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, 6 (11), 9061-72

IL-32α Suppresses Colorectal Cancer Development via TNFR1-mediated Death Signaling

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IL-32α Suppresses Colorectal Cancer Development via TNFR1-mediated Death Signaling

Hyung-Mun Yun et al. Oncotarget.

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-mediated 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.

Keywords: IL-32α; RIP1; TNFR1; colon cancer.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Inhibition of cancer development in IL-32α Tg mice
(A, B) 7-week-old non-Tg and IL-32α-Tg mice were injected intraperitoneally with 10 mg/kg of AOM once a week for 6 weeks. Mice were killed at 32 weeks after AOM injections. Colons were harvested and Tumor weight (A) and volume (B) were monitored. (C) Colon tissues were processed and stained with H&E or analyzed by immunohistochemistry for detection of positive cells for IL-32α and PCNA. The images are representative of three separate experiments performed in triplicate. Scale bars indicate 100 μm. *Significant difference from non-Tg mice (*p < 0.05).
Figure 2
Figure 2. Expression of TNFR1 and cell death in colon cancer tissues of IL-32α Tg mice
(A) TNFR1 and TNFR2 were observed by immunohistochemical analysis as described in Materials and Methods. (B) The expression of TNFR1, TNFR2, apoptotic proteins were detected by Western blotting using specific antibodies in tumor tissue extracts. β-actin protein was used as an loading control. (C) Apoptotic cells were examined by TUNEL staining. (D) Cancer extracts were analyzed by Western blotting as described in Materials and Methods section. Each images and band are representative of three independent mice.
Figure 3
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.
Figure 4
Figure 4. Effects of IL-32α on ROS release and JNK activation in cancer tissues and colon cancer cells
(A) Cells were treated with 30 ng/ml TNFα for 4 hr in SW-pcDNA cells and SW-IL-32α cells. *Significant difference from SW-pcDNA cells (**p < 0.01). (B) SW-IL-32α cells were treated with TNFα for 4 hr in the absence or presence of the NOX inhibitor, DPI (20 μM) for 30 min. ROS levels were determined using ROS detection kit as described in Materials and Methods section. *Significant difference from control or TNFα-treated cell (**p < 0.01). (C) After SW-IL-32α cells were treated with TNFα for 60 min in the absence or presence of DPI (20 μM) for 30 min, cell extracts were analyzed by Western blotting using anti-phospho-JNK and anti-JNK antibodies. (D, E) Cancer extracts were analyzed by Western blotting (D) and ROS detection kit (E) as described in Materials and Methods section. *Significant difference from non-Tg mice (**p < 0.01). Representative results shown in Figure 4 were repeated in triplicate with similar results.
Figure 5
Figure 5. Relationship between IL-32α and TNFR1 in human colon cancer patients
(A) Human normal colon or cancer sections (Stage I–IV) were processed and stained with Hematoxylin or analyzed by immunohistochemistry for detection of positive cells for IL-32α and TNFR1. (B, C) The values are releative fold from human normal colon sections against IL-32α and TNFR1 (C). *Significant difference from normal sections cells (*p < 0.05 and **p < 0.01).
Figure 6
Figure 6. Effect of IL-32α on TNFR1-adaptor complex in colon cancer development
(A, B) SW-pcDNA cells and SW-IL-32α cells were treated with 30 ng/ml TNFα for 15 min. The colon cancer cells (A) and colon tumor tissues (B) from non-Tg and IL-32α Tg mice were lysated, and then immunoprecipitated with anti-TNFR1 antibody. The immunocomplexes were analyzed by immunoblotting with anti-RIP1, anti-TRAF2, anti-TRADD, anti-TNFR1 antibodies. The total protein expression in cell lysates was identified with the specific antibodies. (C) After human patient tissues were permeabilized, TNFR1 (red) was immunostained with mouse anti-TNFR1 followed by Alex555-conjugated secondary antibodies and RIP1 (green) was immunostained with rabbit anti-RIP1 antibody, followed by Alex488-conjugated secondary antibodies. And then sections were stained with DAPI (blue). The right panels show the merged images of the first, second, and third panels.

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