Abstract
BackgroundReactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. In the present research, we examined the pathobiological role of NADPH oxidase (NOX)4-mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology.MethodsNOX4 gene was silenced in vivo and in vitro by NOX4 siRNA transfection with or without atlocollagen. Cell cycle and measurement of ROS were analyzed by flowcytometry. Orthotopic implantation animal model was used in vivo experiment. NOX4 expression in urothelial carcinoma cells was observed by immunohistochemical analysis using surgical specimens of human bladder cancer. Urine cytology was performed after treatment with ROS detection reagents in addition to Papanicolaou staining.ResultsNOX4 was overexpressed in several UC cell lines and the NOX inhibitor, diphenylene iodonium reduced intracellular ROS and induced p16-dependent cell cycle arrest at the G1 phase. Moreover, silencing of NOX4 by siRNA significantly reduced cancer cell growth in vivo as assessed in an orthotopic mouse model. Immunohistochemistry demonstrated high expression of NOX4 in low grade/non-invasive and high grade/invasive UC including precancerous lesions such as dysplasia but not in normal urothelium. Then, we assessed the usefulness of cytological analysis of ROS producing cells in urine (ROS-C). Urine samples obtained from UC cases and normal controls were treated with fluorescent reagents labeling the hydrogen peroxide/superoxide anion and cytological atypia of ROS positive cells were analyzed. As a result, the sensitivity for detection of low grade, non-invasive UC was greatly increased (35% in conventional cytology (C-C) vs. 75% in ROS-C), and the specificity was 95%. Through ROS-C, we observed robust improvement in the accuracy of follow-up urine cytology for cases with previously diagnosed UC, especially in those with low grade/non-invasive cancer recurrence (0% in C-C vs. 64% in ROS-C).ConclusionsThis is the first report demonstrating that ROS generation through NOX4 contributes to an early step of urothelial carcinogenesis and cancer cell survival. In addition, cytology using ROS labeling could be a useful diagnostic tool in human bladder cancer.
Highlights
Reactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression
We show that NOX4 maintained intracellular generation of ROS in both high and low grade and superficial and invasive urothelial carcinoma cells and contributes to cancer cell survival, using in vitro and in vivo experiments and clinicopathological analyses
reverse transcription polymerase chain reaction (RT-PCR) analysis showed that NOX4 was highly expressed in all 3 cell lines
Summary
Reactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. We examined the pathobiological role of NADPH oxidase (NOX)4mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology. Several variants of bladder cancer have been recognized; for example, some reports described an adverse outcome for urothelial carcinomas with squamous, glandular, or trophoblastic differentiation, or nested variants, or invasive micropapillary variants and so on [4]. The current gold standards for diagnosis and surveillance are conventional white light cystoscopy and urine-based cytology. The former enables direct visualization of tumors and histopathological analysis from biopsy samples. Identify a solution that compensates for these disadvantages, cytology will be one of the simplest and most reliable diagnostic and screening tools available with high reproducibility
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
