implicated in gene expression2–4. Addition of methyl groups to the cytosine derivatives in the DNA sequence can render the associated genes transcriptionally inactive5. DNA demethylation can lead to a hypomethylated state, but is counteracted by active DNA methylation to achieve a balanced methylation level6, 7. In neoplasia, the unregulated proliferation of cellular mass, without a sustainable rate of angiogenesis, leads to the development of hypoxic conditions inside the tumor8. In response to the pervasive hypoxic environment, several oncogenic processes occur in the cells, one of which is epigenetic alterations, resulting in an increase in tumor growth and survival of cancer cells9, 10. These alterations include global hypomethylation (primarily of oncogenes rendering them active)11, gene-specific hypermethylation (of CpG islands in the promoter regions of tumor suppressor genes, rendering them inactive), and inducing cell proliferation via dysregulated cell growth3. Ten-eleven translocation (TET) enzymes are a group of Fe2+ and α-ketoglutarate dependent dioxygenases that oxidize the conversion of 5mC to 5hmC and other downstream derivatives12–14. In mammalian cells, TET enzyme is the only characterized factor mediating the active DNA demethylation process12, 13. The activity of TET enzymes that have been shown to catalyze DNA demethylation is also limited by oxygen supply14. Although epigenetic therapy in the laboratory and clinics have largely focused on changes at gene promoters15, 16, epigenetic abnormalities such as DNA 5mC methylation across the genome17 are now being looked upon as diagnostic (tumor staging, outcome prediction, and malignancy) and therapeutic targets (epigenetic drugs). Regulation of the hypoxic microenvironment and epigenetic events are promising steps in anticancer therapies because several hypoxia18–20 and epigenetic15, 21– 23 targeted therapies have shown efficacy in the clinic24–26. Further, this view is also supported by findings on the effect of supplemental oxygen that weaken the hypoxia-driven pathways to improve cancer immunotherapy to promote tumor regression27.
Results
We hypothesize that alteration of DNA hypomethylation in hypoxic
cancer cells can be achieved by the delivery of oxygen to the cellular microenvironment with nanosize oxygen bubbles (Fig. 1a,b). In particular, our approach consists of encapsulating oxygen inside a sodium carboxymethylcellulose polymeric shell (Fig. 1a) to form nanobubbles 100–200 nm in diameter (Fig. 2a,b) by a crosslinking step33.
High resolution TEM micrographs show that the synthesized nanobubbles have a spherical shape (Fig. 2a) and contain an oxygen core
at the center and a ~50 nm carboxymethyl cellulose shell encapsulating
the nanobubble. Dynamic light scattering (DLS) shows that the size distribution of nanobubbles is in the range between 50–200 nm with a normal distribution centered around 70 nm (Fig. 2b). Further, sodium
carboxymethylcellulose is a commercially used, FDA-approved pharmaceutical excipient. Upon uptake, the acidic microenvironment
around and inside the tumor cells34 will cause the nanobubble shells to
disintegrate, thereby increasing the cellular oxygen levels. We expect the
release of oxygen inside the hypoxic cells will destabilize the hypoxia-
adaptive pathways and reprogram the cellular epigenome to attain normal DNA methylation levels, or cause global hypermethylation. The
targeted oxygen delivery is also expected to promote the regression of tumor growth in the hypoxic xenografted MB49 (bladder cancer) and
HeLa (cervical cancer) tumors.
Figure 1
| Oxygen Nanobubble configuration and mechanism of 5mC hypermethylation. (a) Schematic representation of oxygen nanobubble; (b) Oxygen nanobubbles (red arrows) localize within HeLa cells in the cytoplasm as well as the nucleus. Significantly enhanced dark field microscopy images are provided. Scale bar = 10 μm.
Figure 2
| In vitro characterization, hypoxia reprogramming, and imaging of nanobubbles. (a) TEM image of nanobubble with an oxygen compartment at the core surrounded by sodium carboxymethylcellulose shell. Scale bar = 50 nm. (b) Dynamic light scattering (DLS) size distribution of nanobubbles. (c) Ultrasound images of the corresponding signal generated from varying concentrations of nanobubbles (0–300 μg/mL). The contrast generated is due to oxygen trapped inside nanobubbles. Scale bar = 1 mm. (d) Graph displaying averaged mean grey scale intensity corresponding to increasing concentrations of nanobubbles (0–300 μg/mL). The results are mean values from three independent experiments. Error bars represent ± s.d. Note that there is a significant linear relationship between mean ultrasound gray scale intensity and concentration of nanobubbles (see Supplementary Fig. 1 ).
In addition to reoxygenation, we anticipate that oxygen nanobubbles will act as contrast agents for ultrasound imaging. Nanobubbles also possess unique light scattering and absorption characteristics as demonstrated using dark field microscopy (Fig. 1b) and in our prior work33. To test the ultrasound imaging intensity response to increasing concentration of nanobubbles, agarose gel molds were prepared with varying concentration of nanobubbles (Supplementary Fig. 1). B-mode ultrasound images of injected nanobubbles are shown in Fig. 2c and the corresponding mean gray scale intensity measurements are depicted in Fig. 2d. A linear increase in ultrasound grey scale imaging intensity (Fig. 2d and Supplementary Fig. 2) was observed as a function of nanobubble concentration (R2 = 0.95) in the concentration range evaluated (0–300 μg/mL). Further, HeLa cell cultures grown in tissue culture plates were incubated either with oxygen nanobubbles or phosphate buffer saline (PBS). After incubation for 24 h, the cell cultures were imaged using a 256-element 22–55 MHz ultrasound transducer with a center frequency of 40 MHz (for sample preparation and imaging details, see Methods Section). Images show that the spherical nanobubbles are suspended in the media as well as around the HeLa cells adhered to the bottom of the plate (Supplementary Fig. 3b) compared to the HeLa cell culture without nanobubbles (Supplementary Fig. 3a). The ultrasound gray scale imaging intensity in cell cultures with nanobubbles was significantly higher than the control without the addition of nanobubbles (Supplementary Fig. 4). The proposed design allows for customization of its size to accommodate various oxygen carrying capacity capable of generating different ultrasound contrast intensity.
The 5mC levels in the hypoxic regions have been shown to rapidly decrease, independent of the cell proliferation cycle35. In our experiments, DNA was extracted after 48 h of incubation following a factorial experiment design (data not shown) to ensure sufficient time for the methylation changes to take effect35. Colorimetric enzyme-linked immunosorbent assay (ELISA) was used to quantify 5mC levels36 after the exposure of nanobubbles to a hypoxic environment (Fig. 3a,c) and the 5mC levels were further validated using liquid chromatography-mass spectrometry (LC-MS/MS) (Fig. 3b,d). The DNA methylation levels measured from cells exposed to ONBs for different time periods (Fig. 3a,b) showed a distinct decrease (α = 0.05) in the DNA methylation levels in hypoxic cells compared to the control. Further, irrespective of the time of dose (start of treatment at 0 h or 24 h), no significant difference was observed in the methylation levels (Fig. 3a,b and Supplementary Fig. 6). However, in cells treated with nanobubbles, i.e. addition of nanobubbles (0.5 mg/mL) at 0 h and after 24 h of incubation,n a rapid and significant increase in 5mC DNA methylation levels was observed. Under hypoxia, DNA 5mC levels (measured as OD450 absorbance) linearly increased (P < 0.004, R2 = 0.58) corresponding to an increase in oxygen nanobubble concentration (Fig. 3c,d and Supplementary Fig. 5). A significant difference was observed (Fig. 3c,d) between 0, 0.1, and 1 mg/mL of nanobubble concentration (α = 0.05). The trends for different treatment conditions and oxygen nanobubble concentrations were similar and validated by ELISA and LC-MS/MS. Our observations infer that active 5mC levels in hypoxic tumor cells can be increased using oxygen nanobubbles in a dose-dependent manner, in vitro.
