Activated protease receptor-2 induces GATA6 expression to promote survival in irradiated colon cancer cells
Introduction
Radiotherapy is one of the major remedies for the treatment of cancer. Several types of irradiation can be used for this purpose; among them, Ionizing radiation is commonly used in cancer clinic. Ionizing radiation is high-frequency radiation that can remove an electron from an atom or molecule and damage the DNA in cells, which can result in apoptosis of cancer cells, or cause normal cells to become cancer cells [1]. One of the drawbacks in radiotherapy of cancer is the resistance to irradiation [2]. The underlying mechanism is not fully understood yet.
The association between mast cells and the pathogenesis of cancer has been noted in recent years [3]. Mast cells distribute all over the body. The number of mast cells in local tissue is increased during allergic diseases and chronic inflammation [4]. One of the major chemical mediators of mast cells is tryptase; it is released to the micro milieu from mast cells upon activation. Tryptase can cleave and activate the protease-activated receptor-2 (PAR2)2; PAR2 is involved in a number of cell functions as well as several pathologic activities, such as PAR2 has a close relation with the cancer cell growth environment [5]. PAR2 is involved in microRNA-34a mediated colon cancer cell proliferation [6].
Apoptosis is an important mechanism to maintain the homeostasis in the body. Dysfunction of apoptosis is one of the pathological features of cancer [7]. Under physiological conditions, apoptosis is tightly regulated by a number of molecules, such as Fas, Fas ligand and p53 proteins [8]. Among those molecules, p53 is the most important tumor suppressor protein by inducing tumor cell apoptosis [9]. However, how p53 protein is dysfunction during cancer cell growth is unclear.
GATA binding protein 6 (GATA6) in human is encoded by the GATA6 gene. Recent reports suggest that GATA6 is involved in cancer. The levels of GATA6 expression can predict prognosis and hepatic metastasis of colorectal cancer [10]. Silencing GATA6 can inhibit cancer cell growth [11]. Whether GATA6 has a functional relation with p53 protein expression in cancer cells is unclear.
Based on the above information, we hypothesize that mast cell activation interferes with the effect of irradiation on colon cancer cells. In this study, we found that colon cancer cell lines, the HT55 and HT29 cells, expressed PAR2. Exposure to tryptase induced HT55 and HT29 cells to express GATA6 and to resist to the irradiation-induced apoptosis, which was prevented by knockdown of GATA6 in HT55 and HT29 cells.
Section snippets
Reagents
Antibodies of PAR2 (SAM11), GATA6 (H-92), p53 (FL-393), and shRNA kits of GATA6 and p53 were purchased from Santa Cruz Biotech (Shanghai, China). The Annexin V reagent kit was purchased from Sigma Aldrich (Shanghai, China). The DNA purification kit, DNA methylation kit, the reagent of Trizol, reverse transcription kit, SYBR green super mix and gene transfection kits were purchased from Invitrogen (Shanghai, China).
Cell culture
Human colon cancer cell line, HT55 and HT29 cells, was purchased from Sigma
HT55 and HT29 cells express PAR2
In the first attempt, we observed the expression of PAR2 in colon cancer cell lines, HT55 and HT29 cells. As shown by qRT-PCR and Western blotting, the expression of PAR2 was detected in both HT55 and HT29 cells at the mRNA and proteins levels (Fig. 1). The data indicate that HT55 and HT29 cells express PAR2.
Activation of PAR2 promotes the survival of irradiated HT55 and HT29 cells
After irradiation of 8 Gy, more than 30% HT55 and HT29 cells became apoptosis. The presence of tryptase significantly reduced the frequency of apoptotic HT55 and HT29 cells, which did not
Discussion
The irradiation resistance is a great drawback in the treatment of malignant tumor with irradiation. To understand the underlying mechanism is of significance to improve the radiotherapy efficiency for cancer treatment. The present data indicate that the colon cancer cell lines, HT55 and HT29 cells, expresses PAR2; activation of PAR2 interferes with the irradiation-induced HT55 and HT29 cell apoptosis and the expression of GATA6. Inhibition of GATA6 can abolish the PAR2-reduced
Author contribution
W.S.C., F.S., J.L.L., A.F., Y.C.W. and H.Q.X. performed the experiments, collected and analyzed data, and reviewed the manuscript. BX designed the project, supervised the experiments and wrote the manuscript.
Acknowledgments
This work was supported by the Guangdong Province Science and Technology Plan Project and Guangzhou City Medical Science and Technology Key Project (201102A212018).
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Cai W. and Shen F. share the first authorship.