| Background and Objective KAI1/CD82 belongs to tetraspanin superfamily of transmembrane proteins, which are characterized by four membrane-spanning domains and play important roles in the regulation of cell migration, fusion, adhesion, differentiation and proliferation. KAI1 acts as a suppressor of wide-spectrum tumor metastasis during the progression of different solid tumors including prostate, lung and liver cancers. In malignant solid tumors, the presence of KAI1 predicts a better prognosis for patients with cancer, and down-regulation or loss of KAI1 expression is constantly found in the clinically advanced stages. KAI1 over-expression can inhibit cancer cell migration and invasion in vitro and suppress cancer metastasis in animal models.KAI1 can inhibit the cell motility through modulating cell motility-related signaling pathway or signal molecule function. Recently, Bandyopadhyay S et al found that KAI1 interacts with the Duffy antigen receptor for chemokines (DARC) which transmits inhibitory signal of cell proliferation and induces cell senescence by modulating the expression of TBX2 and p21. KAI1 suppresses integrin-induced cell invasion by regulating cell growth factor receptor and its downstream molecules such as c-Met, EGF receptor, Src kinases and FAK. However, the exact molecular mechanism of the suppressor function of KAI1 remains elusive.Ras/ Erk signaling pathway plays a central role in the RTK signal cascades. Sprouty2, a member of sprouty family, has been identified as a negative feedback regulator of RTK signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade. Several lines of evidences suggest sprouty2 has the tumor suppressive function. Consistently, Sprouty2 is downregulated in several types of cancer, including breast, prostate and liver cancers. Overexpression of sprouty2 has shown to suppress migration of cells via inhibition of ERK activation. Sprouty protein can biochemically interact with membrane protein such as Cav-1. Given the similar effect and position in the cytokine signaling, we hypothesize that Sprouty and KAI1 might have a functional overlapping in the suppression of cytokine signaling. Here, we characterized the functional role of Sprouty-2 in the KAI1-induced suppressive effect on migration of HCC.Methods Tranfection of Ad-KAI1 A replication-deficient recombinant adenovirus vector expressing KAI1 (Ad-KAI1) was constructed and expanded. An adenovirus vector carrying green fluorescent protein (GFP) was utilized to transfect SMMC-7721 cells as a negative control. Assay of sphingosine kinase activity Activation of SPK was measured as previously described with slight modifications. Briefly, cells were harvested in buffer and lysed by freeze-thaw. The protein concentration of the cell lysate was determined using bicinchoninic acid. Samples were assayed for SPK activity by incubation with sphingosine and [γ-32P] ATP. Reactions were stopped and the labeled lipids in the organic phase were separated by thin-layer chromatography. Labeled S1P was visualized by autoradiography. Western blotting Cells were washed twice with ice-cold phosphate-buffered saline, and total cell lysates were prepared by rupturing the cells in lysis buffer. Cells were lysed by freeze-thaw. Equal samples of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. After being blocked, the PVDF membrane was incubated with the primary antibody and then with horseradish peroxidase-conjugated secondary antibody. The membrane was developed with enhanced chemiluminescence substrate. Cell migration assay Assessment of cell migration was performed as that recently described with minor modifications. SMMC-7721 cells were dislodged after brief trypsinization. Cell suspension was washed extensively and resuspended in the same medium. Cells were dispersed into the upper chamber of Transwell compartment with 8μm pore size filter. Cells were challenged by HGF or medium control to the lower chamber. Migrated cells were stained with crystal violet and examined by light micrography finally. Data were expressed as the number of migrating cells per visual field. We performed using a wound with a plastic micropipette tip and an in vitro cell migration assay using modified Boyden chambers in order to investigate the effect of the KAI1 -mediated inhibition of cellular motility. The control and stable KAI1 transfectant cells were seeded in a well of 24-well culture plate for the wound-healing assay. The migration of the cells at the wound front was photographed after 1 day using an inverted microscope. RNA interference The siRNA targeting human sprouty2 and control were synthesized by Shanghai Gene Chemical Company. These siRNA were cloned into the lentivirus vector with the GFP as a report gene, respectively. SMMC-7721 cells were transfected with virus carrying sporuty2 siRNA or control virus, according to the recommendation of the manufacturer. GFP positive cells were sorted out by flow cytometry and expanded. The expression of Sprouty2 was monitored by Western blotting analysis.Results Adenovirus-mediated KAI1 gene transfer suppresses HGF-induced SMMC-7721 cells migration SMMC-7721 cells were transfected with Ad-KAI1 or control adenoviral vector. The efficiency of KAI1 expression was proven to be more than 70% by flow cytometry analysis. Then, the HGF-induced migration of these cells was examined. Over-expression of KAI1 could suppress HGF-induced migration of SMMC-7721 cells markedly, and migrated cells are 109.2±6.3 vs 36.7±4.7(P<0.01). The stable KAI1 transfectant clones and the control transfectants were selected for additional functional assays. The wound repairs were significantly inhibited in the KAI1 transfectants as compared with the control transfectant cells. The difference in cellular migration was found to be statistically significant. The ratios of migration are 79.2±9.1% vs 27.2±5.1% respectively. The p values are <0.01. Adenovirus-mediated KAI1 gene transfer cannot influence SMMC-7721 cells cell cycle, apoptosis and cell proliferation. OD values 96h late detected by MTT are 3.33±0.19, 3.09±0.09,2.95±0.13 and 2.71±0.17 respectively. The ratios of apoptosis cells are 5.08±0.97% ,8.05±1.24%,7.15±1.19% and 10.72±2.71%. The ratios of G1 cells are 72.24%,70.55% , 66.70% and 68.63%. Adenovirus-mediated KAI1 gene transfer suppresses SPK activation of hepatocellcular carcinoma cells HGF induces the migration of HCC cells via multiple signals. It has been shown that SPK activation plays a vital role in HGF-induced cell migration. In addition,HGF also activates SPK in SMMC-7721 cells. SPK activation and expression in the SMMC-7721 cells transfected by Ad-KAI1 in different infection titer were investigated. The results show that the protein level of SPK was reduced in SMMC-7721 cells transfected with Ad-KAI1. Meanwhile, the cellular SPK activity was also decreased simultaneously. These data indicate that KAI1 can suppress the HGF-induced activation of SPK and migration of hepatocellcular carcinoma cells. The values of SPK protein are 0.91±0.06,0.64±0.03 and 0.45±0.02, respectively. The values of SPK activation are 0.83±0.04,0.76±0.02 and 0.59±0.06,respectively. HGF activates SPK in SMMC-7721 cells We tested whether HGF could stimulate the SPK activity in SMMC-7721 cells. SMMC-7721 cells were starved overnight in serum-free medium before the addition of HGF. Then, these cells were stimulated with various concentrations of HGF, and the cellular SPK activity was assayed . HGF could activate SPK in a concentration-dependent manner. When the SMMC-7721 cells were treated with HGF at a concentration of 50 ng/ml, the cellular SPK activity reached a maximum value. We then measured the total cellular S1P in SMMC-7721 cells treated with or without HGF. HGF stimulation increased the intracellular S1P level. These data showed that HGF stimulation of SMMC-7721 cells leads to activation of SPK. The values of SPK activation are 1.82±0.09,2.16±0.07 and 3.41±0.11,and the P values are <0.05. HGF could induce the migration of SMMC-7721 cells We examined the migration of SMMC-7721 cells treated with HGF. Our results showed that HGF could induce markedly migration (about 10-fold increase) of SMMC-7721 cells. The number of migration is 19.3±2.9 vs 97.2±7.1 and 115.87±17(P<0.01). DMS abolished the the migration of SMMC-7721 cells We evaluated whether activation of SPK would be involved in the migration of SMMC-7721 cells induced by HGF. The cells were pretreated with DMS, and assayed the HGF-induced migration. DMS nearly completely abolished the migration of SMMC-7721 cells. The number of migration is 115.72±9.7 vs 10.1±2.6. PD98059 and LY294002 abolished the the activation of SPK by HGF To examine the role of PI3K, ERK1/2 in the activation of SPK by HGF in SMMC-7721 cells, we pretreated the cells with PD98059 and LY294002, specific inhibitors of ERK1/2 and PI3K, respectively, and then assayed the HGF-induced SPK activity. LY294002 and PD98059 could completely abolish the activation of SPK induced by HGF. This indicates that HGF activates SPK via PI3K and ERK1/2 pathways. KAI1 upregulated sprouty2 protein level in SMMC-7721 cells Sprouty2 is a negative regulatory molecule in HGF signaling cascades. The changes of sprouty2 expression in SMMC-7721 cells transfected with KAI1 were analyzed by Western blot. Sprouty2 protein was upregulated in KAI1 transfected cells. The upregulation of sprouty2 was consistent with the increased efficiency of KAI1 gene transfer. The values of Sprouty2 protein expression are 1.18±0.04 , 1.56±0.03 and 1.61±0.04. Adenovirus-mediated KAI1 gene transfer suppresses MAPK activation of hepatocellcular carcinoma cells HGF also activates MAPK in SMMC-7721 cells. Phosphorylation of MAPK in the cells transfected by Ad-KAI1 was investigated. The results show that the protein level of phosphorylation-MAPK was reduced in SMMC-7721 cells transfected with Ad-KAI1. These data indicate that KAI1 can suppress the MAPK. The values of p-MAPK are 0.84±0.04 , 0.72±0.06 and 0.44±0.06, respectively. Sprouty2 mediated suppressive effect of KAI1 on HCC cell migration and SPK activation To clarify the role of sprouty2 in KAI1–induced SPK suppression of HCC cells, we employed a specific siRNA to block the expression of sprouty2. SMMC-7721 cells were transfected with the lentiviral vectors carrying sprouty2 siRNA. The transfected cells were selected by flow cytometry and expanded. The sprouty2 protein was down-regulated significantly in transfected SMMC-7721 cells. We also investigated that sprouty2 deletion could abolish the suppressive effect of KAI1 on SPK expression in SMMC-7721 cells. Sprouty2-depleted and control cells were transfected with Ad-KAI1 or control adenovirus, and were stimulated by HGF at concentration of 50ng/ml for migration assay. Number of migrated sprouty2-deleted cells was significant higher than that of control cells.Conclusion KAI1 has been identified to play an important role in suppression of cancer cell metastasis. It attenuates the growth factor signaling in various cancer cells. SPK is a key enzyme catalyzing phosphorylation of sphingomyelin (SPK) to form sphingosine 1-phosphate (S1P) which regulates cellular processes such as growth, differentiation, motility. It plays key role in mediating cytokine-induced cell movement. We found that adenovirus-mediated gene transfer of KAI1 could reduce SPK expression and decrease the SPK cellular activity. This gives the clue that suppression of KAI1 on cell migration is associated with SPK/S1P signaling. Sprouty2, one member of Sprouty family, is known to act downstream in many RTKs pathways. It is found that Sprouty2 expression can be induced by HGF. The overexpressed sprouty2 in the form of negative feedback can regulate HGF signaling. Then it was characterized that KAI1 expression could induce the up-regulation of sprouty2 in HCC cells. The expression of sprouty2 was blocked by a lentivirus vector carrying the specific siRNA. We further identified that depletion of sprouty2 with siRNA attenuated the inhibitory effect of KAI1 in HCC cells, including both SPK expression and cell migration. These data indicated that the mechanism by which KAI1 inhibits tumor metastasis is via the Sprouty2 upregulation. Key words: KAI1; sprouty2; SPK;Migration; Hepatocyte growth factor;... |
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