Overexpression of Klotho suppresses liver cancer progression and induces cell apoptosis by negatively regulating wnt/β-catenin signaling pathway.
BACKGROUND
Klotho is a discovered aging suppressor gene, and its overexpression in mice extends the life span of the animal. Recently, Klotho is also identified as a tumor suppressor gene in variety of tumors; however, the potential role and the antitumor mechanism remain unclarified in liver cancers.


METHODS
RT-PCR and western blotting analysis were used to detect the expression of Klotho, β-catenin, C-myc, and Cyclin D1. MTT assay was used to detect the survival rates of HepG2 and SMMC-7721 cells. Colony formation assay was used to test the proliferation ability in Klotho transfected cells. FACS was used to detect the cell apoptosis rate in different groups.


RESULTS
The results showed that lower expression of Klotho were found in liver cancer cell lines than the immortalized liver cell L02. Also, MTT assay results found that overexpression or recombinant Klotho administration suppressed the proliferation of liver cancer cells HepG2 and SMMC-7721. Moreover, the colony formation assay results showed that the number of colonies was significantly lower in the cells with transfection with pCMV-Klotho than the controls. Thus, functional analysis demonstrated that Klotho expression inhibited the proliferation of liver cancer cells and Klotho worked as an important antitumor gene in tumor progression. Next, the mechanism was partly clarified that Klotho expression induced cell apoptosis in HepG2 and SMMC-7721 cells, and this phenomenon was mainly involved in the Wnt/β-catenin signaling pathway. The western blotting analysis revealed that overexpression or recombinant administration of Klotho obviously decreased the expression levels of β-catenin, C-myc, and Cyclin D1 in HepG2 cells. Most importantly, the antitumor mechanism for Klotho due to that overexpression of Klotho not only decreased the endogenous β-catenin levels but also inhibited the nuclear translocation of β-catenin to delay the cell cycle progression.


CONCLUSIONS
Klotho was a tumor suppressor gene, and overexpression of Klotho suppressed the proliferation of liver cancer cells partly due to negative regulation of Wnt/β-catenin signaling pathway. So, Klotho might be used as a potential target, and the study will contribute to treatment for therapy of liver cancer patients.
Department of Hepatobiliary, Surgery Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province, China.|Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, Shandong Province, China.
Huidong
H
Sun
Department of Hepatobiliary, Surgery Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province, China.
Yanchao
Y
Gao
Department of Gastroenterology, Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province, China.
Kemei
K
Lu
Department of Hepatobiliary, Surgery Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province, China.
Guimei
G
Zhao
Department of Hepatobiliary, Surgery Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province, China.
Xuehua
X
Li
Department of Hepatobiliary, Surgery Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province, China.
Zhu
Z
Li
Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, Shandong Province, China. changhong_1998@163.com.
Hong
H
Chang
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D005434:Flow Cytometry; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000090265:Klotho Proteins; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D051153:Wnt Proteins; D051176:beta Catenin; D005966:Glucuronidase; D000090265:Klotho Proteins