Over Expression Of Klotho Suppresses Liver Cancer Progression And Induces Cell Apoptosis By Negatively Regulating Wnt/?-catenin Signalling Pathway

Background:Liver cancer is one of the most lethal cancers in the world,and its mortality ranks the third in cancer deaths.Each year,about 60 million people worldwide die of liver cancer,and this has a growing trend.Primary liver cancers mainly include two types:hepatocellular carcinoma(HCC)and intrahepatic bile duct cancer.In the developing countries,hepatitis B and hepatitis C are the risk factors responsible for most cases of primary liver cancer.Although the risk factors are preventable,the incidence of liver cancer is actually rising in many developing countries.The most common risk factor in the USA is alcohol abuse,followed by obesity and diabetes.The incidence has gotten over 30,000 cases each year in the USA.With the emergence of the problem,such as an aging population and environmental pollution,the incidence of liver cancer arises rapidly and liver cancer has become the second killer in China.It not only gives the pain and psychological burden for patients but also gives serious economic burden to the family and the community.Thus,it has become the focus of medical research and hotspots in China to investigate the molecular mechanisms of the development and progression of liver cancer,which could give some new clues for the prognosis and treatment on hepatocellular carcinoma.Kuro-o et al found that Klotho-deficient mice developed multiple premature aging syndromes,whereas overexpression of Klotho extended the lifespan of mice.The Klotho gene is located in chromosome 13q12 in human with the size of 50 kb.It encodes a single-pass transmembrane protein,which consists of an extracellular domain,a single transmembrane domain,and an intracellular domain.The intracellular domain is very short and has no clear functions.Membrane Klotho functions as an obligate co-receptor of fibroblast growth factor 23(FGF23)to regulate phosphate homeostasis.The extracellular domain(secreted Klotho)could be released into the serum and functions as a circulating hormone to regulate the activity of oxidative stress,multiple growth factor receptors,and ion channels.The tumor suppressive activity of Klotho was first identified in breast cancer in 2008.In these malignancies,Klotho was elucidated to be a modulator of several signaling pathways,including the FGF signaling,insulin-like growth factor-1 receptor(IGF-1R),and Wnt pathways,which are also involved in the pathogenesis of hematological malignancies.Recent investigations have implicated that Klotho is extensively down regulated in several solid tumors,including cervical cancer,pancreatic cancer,melanoma,and several digestive neoplasm.Recently,the relationship of Klotho expression and cancer progression has been studied;however,the role and mechanism in a variety of cancers remain unclear.Lu et al.had a clinical follow up study of 189 epithelial ovarian cancer patients,which demonstrated that high expression of secreted Klotho was associated with increased risk of disease progression and death and positively correlated with the expression of IGF-I and IGFBP-3 but not with IGF-II.However,Wang et al.reported that Klotho,as a novel tumor suppressor gene,was epigenetically inactivated and silenced through promoter hypermethylation in gastric cancer,and the promoter methylation of Klotho could be used to predict the prognosis of gastric cancer patients.In human colon cancers,expression of Klotho was downregulated and correlated with tumor invasion and Dukes staging,while overexpression of Klotho inhibited cell proliferation and invasion through inhibition of IGFlR-mediated PI3K/AKT pathway in colon cancer cells.Moreover,Klotho inhibited the capacity of cell migration and invasion in cervical cancer,in vitro restoration of Klotho expression in SiHa cells resulting in a decreased cell motility and invasiveness through upregulation of E-cadherin,downregulation of N-cadherin,and reduced expression of MMP-7 and MMP-9.Zhou reported that Klotho effectively inhibited the growth of DLBCL cells.Overexpression of Klotho significantly inhibited cell proliferation and induced cell apoptosis in DLBCL.We also discovered that efficacy of ADR could be enhanced by combination with rhKL in DLBCL cells.Our in vivo investigations demonstrated that upregulation of Klotho,by Klotho transfection,congruously led to inhibitory effect in the tumor growth of xenograft model of DLBCL.Hepatocellular carcinogenesis is a multi-step process that occurs during inflammation and in a progressive manner during tumor progression.It is reported that some inflammatory factors can be reduced the level Klotho in the cycle and Klotho can also be reversed to inhibit the effects of some inflammatory factors(such as TNF-a).In this respect,the abnormal activation of Wnt/?-catenin signaling pathway plays a key role in the pathogenesis of hepatocellular carcinoma.It has been reported that the secreted Klotho protein binds several Wnt ligands and inhibits Wnt signaling by preventing Wnt from binding to its homologous cell surface receptors.Klotho also acts as a Wnt antagonist,and as the stem cells fail,the Klotho gene mutant exhibits an enhanced Wnt signal.Klotho was a new identified aging suppressor gene,which is highly conserved between human and mouse,with 86%amino acid identity.In the present study,we explored the effects and possible mechanisms relating to Klotho in human liver cancer cell lines,and the clarification of the association and mechanism would contribute to treatment for therapy of liver cancer patients.Objective:1.The effect of Klotho expression on the proliferation and apoptosis of HCC cells was detected by RT-PCR and western blotting analysis,MTT assay,colony formation assay and FACS.2.To investigate the relationship between Klotho expression and Wnt/?-catenin signal transduction pathway in HCC cells of groups.Methods:1.Cell proliferation is tested by MTT assay.Generally,the human liver cancer cells HepG2 and SMMC-7721 cells were planted into 48-well plates.Then,the cells were cultured for 8 h and transfected with p CMV6-Klotho and p CMV-6 vector for 24,48,72,and 96 h.Finally,MTT agent was added into the medium,and the purple crystals were dissolved with DMSO.The liquid was transferred into 96-well plates and read on a microplate reader at a test wavelength of 490 nm and a reference wavelength of 570 nm.2.The level of Klotho mRNA in liver cancer cells is tested by RT-PCR,The RNA pure kit(Bioteke,Beijing,China)was used to extract total RNA,and reverse transc?riptase(RT)Super-Script ?(Invitrogen BV,Carlsbad,CA)was used to transcript the cDNA.The primer sequences were as follows:for glyceraldehyde-3-phosphate dehydrogenase(GAPD-H),5-GAAGGTGAAGGTCGGAGTC-3'(sense)and 5'-GAAGATGG-TGATGGGATTTC-3'(antisense)and for Klotho,5'-ACCTGGTG-GCGCACAACC-3'(sense)and 5-TTGGCAAACCAACCTAGTACA-3'.The Klotho PCR reaction condition is that 94 ? 5 min,followed by 30 cycles of 94 ? for 30 s,55 ? for 30 s,and 72 ? for 30 s,and finally,elongation is 72 ? for 10 min.The GAPDH reaction condition is similar to Klotho but for the annealing temperature it is 55 ? for 30s.3.The liver cancer cells were transfected with pCMV6-Klotho and pCMV-6 vector for stable cell lines.For colony formation assay,the cells were plated into 6-well plate.After 8 h,the cells transfected with Klotho or control vector were cultured for 14 days,and the medium was refreshed every 3 days.Then,methanol was used to fix the colonies.They were stained with 1.25%crystal violet and counted under a light microscope.4.Annexin V-FITC/PI dual staining analysis is used for detection of the apoptosis rates in human lung cancer cells according to the kit protocols.Briefly,the liver cancer cells were plated into 6-well plate and transfected with pCMV6-Klotho and pCMV6 vector for 48 h.Then,the cells were washed with ice PBS buffer for three times and resuspended in binding buffer with HEPES-NaOH 10 mM pH 7.4,25 mM CaC12,and 144 mM NaCl.At last,the staining dye of Annexin V and PI were added for incubation in the dark for 30 min on ice,and the cells were subjected to FACS analysis.5.Western blotting analysis were used to detect the expression of Klotho,?-catenin,C-myc,and Cyclin D1 in cytoplasm and nucleus in the cells with transfection.The liver cancer cells were plated into 48-well plates and cultured for 8 h for adherence.Then,the cells were transfected with pCMV6-Klotho and p CMV-6 vector for 48 h.The pCMV6 vector was used as the control.The other treatment was that the cells were treated with rKlotho or BSA at the concentration of 250 ng/mL.BSA was used as the negative control.Next,the cells were washed with ice-cold PBS buffer,and cell lysates were prepared for PAGE.Findings:1.Low level of Klotho is detected in liver cancer cell lines.The endogenous Klotho levels in a panel of four liver cancer cell lines were detected with RT-PCR and western blotting analysis.Low mRNA levels and protein levels were detected in liver cancer cell lines,such as Bel-7404,HepG2,SMMC-7721 etc.,compared with the immortalized liver cell L02.2.HepG2 and SMMC-7721 cells were transfected with expression vector of pCMV6-Klotho for 24,48,72 and 96 h.The OD490 values were obviously decreased in the group transfected with p CMV6-Klotho than those of p CMV6vector,suggest-ing that the cell proliferation was significantly suppressed as higher expression of Klotho.The HepG2 cells and SMMC-7721 cells were treated with recombinant Klotho at the concentration of 300 ng/mL for 24,48,72,and 96 h.The results demo-nstrated that rKlotho administration inhibited cell growth of liver cancer cells HepG2 and SMMC-7721,and higher expression of Klotho,was accompanied with lower proliferation of liver cancer cells.All of the data collectively revealed that Klotho expression inhibited the proliferati-on of liver cancer cells.Overexpression or recombinant Klotho adminis-tration suppresses the proliferation of liver cancer cells.3.Klotho expression suppresses liver cancer cell growth by colony formation assay.The liver cancer cell line HepG2 and SMMC-7721 were transfected with pCMV6-Klotho and control plasmid(pCMV6 vector),and the cells were cultured for 10 days.The number of colonies was significantly lower in the cells with transfection with pCMV Klotho than the controls.Klotho expression inhibits the proliferation of liver cancer cells,and Klotho plays an important antitumor role as a potential tumor suppressor.4.FACS analysis was used by dural staining with PI and Annexin V-FITC.The apoptosis rates in HepG2 cells transfected by pCMV6-Klotho was significantly higher than the control cells(P<0.01),and this was consistent with the situation in SMMC-7721 cells.Klotho expression induces cell apoptosis in human liver cancer cells.5.Wnt/?-catenin signaling pathway is abnormally expressed in liver can-cer cells.The HepG2 cells were transfected with p CMV6-Klotho and control plasmid pCMV6.The expression levels of Klotho,?-catenin,C-myc,and Cyclin D1 were detected by western blot.The HepG2 cells in p CMV6-Klotho transfected group had higher levels of Klotho,accomp-anied by lower expression of ?-catenin,C-myc,and Cyclin D1.Consist-ent with this finding,the HepG2 cells were treated with recombinated Klotho for 48 h,and the results showed increased Klotho expression concomitant with decreased levels of ?-catenin,C-myc,and Cyclin D1.Klotho levels negatively correlate with Wnt/?-catenin signaling pathway in HepG2 cells.Conclusion:Klotho was a tumor suppressor gene and overexpressi-on of Klotho suppressed the proliferation of liver cancer cells partly due to negative regulation of Wnt/?-catenin signalling pathway.So Klotho might be used as a potential target and the study will contribute to treatment for therapy of liver cancer patients.