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Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil.

ABSTRACT: Chemotherapy-induced mucositis (CIM) is a major does limiting side-effect of chemoagents such as 5-fluorouracil (5-FU). Molecules involved in this disease process are still not fully understood. We proposed that the homeostatically regulated genes during CIM may participate in the disease. A cluster of such genes were previously identified by expression gene-array from the mouse jejunum in 5-FU-induced mucositis model. Here, we report that CXCL4 is such a homeostatically regulated gene and serves as a new target for the antibody treatment of CIM. CXCL4 and its receptor CXCR3 were confirmed at both the gene and protein levels to be homeostatically regulated during 5-FU-induced mucositis. Using of CXCL4 neutralizing monoclonal antibody (CXCL4mab) decreased the incidence, severity, and duration of the chemotherapy-induced diarrhea, the major symptom of CIM, in a 5-FU mouse CIM model. Mechanistically, CXCL4mab reduced the apoptosis of the crypt epithelia by suppression of the 5-FU-induced expression of p53 and Bax through its receptor CXCR3. The downstream signaling pathway of CXCL4 in activation of the epithelial apoptosis was identified in an intestinal epithelial cell line (IEC-6). CXCL4 activated the phosphorylation of p38 MAPK, which mediated the stimulated expression of p53 and Bax, and resulted in the ultimate activation of Caspase-8, -9, and -3. Taken together, activation of CXCL4 expression by 5-FU in mice participates in 5-FU-induced intestinal mucositis through upregulation of p53 via activation of p38-MAPK, and CXCL4mab is potentially beneficial in preventing CIM in the intestinal tract.

SUBMITTER: Gao J

PROVIDER: S-EPMC4119083 | biostudies-other | 2014 Aug

REPOSITORIES: biostudies-other

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Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil.

Gao Jing J Gao Jin J Qian Lan L Wang Xia X Wu Mingyuan M Zhang Yang Y Ye Hao H Zhu Shunying S Yu Yan Y Han Wei W

Cancer biology & therapy 20140506 8

Chemotherapy-induced mucositis (CIM) is a major does limiting side-effect of chemoagents such as 5-fluorouracil (5-FU). Molecules involved in this disease process are still not fully understood. We proposed that the homeostatically regulated genes during CIM may participate in the disease. A cluster of such genes were previously identified by expression gene-array from the mouse jejunum in 5-FU-induced mucositis model. Here, we report that CXCL4 is such a homeostatically regulated gene and serve ...[more]

PMID: 24800927

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Project description:Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-treated p53-/- cells is demonstrated to be caspase-dependent, but due to a slow pace, accumulation of mitochondrial reactive oxygen species contributes to necrotic characteristics. The oligomerization status of the p53 target gene DR5 is determined as a significant limiting factor for the initiation of caspase activity in an intracellular TRAIL-dependent manner. Using several experimental approaches, we further conclude that RNA-rather than DNA-related stress follows by caspase activation irrespectively of p53 status. A distinct 5-FU-induced stress mechanism is thereby functionally connected to a successive and discrete cell death signaling pathway. Finally, we provide evidence that silencing of PARP-1 function may be an approach to specifically target p53-deficient cells in 5-FU combinatorial treatment strategies. Together, our results disclose details of impaired cell death signaling engaged as a consequence of 5-FU chemotherapy. Obtained data will contribute to the comprehension of factors restraining 5-FU efficiency, and by excluding DNA as the main stress target in some cell types they propose alternatives to currently used and suggested synergistic treatment regimens.

Project description:The purpose of the present study was to investigate whether bone marrow mesenchymal stem cells (BMMSCs) modified by CXC‑chemokine receptor type 3 (CXCR3) and heme oxygenase‑1 (HO‑1) genes can repair damaged intestinal epithelial cells in vitro, and the role of the p38 mitogen‑activated protein kinase (p38‑MAPK) pathway in this process. A model of intestinal epithelial crypt cell line‑6 (IEC‑6) damage was created, and BMMSCs were transfected with either the CXCR3 and/or HO‑1 gene in vitro. There were nine experimental groups in which the damaged IEC‑6 cells were co‑cultured with differentially‑treated BMMSCs and lymphocytes for 24 h. Reverse transcription‑quantitative polymerase chain reaction analysis, immunohistochemistry and a western blot analysis were performed to detect stem cell transfection, the repair of damaged intestinal epithelial cells and the expression of related molecules in the P38‑MAPK pathway, respectively. Crystal violet staining and live cell imaging were used to detect the chemotaxis of BMMSCs. Flow cytometry was used to detect T lymphocyte activity and the surface markers expressed on BMMSCs. An ELISA was used to quantify cytokine production. The adenovirus (Ad)‑CXCR3/MSCs exhibited the characteristics of stem cells and exhibited chemotaxis. The Ad‑CXCR3/MSCs and Ad‑(CXCR3 + HO)/MSCs exhibited increased expression of tight junction protein zonula occludens‑1 (ZO‑1) and anti‑proliferating cell nuclear antigen in the damaged IEC‑6 cells, and apoptosis of the damaged IEC‑6 cells was decreased. BMMSCs inhibited the phosphorylation of p38, in addition to downstream molecules of the p38MAPK signaling pathway. The Ad‑CXCR3/MSCs and Ad‑(CXCR3 + HO)/MSCs exhibited significantly decreased expression levels of downstream molecules, including phosphorylated (p)‑p38, p‑activated transcription factor 2, p‑C/EBP homologous protein‑10, and p‑myocyte enhancer factor 2C, and target molecules (e.g., apoptotic bodies). The effects of Ad‑(CXCR3 + HO)/MSCs on the repair of the damaged intestinal tract and inhibition of the p38‑MAPK pathway was more marked than those in other groups on day 7 post‑surgery in the rejection model for small bowel transplantation. BMMSCs modified by the CXCR3 and HO‑1 genes exhibited superior ability to repair damaged intestinal epithelial cells and served this role via the p38‑MAPK pathway.

Dataset Information

Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil.

Publications

Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil.

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