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IL-32α suppresses colorectal cancer development via TNFR1-mediated death signaling.

Yun HM, Park KR, Kim EC, Han SB, Yoon do Y, Hong JT - Oncotarget (2015)

Bottom Line: Also, IL-32α increased ROS production to induce prolonged JNK activation.In colon cancer patients, IL-32α and TNFR1 were increased.These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Maxillofacial Tissue Regeneration, School of Dentistry and Research Center for Tooth & Periodontal Regeneration (MRC), Kyung Hee University, Seoul 130-701, Republic of Korea.

ABSTRACT
Inflammation is associated with cancer-prone microenvironment, leading to cancer. IL-32 is expressed in chronic inflammation-linked human cancers. To investigate IL-32α in inflammation-linked colorectal carcinogenesis, we generated a strain of mice, expressing IL-32 (IL-32α-Tg). In IL-32α-Tg mice, azoxymethane (AOM)-induced colon cancer incidence was decreased, whereas expression of TNFR1 and TNFR1-medicated apoptosis was increased. Also, IL-32α increased ROS production to induce prolonged JNK activation. In colon cancer patients, IL-32α and TNFR1 were increased. These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.

No MeSH data available.


Related in: MedlinePlus

Effects of stable expression of IL-32α in SW620 cells on colon cancer cell growth and apoptotic signaling(A) SW620 cells were stably transfected with either the empty pcDNA3.1 vector (SW-pcDNA cells) or the IL-32α expression vector (SW-IL-32α cells), respectively. Cell growth rate was measured by MTT assay during 72 hr (B) Expression of IL-32α and TNFR1 is shown by Western blot analysis. β-actin protein was used as an loading control. (C) Cells were treated with 30 ng/ml TNFα for 24 hr. Cell extracts were analyzed by Western blotting using specific antibodies. (D) The cells were treated with TNFα (30 ng/ml) for the indicated times and assayed to detect phospho-JNK and JNK. The data are represented as relative percentages of the control. *Significant difference from SW-pcDNA cells (*p < 0.05, **p < 0.01, and ***p < 0.001). Representative results shown in Figure 3 were repeated in triplicate with similar results.
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Figure 3: Effects of stable expression of IL-32α in SW620 cells on colon cancer cell growth and apoptotic signaling(A) SW620 cells were stably transfected with either the empty pcDNA3.1 vector (SW-pcDNA cells) or the IL-32α expression vector (SW-IL-32α cells), respectively. Cell growth rate was measured by MTT assay during 72 hr (B) Expression of IL-32α and TNFR1 is shown by Western blot analysis. β-actin protein was used as an loading control. (C) Cells were treated with 30 ng/ml TNFα for 24 hr. Cell extracts were analyzed by Western blotting using specific antibodies. (D) The cells were treated with TNFα (30 ng/ml) for the indicated times and assayed to detect phospho-JNK and JNK. The data are represented as relative percentages of the control. *Significant difference from SW-pcDNA cells (*p < 0.05, **p < 0.01, and ***p < 0.001). Representative results shown in Figure 3 were repeated in triplicate with similar results.

Mentions: Next, it was validated whether IL-32α inhibits colon cancer cell growth. Consistent with IL-32α Tg mice, the stable expression of IL-32α in SW620 cells (SW-IL-32α cells) was significantly inhibited cell growth in a time dependent manner (Figure 3A). It was also found that SW-IL-32α cells have higher TNFR1expression level compared to SW-pcDNA cells (Figure 3B). To confirm the promoting effect of IL-32α on TNFR1-mediated cell death, it was investigated for the expression of apoptotic regulatory proteins in SW-IL-32α cells. As shown in Figure 3C, in the IL-32α-cells stimulated by TNF-α, cleaved caspases-8, cleaved caspases-9, cleaved caspases-3, and cleaved-Bid were more increased compared to TNF-α-stimulated SW-pcDNA cells. Next, it was evaluated for the underlying signal transduction pathway of IL-32α during TNF-α-induced cell growth inhibition. In colon cancer cells, prolonged JNK activation via TNFR1 is involved in growth arrest and cell death. Thus, it was investigated whether the pathways are regulated by IL-32α in TNF-α-mediated cell death. SW-IL-32α cells were treated with TNF-α for 0, 1, 5, 15, 30, and 60 min and the activation of JNK (phospho-JNK) was measured. TNF-α-induced phosphorylation of JNK was significantly increased at 15 min, reached a maximal response at 30 min and returned to a basal level after 60 min incubation in SW-pcDNA cells. While, in SW-IL-32α cells with TNF-α, the activation of JNK (phospho-JNK) was significantly higher, and started to increase significantly at 1 min, reached a maximal response at 30 min, and sustained until 60 min (Figure 3D). In reverse proportion to JNK activation, it was found that NF-κB signaling was significantly inhibited in SW-IL-32α cells (Supplementary Figure 1). These data suggest that IL-32α enhances TNFR1-mediated cell death signaling.


IL-32α suppresses colorectal cancer development via TNFR1-mediated death signaling.

Yun HM, Park KR, Kim EC, Han SB, Yoon do Y, Hong JT - Oncotarget (2015)

Effects of stable expression of IL-32α in SW620 cells on colon cancer cell growth and apoptotic signaling(A) SW620 cells were stably transfected with either the empty pcDNA3.1 vector (SW-pcDNA cells) or the IL-32α expression vector (SW-IL-32α cells), respectively. Cell growth rate was measured by MTT assay during 72 hr (B) Expression of IL-32α and TNFR1 is shown by Western blot analysis. β-actin protein was used as an loading control. (C) Cells were treated with 30 ng/ml TNFα for 24 hr. Cell extracts were analyzed by Western blotting using specific antibodies. (D) The cells were treated with TNFα (30 ng/ml) for the indicated times and assayed to detect phospho-JNK and JNK. The data are represented as relative percentages of the control. *Significant difference from SW-pcDNA cells (*p < 0.05, **p < 0.01, and ***p < 0.001). Representative results shown in Figure 3 were repeated in triplicate with similar results.
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Related In: Results  -  Collection

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Figure 3: Effects of stable expression of IL-32α in SW620 cells on colon cancer cell growth and apoptotic signaling(A) SW620 cells were stably transfected with either the empty pcDNA3.1 vector (SW-pcDNA cells) or the IL-32α expression vector (SW-IL-32α cells), respectively. Cell growth rate was measured by MTT assay during 72 hr (B) Expression of IL-32α and TNFR1 is shown by Western blot analysis. β-actin protein was used as an loading control. (C) Cells were treated with 30 ng/ml TNFα for 24 hr. Cell extracts were analyzed by Western blotting using specific antibodies. (D) The cells were treated with TNFα (30 ng/ml) for the indicated times and assayed to detect phospho-JNK and JNK. The data are represented as relative percentages of the control. *Significant difference from SW-pcDNA cells (*p < 0.05, **p < 0.01, and ***p < 0.001). Representative results shown in Figure 3 were repeated in triplicate with similar results.
Mentions: Next, it was validated whether IL-32α inhibits colon cancer cell growth. Consistent with IL-32α Tg mice, the stable expression of IL-32α in SW620 cells (SW-IL-32α cells) was significantly inhibited cell growth in a time dependent manner (Figure 3A). It was also found that SW-IL-32α cells have higher TNFR1expression level compared to SW-pcDNA cells (Figure 3B). To confirm the promoting effect of IL-32α on TNFR1-mediated cell death, it was investigated for the expression of apoptotic regulatory proteins in SW-IL-32α cells. As shown in Figure 3C, in the IL-32α-cells stimulated by TNF-α, cleaved caspases-8, cleaved caspases-9, cleaved caspases-3, and cleaved-Bid were more increased compared to TNF-α-stimulated SW-pcDNA cells. Next, it was evaluated for the underlying signal transduction pathway of IL-32α during TNF-α-induced cell growth inhibition. In colon cancer cells, prolonged JNK activation via TNFR1 is involved in growth arrest and cell death. Thus, it was investigated whether the pathways are regulated by IL-32α in TNF-α-mediated cell death. SW-IL-32α cells were treated with TNF-α for 0, 1, 5, 15, 30, and 60 min and the activation of JNK (phospho-JNK) was measured. TNF-α-induced phosphorylation of JNK was significantly increased at 15 min, reached a maximal response at 30 min and returned to a basal level after 60 min incubation in SW-pcDNA cells. While, in SW-IL-32α cells with TNF-α, the activation of JNK (phospho-JNK) was significantly higher, and started to increase significantly at 1 min, reached a maximal response at 30 min, and sustained until 60 min (Figure 3D). In reverse proportion to JNK activation, it was found that NF-κB signaling was significantly inhibited in SW-IL-32α cells (Supplementary Figure 1). These data suggest that IL-32α enhances TNFR1-mediated cell death signaling.

Bottom Line: Also, IL-32α increased ROS production to induce prolonged JNK activation.In colon cancer patients, IL-32α and TNFR1 were increased.These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Maxillofacial Tissue Regeneration, School of Dentistry and Research Center for Tooth & Periodontal Regeneration (MRC), Kyung Hee University, Seoul 130-701, Republic of Korea.

ABSTRACT
Inflammation is associated with cancer-prone microenvironment, leading to cancer. IL-32 is expressed in chronic inflammation-linked human cancers. To investigate IL-32α in inflammation-linked colorectal carcinogenesis, we generated a strain of mice, expressing IL-32 (IL-32α-Tg). In IL-32α-Tg mice, azoxymethane (AOM)-induced colon cancer incidence was decreased, whereas expression of TNFR1 and TNFR1-medicated apoptosis was increased. Also, IL-32α increased ROS production to induce prolonged JNK activation. In colon cancer patients, IL-32α and TNFR1 were increased. These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.

No MeSH data available.


Related in: MedlinePlus