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. 2017 May 23;8(21):35033-35047.
doi: 10.18632/oncotarget.16591.

A potential anti-tumor effect of leukotriene C4 through the induction of 15-hydroxyprostaglandin dehydrogenase expression in colon cancer cells

Lubna M Mehdawi  1 , Shakti Ranjan Satapathy  1 , Annika Gustafsson  2 , Kent Lundholm  2 , Maria Alvarado-Kristensson  3 , Anita Sjölander  1
Affiliations

A potential anti-tumor effect of leukotriene C4 through the induction of 15-hydroxyprostaglandin dehydrogenase expression in colon cancer cells

Lubna M Mehdawi et al. Oncotarget. .

Abstract

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2, which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2, and is often down-regulated in cancer. Interestingly, CRC patients expressing high levels of the cysteinyl leukotriene 2 (CysLT2) receptor have a good prognosis; therefore, we investigated a potential link between CysLT2 signaling and the tumor suppressor 15-PGDH in colon cancer cells.We observed a significant up-regulation of 15-PGDH after treatment with LTC4, a CysLT2 ligand, in colon cancer cells at both the mRNA and protein levels, which could be reduced by a CysLT2 antagonist or a JNK inhibitor. LTC4 induced 15-PGDH promoter activity via JNK/AP-1 phosphorylation. Furthermore, we also observed that LTC4, via the CysLT2/JNK signaling pathway, increased the expression of the differentiation markers sucrase-isomaltase and mucin-2 in colon cancer cells and that down-regulation of 15-PGDH totally abolished the observed increase in these markers.In conclusion, the restoration of 15-PGDH expression through CysLT2 signaling promotes the differentiation of colon cancer cells, indicating an anti-tumor effect of CysLT2 signaling.

Keywords: 15-PGDH; CysLTR2; LTC4 signaling; anti-tumor; colon cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1. Expression of COX-2, 15-PGDH, and the CysLT2 receptor in colon cancer patients
Quantification of the mRNA expression of (A) COX-2, (C) 15-PGDH, and (D) the CysLT2 receptor from 23 colon cancer patients by qPCR. (B, E) Immunohistochemistry staining with an antibody against the COX-2 ((B), 1:200 dilution) or the CysLT2 receptor ((E), 1:50 dilution); representative protein expression images from colorectal adenocarcinoma tissue and matched normal control tissue from one patient are shown. (F) Showing representative negative control immunohistochemistry staining. The intensity of the CysLT2 staining from all 22 patients was scored and presented in the graph. The data are presented as the percent of control (mucosa) and represent the mean ± standard error of mean (SEM). Statistical analysis was performed using an unpaired t-test; *P≤0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2. LTC4 up-regulates both the protein and mRNA levels of 15-PGDH in HT-29 cells
(A) Western blot and densitometric analyses of LTC4-induced 15-PGDH protein expression. Cells were treated with 20, 40 or 80 nM LTC4 for 24 h, and the up-regulation of 15-PGDH was detected using a 15-PGDH antibody (1:5000 dilution). (B) Western blot and densitometric analyses of LTC4-induced 15-PGDH up-regulation after the cells were stimulated with 40 nM LTC4 for the indicated periods of time. (C) The cells were treated with 1 μM AP100984 (CysLT2 receptor antagonist) for 30 min prior to stimulation with or without 40 nM LTC4 for 24 h. The cells were lysed, subjected to SDS-PAGE and immunoblotting with a 15-PGDH antibody and subsequently re-incubated with an antibody against β-actin (1:1000 dilution) to ensure equal loading. (D) Confocal microscopy immunofluorescence images showing the expression of 15-PGDH, with antibody dilution of 1:200 (15-PGDH is shown in green; DAPI is in blue and was used at a 1:1000 dilution), after 24 h of stimulation with LTC4 in HT-29 cells. The objective used was 63x, and the scale bar is 50 μm. (E) mRNA analysis of the effect of LTC4 on COX-2 mRNA after 12 or 24 h of stimulation. The data are presented as the percent of untreated control cells and represent the mean ± SEM of at least three separate experiments. Statistical analysis was performed using an unpaired t-test; *P≤0.05, **P<0.01, ***P<0.001.
Figure 3
Figure 3. LTC4 induced 15-PGDH promoter activity in HT-29 cells
(A) Schematic cartoon of both 15-PGDH promoters showing the different potential binding sites for the transcription factor AP-1 and the luciferase activity of 15-PGDH in cells seeded onto 12-well plates, transfected with the 15-PGDH promoter construct (-1024 bp) for 24 h, and subsequently pretreated with or without 50 μM PD 98059, 50 μM LY294002, or 10 μM JNK inhibitor I for 30 min prior to stimulation with 40 nM LTC4 for 24 h. (B) The cells were transfected with a 15-PGDH promoter construct (-388 bp) and pretreated with or without 10 μM JNK inhibitor I for 30 min prior to stimulation with 40 nM LTC4. (C) Western blot and densitometric analyses of LTC4-induced JNK phosphorylation in HT-29 cells at the indicated time-points. (D) The cells were treated with JNK inhibitor I 30 min prior to stimulation with or without 40 nM LTC4 for 2 h and then lysed, and the lysates were subjected to SDS-PAGE and immunoblotting with an antibody against p-JNK. The membrane was re-probed with an antibody against total JNK (both the p-JNK and total JNK antibodies were diluted 1:1000) and subsequently incubated with an antibody against GAPDH to ensure equal loading. (E) Western blot analyses of nuclear fractions of HT-29 cells incubated with JNK inhibitor I 30 min prior to stimulation or not with LTC4; the blots show phosphorylated AP-1 (AP-1; re-probed for total AP-1/Jun; both diluted 1:1000) and c-MYC (1:1000) and were re-probed for Lamin-B expression to ensure equal loading. (F) Analysis of the effect of LTC4 on 15-PGDH mRNA expression in HT-29 cells pretreated with or without the CysLT2 antagonist AP100984 or JNK inhibitor I. The data are presented as the percent of untreated control cells and represent the mean ± SEM of at least three separate experiments. Statistical analysis was conducted using an unpaired t-test and ANOVA; *P≤0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4. Effect of LTC4 on 15-PGDH expression in the colon cancer cell line Caco-2
(A) Quantification by qPCR of the mRNA expression of 15-PGDH following treatment with 40 nM LTC4 for 48 h in the presence or absence of AP100984 (a CysLT2 receptor antagonist, 1 μM) and JNK inhibitor I (10 μM). (B) Western blot showing 15-PGDH expression (antibody dilution, 1:5000) and densitometric analysis of LTC4-induced 15-PGDH up-regulation before or after the cells were stimulated with 40 nM LTC4 in the presence or absence of AP100984 and JNK inhibitor I for 48 h. (C) Confocal microscopy immunofluorescence images showing the expression of 15-PGDH (in green; antibody dilution, 1:200; DAPI in blue) after 24 h of stimulation with LTC4 in Caco-2 cells. The objective used was 63x. The data are presented as the percent of untreated control cells and represent the mean ± SEM of at least three separate experiments. Statistical analysis was conducted using an unpaired t-test; *P≤0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5. Effect of LTC4 on the expression of the differentiation markers SI and Mucin-2 in colon cancer cells
(A) Western blot and densitometric analyses of SI expression after stimulation with 40 nM LTC4 for 24, 48 and 72 h in HT-29 cells. The SI antibody dilution was 1:1000. (B) Quantification by qPCR of the mRNA expression of Mucin-2. HT-29 cells were stimulated with 40 nM LTC4 after pretreatment with AP100984 (1 μM) or JNK inhibitor I (JNKI1, 10 μM) 30 min prior to stimulation. (C) Confocal microscopy immunofluorescence images showing the expression of Mucin-2 after 48 h of stimulation with LTC4 in HT-29 cells. (D and E) Quantification by qPCR of SI and Mucin-2 mRNA expression in Caco-2 cells. Caco-2 cells were stimulated with 40 nM LTC4 and pretreated with AP100984 and JNKI1 30 min prior to stimulation for 48 h. (F) Confocal microscopy immunofluorescence images showing the expression of Mucin-2 (C, F in red; antibody dilution, 1:500; DAPI in blue dilution 1:1000) after 48 h of stimulation with LTC4 in Caco-2 cells. The objective used was 63x, and the scale bar is 50 μm. The data are presented as the percent of untreated control cells and represent the mean ± SEM of at least three separate experiments. Statistical analysis was conducted using an unpaired t-test and ANOVA; *P≤0.05, **P<0.01, ***P<0.001.
Figure 6
Figure 6. Effect of 15-PGDH down-regulation on the cell differentiation markers SI and Mucin-2
(A) A representative western blot and densitometric analysis of LTC4-induced SI protein expression after transfection with a scrambled control siRNA or 15-PGDH siRNA in HT-29 cells are shown. The cells were treated with or without 40 nM LTC4 for 48 h, and the change in the SI protein level was detected using an SI-specific antibody (1:1000 dilution) and 15-PGDH was detected using a 15-PGDH antibody (1:5000 dilution). The membrane was re-probed with an antibody against β–actin to ensure equal loading. (B) A representative western blot and densitometric analysis performed as in (A) shown here for Caco-2 cells. (C, D) Representative confocal microscopy immunofluorescence images from cells transfected with a scrambled control siRNA or 15-PGDH siRNA with or without stimulation with 40 nM LTC4 for 48 h. The expression of Mucin-2 (in red; antibody dilution, 1:500; DAPI in blue, dilution 1:1000) (C) in HT-29 cells and (D) Caco-2 cells is shown. The objective used was 63x, and the scale bar is 50 μm. The data are presented as the percent of control cells and represent the mean ± SEM of at least three separate experiments. Statistical analysis was conducted using an unpaired t-test; *P≤0.05, **P<0.01.
Figure 7
Figure 7. Schematic representation of the induction of 15-PGDH promoter activity by LTC4 via the CysLT2 receptor signaling pathway
LTC4, leukotriene C4; CysLTR2, cysteinyl leukotriene 2 receptor; JNKI1, c-Jun N-terminal kinase (JNK) inhibitor; AP-1 transcription factor.
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