Expression And Significance Of PAK4 In Gastric Cancer

Tumor is among the most severe human diseases around the world threateningpublic health with dead rate keeping on increasing. Therefore, the study on thepathogenesis , diagnosis and therapy of tumor is a major subject in our county. Byexploring the molecule mechanism of the key target proteins in cell signalingtransduction pathways, we know more about the genesis and development of themalignant tumor. Now most researchers are searching for the target proteins which areessential in the tumor therapy. So we may cure patients with cancer by targeting theproteins. p21-activated kinase, one of the evolutionarily conserved families ofserine/threonine protein kinases, is a down-stream effector of small GTPase Cdc42/Rac,which have been implicated in a wide range of biological activities. Until now, PAKfamilies has 6 members which contain an N-terminal regulatory domain and ac-terminal catalytic domain. PAKs are now categorized into two subgroups based onarchitectrual similarities. The GroupⅠcontains PAK1, PAK2, PAK3, GroupⅡcontains PAK4, PAK5, PAK6. PAK4 is the most recently identified member of the PAKfamily of serine/threonine kinases. PAK4 differs from other members of the PAKfamily in sequence and in many of its functions. PAK4 is a member of the groupⅡfamily of p21 activated kinases (PAKs). Its expression is elevated in many cancer celllines and activated PAK4 is highly transforming, suggesting that it plays an importantrole in tumorigenesis. 1. Materials(1) For immunostaining of Pak4, archival paraffin blocks of gastric specimens of95 gastric cancer patients were collected from the Gstric Cancer Lab in our hospitalfrom 2003～2005. There were 72 male and 23 female cases aged 29-80 years with anaverage age of 54 years.(2) Gastric cancer tissues and adjacent non-carcinomatous gastric mucosa from 40patients For Western blotting, fresh surgical gastric cancer and adjacent normal tissueswere obtained from 40 patients who underwent surgery at the Department of oncologyin our hospital.(3) Gastric cancer cell lines Gastric cancer cell lines MKN45, AGS, SGC7901,MKN1, N87, MGC823, MGC803, GES-1 were preserved in Cell Lab of China medicalmedical University.2. Methods(1) Immunohistochemical method was employed for PAK4 localization in 95tissue samples obtained from gastric cancer patients undergoing surgical treatment.Immunohistochemical test were performed by S-P method. The S-P Kit wasproduced by FuZhou Maixin Biological Corp, China.The primary anti-human wasmonoclonal mouse antibody against PAK4(1:50), the negative control was prepared inthe PBS, The positive were defined as brown to yellow staining in cytoplasm. Thepositive cells were classified into 3 degrees according to the number and color intensityof the cells. Weak positive (+), positive cells＜25%; intense positive (+++), positivecell number＞50%; positive cell number were between weak positive and intensepositive(++). The section having no obvious positive cells was defined negative.(2) Western blot was used to measure the expression of PAK4 in gastric cancertissues and adjacent non-carcinomatous gastric mucosa from 40 patients, gastric cancer cell lines.Tissue samples were lysed in 200μl RIPA buffer. The protein concentration wasmeasured according to the Bradford method; polyacrylamide gel eletrophoresis; after12 hour of transferring to PVDF membrane, the blot was probe with mouse monoclonalanti-human PAK4 (1:2000, BD, USA), and murine monoclonal anti-human GADPHanti-body(1:10000, SIGMA, USA), incubated 12h in 4℃. The secondary anti-body wasgoat anti-murine monoclon antibody, challenged with the appropriate second antibodyconjugated with peroxidase-conjugated affinipure goat anti-mouse IgG for 2h at roomtemperature, Ecl (PIERCE, USA) was used for detaction.The positive blot werescanned and then measured for intensity by using the Bandscan 5.0.Results(1) Immunohistochemical method indicated that positive staining of PAK4 ingastric cancer tissues was demonstrated in 67.37％of 95 cases. 62 cases(62/64, 96.88％)were positive for PAK4 staining which was located in the cytoplasm, only 2 cases werein the cell membrane. Expression of PAK4 was significantly associated with both TNMstage (x~2=10.426, P＜0.01) and lymph node metastasis (x~2=4.912, P＜0.05).(2) Western blot showed that PAK4 expression of gastric cancer tissues wasobviously higher than that of adjacent non-carcinomatous gastric mucosa (t=-5.978,P＜0.01). Expression of PAK4 was gradually increased paralleling with TNM stages.Expression of PAK4 could be detected in 7 human gastric cancer cell lines. Ascompared with GES-1, expression of PAK4 is higher in MKN45, AGS, N87, BGC823,while expression of PAK4 is lower in MGC803, MKN-1, SGC7901. ConclusionThese results suggest that PAK4 may play an important role in the developmentand metastasis of human gastric cancer.