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, 2019, 6712536
eCollection

TRPV1 Induced Apoptosis of Colorectal Cancer Cells by Activating Calcineurin-NFAT2-p53 Signaling Pathway

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TRPV1 Induced Apoptosis of Colorectal Cancer Cells by Activating Calcineurin-NFAT2-p53 Signaling Pathway

Nengyi Hou et al. Biomed Res Int.

Abstract

Background/aims: TRPV1 is a nonselective Ca2+ channel which has recently been observed in many cancers, while its effect on cell proliferation, apoptosis, metabolism, and cancer development in colorectal cancer (CRC) is still unclear. In this study, we hypothesized that TRPV1 is a tumor suppressor in CRC development as well as the underlying mechanism.

Methods: Immunohistochemistry assay was applied to detect the expression of TRPV1 protein in CRC tissues. HCT116 cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. Cellular Ca2+ concentration was measured by Fluo-4/AM-based flow cytometer. Apoptosis-related proteins were measured by Western blotting.

Results: In this study, we found that TRPV1 expression was significantly decreased in CRC tissues, compared with CRC-adjacent tissues and normal tissues, respectively. Then, we found that the TRVP1 agonist capsaicin treatment inhibited CRC growth and induced apoptosis by activating P53. Subsequent mechanistic study revealed that the TRPV1 induced cytosolic Ca2+ influx to regulate cell apoptosis and p53 activation through calcineurin.

Conclusions: This study suggests that TRPV1 served as a tumor suppressor in CRC and contributed to the development of novel therapy of CRC.

Figures

Figure 1
Figure 1
Decrease of TRPV1 expression level in CRC. (a) Immunohistochemical assay was performed to detect the expression of TRPV1 in CRC tissues and adjacent tissues. (b) The expression change of TRPV1 was statistically analyzed (CRC tissuesn=10, CRC-adjacent tissuesn=10, and normal tissuesn=10). The result was shown as means ± standard deviation.  p < 0.05 and  ∗∗p < 0.01 versus normal group. ##p < 0.01 versus CRC-adjacent tissues group.
Figure 2
Figure 2
TRPV1 promoted CRC cell apoptosis through activating p53. HCT116 cells were incubated with capsaicin (50 μM) in the absence or presence of Pifithrin-α (20 μM). (a) Cell viability was determined by CCK-8 assay following indicated treatment. (b) Cell apoptosis were detected by flow cytometry. (c) The expression levels of apoptosis-related protein were examined by Western blotting. (d) The relative intensity was shown as a bar graph. The result was shown as means ± standard deviation.  p < 0.05 and  ∗∗p < 0.01 versus control group. ##p < 0.01 and #p < 0.05 versus capsaicin group.
Figure 3
Figure 3
Increased cytosolic Ca 2+ influx and NFAT protein induced by TRPV1 overexpression. (a) Measurement of Ca2+ influx by staining with Fluo-4 AM in HCT116 cells following treated with capsaicin (50 μM) and then detected by using flow cytometer. (b) The expression levels of p-NFAT2 and NFAT2 protein were examined by Western blotting, and the relative intensity was shown as a bar graph. (c) The expression level of NFAT2 protein was examined by immunofluorescence assay; images were observed by fluorescence microscopy (magnification, ×400). The result was shown as means ± standard deviation.  p < 0.05 and  ∗∗p < 0.01.
Figure 4
Figure 4
TRPV1 induced cell apoptosis through activating calcineurin. HCT116 cells were incubated with capsaicin (50 μM) in the absence or presence of FK506 (4 μM). (a) The expression level of NFAT2 protein was examined by immunofluorescence assay; images were observed by fluorescence microscopy (magnification, ×400). (b) Cell apoptosis were detected by flow cytometry. (c) The expression levels of apoptosis-related protein were examined by Western blotting. (d) The relative intensity was shown as a bar graph. The result was shown as means ± standard deviation.  p < 0.05 and  ∗∗p < 0.01 versus control group. ##p < 0.01 and #p < 0.05 versus capsaicin group.

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