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. 2019 Jan;110(1):235-244.
doi: 10.1111/cas.13861. Epub 2018 Dec 4.

Vasoactive intestinal peptide increases apoptosis of hepatocellular carcinoma by inhibiting the cAMP/Bcl-xL pathway

Masaki Hara  1 , Yuko Takeba  2 , Taroh Iiri  2 , Yuki Ohta  2 , Masanori Ootaki  2 , Minoru Watanabe  3 , Daiki Watanabe  2 , Satoshi Koizumi  4 , Takehito Otsubo  4 , Naoki Matsumoto  2
Affiliations

Vasoactive intestinal peptide increases apoptosis of hepatocellular carcinoma by inhibiting the cAMP/Bcl-xL pathway

Masaki Hara et al. Cancer Sci. 2019 Jan.

Abstract

Vasoactive intestinal peptide (VIP) is a modulator of inflammatory responses. VIP receptors are expressed in several tumor types, such as colorectal carcinoma. The study described herein was conducted to confirm the presence of VIP and its receptors (VPAC1 and VPAC2) in surgically resected hepatocellular carcinoma (HCC) tissues and in the HCC cell line Huh7. The mechanism responsible for apoptosis of HCC cells was then examined because VIP treatment (10-10 M) significantly suppressed proliferation of Huh7 cells. In examining apoptosis-related proteins, we found caspase-3 to be significantly increased and Bcl-xL and cyclic AMP (cAMP) response element-binding protein (CREB) to be significantly decreased in Huh7 cells cultured with VIP. Furthermore, the CREB level and phosphorylation were reduced. These effects were reversed by the addition of VIP receptor antagonist or cAMP antagonist Rp-cAMPS. Pretreatment with cAMP analogue blocked the increased apoptosis, suggesting that VIP induces apoptosis via a PKA-independent signaling mechanism. Our data indicate that VIP prevents the progression of HCC by apoptosis through the cAMP/Bcl-xL pathway.

Keywords: Bcl-xL; CREB; apoptosis; hepatocellular carcinoma; vasoactive intestinal peptide.

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Figures

Figure 1
Figure 1
A, Immnohistochemical staining for vasoactive intestinal peptide (VIP), VIP G protein‐coupled receptor (VPAC)1 and VPAC2 in noncancer liver, liver metastases of colorectal cancer and hepatocellular carcinoma (HCC) of various etiologies, and adjacent nontumorous tissues (from at least 10 cm away from the HCC). The data represent 3 individual patients. Original magnification is ×200. B, Immunohistochemical staining for VIP, VPAC1 and VPAC2 in non‐HBV‐related HCC, non‐HCV‐related HCC and Huh7 cells. Original magnification of HCC tissues is ×200, and that of Huh7 cells is ×400. The nontumorous tissues were free of tumor cells. HBV+, hepatitis B virus‐positive; HCV+, hepatitis C virus‐positive
Figure 2
Figure 2
Graphical representations of proliferation and apoptosis of hepatocellular carcinoma (HCC) cells treated with vasoactive intestinal peptide (VIP) at various concentrations. A, Huh 7 or HepG2 Cell proliferations are expressed as the ratio of the optical density of VIP‐treated cells to that of untreated (control) cells. The data represent 13 independent experiments. Values are mean ± SEM, *P < .05, **P < .01 vs control. B, Proliferation of Huh7 cells (4 × 104) treated with VIP was evaluated for 24‐h periods. The data represent 7 independent experiments, and values are mean ± SEM. **P < .01 vs control. C, Apoptosis of Huh7 cells treated with VIP. Apoptosis is shown relative to that of control cells and the data represent 9 independent experiments and expressed as mean ± SEM percentages. **P < .01 vs control. D, Apoptosis in the 2 types of primary cells. Apoptosis is shown relative to that of control cells, and the data represent 7 independent experiments. No significant difference was found between the 2 groups
Figure 3
Figure 3
Graphical representations of ELISA‐determined expression of cAMP, cAMP response element‐binding protein (CREB) and phosphorylated CREB (pCREB) in Huh7 cells cultured with vasoactive intestinal peptide (VIP) at various concentrations for 15 min. A, Concentrations of intracellular cAMP in Huh7 cells (2 × 104) treated with VIP for 15 min. Huh7 cells were 80% confluent at the time of treatment. The data represent 6 independent experiments and expressed as mean ± SEM. *P < .05 vs control. B, Western blot analysis showed that VIP significantly decreased CREB protein levels in the nucleus of Huh7 cells treated with VIP. The data represent 5 independent experiments and expressed as mean ± SEM. *P < .05 vs control. C, pCREB was measured using ELISA in VIP‐induced Huh7 cells. VIP‐treated Huh7 cells for 15 min after VIP addition. The data represent 8 independent experiments. Data are expressed as mean ± SEM, **P < .01 vs the control. D, Effects of [D‐p‐Cl‐Phe6, Leu17]‐VIP on the concentration of cAMP in Huh7 cells. Huh7 cells were precultured with [D‐p‐Cl‐Phe6, Leu17]‐VIP for 1 . The data represent 7 independent experiments and expressed as mean ± SEM. *P < .05 vs the control
Figure 4
Figure 4
Graphical representations and results of western blotting of protein levels of caspase‐3, Bcl‐xL, nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB p65), TNF receptor type 1‐associated death domain protein (TRADD) and cyclin‐D in Huh7 cells treated with vasoactive intestinal peptide (VIP) at various concentrations for 24 h. A, B, Caspase‐3 and Bcl‐xL protein levels in Huh7 cells treated with VIP for 24 h. VIP (10−10 M) significantly increased caspase‐3 protein levels, and VIP significantly decreased Bcl‐xL protein levels. α‐tubulin was used as a control. The data represent 7 independent experiments. Values are mean ± SEM, *P < .05 vs control. C, Expression of Bax and Bad in Huh7 cells treated with VIP. D, Expression of NFκB, TNF receptor type 1‐associated death domain protein (TRADD) and cyclin‐D in Huh7 cells treated with VIP did not differ significantly
Figure 5
Figure 5
Graphical representations of the effects of [D‐p‐Cl‐Phe6, Leu17]‐VIP and Rp‐cAMPS on cell proliferation (A) and Bcl‐xL protein levels (B) in Huh7 cells. Huh7 cells were pretreated with [D‐p‐Cl‐Phe6, Leu17]‐VIP or Rp‐cAMPS for 1 h before 24‐h incubation with vasoactive intestinal peptide (VIP). Cell proliferation was evaluated by MTS assay. The data represent 18 independent experiments. Bcl‐xL protein levels were evaluated by western blotting. The data represent 7 independent experiments, and values are shown as mean ± SEM. *P < .05 vs control
Figure 6
Figure 6
Graphical representations of the effects of [D‐p‐Cl‐Phe6, Leu17]‐VIP and Rp‐cAMPS on vasoactive intestinal peptide (VIP)‐induced apoptosis in Huh7 cells treated with VIP. A, Huh7 cells were precultured with [D‐p‐Cl‐Phe6, Leu17]‐VIP or Rp‐cAMPS for 1 h. The data represent 7 independent experiments. Apoptosis levels in Huh7 cells precultured with Rp‐cAMPS or dibutyryl cAMP. B, The data represent 7 independent experiments and shown as mean ± SEM. *P < .05 vs control
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