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Connexin32 regulates hepatoma cell metastasis and proliferation via the p53 and Akt pathways.

Zhao B, Zhao W, Wang Y, Xu Y, Xu J, Tang K, Zhang S, Yin Z, Wu Q, Wang X - Oncotarget (2015)

Bottom Line: The expression of connexin32 (Cx32) is frequently downregulated in HCC tissues.The reduction of Cx32 in HCC tissues was significantly associated with increased vascular invasion, increased tumor size, and poor survival.Additionally, Cx32 negatively regulated the phosphorylation of Akt and the expression of the cell cycle regulation protein cyclin D1, thereby inhibiting the proliferation of HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma, Xiamen University Affiliated Zhongshan Hospital, Xiamen, Fujian, China.

ABSTRACT
Hepatocellular carcinoma (HCC) progresses rapidly and is frequently associated with vascular invasion, metastasis, recurrence, and poor prognosis. The expression of connexin32 (Cx32) is frequently downregulated in HCC tissues. In this study, the role of Cx32 in HCC metastasis and proliferation was investigated. The reduction of Cx32 in HCC tissues was significantly associated with increased vascular invasion, increased tumor size, and poor survival. In vitro assays revealed that Cx32 not only suppressed the invasion and migration of HCC cells, but also repressed HCC cell proliferation. Subsequent investigations revealed that Cx32 directly enhanced the acetylation and transcriptional activity of p53, thus upregulating the expression of the tumor metastasis suppressor protein KAI1/CD82, which is a p53 target gene. Additionally, Cx32 negatively regulated the phosphorylation of Akt and the expression of the cell cycle regulation protein cyclin D1, thereby inhibiting the proliferation of HCC cells. Our in vivo nude mice model further confirmed that Cx32 is able to suppress HCC tumor growth and metastasis in nude mice. Our results imply that Cx32 downregulation contributes to the proliferation and metastasis of HCC, and the restoration of Cx32 expression may be a promising strategy for HCC therapy.

No MeSH data available.


Related in: MedlinePlus

Cx32 exerts its anti-metastatic function via the p53-CD82 pathway(A) The protein level of CD82 was downregulated in shCx32 HepG2 cells. (B) Cx32 upregulated the expression of CD82 in a p53-dependent manner. Cx32, p53, or p53 mutant (K373/382R) expression vectors were transfected into Hep3B or SMMC-7721 cells, as indicated, and the cells were monitored for the expression of CD82 by western blot. (C) Cx32 induced p53 transcriptional activity. The p53-Luciferase reporter and β-galactosidase (β-gal) gene expression vectors, together with p53, Cx32 expression vectors, Cx32 siRNA, or siCtrl, as indicated, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the co-transfected β-gal activity. The bars represent the mean ± SEM from three independent experiments. (D) Cx32 positively regulated p53 acetylation. Cx32 knockdown HepG2 cells and control cells were collected, and SMMC-7721 cells were non-transfected or transfected with the Cx32 expression vector for 48 h. Each cell lysate was then subjected to western blot analysis using an anti-Acetylated-p53 (K373/382), -Cx32, or -p53 antibody. (E) Cx32 prolongs the half-life of p53. Cx32 and Myc-p53 were transfected into 293T cells and then treated with CHX (100 μg/ml) for the indicated times. p53 expression level was determined by western blotting using an anti-Myc antibody. The levels of p53 protein were quantified by densitometry. (F) Cx32 inhibited HDAC1 expression in a dose-dependent manner. (G) Cx32 inhibited the interaction of p53 and HDAC1. GFP-Cx32 was transfected into SMMC-7721 cells, as indicated. The loading of HDAC1 was normalized before immunoprecipitation, and the cell lysate was then immunoprecipitated using an anti-p53 antibody. The immunoprecipitates were examined by western blotting using an anti-HDAC1 antibody. The input represented 10% of the cell lysates used in the co-IP experiment. (H) p53 siRNA attenuated the anti-migratory function of Cx32. SMMC-7721 cells were transfected with Cx32, siCtrl, or si-p53, as indicated. Twenty-four hours later, cells were added to transwell chambers and incubated for 20 h, followed by staining with crystal violet; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 3: Cx32 exerts its anti-metastatic function via the p53-CD82 pathway(A) The protein level of CD82 was downregulated in shCx32 HepG2 cells. (B) Cx32 upregulated the expression of CD82 in a p53-dependent manner. Cx32, p53, or p53 mutant (K373/382R) expression vectors were transfected into Hep3B or SMMC-7721 cells, as indicated, and the cells were monitored for the expression of CD82 by western blot. (C) Cx32 induced p53 transcriptional activity. The p53-Luciferase reporter and β-galactosidase (β-gal) gene expression vectors, together with p53, Cx32 expression vectors, Cx32 siRNA, or siCtrl, as indicated, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the co-transfected β-gal activity. The bars represent the mean ± SEM from three independent experiments. (D) Cx32 positively regulated p53 acetylation. Cx32 knockdown HepG2 cells and control cells were collected, and SMMC-7721 cells were non-transfected or transfected with the Cx32 expression vector for 48 h. Each cell lysate was then subjected to western blot analysis using an anti-Acetylated-p53 (K373/382), -Cx32, or -p53 antibody. (E) Cx32 prolongs the half-life of p53. Cx32 and Myc-p53 were transfected into 293T cells and then treated with CHX (100 μg/ml) for the indicated times. p53 expression level was determined by western blotting using an anti-Myc antibody. The levels of p53 protein were quantified by densitometry. (F) Cx32 inhibited HDAC1 expression in a dose-dependent manner. (G) Cx32 inhibited the interaction of p53 and HDAC1. GFP-Cx32 was transfected into SMMC-7721 cells, as indicated. The loading of HDAC1 was normalized before immunoprecipitation, and the cell lysate was then immunoprecipitated using an anti-p53 antibody. The immunoprecipitates were examined by western blotting using an anti-HDAC1 antibody. The input represented 10% of the cell lysates used in the co-IP experiment. (H) p53 siRNA attenuated the anti-migratory function of Cx32. SMMC-7721 cells were transfected with Cx32, siCtrl, or si-p53, as indicated. Twenty-four hours later, cells were added to transwell chambers and incubated for 20 h, followed by staining with crystal violet; *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: To understand the mechanisms underlying the changes in the invasive and migratory abilities of HCC cells, the levels of several invasion-related proteins were compared between the control HepG2 cells and the Cx32-depleted HepG2 cells, using western blot analysis. Downregulation of Cx32 did not cause a significant change in the expression of matrix metallopeptidase 2 (MMP2) or in that of VEGF, but did result in a significant decrease in the expression of KAI1/CD82, a tumor metastasis suppressor protein (Fig. 3A). The expression of CD82 in SMMC-7721 cells was increased following Cx32 overexpression (Fig. 3B). However, in the p53- Hep3B cells, overexpression of Cx32 failed to alter the expression of CD82 (Fig. 3B). Therefore, we believe that the upregulation of CD82 expression by Cx32 is p53-dependent because KAI1/CD82 transcription is also upregulated by p53 [21]. To confirm this notion, p53 was co-expressed in Hep3B cells; under these conditions, Cx32 was able to upregulate CD82 expression (Fig. 3B).


Connexin32 regulates hepatoma cell metastasis and proliferation via the p53 and Akt pathways.

Zhao B, Zhao W, Wang Y, Xu Y, Xu J, Tang K, Zhang S, Yin Z, Wu Q, Wang X - Oncotarget (2015)

Cx32 exerts its anti-metastatic function via the p53-CD82 pathway(A) The protein level of CD82 was downregulated in shCx32 HepG2 cells. (B) Cx32 upregulated the expression of CD82 in a p53-dependent manner. Cx32, p53, or p53 mutant (K373/382R) expression vectors were transfected into Hep3B or SMMC-7721 cells, as indicated, and the cells were monitored for the expression of CD82 by western blot. (C) Cx32 induced p53 transcriptional activity. The p53-Luciferase reporter and β-galactosidase (β-gal) gene expression vectors, together with p53, Cx32 expression vectors, Cx32 siRNA, or siCtrl, as indicated, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the co-transfected β-gal activity. The bars represent the mean ± SEM from three independent experiments. (D) Cx32 positively regulated p53 acetylation. Cx32 knockdown HepG2 cells and control cells were collected, and SMMC-7721 cells were non-transfected or transfected with the Cx32 expression vector for 48 h. Each cell lysate was then subjected to western blot analysis using an anti-Acetylated-p53 (K373/382), -Cx32, or -p53 antibody. (E) Cx32 prolongs the half-life of p53. Cx32 and Myc-p53 were transfected into 293T cells and then treated with CHX (100 μg/ml) for the indicated times. p53 expression level was determined by western blotting using an anti-Myc antibody. The levels of p53 protein were quantified by densitometry. (F) Cx32 inhibited HDAC1 expression in a dose-dependent manner. (G) Cx32 inhibited the interaction of p53 and HDAC1. GFP-Cx32 was transfected into SMMC-7721 cells, as indicated. The loading of HDAC1 was normalized before immunoprecipitation, and the cell lysate was then immunoprecipitated using an anti-p53 antibody. The immunoprecipitates were examined by western blotting using an anti-HDAC1 antibody. The input represented 10% of the cell lysates used in the co-IP experiment. (H) p53 siRNA attenuated the anti-migratory function of Cx32. SMMC-7721 cells were transfected with Cx32, siCtrl, or si-p53, as indicated. Twenty-four hours later, cells were added to transwell chambers and incubated for 20 h, followed by staining with crystal violet; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 3: Cx32 exerts its anti-metastatic function via the p53-CD82 pathway(A) The protein level of CD82 was downregulated in shCx32 HepG2 cells. (B) Cx32 upregulated the expression of CD82 in a p53-dependent manner. Cx32, p53, or p53 mutant (K373/382R) expression vectors were transfected into Hep3B or SMMC-7721 cells, as indicated, and the cells were monitored for the expression of CD82 by western blot. (C) Cx32 induced p53 transcriptional activity. The p53-Luciferase reporter and β-galactosidase (β-gal) gene expression vectors, together with p53, Cx32 expression vectors, Cx32 siRNA, or siCtrl, as indicated, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the co-transfected β-gal activity. The bars represent the mean ± SEM from three independent experiments. (D) Cx32 positively regulated p53 acetylation. Cx32 knockdown HepG2 cells and control cells were collected, and SMMC-7721 cells were non-transfected or transfected with the Cx32 expression vector for 48 h. Each cell lysate was then subjected to western blot analysis using an anti-Acetylated-p53 (K373/382), -Cx32, or -p53 antibody. (E) Cx32 prolongs the half-life of p53. Cx32 and Myc-p53 were transfected into 293T cells and then treated with CHX (100 μg/ml) for the indicated times. p53 expression level was determined by western blotting using an anti-Myc antibody. The levels of p53 protein were quantified by densitometry. (F) Cx32 inhibited HDAC1 expression in a dose-dependent manner. (G) Cx32 inhibited the interaction of p53 and HDAC1. GFP-Cx32 was transfected into SMMC-7721 cells, as indicated. The loading of HDAC1 was normalized before immunoprecipitation, and the cell lysate was then immunoprecipitated using an anti-p53 antibody. The immunoprecipitates were examined by western blotting using an anti-HDAC1 antibody. The input represented 10% of the cell lysates used in the co-IP experiment. (H) p53 siRNA attenuated the anti-migratory function of Cx32. SMMC-7721 cells were transfected with Cx32, siCtrl, or si-p53, as indicated. Twenty-four hours later, cells were added to transwell chambers and incubated for 20 h, followed by staining with crystal violet; *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: To understand the mechanisms underlying the changes in the invasive and migratory abilities of HCC cells, the levels of several invasion-related proteins were compared between the control HepG2 cells and the Cx32-depleted HepG2 cells, using western blot analysis. Downregulation of Cx32 did not cause a significant change in the expression of matrix metallopeptidase 2 (MMP2) or in that of VEGF, but did result in a significant decrease in the expression of KAI1/CD82, a tumor metastasis suppressor protein (Fig. 3A). The expression of CD82 in SMMC-7721 cells was increased following Cx32 overexpression (Fig. 3B). However, in the p53- Hep3B cells, overexpression of Cx32 failed to alter the expression of CD82 (Fig. 3B). Therefore, we believe that the upregulation of CD82 expression by Cx32 is p53-dependent because KAI1/CD82 transcription is also upregulated by p53 [21]. To confirm this notion, p53 was co-expressed in Hep3B cells; under these conditions, Cx32 was able to upregulate CD82 expression (Fig. 3B).

Bottom Line: The expression of connexin32 (Cx32) is frequently downregulated in HCC tissues.The reduction of Cx32 in HCC tissues was significantly associated with increased vascular invasion, increased tumor size, and poor survival.Additionally, Cx32 negatively regulated the phosphorylation of Akt and the expression of the cell cycle regulation protein cyclin D1, thereby inhibiting the proliferation of HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma, Xiamen University Affiliated Zhongshan Hospital, Xiamen, Fujian, China.

ABSTRACT
Hepatocellular carcinoma (HCC) progresses rapidly and is frequently associated with vascular invasion, metastasis, recurrence, and poor prognosis. The expression of connexin32 (Cx32) is frequently downregulated in HCC tissues. In this study, the role of Cx32 in HCC metastasis and proliferation was investigated. The reduction of Cx32 in HCC tissues was significantly associated with increased vascular invasion, increased tumor size, and poor survival. In vitro assays revealed that Cx32 not only suppressed the invasion and migration of HCC cells, but also repressed HCC cell proliferation. Subsequent investigations revealed that Cx32 directly enhanced the acetylation and transcriptional activity of p53, thus upregulating the expression of the tumor metastasis suppressor protein KAI1/CD82, which is a p53 target gene. Additionally, Cx32 negatively regulated the phosphorylation of Akt and the expression of the cell cycle regulation protein cyclin D1, thereby inhibiting the proliferation of HCC cells. Our in vivo nude mice model further confirmed that Cx32 is able to suppress HCC tumor growth and metastasis in nude mice. Our results imply that Cx32 downregulation contributes to the proliferation and metastasis of HCC, and the restoration of Cx32 expression may be a promising strategy for HCC therapy.

No MeSH data available.


Related in: MedlinePlus