Hyperbaric Oxygen Increases Sensitivity to Chemotherapy in Acute Leukemia
Authors: Lin Tara L. , Wise Amanda , Berry Abigale , Fontes Joseph , Kasturi Partha , Lipe Brea C. , Aljitawi Omar S. 
Published: October 2019

Abstract
Introduction: Chemotherapy resistance remains a significant challenge in the treatment of adults with Acute Myeloid Leukemia (AML). Although new targets and novel agents are under clinical investigation, the majority of AML patients are treated with conventional induction chemotherapy consisting of an anthracycline and cytarabine, with little modification in over 40 years. Insights into the biology of resistance to cytarabine and anthracyclines may suggest novel strategies in the treatment of AML. Hyperbaric oxygen (HBO) treatment is commonly used in the treatment of decompression sickness from scuba diving, wound healing and infections. 100% oxygen is administered at increased pressure. Limited published data suggest a role for HBO to cause in vitro apoptosis of leukemia cell lines, but no mechanism has been described. We examined the role of HBO in AML sensitivity to chemotherapy, reactive oxygen species (ROS) generation, glycolytic pathway activation and gene expression.
Methods: Human AML cell lines HL-60 and MV-411 were treated with escalating doses of cytarabine or daunorubicin as single agents followed by HBO treatment for 2 hours or normobaric conditions. HBO treatment consisted of exposure to 100% oxygen at 2.5 atmosphere absolutes for a total of 2 hours in a specifically designed in vitro HBO chamber. At 24 hours after chemotherapy dosing, leukemia cells were analyzed for proliferation using Cell Titer 96 Aqueous MTS assay (Promega). Apoptosis was measured using Annexin V-FITC and analyzed by flow cytometry. In order to understand potential mechanisms of HBO activity on leukemia cells, studies of HBO-treated cells without use of chemotherapy were also performed. CellROX Oxidative Stress assay (Life Technologies) was performed at 24 hours. CellROX Green and Deep Red reagents measured ROS level in the DNA and cytoplasm. RNA was isolated and real-time PCR was performed using CFX384 Real-time PCR system using primer sets specific for human hexokinase2 and phosphofructokinase to examine the effects of HBO on glycolysis. Gene expression profiling was performed using the Human Transcriptome Array 2.0 (Affymetrix) and examined differences in gene expression following HBO treatment of HL-60 cells and bone marrow from an AML patient.
Results: HBO-treated leukemia cells had increased sensitivity to cytarabine (Table 1, Figure 1) (MV411 response to cytarabine: IC50 2649nM without HBO treatment, 1921nM with HBO, p=0.02; HL60 response to cytarabine: linear response without HBO, IC50 1625nM with HBO, p<0.035). Decreased proliferation was also seen with HBO-treated cells in response to daunorubicin but was not statistically significant (Table 1). Similarly, apoptosis from cytarabine in HL-60 cells was increased with HBO treatment (p<0.02), but the results with daunorubicin were not significant. Significant increases in ROS levels were observed at 24 hours post-HBO for both cell lines when compared to non-treated cells (HL60 DNA p<0.0001, HL60 cytoplasm p<0.01, MV411 DNA p<0.01, MV411 cytoplasm p<0.01). Glycolysis pathway components human hexokinase 2 and phosphofructokinase were upregulated in HL60 and MV411 cells treated with HBO compared to controls. Gene expression profiling showed that approximately 200 genes in coding regions were upregulated in HBO-treated HL-60 cells; specific piRNA clusters were downregulated in both AML cell line HL60 and a bone marrow sample from a patient with relapsed AML when treated with HBO.
Conclusions: HBO treatment significantly increases the sensitivity to cytarabine AML cell lines in vitro. Increased ROS, upregulation of glycolysis-related enzymes and changes in gene expression profiling provide insight into the potential mechanisms of HBO enhanced chemotherapy sensitivity. Given the safety and commonplace use of HBO for other indications, and the significant challenge of chemotherapy resistance, it is important to pursue further studies to understand the biology of HBO treatment in AML.
Table 1. IC50 (nM) of chemotherapy agents in AML cell lines with and without co-treatment with HBO. Cytarabine Daunorubicin no HBO HBO p-value no HBO HBO p-value MV411 2649 1921 0.0214 709.1 641.1 ns HL60 * 1625 0.0346 639 406.5 ns * linear response, no IC50 available
Figure 1. Increased sensitivity to cytarabine in AML cell lines following treatment with HBO. Figure 1. Increased sensitivity to cytarabine in AML cell lines following treatment with HBO.

Disclosures
No relevant conflicts of interest to declare.