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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 suppresses HCC cell proliferation through inhibition of the Akt signaling pathway(A, B) EdU assay analysis of the Cx32 knockdown (A) and overexpression (B) effect on the proliferation of HepG2 and SMMC-7721 cells. Cells were cultured in 24-well microtiter plates after transfection or non-transfection. EdU (100 μM) was added, and the cells were cultured for another 2 h before EdU and Hoechst staining. The total cell number and EdU-positive cell number were counted in five random fields; ** p < 0.01. (C) The expression of PCNA, Akt, and cell cycle regulatory proteins in Cx32-overexpression or knockdown HCC cells and control cells. (D) The PI3K inhibitor attenuated the inhibitory function of Cx32 on PCNA and cyclin D1 protein levels, as examined by western blotting. Data were measured as relative density (RD) of p-Akt/Akt, Cyclin D1/Actin, and PCNA/Actin. The first control lane was defined as 1.
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Figure 4: Cx32 suppresses HCC cell proliferation through inhibition of the Akt signaling pathway(A, B) EdU assay analysis of the Cx32 knockdown (A) and overexpression (B) effect on the proliferation of HepG2 and SMMC-7721 cells. Cells were cultured in 24-well microtiter plates after transfection or non-transfection. EdU (100 μM) was added, and the cells were cultured for another 2 h before EdU and Hoechst staining. The total cell number and EdU-positive cell number were counted in five random fields; ** p < 0.01. (C) The expression of PCNA, Akt, and cell cycle regulatory proteins in Cx32-overexpression or knockdown HCC cells and control cells. (D) The PI3K inhibitor attenuated the inhibitory function of Cx32 on PCNA and cyclin D1 protein levels, as examined by western blotting. Data were measured as relative density (RD) of p-Akt/Akt, Cyclin D1/Actin, and PCNA/Actin. The first control lane was defined as 1.

Mentions: To investigate the role of Cx32 in HCC cell proliferation, an EdU assay was performed in stable Cx32-knockdown HepG2 cells and control HepG2 cells. As shown in Fig. 4A, Cx32-knockdown HepG2 cells (sh-Cx32) had a significantly higher positive rate for EdU incorporation than shCtrl HepG2 cells did (39.6% vs. 24.5%, respectively, p = 0.0025). Similarly, Cx32 overexpression in SMMC-7721 cells significantly suppressed cell proliferation (from 30% to 19.6% EdU-positive cells, respectively, p = 0.0078; Fig. 4B). The expression of the proliferation marker proliferating cell nuclear antigen (PCNA) was also decreased following Cx32 overexpression, and was induced in Cx32-knockdown cells, as determined by western blot analysis (Fig. 4C). These results demonstrate the suppressing effect of Cx32 on HCC cell proliferation. Surprisingly, the expression of the cell cycle inhibitor p21Cip1/Waf1 was also decreased in the Cx32-overexpressing SMMC-7721 cells. p21 is a p53 target gene, and Cx32 was shown to positively regulate the transcriptional activity of p53 (Fig. 3C); however, here it negatively regulated p21 expression. Therefore, we concluded that the effect of Cx32 on p21 expression was p53-independent and did not occur at the transcriptional level; thus, p21 might not be involved in the regulation of HCC proliferation by Cx32.


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 suppresses HCC cell proliferation through inhibition of the Akt signaling pathway(A, B) EdU assay analysis of the Cx32 knockdown (A) and overexpression (B) effect on the proliferation of HepG2 and SMMC-7721 cells. Cells were cultured in 24-well microtiter plates after transfection or non-transfection. EdU (100 μM) was added, and the cells were cultured for another 2 h before EdU and Hoechst staining. The total cell number and EdU-positive cell number were counted in five random fields; ** p < 0.01. (C) The expression of PCNA, Akt, and cell cycle regulatory proteins in Cx32-overexpression or knockdown HCC cells and control cells. (D) The PI3K inhibitor attenuated the inhibitory function of Cx32 on PCNA and cyclin D1 protein levels, as examined by western blotting. Data were measured as relative density (RD) of p-Akt/Akt, Cyclin D1/Actin, and PCNA/Actin. The first control lane was defined as 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496344&req=5

Figure 4: Cx32 suppresses HCC cell proliferation through inhibition of the Akt signaling pathway(A, B) EdU assay analysis of the Cx32 knockdown (A) and overexpression (B) effect on the proliferation of HepG2 and SMMC-7721 cells. Cells were cultured in 24-well microtiter plates after transfection or non-transfection. EdU (100 μM) was added, and the cells were cultured for another 2 h before EdU and Hoechst staining. The total cell number and EdU-positive cell number were counted in five random fields; ** p < 0.01. (C) The expression of PCNA, Akt, and cell cycle regulatory proteins in Cx32-overexpression or knockdown HCC cells and control cells. (D) The PI3K inhibitor attenuated the inhibitory function of Cx32 on PCNA and cyclin D1 protein levels, as examined by western blotting. Data were measured as relative density (RD) of p-Akt/Akt, Cyclin D1/Actin, and PCNA/Actin. The first control lane was defined as 1.
Mentions: To investigate the role of Cx32 in HCC cell proliferation, an EdU assay was performed in stable Cx32-knockdown HepG2 cells and control HepG2 cells. As shown in Fig. 4A, Cx32-knockdown HepG2 cells (sh-Cx32) had a significantly higher positive rate for EdU incorporation than shCtrl HepG2 cells did (39.6% vs. 24.5%, respectively, p = 0.0025). Similarly, Cx32 overexpression in SMMC-7721 cells significantly suppressed cell proliferation (from 30% to 19.6% EdU-positive cells, respectively, p = 0.0078; Fig. 4B). The expression of the proliferation marker proliferating cell nuclear antigen (PCNA) was also decreased following Cx32 overexpression, and was induced in Cx32-knockdown cells, as determined by western blot analysis (Fig. 4C). These results demonstrate the suppressing effect of Cx32 on HCC cell proliferation. Surprisingly, the expression of the cell cycle inhibitor p21Cip1/Waf1 was also decreased in the Cx32-overexpressing SMMC-7721 cells. p21 is a p53 target gene, and Cx32 was shown to positively regulate the transcriptional activity of p53 (Fig. 3C); however, here it negatively regulated p21 expression. Therefore, we concluded that the effect of Cx32 on p21 expression was p53-independent and did not occur at the transcriptional level; thus, p21 might not be involved in the regulation of HCC proliferation by Cx32.

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