Abstract 2620: The relationships between necroptosis, apoptosis, and inflammation in human colorectal tissue
Authors: Timothy Su, Chang Yu, Martha J. Shrubsole, Xiangzhu Zhu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai*
Journal: Cancer Research (2016)

Abstract Background: Formerly, apoptosis was considered the only form of regulated cell death and evading apoptosis was considered a hallmark of cancer. However, in recent years, necroptosis has been discovered as a new pathway of regulated cell death. Animal studies revealed very critical roles of necroptosis in many human inflammatory diseases and tumors. Early studies examining apoptosis by using TdT-mediated dUTP nick end labelling (TUNEL) assays cannot distinguish between apoptotic and necroptotic cells. Bax, which is a pro-apoptoic member of Bcl-2 family, is essential for mitochondrion-mediated apoptosis. Very recently, mixed lineage kinase domain-like (MLKL) is identified to be necessary for the execution of necroptosis. Cyclooxygenase (Cox), including Cox-1 and Cox-2, are the key enzymes responsible for the conversion of arachidonic acid to prostaglandins, which are important mediators of human physiology and pathophysiology, particularly inflammation. High expression of Cox-2 plays a critical role in the development of colorectal neoplasms. No study has examined how necroptosis biomarker (i.e. MLKL) and pro-apoptosis biomarker (i.e. Bax) are related to inflammation biomarker (Cox-2), TUNEL and cell proliferation biomarker (Ki-67). Methods: In our ongoing randomized trial (R01CA149633) “Personalized Prevention of Colorectal Cancer Trial (PPCCT)” conducted among colorectal polyp patients or those at high risk of colorectal cancer, we newly investigated the relationships between markers in these pathways using rectal biopsies (n = 110) collected prior to intervention in the trial. The formalin-fixed and paraffin-embedded tissue from colorectal biopsy was serially sectioned, and three levels of the serial sections spaced 50 μm apart. were mounted on to one slide for each tissue block. The protein expression levels of Cox-2, Bax, pMLKL, and Ki-67 were detected immunohistochemically following EnVision+ System-HRP (DAB) rabbit or mouse kit (DAKO). Apoptotic cells in situ were detected with DeadEnd™ Colorimetric TUNEL System (Promega). Results: Cox-2 expression in both epithelium and stroma had strong positive correlation with Bax expression in the same zone (p-trend = 0.00186 and 0.00004, respectively) but was not significantly correlated with pMLKL. The positive associations were stronger in the upper zone than the bottom of the crypts. The upper zone also showed more TUNEL positive cells than the bottom zone, and had significant positive correlation with pMLKL intensity (r = 0.458, p = 0.005). Bax expression was not significantly related to MLKL expression. Conclusion: Cox-2 expression in non-tumoral colonic tissue may activate apoptotic pathway through Bax upregulation, but is not correlated with necroptotic pathway marker MLKL. In the epithelium, the necroptotic pathway may be more common in the upper zone of colonic cryps than the bottom zone,and may more strongly contribute to TUNEL than apoptosis pathway. Citation Format: Timothy Su, Chang Yu, Martha J. Shrubsole, Xiangzhu Zhu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai*. The relationships between necroptosis, apoptosis, and inflammation in human colorectal tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2620.