Project Title

Expression of Oxidized Protein Hydrolase in Bladder Cancers

Authors' Affiliations

Noah P. Rutherford, Department of Pediatrics, Quillen College of Medicine, East Tennessee State University William L. Stone, Department of Pediatrics, Quillen College of Medicine, East Tennessee State University Tesha E. Blair, Department of Pediatrics, Quillen College of Medicine, East Tennessee State University Bel Krishna Thakuri, Department of Pediatrics, Quillen College of Medicine, East Tennessee State University Marianne Brannon, Department of Pediatrics, Quillen College of Medicine, East Tennessee State University

Location

White Top Mtn

Start Date

4-12-2019 9:00 AM

End Date

4-12-2019 2:30 PM

Poster Number

118

Faculty Sponsor’s Department

Pediatrics

Name of Project's Faculty Sponsor

Dr. William Stone

Type

Poster: Competitive

Classification of First Author

Undergraduate Student

Project's Category

Cancer or Carcinogenesis, Proteomics

Abstract Text

The National Cancer Institution reported over 80,000 diagnoses of bladder cancer (BCa) in the United States in 2018. Despite these numbers, minimal research toward developing new diagnostic techniques and treatment options are underway. Evidence suggests a significant increase in non-specific a-naphthyl acetateesterase levels in BCa patient’s urine. There has been little research focused on identification of the esterase present. It is also suggested that elevated oxidative stress resulting in production of reactive oxygen species (ROS) is common in tumorigenic bladder cells as a result of increased metabolic activity. Oxidized protein hydrolase (OPH) is an 80kD serine protease, previously found to be elevated in many other types of cancer. OPH degrades proteins damaged by ROS and also exhibits a highly specific esterase activity toward (AcApNA) N-acetyl-alanyl-p-nitroanilide and ANAA (α-naphthyl N-acetylalaninate) containing substrate. Investigation of OPH expression in BCa could result in development of new diagnostic techniques and possible application toward prodrugs targeting cells with elevated ROS and/or OPH. Due to lack of commercial OPH, a positive control for this protein is needed for testing. To do this E. coli(BL-21 DE-3) were cultured and inserted with pET-21a (+) plasmids containing a human OPH gene insert prior to a His7 tag. After being selectively grown on ampicillin media, the bacteria were induced by IPTG and digested using lysozyme. The soluble rOPH suspended in the supernatant was separated from the pellet by centrifugation and further purified using Ni-NTA resin chromatography columns specific for the His7 tag sequence. The UM-UC-3 bladder cancer cell line, commonly used in published research to screen efficiency of chemotherapeutics, were cultured in accordance to ATCC. These cells were then compared against none tumorigenic bladder cancer cells and rOPH in a series of tests. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) were transferred for western blot analysis using antibodies specific for human OPH to investigate the expression levels present in cells. Native-PAGE electrophoresis showed OPH esterase activity across these cells using S-ANAA substrate as a specific esterase colorimetric stain. With these results, possible treatment options can be investigated with use of novel prodrug chemotherapy specifically targeting OPH in BCa cells, ultimately leading to apoptosis in effected cells. These events may also lead to possible biomarkers used for easier and earlier diagnosis of BCa across various spectrums.

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Apr 12th, 9:00 AM Apr 12th, 2:30 PM

Expression of Oxidized Protein Hydrolase in Bladder Cancers

White Top Mtn

The National Cancer Institution reported over 80,000 diagnoses of bladder cancer (BCa) in the United States in 2018. Despite these numbers, minimal research toward developing new diagnostic techniques and treatment options are underway. Evidence suggests a significant increase in non-specific a-naphthyl acetateesterase levels in BCa patient’s urine. There has been little research focused on identification of the esterase present. It is also suggested that elevated oxidative stress resulting in production of reactive oxygen species (ROS) is common in tumorigenic bladder cells as a result of increased metabolic activity. Oxidized protein hydrolase (OPH) is an 80kD serine protease, previously found to be elevated in many other types of cancer. OPH degrades proteins damaged by ROS and also exhibits a highly specific esterase activity toward (AcApNA) N-acetyl-alanyl-p-nitroanilide and ANAA (α-naphthyl N-acetylalaninate) containing substrate. Investigation of OPH expression in BCa could result in development of new diagnostic techniques and possible application toward prodrugs targeting cells with elevated ROS and/or OPH. Due to lack of commercial OPH, a positive control for this protein is needed for testing. To do this E. coli(BL-21 DE-3) were cultured and inserted with pET-21a (+) plasmids containing a human OPH gene insert prior to a His7 tag. After being selectively grown on ampicillin media, the bacteria were induced by IPTG and digested using lysozyme. The soluble rOPH suspended in the supernatant was separated from the pellet by centrifugation and further purified using Ni-NTA resin chromatography columns specific for the His7 tag sequence. The UM-UC-3 bladder cancer cell line, commonly used in published research to screen efficiency of chemotherapeutics, were cultured in accordance to ATCC. These cells were then compared against none tumorigenic bladder cancer cells and rOPH in a series of tests. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) were transferred for western blot analysis using antibodies specific for human OPH to investigate the expression levels present in cells. Native-PAGE electrophoresis showed OPH esterase activity across these cells using S-ANAA substrate as a specific esterase colorimetric stain. With these results, possible treatment options can be investigated with use of novel prodrug chemotherapy specifically targeting OPH in BCa cells, ultimately leading to apoptosis in effected cells. These events may also lead to possible biomarkers used for easier and earlier diagnosis of BCa across various spectrums.