Reg IV Regulates Normal Intestinal and Colorectal Cancer Cell Susceptibility to Radiation-Induced Apoptosis

doi:10.1053/j.gastro.2009.10.050

Gastroenterology

Volume 138, Issue 2, February 2010, Pages 616-626.e2

https://doi.org/10.1053/j.gastro.2009.10.050 Get rights and content

Background & Aims

Regenerating (Reg) gene IV is predominantly expressed in gastrointestinal cells and highly up-regulated in many gastrointestinal malignancies, including colorectal cancer (CRC). Human CRC cells expressing higher levels of Reg IV gene and its protein product (Reg IV) are resistant to conventional therapies, including irradiation (IR). However, the underlying mechanism is not well defined.

Methods

A murine model of IR-induced intestinal injury and in vitro and in vivo models of human CRC were used to determine the role of Reg IV in regulation of normal intestinal and colorectal cancer cell susceptibility to IR-induced apoptosis.

Results

Treatments of recombinant human Reg IV (rhR4) protein protected normal intestinal crypt cells from IR-induced apoptosis by increasing the expression of antiapoptotic genes Bcl-2, Bcl-XL, and survivin. However, overexpression of Reg IV in human CRC cells was associated with increased resistance to IR-induced apoptosis. Therefore, we used antagonism of Reg IV as a tool to increase CRC cell susceptibility to IR-induced cell death. Two complementary approaches using specific monoclonal antibodies and small interfering RNAs were tested in both in vitro and in vivo models of human CRC. Both approaches resulted in increased apoptosis and decreased cell proliferation, leading to decreased tumor growth and increased animal survival. Furthermore, these approaches increased CRC cell susceptibility to IR-induced apoptosis.

Conclusions

These results implicate Reg IV as an important modulator of gastrointestinal cell susceptibility to IR; hence, it is a potential target for adjunctive treatments for human CRC and other gastrointestinal malignancies.

Section snippets

Cell Lines and Culture

The human CRC cell lines HCT116, SW480, and HT29 (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle medium (Cambrex, Walkersville, MD) containing 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT).

Anti–Reg IV Specific Polyclonal Antibody and mAbs

Armenian hamster mAbs (2H6 and 3E5) and rabbit polyclonal antibody (α-Reg IV 4261) against human Reg IV protein as well as recombinant human Reg IV protein (rhR4) were produced and characterized with the help of the Hybridoma Center at the Washington

Reg IV Inhibits IR-Induced Intestinal Cell Apoptosis

We previously showed that Reg IV regulates apoptosis in human CRC cells.6, 12 However, its role in normal GI cells was not established. In the present study, we used a murine model of IR-induced intestinal injury to determine the role of Reg IV in normal GI cell apoptosis. Following immunodetection of Reg IV–positive cells in distal jejunum sections using an α-Reg IV 4261 antibody, IR exposures led to a dose-dependent decrease in Reg IV expression (Figure 1A). Furthermore, IR (6 Gy) exposure to

Discussion

The normal GI epithelial architecture is frequently disrupted following injury. The homeostasis in a normal GI epithelial cell population is maintained by their continuous replacement with replicating undifferentiated epithelial cells (transit cells) located in the crypts, followed by their subsequent differentiation and migration away from the zone of replication.24, 25 Crypt stem cells play a central role in maintaining GI architecture both in normal and injury states.23, 26 Stem cells

Acknowledgments

Repository URL and data accession numbers: hybridoma cell lines 2H6.3.1 and 3E5.4.1 (http://pathology.wustl.edu/research/hybridoma.php; fusion no. 4465), Reg IV mRNA (GenBank accession no. NM_032044).

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The vitamin A metabolite all-trans retinoic acid (ATRA; tretinoin) has anticancer potential. However, lack of clinical success has prevented its approval for solid tumours. Herein, we propose combining short-term low-dose ATRA with fimaporfin-based photodynamic therapy (ATRA+PDT) for the improved treatment of solid cancers. Compared to monotherapies, ATRA+PDT induced synergistic cytotoxic responses including promotion of apoptosis in colon and breast carcinoma cell lines. Neither enhanced activity of alkaline phosphatase (ALP) nor increased expression of CD133 was detected after ATRA treatment indicating that the improved therapeutic effect of ATRA+PDT is independent of the differentiation state of the cancer cells. In the human colorectal adenocarcinoma cell line HT-29, the effect of ATRA+PDT on gene expression was evaluated by RNA sequencing (RNA-seq). We identified 1129 differentially expressed genes (DEGs) after ATRA+PDT compared to PDT. Ingenuity Pathway Analysis (IPA) predicted the unfolded protein response (UPR), interferon (IFN) signaling and retinoic acid-mediated apoptosis signaling as strongly activated canonical pathways after ATRA+PDT compared to PDT. A validation of the RNA-sec data by RT-qPCR revealed that ATRA+PDT elevated mRNA expression of early growth response 1 (EGR1) and strongly the stress-induced activating transcription factor 3 (ATF3), of which was confirmed on the protein level. In addition, ATRA+PDT abolished mRNA expression of regenerating islet-derived protein 4 (REG4). During the first 20 days post-ATRA+PDT, we obtained significant anti-tumour responses in HT-29 xenografts, including complete responses in 2/5 mice. In conclusion, ATRA+PDT represent a novel combination therapy for solid tumours that should be further tested in immunocompetent preclinical models.
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Preoperative radiotherapy (RT) plays an important role in the management of locally advanced rectal cancer (RC). Tumor regression after RT shows marked variability, and robust molecular methods are needed to help predict likely response. The aim of this study was to review the current published literature and use Gene Ontology (GO) analysis to define key molecular biomarkers governing radiation response in RC.
A systematic review of electronic bibliographic databases (Medline, Embase) was performed for original articles published between 2000 and 2015. Biomarkers were then classified according to biological function and incorporated into a hierarchical GO tree. Both significant and nonsignificant results were included in the analysis. Significance was binarized on the basis of univariate and multivariate statistics. Significance scores were calculated for each biological domain (or node), and a direct acyclic graph was generated for intuitive mapping of biological pathways and markers involved in RC radiation response.
Seventy-two individual biomarkers across 74 studies were identified. On highest-order classification, molecular biomarkers falling within the domains of response to stress, cellular metabolism, and pathways inhibiting apoptosis were found to be the most influential in predicting radiosensitivity.
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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by National Institutes of Health grants DK060106 and P30 DK52574 (to B.K.D.) and grants DK62265 and CA109269 (to S.A.).

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Basic—Alimentary TractReg IV Regulates Normal Intestinal and Colorectal Cancer Cell Susceptibility to Radiation-Induced Apoptosis

Background & Aims

Methods

Results

Conclusions

Section snippets

Cell Lines and Culture

Anti–Reg IV Specific Polyclonal Antibody and mAbs

Reg IV Inhibits IR-Induced Intestinal Cell Apoptosis

Discussion

Acknowledgments

Biochim Biophys Acta

Biochim Biophys Acta

Gastroenterology

Protein Expr Purif

Surgery

Curr Opin Cell Biol

Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone

Endocrinology

Expression of the regenerating gene family in inflammatory bowel disease mucosa: Reg Ialpha upregulation, processing, and antiapoptotic activity

J Investig Med

Structural organization and chromosomal localization of a human gene (HIP/PAP) encoding a C-type lectin overexpressed in primary liver cancer

Eur J Biochem

Dysregulation of Reg gene expression occurs early in gastrointestinal tumorigenesis and regulates anti-apoptotic genes

Cancer Biol Ther