Contribution of Dual Oxidase 2 (DUOX2) to Hyper-Radiosensitivity in Human Gastric Cancer Cells

Abstract

Whole-abdominal radiotherapy (WART) is a primary method for managing gastrointestinal cancers that have disseminated into intra-abdominal tissues. While effective, this approach is limited because of the increased toxicity to normal tissue associated with combined WART and full-dose chemotherapy regimens. Recent studies have demonstrated a survival advantage in a novel treatment paradigm that allows for the safe use of full-dose systemic chemotherapy in combination with low-dose fractionated radiotherapy (LDFRT). Traditionally, radiation doses greater than 120 cGy have been used in radiotherapy because lower doses were thought to be ineffective for tumor therapy. However, we now know that LDFRT can produce hyper-radiosensitivity (HRS), a phenomenon where cells undergo apoptosis at radiation doses as low as 15 cGy, in a number of proliferating cells. The objectives of our current study were to determine whether LDFRT can induce HRS in gastrointestinal cancer cells and to identify biomarkers of chemopotentiation by LDFRT. Our data indicate that three consecutive daily fractions of 15 cGy produced HRS in gastric cancer cells and potentiated a modified regimen of docetaxel, cisplatin and 5′-fluorouracil (mDCF). Colony survival assays indicated that 15 cGy was sufficient to kill 90% of the cells when LDFRT was combined with mDCF whereas a dose almost 10 times higher (135 cGy) was needed to achieve the same rate when using conventional radiotherapy alone. RT2 PCR Profiler™ array analysis indicated that this combined regimen upregulated dual oxidase 2 (DUOX2), an enzyme functioning in the production of hydrogen peroxide, without upregulating genes involved in DNA repair. Moreover, downregulation of DUOX2 increased radioresistance at every radiation dose tested. In addition, our data indicate that reactive oxygen species (ROS) increase up to 3.5-fold in cells exposed to LDFRT and mDCF. Furthermore, inhibition of NADPH oxidase abrogated the killing efficiency of this combined regimen. Taken together these data suggest that chemopotentiation by LDFRT in gastric cancer cells may be due, at least in part, to increased ROS production (DUOX2) without upregulation of the DNA repair machinery. These data thus provide a rationale for further explorations of potential clinical applications of LDFRT, such as in WART, as a chemopotentiator for advanced and metastatic gastric cancers.

DUOX2, a New Biomarker for Disseminated Gastric Cancer’s Response to Low Dose Radiation in Mice

“…Human gastric cancer cell line NCI-N87 was obtained from American Type Culture Collection (Manassas, VA, USA) and grown as described in [18]. Normal human gastric epithelial cells GES-1 were obtained from Dr. Dawit Kidane-Mulat (University of Texas) and grown in RPMI 10% FBS [21].…”

“…Detection of GFP expression and Western blots were performed to determine infection efficiency. Chemotherapy and cells irradiation were performed as described in [18], except that irradiation was performed twice a day with 6 h intervals between the fractions. The primary mouse gastric cancer cell line NCC-S1 (S1) and its derivative NCC-S1M (S1M) were obtained from Hark Kyun Kim, National cancer center, Republic of Korea.…”

“…The mouse gastric cancer cells were maintained and grown in RPMI, 10% FBS. Western blots were performed as described in [18] with the exception that DUOX2 proteins were extracted with the Membrane Protein Extraction Kit Mem-PER TM Plus according to the manufacturer's recommendations (Cat no: 89842, Thermo Scientific, Waltham, MA, USA). For Stat1 expression, nuclear protein extracts were used, the blots were hybridized with Stat1 rabbit polyclonal antibody (Cat no: 9172, Cell Signaling Technology, Danvers, MA, USA) stripped and rehybridized with phospho specific Stat1 antibodies (Cell Signaling Technology, Phospho-Stat1 (Ser727) #9177; Cell Signaling Technology, Phospho-Stat1 (Tyr701) #9167)).…”

“…In the case of head and neck cancer, the status of p16 has been described as an important predictor of chemopotentiation by LDFRT [16], but this does not seem to be specific to LDFRT since p16 is also a predictor of the standard of care response [17]. In an effort to identify potential biomarkers for stomach cancer cells' response to chemopotentiation by LDFRT, we recently identified DUOX2, an enzyme involved in hydrogen peroxide (H 2 O 2 ) production, as an important mediator of hyper-radiosensitivity (HRS) and contributor to chemopotentiation by LDFRT in these cells [18]. In fact, the combined regimen of LDFRT and chemotherapy upregulated Reactive Oxygen Species (ROS) by more than 3 fold, and DUOX2 downregulation abolished HRS [18].…”

“…In an effort to identify potential biomarkers for stomach cancer cells' response to chemopotentiation by LDFRT, we recently identified DUOX2, an enzyme involved in hydrogen peroxide (H 2 O 2 ) production, as an important mediator of hyper-radiosensitivity (HRS) and contributor to chemopotentiation by LDFRT in these cells [18]. In fact, the combined regimen of LDFRT and chemotherapy upregulated Reactive Oxygen Species (ROS) by more than 3 fold, and DUOX2 downregulation abolished HRS [18]. ROS, including H 2 O 2 , can either promote cell survival or cell death depending on their intracellular concentration and localization [19].…”