KAI1通过调控Sprouty2和SPK抑制HGF诱导肝癌细胞迁移的实验研究
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摘要
研究背景及目的
原发性肝细胞癌(hepatocellular carcinoma,HCC)是我国最常见的恶性肿瘤之一,每年新发病例数占全世界新发病例数的比例高达42.5%,居各种肿瘤发病率的第二位; HCC的死亡率约为20/100,000,已成为农村第一位和城市第二位的癌症死因。HCC具有转移早、临床症状出现晚的特点,70%患者确诊时已属晚期,丧失了手术时机,即使直径小于5cm的小肝癌,根治性切除后的3年复发率也高达40-50%,追其原因,肿瘤细胞的转移是阻碍进一步提高疗效的最大障碍。
转移是恶性肿瘤的共同生物学特征,亦是导致肿瘤患者临床治疗预后差,易复发的主要原因;但由于肿瘤细胞转移及其调控的复杂性,至今我们对其发生的确切机制仍不清楚。因此,从分子水平阐明肿瘤转移机制可能为临床肿瘤的诊治提供新途径。KAI1/CD82基因于1995年首次被证实与肿瘤细胞的转移有关,其编码的跨膜糖蛋白具有调节细胞运动、黏附和抑制肿瘤细胞迁移、浸润的作用。在肝癌中,临床和实验证据都表明KAI1表达下调与转移灶的形成及患者预后不良密切相关,KAI1能使癌细胞转移能力明显减弱。目前,关于KAI1能够抑制肿瘤转移的认识已得到学者们普遍认同,但其调控肿瘤转移的具体作用机制仍不清楚。国外对KAI1基因作用机理的研究近年有逐渐增多的趋势,研究思路主要倾向于探讨KAI1对与肿瘤转移相关的信号分子和信号通路的影响,并已取得了一些初步的成果,但有关KAI1抑制癌细胞迁移的作用机制仍然不十分清楚。
我们课题组最近发现KAI1可下调胰腺癌细胞内信号分子鞘氨醇激酶(SPK)的活性,为我们研究KAI1抑制肿瘤转移的信号转导机制奠定了初步的基础。SPK是调节细胞内神经酰胺(Ceramide,Cer)和1-磷酸鞘氨醇(sphingosine1-phospate,S1P)水平的重要限速酶,可通过酶促反应维持二者的动态平衡,从而维持细胞的生理功能。Cer是一种负调控因子,抑制细胞生长、迁移、促进细胞凋亡;S1P是Cer的进一步代谢产物,是正调控因子,促进细胞增殖、分化、迁移和抑制凋亡。Cer和S1P——这两个鞘磷脂代谢产物的动态平衡决定了细胞状态的发展方向。近年来,研究表明SPK的活性受多种与肿瘤发生和转移密切相关的生长因子的调节,如血管内皮生长因子(VEGF)等,都可以通过MAPK等信号通路使SPK磷酸化,激活SPK,进而调节细胞内Cer和S1P的动态平衡发展方向,最终影响肿瘤细胞的增殖、凋亡以及迁移等生物学功能。因此,SPK也被认为是与肿瘤细胞生物学特性密切相关的生长因子信号传导途径中的一个重要信号分子。
Sprouty2是MAPK信号通路的抑制因子,发挥抑制细胞增殖、分化及迁移等生物学效应。肿瘤细胞大多伴有该信号通路的异常,作为负反馈抑制因子——Sprouty2也被认为有可能是一种抑癌基因,且在前列腺癌和乳腺癌等肿瘤细胞中已经证实了这一点。那么Sprouty2和SPK——这两个与肿瘤转移密切相关的信号分子是否参与了KAI1对肝癌细胞转移的抑制作用呢?本课题旨在探明KAI1对上述两个蛋白表达及酶活性的影响,以及KAI1、SPK和Sprouty2蛋白三者之间的作用关系,进一步探讨KAI1抑制肿瘤转移的信号转导途径。
研究方法
本研究采用肝癌细胞系SMMC-7721细胞为研究对象。应用本室构建的携带KAI1基因的复制缺陷型腺病毒载体(Ad-KAI1),将该基因导入肝癌细胞,采用Transwell、划痕实验检测KAI1对HGF诱导肝癌细胞迁移的影响;应用MTT、流式细胞仪检测KAI1对SMMC-7721细胞增殖能力、凋亡以及细胞周期等生物学特性的作用;Western Blot检测KAI1对SPK蛋白表达的影响;同位素掺入放射自显影法检测对酶活性的作用;Transwell检测HGF诱导肝癌细胞迁移,以及同位素掺入放射自显影检测HGF对SPK的激活作用;用PI-3K、MAPK信号通路的特异性阻滞剂PD98059和LY294002预处理SMMC-7721细胞,观察是否能阻断HGF对SPK的激活作用,阐明HGF通过何种信号通路激活SPK;加入SPK特异性阻断剂DMS,观察是否能阻断HGF诱导肝癌细胞迁移,以证实SPK在HGF诱导迁移信号通路中的作用。Western Blot检测SMMC-7721细胞转染KAI1后Sprouty2蛋白的表达情况和MAPK的磷酸化水平;构建携带靶向Sprouty2 siRNA序列的慢病毒表达载体,PCR鉴定阳性克隆,感染SMMC-7721细胞,通过挑克隆和流式细胞分选仪筛选分离Sprouty2 RNA干涉阳性细胞株,以Western Blot检测Sprouty2干涉后的表达水平;观察Sprouty2功能沉默后,KAI1对SPK以及对HGF诱导肝癌SMMC-7721细胞转移的抑制作用是否能够被阻断或明显减弱。
研究结果
(1) KAI1抑制HGF诱导的人肝癌SMMC-7721细胞迁移。转染Ad-KAI1后,HGF诱导的细胞迁移受到明显的抑制,其未转染KAI1的迁移细胞数值为109.2±6.3,转染的迁移细胞数值为36.7±4.7,两者差异具有统计学意义(P<0.01),划痕实验结果也提示转染KAI1的SMMC-7721细胞迁移能力明显下降,未转染Ad-KAI1的细胞迁移了79.2±9.1%,而转染Ad-KAI1的细胞仅迁移27.2±5.1%,差异具有显著性(P<0.05);
(2) KAI1对SMMC-7721细胞增殖、凋亡和细胞周期无明显影响。转染KAI1后细胞增殖受到轻度抑制,96h后测MTT相对OD值分别为:对照组3.33±0.19,50MOI组3.09±0.09,100MOI组2.95±0.13,150MOI组2.71±0.17,但差异不显著;转染KAI1后细胞凋亡变化不明显,四组的细胞凋亡比例分别为:5.08±0.97%,8.05±1.24%,7.15±1.19%和10.72±2.71%;细胞周期亦未见明显影响,结果显示:G1期细胞比例分别为:72.24%,70.55%,66.70%和68.63%;
(3)转染KAI1后SMMC-7721细胞内SPK的蛋白表达及酶活性均受到明显抑制,将未转染KAI1的对照组设值为1,转染KAI1各组细胞SPK蛋白表达相对值分别为:50MOI组0.91±0.06,100MOI组0.64±0.03和150MOI组0.45±0.02,SPK酶活性的相对值分别为;0.83±0.04,0.76±0.02和0.59±0.06,KAI1对SPK的抑制作用存在剂量效应关系;
(4) HGF激活SPK的酶活性。将未加HGF刺激的对照组细胞SPK酶活性设值为1,加入不同浓度HGF刺激的细胞SPK酶活性相对值分别为:1.82±0.09,2.16±0.07;3.41±0.11,升高明显,差异显著(P<0.05),且HGF对SPK的激活存在剂量效应关系;
(5) HGF刺激诱导细胞迁移。未加HGF诱导的细胞迁移数只有19.3±2.9,25ng/mlHGF诱导迁移细胞数为97.2±7.1,50ng/mlHGF诱导迁移细胞数为115.87±17,差异具有统计学意义(P<0.01),诱导迁移作用呈明显的剂量效应依赖关系;
(6) DMS阻断HGF诱导SMMC-7721细胞迁移。将肝癌细胞用SPK特异性阻滞剂DMS预处理,加入HGF诱导迁移,未加DMS的细胞迁移数为115.72±9.7,而加入DMS预处理的SMMC-7721细胞的迁移数目明显减少,为10.1±2.6,二者比较有显著性差异(P<0.01),DMS阻断HGF诱导迁移的作用;
(7) PI-3K、MAPK信号通路阻滞剂对HGF激活SPK的影响。将SMMC-7721细胞用PI-3K、MAPK信号通路阻滞剂PD98059和LY294002预处理,加入HGF,未加处理的对照组SPK酶活性为2.82±0.18,加入PD98059和LY294002预处理的细胞SPK酶活性分别为0.49±0.03和0.38±0.07;
(8) KAI1上调Sprouty2表达。转染Ad-KAI1上调SMMC-7721细胞中Sprouty2蛋白的表达,将未转染Ad-KAI1的对照组Sprouty2蛋白表达量设为1,转染各组相对值分别为:1.18±0.04,1.56±0.03和1.61±0.04, Ad-KAI1的感染强度与Sprouty 2的蛋白表达呈剂量依赖的正相关;
(9) KAI1抑制MAPK磷酸化。转染Ad-KAI1抑制MAPK的磷酸化,将未转染Ad-KAI1的对照组MAPK磷酸化表达量设为1,其余转染的各组相对值分别为:0.84±0.04,0.72±0.06和0.44±0.06;
(10)设计构建的靶向Sprouty2的siRNA慢病毒表达载体。PCR扩增鉴定阳性克隆,在与理论值相符的300 bp位置扩增出条带,证明重组慢病毒载体带有目的基因,将该表达载体转染细胞,Western Blot证实其对SMMC-7721细胞中Sprouty2的表达确有干涉作用,将未干涉的对照组Sprouty2蛋白表达量设为1,Sprouty2干涉细胞的蛋白表达量为0.35±0.01,差异显著(P<0.01);
(11)沉默Sprouty2对KAI1抑制SPK的影响。采用RNAi沉默Sprouty2功能,发现未干涉Sprouty2的细胞KAI1使SPK活性降低达到60%左右,使SPK表达下降50%左右,而Sprouty2功能被干涉沉默后,KAI1对SPK活性的抑制率只有20%左右,对SPK表达的抑制率也只有10%,差异有统计学意义(酶活性:P<0.01;蛋白表达:P<0.05);
(12)沉默Sprouty2对KAI1抑制癌细胞迁移的影响。在未干涉Sprouty2的细胞,KAI1能够明显抑制HGF诱导SMMC-7721细胞迁移,未转染KAI1迁移细胞数为156.50±9.66,转染KAI1的迁移细胞数只有41.25±2.86,差异显著(P<0.01);而Sprouty2功能被沉默后,KAI1对HGF诱导细胞迁移的抑制减弱,未转染KAI1迁移细胞数为161.41±4.85,转染KAI1的迁移细胞数也达到120.23±11.98,无明显差异(P>0.05)。
研究结论
(1) KAI1显著抑制HGF诱导的肝癌SMMC-7721细胞迁移,对该细胞系的细胞增殖、细胞周期和凋亡的影响不明显;
(2) KAI1抑制SMMC-7721细胞鞘氨醇激酶(SPK)蛋白表达和酶活性;
(3) HGF通过PI3K、MAPK通路激活SPK来传递诱导SMMC-7721细胞迁移的信号,SPK是HGF诱导肝癌细胞迁移信号通路中的重要信号分子;
(4) KAI1通过调控SMMC-7721细胞SPK的蛋白表达和酶活性,实现其抑制HGF诱导细胞迁移的作用;
(5) KAI1能够上调SMMC-7721细胞的Sprouty2蛋白表达;
(6) Sprouty2蛋白表达的上调参与了KAI1抑制SPK及KAI1抑制肝癌细胞迁移的信号转导机制。
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;
原发性肝细胞癌(hepatocellular carcinoma,HCC)是我国最常见的恶性肿瘤之一,每年新发病例数占全世界新发病例数的比例高达42.5%,居各种肿瘤发病率的第二位; HCC的死亡率约为20/100,000,已成为农村第一位和城市第二位的癌症死因。HCC具有转移早、临床症状出现晚的特点,70%患者确诊时已属晚期,丧失了手术时机,即使直径小于5cm的小肝癌,根治性切除后的3年复发率也高达40-50%,追其原因,肿瘤细胞的转移是阻碍进一步提高疗效的最大障碍。
转移是恶性肿瘤的共同生物学特征,亦是导致肿瘤患者临床治疗预后差,易复发的主要原因;但由于肿瘤细胞转移及其调控的复杂性,至今我们对其发生的确切机制仍不清楚。因此,从分子水平阐明肿瘤转移机制可能为临床肿瘤的诊治提供新途径。KAI1/CD82基因于1995年首次被证实与肿瘤细胞的转移有关,其编码的跨膜糖蛋白具有调节细胞运动、黏附和抑制肿瘤细胞迁移、浸润的作用。在肝癌中,临床和实验证据都表明KAI1表达下调与转移灶的形成及患者预后不良密切相关,KAI1能使癌细胞转移能力明显减弱。目前,关于KAI1能够抑制肿瘤转移的认识已得到学者们普遍认同,但其调控肿瘤转移的具体作用机制仍不清楚。国外对KAI1基因作用机理的研究近年有逐渐增多的趋势,研究思路主要倾向于探讨KAI1对与肿瘤转移相关的信号分子和信号通路的影响,并已取得了一些初步的成果,但有关KAI1抑制癌细胞迁移的作用机制仍然不十分清楚。
我们课题组最近发现KAI1可下调胰腺癌细胞内信号分子鞘氨醇激酶(SPK)的活性,为我们研究KAI1抑制肿瘤转移的信号转导机制奠定了初步的基础。SPK是调节细胞内神经酰胺(Ceramide,Cer)和1-磷酸鞘氨醇(sphingosine1-phospate,S1P)水平的重要限速酶,可通过酶促反应维持二者的动态平衡,从而维持细胞的生理功能。Cer是一种负调控因子,抑制细胞生长、迁移、促进细胞凋亡;S1P是Cer的进一步代谢产物,是正调控因子,促进细胞增殖、分化、迁移和抑制凋亡。Cer和S1P——这两个鞘磷脂代谢产物的动态平衡决定了细胞状态的发展方向。近年来,研究表明SPK的活性受多种与肿瘤发生和转移密切相关的生长因子的调节,如血管内皮生长因子(VEGF)等,都可以通过MAPK等信号通路使SPK磷酸化,激活SPK,进而调节细胞内Cer和S1P的动态平衡发展方向,最终影响肿瘤细胞的增殖、凋亡以及迁移等生物学功能。因此,SPK也被认为是与肿瘤细胞生物学特性密切相关的生长因子信号传导途径中的一个重要信号分子。
Sprouty2是MAPK信号通路的抑制因子,发挥抑制细胞增殖、分化及迁移等生物学效应。肿瘤细胞大多伴有该信号通路的异常,作为负反馈抑制因子——Sprouty2也被认为有可能是一种抑癌基因,且在前列腺癌和乳腺癌等肿瘤细胞中已经证实了这一点。那么Sprouty2和SPK——这两个与肿瘤转移密切相关的信号分子是否参与了KAI1对肝癌细胞转移的抑制作用呢?本课题旨在探明KAI1对上述两个蛋白表达及酶活性的影响,以及KAI1、SPK和Sprouty2蛋白三者之间的作用关系,进一步探讨KAI1抑制肿瘤转移的信号转导途径。
研究方法
本研究采用肝癌细胞系SMMC-7721细胞为研究对象。应用本室构建的携带KAI1基因的复制缺陷型腺病毒载体(Ad-KAI1),将该基因导入肝癌细胞,采用Transwell、划痕实验检测KAI1对HGF诱导肝癌细胞迁移的影响;应用MTT、流式细胞仪检测KAI1对SMMC-7721细胞增殖能力、凋亡以及细胞周期等生物学特性的作用;Western Blot检测KAI1对SPK蛋白表达的影响;同位素掺入放射自显影法检测对酶活性的作用;Transwell检测HGF诱导肝癌细胞迁移,以及同位素掺入放射自显影检测HGF对SPK的激活作用;用PI-3K、MAPK信号通路的特异性阻滞剂PD98059和LY294002预处理SMMC-7721细胞,观察是否能阻断HGF对SPK的激活作用,阐明HGF通过何种信号通路激活SPK;加入SPK特异性阻断剂DMS,观察是否能阻断HGF诱导肝癌细胞迁移,以证实SPK在HGF诱导迁移信号通路中的作用。Western Blot检测SMMC-7721细胞转染KAI1后Sprouty2蛋白的表达情况和MAPK的磷酸化水平;构建携带靶向Sprouty2 siRNA序列的慢病毒表达载体,PCR鉴定阳性克隆,感染SMMC-7721细胞,通过挑克隆和流式细胞分选仪筛选分离Sprouty2 RNA干涉阳性细胞株,以Western Blot检测Sprouty2干涉后的表达水平;观察Sprouty2功能沉默后,KAI1对SPK以及对HGF诱导肝癌SMMC-7721细胞转移的抑制作用是否能够被阻断或明显减弱。
研究结果
(1) KAI1抑制HGF诱导的人肝癌SMMC-7721细胞迁移。转染Ad-KAI1后,HGF诱导的细胞迁移受到明显的抑制,其未转染KAI1的迁移细胞数值为109.2±6.3,转染的迁移细胞数值为36.7±4.7,两者差异具有统计学意义(P<0.01),划痕实验结果也提示转染KAI1的SMMC-7721细胞迁移能力明显下降,未转染Ad-KAI1的细胞迁移了79.2±9.1%,而转染Ad-KAI1的细胞仅迁移27.2±5.1%,差异具有显著性(P<0.05);
(2) KAI1对SMMC-7721细胞增殖、凋亡和细胞周期无明显影响。转染KAI1后细胞增殖受到轻度抑制,96h后测MTT相对OD值分别为:对照组3.33±0.19,50MOI组3.09±0.09,100MOI组2.95±0.13,150MOI组2.71±0.17,但差异不显著;转染KAI1后细胞凋亡变化不明显,四组的细胞凋亡比例分别为:5.08±0.97%,8.05±1.24%,7.15±1.19%和10.72±2.71%;细胞周期亦未见明显影响,结果显示:G1期细胞比例分别为:72.24%,70.55%,66.70%和68.63%;
(3)转染KAI1后SMMC-7721细胞内SPK的蛋白表达及酶活性均受到明显抑制,将未转染KAI1的对照组设值为1,转染KAI1各组细胞SPK蛋白表达相对值分别为:50MOI组0.91±0.06,100MOI组0.64±0.03和150MOI组0.45±0.02,SPK酶活性的相对值分别为;0.83±0.04,0.76±0.02和0.59±0.06,KAI1对SPK的抑制作用存在剂量效应关系;
(4) HGF激活SPK的酶活性。将未加HGF刺激的对照组细胞SPK酶活性设值为1,加入不同浓度HGF刺激的细胞SPK酶活性相对值分别为:1.82±0.09,2.16±0.07;3.41±0.11,升高明显,差异显著(P<0.05),且HGF对SPK的激活存在剂量效应关系;
(5) HGF刺激诱导细胞迁移。未加HGF诱导的细胞迁移数只有19.3±2.9,25ng/mlHGF诱导迁移细胞数为97.2±7.1,50ng/mlHGF诱导迁移细胞数为115.87±17,差异具有统计学意义(P<0.01),诱导迁移作用呈明显的剂量效应依赖关系;
(6) DMS阻断HGF诱导SMMC-7721细胞迁移。将肝癌细胞用SPK特异性阻滞剂DMS预处理,加入HGF诱导迁移,未加DMS的细胞迁移数为115.72±9.7,而加入DMS预处理的SMMC-7721细胞的迁移数目明显减少,为10.1±2.6,二者比较有显著性差异(P<0.01),DMS阻断HGF诱导迁移的作用;
(7) PI-3K、MAPK信号通路阻滞剂对HGF激活SPK的影响。将SMMC-7721细胞用PI-3K、MAPK信号通路阻滞剂PD98059和LY294002预处理,加入HGF,未加处理的对照组SPK酶活性为2.82±0.18,加入PD98059和LY294002预处理的细胞SPK酶活性分别为0.49±0.03和0.38±0.07;
(8) KAI1上调Sprouty2表达。转染Ad-KAI1上调SMMC-7721细胞中Sprouty2蛋白的表达,将未转染Ad-KAI1的对照组Sprouty2蛋白表达量设为1,转染各组相对值分别为:1.18±0.04,1.56±0.03和1.61±0.04, Ad-KAI1的感染强度与Sprouty 2的蛋白表达呈剂量依赖的正相关;
(9) KAI1抑制MAPK磷酸化。转染Ad-KAI1抑制MAPK的磷酸化,将未转染Ad-KAI1的对照组MAPK磷酸化表达量设为1,其余转染的各组相对值分别为:0.84±0.04,0.72±0.06和0.44±0.06;
(10)设计构建的靶向Sprouty2的siRNA慢病毒表达载体。PCR扩增鉴定阳性克隆,在与理论值相符的300 bp位置扩增出条带,证明重组慢病毒载体带有目的基因,将该表达载体转染细胞,Western Blot证实其对SMMC-7721细胞中Sprouty2的表达确有干涉作用,将未干涉的对照组Sprouty2蛋白表达量设为1,Sprouty2干涉细胞的蛋白表达量为0.35±0.01,差异显著(P<0.01);
(11)沉默Sprouty2对KAI1抑制SPK的影响。采用RNAi沉默Sprouty2功能,发现未干涉Sprouty2的细胞KAI1使SPK活性降低达到60%左右,使SPK表达下降50%左右,而Sprouty2功能被干涉沉默后,KAI1对SPK活性的抑制率只有20%左右,对SPK表达的抑制率也只有10%,差异有统计学意义(酶活性:P<0.01;蛋白表达:P<0.05);
(12)沉默Sprouty2对KAI1抑制癌细胞迁移的影响。在未干涉Sprouty2的细胞,KAI1能够明显抑制HGF诱导SMMC-7721细胞迁移,未转染KAI1迁移细胞数为156.50±9.66,转染KAI1的迁移细胞数只有41.25±2.86,差异显著(P<0.01);而Sprouty2功能被沉默后,KAI1对HGF诱导细胞迁移的抑制减弱,未转染KAI1迁移细胞数为161.41±4.85,转染KAI1的迁移细胞数也达到120.23±11.98,无明显差异(P>0.05)。
研究结论
(1) KAI1显著抑制HGF诱导的肝癌SMMC-7721细胞迁移,对该细胞系的细胞增殖、细胞周期和凋亡的影响不明显;
(2) KAI1抑制SMMC-7721细胞鞘氨醇激酶(SPK)蛋白表达和酶活性;
(3) HGF通过PI3K、MAPK通路激活SPK来传递诱导SMMC-7721细胞迁移的信号,SPK是HGF诱导肝癌细胞迁移信号通路中的重要信号分子;
(4) KAI1通过调控SMMC-7721细胞SPK的蛋白表达和酶活性,实现其抑制HGF诱导细胞迁移的作用;
(5) KAI1能够上调SMMC-7721细胞的Sprouty2蛋白表达;
(6) Sprouty2蛋白表达的上调参与了KAI1抑制SPK及KAI1抑制肝癌细胞迁移的信号转导机制。
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;
引文
1. Ikail I, Itai Y, Okita K, Omata M, Kojiro M, Kobayashi K, Nakanuma Y, Futagawa S, Makuuchi M, Yamaoka Y. Report of the 15th follow-up survey of primary liver cancer. Hepatol Res, 2004;28(1):21-29
2. Parkin DM. Global cancerr statistics in the year 2000. Lancet oncol, 2001;2(9):533-43.
3. EI-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United states, N Engl J Med, 1999; 340(7):745-50.
4. Ling Y, Donald M, Parkin D, Chen Y. Time trends in cancer motality in China: 1987-1999. Int J cancer, 2003;106(4):771-783.
5. Li LD, Liu FZ, Zhang SWl. Distribution analysis of cancer mortality in China during 1990-1992. Chin J oncol, 1996;18(4):403-407.
6. Zhou XD. Recurrence and metastasis of hepatocellular carcinoma: progress and prospects. Hepatobiliary Pancreat Dis Int, 2002; l(l): 35-41.
7. 李坚, 王洪林. 肝癌肝移植术后肝癌复发的研究进展. 中华外科杂志,2005; 43(11):753-756.
8. Dong JT, Lamb PW, Rinker-Schaeffer CW,Vukanovic J, Ichikawa T, Isaacs JT, Barrett JC. KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science. 1995;268 (5212): 884–886.
9. Guo XZ, Friess H, Di Mola FF, Heinicke JM, Abou-Shady M, Graber HU, Baer HU, Zimmermann A, Korc M, Büchler MW. KAI1, a new metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma. Hepatology 1998;28:1481-1486
10. Guo XZ, Friess H, Graber HU, Kashiwagi M, Zimmermann A, Korc M,Büchler MW. KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res 1996; 56: 4876-4880.
11. 徐建华,郭晓钟,任丽楠,赵家钧,崔忠敏,刘民培.KAI1 基因抗胰腺癌细胞转移的研究. 中华消化杂志 2004;24:541-544
12. Dong JT, Suzuki H, Pin SS, Bova GS, Schalken JA, Isaacs WB, Barrett JC, Isaacs JT. Down-regulation of the KAI1 metastsis suppressor gene during the progression of human prostatic cancer infre quently involves gene mutation or allelic loss.Cancer Res, 1996; 56(19):4387-4390.
13. Dong JT, Isaacs WB, Barrett JC, Isaacs JT. Genomic organization ofthe human KAI1 metastasis suppressor gene. Genomics, 1997; 41(18):25-32.
14. Gao AC, Lou W, Dong JT, Barrett JC, Danielpour D, Isaacs JT. Defining regulatory elementsin the human KAI1/CD82 metastasis suppressor gene.Prostate, 2003; 57(4):256-260.
15. Jackson P, Millar D,Kingsley E, Yardley G, Ow K, Clark S, Russell PJ.. Methylation of a CpG island within the promoter region of the KAI1/CD82 metastasis suppressor gene is not responsible for down-regulation of KAI1/CD82 expression in invasive cancers or cancer cell lines. Cancer Lett, 2000;157(2):169-176.
16. M. Nagira, T. Imai, I. Ishikawa, K.I. Uwabe, O. Yoshie, Mouse homologue of C33 antigen (CD82), a member of the transmembrane 4 superfamily: complementary DNA, genomic structure, and expression. Cell. Immunol. 1994;157 (1):144–157.
17. C.S. Stipp, T.V. Kolesnikova, M.E. Hemler, Functional domains in tetraspanin proteins. Trends Biochem. Sci. 2003; 28(2): 106–112.
18. Fedor B , Elena O. Characterization of integrin-tetraspaninadhesioncomplexes:role of tetraspanins in integrin signaling. Cell Bio,1999;146:477-492.
19. Naotaka S,Kenichi H,Hironori Y,Koyanagi M, Itoh N, Nishikawa J, Tanaka K. l.Verexpression of CD82 on human T cells enhances LFA-1/ICAM-1 mediated cellcelIadhesion:functionaI association between CD82 and LFA-1 inT cell activation.Eur J Immunol,1999;29:4081-4091.
20. Delaguillaumie A, Lagaudrière-Gesbert C, Popoff MR, Conjeaud H. Rho GTPases link cytoskeletal rearrangements and activation processes induced byvia the tetraspanin CD82 in T lymphocytes.J Cell Sci,2002;115:433-443.
21. Cecile LG,Sophie LB,Emmanuel W. The tetraspaninprotein CD82 associates with both free HLA class I heavy chainand heterodimeric 62-microglobulin complexes.Immunol,1997;158:2790-2797.
22. Zhang XA,He B,Zhou B,Liu L. Requirement of the pl30-CAs-Crk coucell migration. J Biol Chem,2003;278(29):27319-27328.
23. Hang XA,LaBe WS,Charrin S,Rubinstein E, Liu L. EWI2/PGRL associates with themetastasis suppressor KAI1/CD82 and inhibits the migration of prostate cancer cells.Cancer Res,2003;63(101):2665-2674.
24. Ono M,Handa K,Withers DA,Hakomori S.Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogne CD9 with concurrent glycosylation.Cancer Res. 1999; 59(10):2335-39.
25. Ono M,Handa K,Withers DA,Hakomori S. Glyeosylation efect on membrane domain(GEM)involved in cell adhesion and motility :prelimirary note on function CD82 glycosylation complex in IdID 14 cel1.Biochem Biophys Res Commun。2000; 279(3):744-750.
26. Zhou B,Liu L,Muralidhar R,Zhang XA. The Palmitoylation of Metastasis suppressor KAI1/CD82 Is Important for Its Motility and Invasiveness Inhibitory Activity.Cancer Research. 2004;64(23):7455-63.
27.Bouras T, Frauman AG.. Expression of the prostate cancermetastasis suppressor gene KAI1 in primary prostate cancers;a biphasic relationship with grade.J Paihol. 1999; 188(4):382-388.
28.HU WL,LI YQ, He HX, Li QR, Tian Y, Lai RQ, Mei H. KAI1/CD82 gene expression in benign prostatic hpperplasia and late-stage prostate cancer in Chinese.Asian Journal of Andrology. 2000; 2(3):221-224.
29. Kawana Y; Komiya A; Ueda T; Nihei N; Kuramochi H; Suzuki H; Yatani R; Imai T; Dong JT; Imai T; Yoshie O; Barrett JC; Isaacs JT; Shimazaki J; Ito H; Ichikawa T. Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer, Prostate 1997;36 (32): 205–213.
30. Dong LP, Cao FY, Zhang YF. The relationship between gastric cancer and KAI1/CD82 exprossion. Henan Journal of Oncology, 2004;17(6):397-399.
31. Tsutsumi S, Shimura T, Morinaga N, Mochiki E, Asao T, Kuwano H. Loss of KAI1 expression in gastric cancer. Hepato-Gastroenterology,2005;52(61):281-284.
32. Guo XZ, Friess H, Maurer C, Berberat P, Tang WH, Zimmermann A, Naef M, Graber HU, Korc M, Buchler MW. KAII is unchanged in metastatic and nonmetastatic esophageal and gastric cancers. Cancer Res, 1998; 58(4): 753-758
33. Friess H,Guo XZ,Berberat P. KAI1 facilitates the formation of metastasis in pancreatic cancer. Gut,1997;41(4S):76A.
34. Guo XZ, Xu JH, Liu MP, Kleeff J, Ho CK, Ren LN, Li HY, K?ninger J, Cui ZM, Wang D, Wu CY, Zhao JJ, Friess H. KAII inhibitsanchorage-dependent and –independent pancreatic cancer cell growth. Onco Rep. 2005; 14: 59-66
35. Guo XZ, Friess H, Shao XD, Liu MP, Xia YT, Xu JH. KAII gene is differently expressed in papillary and pancreatic cancer influence on metastasis. WJG, 2000;6(6):866-871
36. 徐建华,任丽楠,邵丽春,郭晓钟,刘民培. KAIl 基因抑制人胰腺癌细胞转移的体内实验. 胰腺病学 2006:6(1):8-11。
37. Yang GZ,Ding YQ,Liu L,et al.Changges of biological characteristics of colon carcinoma cell line LoVo induced by KAI 1/CD82 transfection. Zhonghua Bing Li Xue Za Zhi,2004;33(1):49-52.
38. L L, L ZG, Y GZ, Ding YQ. Effect of tumor suppressor gene KAI1 on the biological behaviors of human colorectal carcinoma. J First Mil Med Univ, 2003;23(6):587-590.
39. 董良鹏, 曹风雨, 张豫峰, 陈壬寅. KAI1/CD82 的表达与大肠腺癌的相关性分析.河南肿瘤学杂志. 2004;17(6):397-399
40. Lombardi DP, Geradts J, Foley JF, Chiao C, Lamb PW, Barrett JC. Loss of KAI1 expression in the progression of colorectal cancer. Cancer Res. 1999; 59(16): 5724–5731.
41. Adachi M, Taki T, Ieki Y, Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer. Cancer Res. 1996;56(8):1751-1715. .
42. Tagawa K, Arihiro K, Takeshima Y, Hiyama E, Yamasaki M, Inai K. Down-regulation of KAI1 messenger RNA expression is not associated with loss of heterozygosity of the KAI1 gene region in lung adenocarcinoma. J Cancer Res, 1999; 90(9):970-976.
43. Higashiyama M, Kodama K,Yodouchi H, et al. KAI1/CD82 expression innonsmall cell lung carcinoma is a novel, favorable prognostic factor:an immunohistochemical analysis.Cancer Res, 1998; 83(3):466-474.
44. Houle CD,Ding XY,Foley JF,Afshari CA, Barrett JC, Davis BJ. Loss of expression and altered localization of KAl1/CD82 and C199 protein are associated with epithelialovarian cancer progression.Gynec Oncol,2002;86:69-78.
45. Schindl M,Birner P,Bachtiary B,Plank C, Breitenecker G, Kowalski H, Oberhuber G.. The impact of expression ofthe metastasis suppressor protein KAI1/CD82 on prognosis in inva : sive squamous cell cervical cancer.Anticancer Res,2000;20(6B):45516-45517.
46. Liu FS, Chen JT, Dong JT, Hsieh YT, Lin AJ, Ho ES et al. KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma. Am J Pathol 2001;159 (5): 1629–1634.
47. Liu FS,Dong JT,Chen JT, Hsieh YT, Ho ES, Hung MJ, Lu CH, Chiou LC. KAI1/CD82 metastasis suppressor protein is down regulated during the progresion of human endometrial cancer. Clin Cancer Res,2003;9(4):1393-1398.
48. Chen Z,Mustafa T,Trojanowicz B,Brauckhoff M, Gimm O, Schmutzler C, K?hrle J, Holzhausen HJ, Kehlen A, Klonisch T, Finke R, Dralle H, Hoang-Vu C..CD82 and CD63 in thyroidcancer. Int J Mol Med,2004;14:517-527.
49. Imai Y,Sasaki T,Shinagawa Y,Akimoto K, Fujibayashi T. Expression of metastsis suppressorgen(eKAI1/CD82)in oral squamous cell carcinoma and its clinco pathological sign ificance. Oral Onco1,2002;38:557-561.
50. Shu Y,Chen HY,Tan,Y. Expression and clinical value of KAI1/CD82 in carcinoma of larynx. Lin Chuang Er Bi Yan Hou Ke Za Zhi,2005;19(23):1065-1067.
51. AmirthaLakshmi S,Pushparaj V,Krishnamurthy,Biswas J, Krishnakumar S, Shanmugam MP. Tetraspanin protein KAI1 expression in retinoblastoma. British Journal of Ophthalmology,2004;88(4):593-595.
52.Jing S,Arima K,Hasegawa M,Franco OE, Umeda Y, Yanagawa M, Sugimura Y, Kawamura J. Decreased expression of KAI1 metastasis suppressor gene is a recurrence predictor in primary pTa and pT1 urothelial bladder carcinoma. International Journal of Urology,2004;11(2):74-82.
53. Yu Y, Yang JL, Markovic B, Jackson P, Yardley G, Barrett J, Russell PJ. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res .1997;3(19): 1045–1049
54.Huang CI,Kohno N,Ogawa E,Adachi M, Taki T, Miyake M. Correlaation of reduction in MPR-1/CD9 and KAI1/CD82 expression with recurrences in breast patiengts.Am J Pathol,1998;153(3):973-983.
55.X. Yang, L. Wei, C. Tang, R. Slack, E. Montgomery, M. Lippman, KAI1 protein is down-regulated during the progression of human breast cancer, Clin Cancer Res. 2000;6 (9): 3424–3429.
56. X. Yang, L Wei, C. Tang, R. Slack, S. Mueller, M.E. Lippman, Overexpression of KAI1 suppresses in vitro invasiveness and in vivo metastasis in breast cancer cells, Cancer Res. 2001;61 (13): 5284–5288.
57. Burchert, Aotter, Menssenl. CD82(KAI1),a member of the tetraspan family , is expressed on early haemopoietic progenitor cells and up-regulated in distinct human leukaemias. British Journal of Haematology,1999;107(3):494-504.
58. HC Sun, ZY Tang, G. Zhou, XM Li, KAI1 gene expression in hepatocellular carcinoma and its relationship with intrahepatic metastases,J. Exp. Clin. Cancer Res. 1998;17 (15) 307–311.
59.Zhang WG, Wang Y, Chen H, Cheng YX, Ma XR, Yang Y, Wang YL.The significance of transmembrane 4 superfamily(TM4SF)expression in human hepatocellular carcinoma(HCC). Chin J Cli Med Pra, 2004; 3(3):197-199.
60.郑伟红,易继林,李兴睿,冯亮,董朝富. KAI1 基因在肝癌中的表达及临床意义. 临床和实验医学杂志;2006;5(9):1285-1289
61.彭志红, 唐波, 杨建民, 司遂海 , 陈文生 , 房殿春 , 罗元辉 , PENG Zhi-hong , TANG Bo , YANG Jian-min , SI Sui-hai , CHEN Wen-sheng , FANG Dian-chun , LUO Yuan-hui. KAI1 基因对MHCC97-H肝癌细胞粘附力的影响. 第三军医大学学报,2005;27(23):2311-2313
62.司遂海,杨建民,彭志红, 罗元辉, 周平. KAI1 基因对肝癌 MHCC97-H细胞生长及侵袭能力的影响. 中华肝脏病杂志;2005:13(8);615-617
63. Mashimo T,Watabe M,Hirota S,Hosobe S, Miura K, Tegtmeyer PJ, Rinker-Shaeffer CW, Watabe K. The expression of the KAI1 gene,a tumor metastasis suppressor,is directly activated by P53.Proc Natl Acad Sci USA,1998;95(19):1130-1131.
64. Duriez C,Falette N,Cortes U,Moyret-Lalle C, Puisieux A. Absence of p53 dependent induction of the metastatic suppressor KAI1 gene after DNA damage. Oncogene, 2000; 19(20):2461-2464.
65.Tomoyuki M,Misako W,Shigeru H,Hosobe S, Miura K, Tegtmeyer PJ, Rinker-Shaeffer CW, Watabe K. The expression of KAI1 gene,a tumor metastasis suppressor,is directly activatedby P53 . Proc Natl Acad Sci USA,1998;95:11307-11311.
66. Mashimo T,Bandyopadhyay S,Goodarzi G,Watabe M, Pai SK, Gross SC, Watabe K. Activation of the tumor metastasis suppressor gene KAI1,by etoposide ismediated by p53 and c-Jun genes.Biochem Biophys ResCommun,2000;274:370-376.
67.Marreiros A, Dudgeon K, Dao V. KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells. Oncogene. 2005;24(4):637-649.
68.Cyril D,Nicole FU,lrich C,Moyret-Lalle C, Puisieux A. Absence of p53-dependent induction of the metastatic suppressor KAI1 gene after DNA damage.Oncogene,2000;19:2461-2464.
69.Tsutomu Shinohara, Toyokazu Miki, Naoki Nishimura, Nokihara H, Hamada H, HMukaida N, Sone S. Nuclear factor-kappaB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53. Cancer Res, 2001;61(2):673-678.
70.Laguudriere GC. Lebel BS. Hubeau C. Signaling through the Tetraspanin CD82 triggers is association with the cytoskeleton leading to sustained morphological change and T cellactivation.Eur J Immunol.1998; 28(12):4332-44.
71. Delaguiuaumie A. Lagaudrlere GC. Popoff MR. Rho GTPases link cytoskeleton rearrangemants and activation processes induced via the tetraspanin CD82 in T lymphocytes.J Cell Sci. 2002;15(2):433-443
72.Bandyopadhyay S, Zhan R, Chaudhuri A, Watabe M, Pai SK, Hirota S, Hosobe S, Tsukada T, Miura K, Takano Y, Saito K, Pauza ME, Hayashi S, Wang Y, Mohinta S, Mashimo T, Iiizumi M, Furuta E, Watabe K. Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression.Nat Med. 2006;12(8):933-8.
73.Zijlstra A, Quigley JP. The DARC side of metastasis: shining a light on KAI1-mediated metastasis suppression in the vascular tunnel.Cancer Cell. 2006 ;10(3):177-8.
74.Iiizumi M, Bandyopadhyay S, Watabe K. Interaction of Duffy antigen receptor for chemokines and KAI1: a critical step in metastasis suppression.Cancer Res. 2007; 67(4):1411-4.
75. Zhang XA,He B,Zhou B,Liu L.Requirement ofthe pl30-CAs-Crk coucell migration.J Biol Chem,2003;278(29):27319-27328.
76. Jee B, Jin K, Hahn JH, Song HG, Lee H. Metastasis-suppressor KAII/CD82 induces homotypic aggregation of human prostate cancer cells through Src dependent pathway.Exp Mol Med, 2003; 35(1):30-37
77. Sridhar SC, Miranti CK. Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene. 2006;25(16):2367-78.
78. Wang XY, Liu T, Zhu CZ. Expression of KAI1, MRP-1, and FAK proteins in lung cancer detected by high-density tissue microarray. Ai Zheng. 2005 ;24(9):1091-5.
79. Takahashi M, Sugiura T, Abe M, Ishii K, Shirasuna K. Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration. Int J Cancer. 2007; 121(9):1919-29.
80.Zhang XA. Lane WS. Chanin S. Rubinstein E, Liu L. EW12/PGRL associates with the metastasis suppressor KAII/CD82 and inhibits the migration of prostate cancer cel1.Cancer research,2003;63(10):2665-74.
81. Jee JH , Park SR , Chay KO . KAII COOH-tenninal interacting tetraspanin(K1TENIN),a member of the trtraslmnin family,interacts with KAI1 a tumor metastasis suppressor , and enhances metastasis of cancer,Cancer research,2004; 64(12):4235-4243.
82. Rowe A, Jackson P. Expression of KITENIN, a KAI1/CD82 binding protein and metastasis enhancer, in bladder cancer cell lines: relationship toKAI1/CD82 levels and invasive behaviour. Oncol Rep. 2006 ;16(6):1267-72.
83.Jee BK, Park KM, Surendran S, Lee WK, Han CW, Kim YS, Lim Y. KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line. Biochem Biophys Res Commun. 2006; 342(2):655-61.
84.Jee BK, Lee JY, Lim Y, Lee KH, Jo YH. Effect of KAI1/CD82 on the beta1 integrin maturation in highly migratory carcinoma cells. Biochem Biophys Res Commun. 2007; 359(3):703-8.
85. Bass R, Werner F, Odintsova E, Regulation of urokinase receptor proteotic function by the tetraspanin CD82. J Biol Chem. 2005 ;280(15):14811-8.
86. Odintsova E,Sugiura T,Berditchevski F.Attenuation of EGF receptor signalingby a metastasis suppressor,the tetraspanin CD82/KAI-1. Curr Biol, 2000; 10(16): 1009-1012
87.Sehoenfeld N. Bauer MA.Grimim S. Bauer MA, Allen B, Al-Sebaei MO, Kakar S, Leone CW, Morgan EF, Gerstenfeld LC, Einhorn TA, Graves DT. Th e metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates.FASEB Journa1.2004; 18(1):158-160.
88.郭晓钟,张巍巍,王立生, 鲁茁壮, 王华, 徐建华, 田宏. Kai1 基因抑制胰腺癌细胞鞘氨醇激酶活性及其迁移. 中华内科杂志.2006: 45(9); 752-754.
89.Spiegel S, Milstien S. Sphingosine-1-Phosphate, a Key Cell Signaling Molecule. The Journal of Biological Chemistry. 2002: 277(29); 25851–25854,
90. Maceyka M, Payne SG, Milstien S, Spiegel S. Sphingosine kinase,sphingosine-1-phosphate, and apoptosis. Biochim Biophys Acta. 2002; 1585 (2-3):193-201
91. Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, Milstien S, Kohama T, Spiegel S. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. Biol Chem, 2000; 275(26):19513-20
92. Kohama T, Olivera A, EdsallL, Nagiec MM, Dickson R, Spiegel S. Molecular cloning and functional characterization of murine sphingosine kinase.J Biol Chem, 1998; 273(37): 23722-8
93. Le Stunff H, Galve-Roperh I, Peterson C, Milstien S, Spiegel S. Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis. J Cell Biol 2002; 158(6):1039-1049.
94.Sadahira Y, Ruan F, Hakomori S, Igarashi Y. Sphingosine 1 - phosphate ,a specific endogenous signaling molecule controlling cell ,motility and tumor cell invasiveness. Proc Natl Acad Sci USA , 1992; 89 (20) :9886 - 9690.
95.Paik J H ,Chae S ,Lee MJ , Cowan AE, Han DK, Proia RL, Hla T. Sphingosine -1- phosphate induced endothelial cell migration requires the expression of EDG- 1and EDG - 3 receptors and Rho - dependent activation of alphavbeta3 - and betal - containing integrins . J Biol Chem ,2001; 276 (15) :11830 - 11837.
96.Lee MJ , Thangada S , Paik J H , Sapkota GP, Ancellin N, Chae SS, Wu M, Morales-Ruiz M, Sessa WC, Alessi DR, Hla T. Akt - mediated phosphorylation of the G protein - coupled receptor EDG- 1 is required for endothelial cell chemotaxis . Mol Cell ,2001; 8 (3) :693 - 704.
97.Pu Xia, Jennifer R. Gamble,An oncogenic role of sphingosine kinase Current Biology. 2000; 10(23):1527-1530
98. Edsall LC, Van Brocklyn JR, Cuvillier O, Kleuser B, Spiegel S. N, N-Dimethylsphingosine is a potent compettitive inhibitor of sphingosine kinase but not of protein kinase C: Modulation of cellular levels of sphingosine-1-phosphate and ceramide. Biochemistry, 1998: 37 ( 37 ) : 128922-128981
99. 郭强, 李荣, 李庆芳. 高表达鞘氨醇激酶对胃癌细胞生物学特性的影响. 解放军医学杂志.2006; 31(12):1149-1151
100. S. Eigenbrod, R. Derwand, V. Jakl.Sphingosine Kinase and Sphingosine-1- Phosphate Regulate Migration, Endocytosis and Apoptosis of Dendritic Cells Immunological Investigations, 2006; 35:149–165
101. Fang Wang, James R. Van Brocklyn. Sphingosine-1-phosphate Inhibits Motility of Human Breast Cancer Cells Independently of Cell Surface Receptors. Cancer Res. 1999;59(11): 6185-6191
102. Sukumar Sarkara,1, Michael Maceykaa,1, Nitai C. Haita Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells. FEBS Letters. 2005; 579(9): 5313–5317
103. Moehler TM, Ho AD, Barogie B, Barlogie B. Angiogenesis in hematologic malignancies. Critical Reviews in Oncology Hematology 2003; 45(3):227-244
104. Krump-Konvalinkova V, Yasuda S, Rubic T, Makarova N, Mages J, Erl W, Vosseler C, Kirkpatrick CJ, Tigyi G, Siess W. Stable knock-down of the Sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells. Arterioscler Thromb Vasc Biol. 2005;25(3):546-552
105. Hla T. Signaling and biological actions of sphingosine-1-phosphate. Pharmacol Res. 2003; 47(5):401-7
106. Yabu T, Tomimoto H, Taguchi Y, Yamaoka S, Igarashi Y, Okazaki T. Thalidomide-induced antiangiogenic action is mediated by ceramide through depletion of VEGF receptors, and is antagonized by sphingosine-1-phosphate. Blood. 2005; 106(1):125-134
107. Pitson SM, Moretti PA, Zebol JR, Lynn HE, Xia P, Vadas MA, Wattenberg BW. Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J. 2003; 22(20):5491-5500
108. Le Scolan E, Pchejetski D, Banno Y, Denis N, Mayeux P, Vainchenker W, Levade T, Moreau-Gachelin F. Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression. Blood. 2005; 106(5):1808-1816
109. 孙慧燕, 段海峰, 贾向旭, 李庆芳 , 刘玉和 , 吴祖泽 , 王立生. IL-6对多发性骨髓瘤细胞鞘氨醇激酶的激活作用.军事医学科学院院刊,2004;28:130-132
110. Cuvillier O, Levade T. Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria. Blood. 2001; 98(9):2828-2830
111. Greenhalgh CJ, Miller ME, Hilton DJ, Lund PK. Suppressors of cytokine signaling: Relevance to gastrointestinal function and disease. Gastroenterology. 2002;123(6) :2064- 2081
112. C Zhang, D Chaturvedi, L Jaggar, D Magnuson, JM Lee, TB Patel. Regulation of vascular smooth muscle cell proliferation and migration by human sprouty 2, Arterioscler. Thromb. Vasc. Biol. 2005; 25 (21) ;533–538.
113. N Hacohen, S Kramer, D Sutherland, Y Hiromi, MA Krasnow. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branchingof the Drosophila airways, Cell. 1998; 92 (13) 253–263.
114. T. Casci, J. Vinos, M. Freeman, Sprouty, an intracellular inhibitor of Ras signaling, Cell ;1999;96(5) ;655–665.
115. G. Minowada, L.A. Jarvis, C.L. Chi, A. Neubuser, X. Sun, N. Hacohen, Krasnow MA, Martin GR. Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed, Development , 1999;126 (20): 4465–4475.
116. Lo TL, Yusoff P, Fong CW, Guo K, McCaw BJ, Phillips WA, Yang H, Wong ES, Leong HF, Zeng Q, Putti TC, Guy GR.The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer. Cancer Res. 2004; 64(17): 6127–6136.
117. K Ozaki, R Kadomoto, K Asato, S Tanimura, N Itoh, M Kohno. ERK pathway positively regulates the expression of Sprouty genes, Biochem. Biophys. Res. Commun. 2001; 285 (34) 1084–1088.
118. Sasaki A, Taketomi T, Kato R, Saeki K, Nonami A, Sasaki M, Kuriyama M, Saito N, Shibuya M, Yoshimura A. Mammalian Sprouty4 suppresses Rasindependent ERK activation by binding to Raf1, Nat. Cell Biol. 2003; 5 (3) 427–432.
119. M.A. Impagnatiello, S. Weitzer, G. Gannon, A. Compagni, M. Cotton, G. Christofori, Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signalling in endothelial cells, J. Cell Biol. 2001;152 (17) 1087– 1098.
120. Lee CC, Putnam AJ, Miranti CK, Gustafson M, Wang LM, Vande Woude GF, Gao CF. Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis, Oncogene 2004; 23 (12)5193–5202.
121. Tsavachidou D, Coleman ML, Athanasiadis G. SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant. Cancer Res. 2004; 64: 5556- 5559
122. Sutterlüty H, Mayer CE, Setinek U, Attems J, Ovtcharov S, Mikula M, Mikulits W, Micksche M, Berger W.Down-regulation of Sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms. Mol Cancer Res. 2007; 5(5:509-20
123. M.A. Cabrita, F. J?ggi, S.P. Widjaja, G. Christofori, A functional interaction between sprouty proteins and caveolin-1, J. Biol. Chem. 2006; 281:29201-29212
124. 朱悦.王东煜.木村春彦。h-Spry2 基因在部分人类肿瘤中的表达和突变研究.中华实验外科杂志. 2002 :19(1) ;56-57
125. T.L. Lo, P. Yusoff, C.W. Fong, K. Guo, B.J. McCaw,W.A. Phillips, et al., The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer, Cancer Res. 2004;64(14) :6127–6136.
126. B. Kwabi-Addo, J. Wang, H. Erdem, A. Vaid, P. Castro, G. Ayala. The expression of Sprouty2, an inhibitor of fibroblast growth factor signal transduction, is decreased in human prostate cancer, Cancer Res. 2004 (21): 4728–4735.
127. McKie AB, Douglas DA, Olijslagers S, Graham J, Omar MM, Heer R, Gnanapragasam VJ, Robson CN, Leung HY. Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer, Oncogene.2005;24 (14) :2166–2174.
128. Fong CW, Chua MS, McKie AB, Ling SH, Mason V, Li R, Yusoff P, Lo TL, Leung HY, So SK, Guy GR. Sprouty 2, an inhibitor of mitogen- activated protein kinase signaling, is down-regulated in hepatocellular carcinoma. Cancer Res. 2006; 66(4): 2048-2058.
129. Miyoshi K, Wakioka T, Nishinakamura H, Kamio M, Yang L, Inoue M, Hasegawa M, Yonemitsu Y, Komiya S, Yoshimura A. The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization, Oncogene. 2004;23 (17) : 5567–5576.
130. Ogawa K, Nakanishi H, Takeshita F, Futakuchi M, Asamoto M, Imaida K, Tatematsu M, Shirai T. Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice. Int J Cancer. 2001;91(6):797-802
131.Yang JM, Peng ZH, Si SH, Liu WW, Luo YH, Ye ZY. KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97-H cells in vitro and in animal models. Liver Int. 2008; 28(1):132-9.
132. Si SH, Yang JM, Peng ZH, Luo YH, Zhou P. Influence of KAI1 gene on the growth and invasion of human hepatocellular carcinoma MHCC97-H cells.Zhonghua Gan Zang Bing Za Zhi. 2005; 13(8):615-6.
133. Mark D. Stenrlieht H. How matrix metalloproteniases regulate cell behaviour. Annu Rev Cell Dev Biol,2001; 17(2):463-469.
134. Brand K, Baker AH, Perez A, Possling A, Sacharjat M, Geheeb M, Arnold W. Treatment of colorectal liver metastases by adenoviral transfer of tissue inhibitor of metallopreteinases-2 into the liver tissue. Cancer Research, 2000; 60(20):5723-5730.
135. Zacchigna S, Zentilin L, Morini M, Dell'Eva R, Noonan DM, Albini A, Giacca M. AAV-mediated gene transfer of tissue inhibitor ofmetalloproteinases-1 inhibits vascular tumor growth and angiogenesis in vivo. Cancer Gene Ther, 2004; 11(1):73-80.
136. WM Liu, XA Zhang. KAI1/CD82, a tumor metastasis suppressor. Cancer Letters, 2006; 240 (2):183–194
2. Parkin DM. Global cancerr statistics in the year 2000. Lancet oncol, 2001;2(9):533-43.
3. EI-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United states, N Engl J Med, 1999; 340(7):745-50.
4. Ling Y, Donald M, Parkin D, Chen Y. Time trends in cancer motality in China: 1987-1999. Int J cancer, 2003;106(4):771-783.
5. Li LD, Liu FZ, Zhang SWl. Distribution analysis of cancer mortality in China during 1990-1992. Chin J oncol, 1996;18(4):403-407.
6. Zhou XD. Recurrence and metastasis of hepatocellular carcinoma: progress and prospects. Hepatobiliary Pancreat Dis Int, 2002; l(l): 35-41.
7. 李坚, 王洪林. 肝癌肝移植术后肝癌复发的研究进展. 中华外科杂志,2005; 43(11):753-756.
8. Dong JT, Lamb PW, Rinker-Schaeffer CW,Vukanovic J, Ichikawa T, Isaacs JT, Barrett JC. KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science. 1995;268 (5212): 884–886.
9. Guo XZ, Friess H, Di Mola FF, Heinicke JM, Abou-Shady M, Graber HU, Baer HU, Zimmermann A, Korc M, Büchler MW. KAI1, a new metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma. Hepatology 1998;28:1481-1486
10. Guo XZ, Friess H, Graber HU, Kashiwagi M, Zimmermann A, Korc M,Büchler MW. KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res 1996; 56: 4876-4880.
11. 徐建华,郭晓钟,任丽楠,赵家钧,崔忠敏,刘民培.KAI1 基因抗胰腺癌细胞转移的研究. 中华消化杂志 2004;24:541-544
12. Dong JT, Suzuki H, Pin SS, Bova GS, Schalken JA, Isaacs WB, Barrett JC, Isaacs JT. Down-regulation of the KAI1 metastsis suppressor gene during the progression of human prostatic cancer infre quently involves gene mutation or allelic loss.Cancer Res, 1996; 56(19):4387-4390.
13. Dong JT, Isaacs WB, Barrett JC, Isaacs JT. Genomic organization ofthe human KAI1 metastasis suppressor gene. Genomics, 1997; 41(18):25-32.
14. Gao AC, Lou W, Dong JT, Barrett JC, Danielpour D, Isaacs JT. Defining regulatory elementsin the human KAI1/CD82 metastasis suppressor gene.Prostate, 2003; 57(4):256-260.
15. Jackson P, Millar D,Kingsley E, Yardley G, Ow K, Clark S, Russell PJ.. Methylation of a CpG island within the promoter region of the KAI1/CD82 metastasis suppressor gene is not responsible for down-regulation of KAI1/CD82 expression in invasive cancers or cancer cell lines. Cancer Lett, 2000;157(2):169-176.
16. M. Nagira, T. Imai, I. Ishikawa, K.I. Uwabe, O. Yoshie, Mouse homologue of C33 antigen (CD82), a member of the transmembrane 4 superfamily: complementary DNA, genomic structure, and expression. Cell. Immunol. 1994;157 (1):144–157.
17. C.S. Stipp, T.V. Kolesnikova, M.E. Hemler, Functional domains in tetraspanin proteins. Trends Biochem. Sci. 2003; 28(2): 106–112.
18. Fedor B , Elena O. Characterization of integrin-tetraspaninadhesioncomplexes:role of tetraspanins in integrin signaling. Cell Bio,1999;146:477-492.
19. Naotaka S,Kenichi H,Hironori Y,Koyanagi M, Itoh N, Nishikawa J, Tanaka K. l.Verexpression of CD82 on human T cells enhances LFA-1/ICAM-1 mediated cellcelIadhesion:functionaI association between CD82 and LFA-1 inT cell activation.Eur J Immunol,1999;29:4081-4091.
20. Delaguillaumie A, Lagaudrière-Gesbert C, Popoff MR, Conjeaud H. Rho GTPases link cytoskeletal rearrangements and activation processes induced byvia the tetraspanin CD82 in T lymphocytes.J Cell Sci,2002;115:433-443.
21. Cecile LG,Sophie LB,Emmanuel W. The tetraspaninprotein CD82 associates with both free HLA class I heavy chainand heterodimeric 62-microglobulin complexes.Immunol,1997;158:2790-2797.
22. Zhang XA,He B,Zhou B,Liu L. Requirement of the pl30-CAs-Crk coucell migration. J Biol Chem,2003;278(29):27319-27328.
23. Hang XA,LaBe WS,Charrin S,Rubinstein E, Liu L. EWI2/PGRL associates with themetastasis suppressor KAI1/CD82 and inhibits the migration of prostate cancer cells.Cancer Res,2003;63(101):2665-2674.
24. Ono M,Handa K,Withers DA,Hakomori S.Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogne CD9 with concurrent glycosylation.Cancer Res. 1999; 59(10):2335-39.
25. Ono M,Handa K,Withers DA,Hakomori S. Glyeosylation efect on membrane domain(GEM)involved in cell adhesion and motility :prelimirary note on function CD82 glycosylation complex in IdID 14 cel1.Biochem Biophys Res Commun。2000; 279(3):744-750.
26. Zhou B,Liu L,Muralidhar R,Zhang XA. The Palmitoylation of Metastasis suppressor KAI1/CD82 Is Important for Its Motility and Invasiveness Inhibitory Activity.Cancer Research. 2004;64(23):7455-63.
27.Bouras T, Frauman AG.. Expression of the prostate cancermetastasis suppressor gene KAI1 in primary prostate cancers;a biphasic relationship with grade.J Paihol. 1999; 188(4):382-388.
28.HU WL,LI YQ, He HX, Li QR, Tian Y, Lai RQ, Mei H. KAI1/CD82 gene expression in benign prostatic hpperplasia and late-stage prostate cancer in Chinese.Asian Journal of Andrology. 2000; 2(3):221-224.
29. Kawana Y; Komiya A; Ueda T; Nihei N; Kuramochi H; Suzuki H; Yatani R; Imai T; Dong JT; Imai T; Yoshie O; Barrett JC; Isaacs JT; Shimazaki J; Ito H; Ichikawa T. Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer, Prostate 1997;36 (32): 205–213.
30. Dong LP, Cao FY, Zhang YF. The relationship between gastric cancer and KAI1/CD82 exprossion. Henan Journal of Oncology, 2004;17(6):397-399.
31. Tsutsumi S, Shimura T, Morinaga N, Mochiki E, Asao T, Kuwano H. Loss of KAI1 expression in gastric cancer. Hepato-Gastroenterology,2005;52(61):281-284.
32. Guo XZ, Friess H, Maurer C, Berberat P, Tang WH, Zimmermann A, Naef M, Graber HU, Korc M, Buchler MW. KAII is unchanged in metastatic and nonmetastatic esophageal and gastric cancers. Cancer Res, 1998; 58(4): 753-758
33. Friess H,Guo XZ,Berberat P. KAI1 facilitates the formation of metastasis in pancreatic cancer. Gut,1997;41(4S):76A.
34. Guo XZ, Xu JH, Liu MP, Kleeff J, Ho CK, Ren LN, Li HY, K?ninger J, Cui ZM, Wang D, Wu CY, Zhao JJ, Friess H. KAII inhibitsanchorage-dependent and –independent pancreatic cancer cell growth. Onco Rep. 2005; 14: 59-66
35. Guo XZ, Friess H, Shao XD, Liu MP, Xia YT, Xu JH. KAII gene is differently expressed in papillary and pancreatic cancer influence on metastasis. WJG, 2000;6(6):866-871
36. 徐建华,任丽楠,邵丽春,郭晓钟,刘民培. KAIl 基因抑制人胰腺癌细胞转移的体内实验. 胰腺病学 2006:6(1):8-11。
37. Yang GZ,Ding YQ,Liu L,et al.Changges of biological characteristics of colon carcinoma cell line LoVo induced by KAI 1/CD82 transfection. Zhonghua Bing Li Xue Za Zhi,2004;33(1):49-52.
38. L L, L ZG, Y GZ, Ding YQ. Effect of tumor suppressor gene KAI1 on the biological behaviors of human colorectal carcinoma. J First Mil Med Univ, 2003;23(6):587-590.
39. 董良鹏, 曹风雨, 张豫峰, 陈壬寅. KAI1/CD82 的表达与大肠腺癌的相关性分析.河南肿瘤学杂志. 2004;17(6):397-399
40. Lombardi DP, Geradts J, Foley JF, Chiao C, Lamb PW, Barrett JC. Loss of KAI1 expression in the progression of colorectal cancer. Cancer Res. 1999; 59(16): 5724–5731.
41. Adachi M, Taki T, Ieki Y, Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer. Cancer Res. 1996;56(8):1751-1715. .
42. Tagawa K, Arihiro K, Takeshima Y, Hiyama E, Yamasaki M, Inai K. Down-regulation of KAI1 messenger RNA expression is not associated with loss of heterozygosity of the KAI1 gene region in lung adenocarcinoma. J Cancer Res, 1999; 90(9):970-976.
43. Higashiyama M, Kodama K,Yodouchi H, et al. KAI1/CD82 expression innonsmall cell lung carcinoma is a novel, favorable prognostic factor:an immunohistochemical analysis.Cancer Res, 1998; 83(3):466-474.
44. Houle CD,Ding XY,Foley JF,Afshari CA, Barrett JC, Davis BJ. Loss of expression and altered localization of KAl1/CD82 and C199 protein are associated with epithelialovarian cancer progression.Gynec Oncol,2002;86:69-78.
45. Schindl M,Birner P,Bachtiary B,Plank C, Breitenecker G, Kowalski H, Oberhuber G.. The impact of expression ofthe metastasis suppressor protein KAI1/CD82 on prognosis in inva : sive squamous cell cervical cancer.Anticancer Res,2000;20(6B):45516-45517.
46. Liu FS, Chen JT, Dong JT, Hsieh YT, Lin AJ, Ho ES et al. KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma. Am J Pathol 2001;159 (5): 1629–1634.
47. Liu FS,Dong JT,Chen JT, Hsieh YT, Ho ES, Hung MJ, Lu CH, Chiou LC. KAI1/CD82 metastasis suppressor protein is down regulated during the progresion of human endometrial cancer. Clin Cancer Res,2003;9(4):1393-1398.
48. Chen Z,Mustafa T,Trojanowicz B,Brauckhoff M, Gimm O, Schmutzler C, K?hrle J, Holzhausen HJ, Kehlen A, Klonisch T, Finke R, Dralle H, Hoang-Vu C..CD82 and CD63 in thyroidcancer. Int J Mol Med,2004;14:517-527.
49. Imai Y,Sasaki T,Shinagawa Y,Akimoto K, Fujibayashi T. Expression of metastsis suppressorgen(eKAI1/CD82)in oral squamous cell carcinoma and its clinco pathological sign ificance. Oral Onco1,2002;38:557-561.
50. Shu Y,Chen HY,Tan,Y. Expression and clinical value of KAI1/CD82 in carcinoma of larynx. Lin Chuang Er Bi Yan Hou Ke Za Zhi,2005;19(23):1065-1067.
51. AmirthaLakshmi S,Pushparaj V,Krishnamurthy,Biswas J, Krishnakumar S, Shanmugam MP. Tetraspanin protein KAI1 expression in retinoblastoma. British Journal of Ophthalmology,2004;88(4):593-595.
52.Jing S,Arima K,Hasegawa M,Franco OE, Umeda Y, Yanagawa M, Sugimura Y, Kawamura J. Decreased expression of KAI1 metastasis suppressor gene is a recurrence predictor in primary pTa and pT1 urothelial bladder carcinoma. International Journal of Urology,2004;11(2):74-82.
53. Yu Y, Yang JL, Markovic B, Jackson P, Yardley G, Barrett J, Russell PJ. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res .1997;3(19): 1045–1049
54.Huang CI,Kohno N,Ogawa E,Adachi M, Taki T, Miyake M. Correlaation of reduction in MPR-1/CD9 and KAI1/CD82 expression with recurrences in breast patiengts.Am J Pathol,1998;153(3):973-983.
55.X. Yang, L. Wei, C. Tang, R. Slack, E. Montgomery, M. Lippman, KAI1 protein is down-regulated during the progression of human breast cancer, Clin Cancer Res. 2000;6 (9): 3424–3429.
56. X. Yang, L Wei, C. Tang, R. Slack, S. Mueller, M.E. Lippman, Overexpression of KAI1 suppresses in vitro invasiveness and in vivo metastasis in breast cancer cells, Cancer Res. 2001;61 (13): 5284–5288.
57. Burchert, Aotter, Menssenl. CD82(KAI1),a member of the tetraspan family , is expressed on early haemopoietic progenitor cells and up-regulated in distinct human leukaemias. British Journal of Haematology,1999;107(3):494-504.
58. HC Sun, ZY Tang, G. Zhou, XM Li, KAI1 gene expression in hepatocellular carcinoma and its relationship with intrahepatic metastases,J. Exp. Clin. Cancer Res. 1998;17 (15) 307–311.
59.Zhang WG, Wang Y, Chen H, Cheng YX, Ma XR, Yang Y, Wang YL.The significance of transmembrane 4 superfamily(TM4SF)expression in human hepatocellular carcinoma(HCC). Chin J Cli Med Pra, 2004; 3(3):197-199.
60.郑伟红,易继林,李兴睿,冯亮,董朝富. KAI1 基因在肝癌中的表达及临床意义. 临床和实验医学杂志;2006;5(9):1285-1289
61.彭志红, 唐波, 杨建民, 司遂海 , 陈文生 , 房殿春 , 罗元辉 , PENG Zhi-hong , TANG Bo , YANG Jian-min , SI Sui-hai , CHEN Wen-sheng , FANG Dian-chun , LUO Yuan-hui. KAI1 基因对MHCC97-H肝癌细胞粘附力的影响. 第三军医大学学报,2005;27(23):2311-2313
62.司遂海,杨建民,彭志红, 罗元辉, 周平. KAI1 基因对肝癌 MHCC97-H细胞生长及侵袭能力的影响. 中华肝脏病杂志;2005:13(8);615-617
63. Mashimo T,Watabe M,Hirota S,Hosobe S, Miura K, Tegtmeyer PJ, Rinker-Shaeffer CW, Watabe K. The expression of the KAI1 gene,a tumor metastasis suppressor,is directly activated by P53.Proc Natl Acad Sci USA,1998;95(19):1130-1131.
64. Duriez C,Falette N,Cortes U,Moyret-Lalle C, Puisieux A. Absence of p53 dependent induction of the metastatic suppressor KAI1 gene after DNA damage. Oncogene, 2000; 19(20):2461-2464.
65.Tomoyuki M,Misako W,Shigeru H,Hosobe S, Miura K, Tegtmeyer PJ, Rinker-Shaeffer CW, Watabe K. The expression of KAI1 gene,a tumor metastasis suppressor,is directly activatedby P53 . Proc Natl Acad Sci USA,1998;95:11307-11311.
66. Mashimo T,Bandyopadhyay S,Goodarzi G,Watabe M, Pai SK, Gross SC, Watabe K. Activation of the tumor metastasis suppressor gene KAI1,by etoposide ismediated by p53 and c-Jun genes.Biochem Biophys ResCommun,2000;274:370-376.
67.Marreiros A, Dudgeon K, Dao V. KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells. Oncogene. 2005;24(4):637-649.
68.Cyril D,Nicole FU,lrich C,Moyret-Lalle C, Puisieux A. Absence of p53-dependent induction of the metastatic suppressor KAI1 gene after DNA damage.Oncogene,2000;19:2461-2464.
69.Tsutomu Shinohara, Toyokazu Miki, Naoki Nishimura, Nokihara H, Hamada H, HMukaida N, Sone S. Nuclear factor-kappaB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53. Cancer Res, 2001;61(2):673-678.
70.Laguudriere GC. Lebel BS. Hubeau C. Signaling through the Tetraspanin CD82 triggers is association with the cytoskeleton leading to sustained morphological change and T cellactivation.Eur J Immunol.1998; 28(12):4332-44.
71. Delaguiuaumie A. Lagaudrlere GC. Popoff MR. Rho GTPases link cytoskeleton rearrangemants and activation processes induced via the tetraspanin CD82 in T lymphocytes.J Cell Sci. 2002;15(2):433-443
72.Bandyopadhyay S, Zhan R, Chaudhuri A, Watabe M, Pai SK, Hirota S, Hosobe S, Tsukada T, Miura K, Takano Y, Saito K, Pauza ME, Hayashi S, Wang Y, Mohinta S, Mashimo T, Iiizumi M, Furuta E, Watabe K. Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression.Nat Med. 2006;12(8):933-8.
73.Zijlstra A, Quigley JP. The DARC side of metastasis: shining a light on KAI1-mediated metastasis suppression in the vascular tunnel.Cancer Cell. 2006 ;10(3):177-8.
74.Iiizumi M, Bandyopadhyay S, Watabe K. Interaction of Duffy antigen receptor for chemokines and KAI1: a critical step in metastasis suppression.Cancer Res. 2007; 67(4):1411-4.
75. Zhang XA,He B,Zhou B,Liu L.Requirement ofthe pl30-CAs-Crk coucell migration.J Biol Chem,2003;278(29):27319-27328.
76. Jee B, Jin K, Hahn JH, Song HG, Lee H. Metastasis-suppressor KAII/CD82 induces homotypic aggregation of human prostate cancer cells through Src dependent pathway.Exp Mol Med, 2003; 35(1):30-37
77. Sridhar SC, Miranti CK. Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene. 2006;25(16):2367-78.
78. Wang XY, Liu T, Zhu CZ. Expression of KAI1, MRP-1, and FAK proteins in lung cancer detected by high-density tissue microarray. Ai Zheng. 2005 ;24(9):1091-5.
79. Takahashi M, Sugiura T, Abe M, Ishii K, Shirasuna K. Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration. Int J Cancer. 2007; 121(9):1919-29.
80.Zhang XA. Lane WS. Chanin S. Rubinstein E, Liu L. EW12/PGRL associates with the metastasis suppressor KAII/CD82 and inhibits the migration of prostate cancer cel1.Cancer research,2003;63(10):2665-74.
81. Jee JH , Park SR , Chay KO . KAII COOH-tenninal interacting tetraspanin(K1TENIN),a member of the trtraslmnin family,interacts with KAI1 a tumor metastasis suppressor , and enhances metastasis of cancer,Cancer research,2004; 64(12):4235-4243.
82. Rowe A, Jackson P. Expression of KITENIN, a KAI1/CD82 binding protein and metastasis enhancer, in bladder cancer cell lines: relationship toKAI1/CD82 levels and invasive behaviour. Oncol Rep. 2006 ;16(6):1267-72.
83.Jee BK, Park KM, Surendran S, Lee WK, Han CW, Kim YS, Lim Y. KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line. Biochem Biophys Res Commun. 2006; 342(2):655-61.
84.Jee BK, Lee JY, Lim Y, Lee KH, Jo YH. Effect of KAI1/CD82 on the beta1 integrin maturation in highly migratory carcinoma cells. Biochem Biophys Res Commun. 2007; 359(3):703-8.
85. Bass R, Werner F, Odintsova E, Regulation of urokinase receptor proteotic function by the tetraspanin CD82. J Biol Chem. 2005 ;280(15):14811-8.
86. Odintsova E,Sugiura T,Berditchevski F.Attenuation of EGF receptor signalingby a metastasis suppressor,the tetraspanin CD82/KAI-1. Curr Biol, 2000; 10(16): 1009-1012
87.Sehoenfeld N. Bauer MA.Grimim S. Bauer MA, Allen B, Al-Sebaei MO, Kakar S, Leone CW, Morgan EF, Gerstenfeld LC, Einhorn TA, Graves DT. Th e metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates.FASEB Journa1.2004; 18(1):158-160.
88.郭晓钟,张巍巍,王立生, 鲁茁壮, 王华, 徐建华, 田宏. Kai1 基因抑制胰腺癌细胞鞘氨醇激酶活性及其迁移. 中华内科杂志.2006: 45(9); 752-754.
89.Spiegel S, Milstien S. Sphingosine-1-Phosphate, a Key Cell Signaling Molecule. The Journal of Biological Chemistry. 2002: 277(29); 25851–25854,
90. Maceyka M, Payne SG, Milstien S, Spiegel S. Sphingosine kinase,sphingosine-1-phosphate, and apoptosis. Biochim Biophys Acta. 2002; 1585 (2-3):193-201
91. Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, Milstien S, Kohama T, Spiegel S. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. Biol Chem, 2000; 275(26):19513-20
92. Kohama T, Olivera A, EdsallL, Nagiec MM, Dickson R, Spiegel S. Molecular cloning and functional characterization of murine sphingosine kinase.J Biol Chem, 1998; 273(37): 23722-8
93. Le Stunff H, Galve-Roperh I, Peterson C, Milstien S, Spiegel S. Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis. J Cell Biol 2002; 158(6):1039-1049.
94.Sadahira Y, Ruan F, Hakomori S, Igarashi Y. Sphingosine 1 - phosphate ,a specific endogenous signaling molecule controlling cell ,motility and tumor cell invasiveness. Proc Natl Acad Sci USA , 1992; 89 (20) :9886 - 9690.
95.Paik J H ,Chae S ,Lee MJ , Cowan AE, Han DK, Proia RL, Hla T. Sphingosine -1- phosphate induced endothelial cell migration requires the expression of EDG- 1and EDG - 3 receptors and Rho - dependent activation of alphavbeta3 - and betal - containing integrins . J Biol Chem ,2001; 276 (15) :11830 - 11837.
96.Lee MJ , Thangada S , Paik J H , Sapkota GP, Ancellin N, Chae SS, Wu M, Morales-Ruiz M, Sessa WC, Alessi DR, Hla T. Akt - mediated phosphorylation of the G protein - coupled receptor EDG- 1 is required for endothelial cell chemotaxis . Mol Cell ,2001; 8 (3) :693 - 704.
97.Pu Xia, Jennifer R. Gamble,An oncogenic role of sphingosine kinase Current Biology. 2000; 10(23):1527-1530
98. Edsall LC, Van Brocklyn JR, Cuvillier O, Kleuser B, Spiegel S. N, N-Dimethylsphingosine is a potent compettitive inhibitor of sphingosine kinase but not of protein kinase C: Modulation of cellular levels of sphingosine-1-phosphate and ceramide. Biochemistry, 1998: 37 ( 37 ) : 128922-128981
99. 郭强, 李荣, 李庆芳. 高表达鞘氨醇激酶对胃癌细胞生物学特性的影响. 解放军医学杂志.2006; 31(12):1149-1151
100. S. Eigenbrod, R. Derwand, V. Jakl.Sphingosine Kinase and Sphingosine-1- Phosphate Regulate Migration, Endocytosis and Apoptosis of Dendritic Cells Immunological Investigations, 2006; 35:149–165
101. Fang Wang, James R. Van Brocklyn. Sphingosine-1-phosphate Inhibits Motility of Human Breast Cancer Cells Independently of Cell Surface Receptors. Cancer Res. 1999;59(11): 6185-6191
102. Sukumar Sarkara,1, Michael Maceykaa,1, Nitai C. Haita Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells. FEBS Letters. 2005; 579(9): 5313–5317
103. Moehler TM, Ho AD, Barogie B, Barlogie B. Angiogenesis in hematologic malignancies. Critical Reviews in Oncology Hematology 2003; 45(3):227-244
104. Krump-Konvalinkova V, Yasuda S, Rubic T, Makarova N, Mages J, Erl W, Vosseler C, Kirkpatrick CJ, Tigyi G, Siess W. Stable knock-down of the Sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells. Arterioscler Thromb Vasc Biol. 2005;25(3):546-552
105. Hla T. Signaling and biological actions of sphingosine-1-phosphate. Pharmacol Res. 2003; 47(5):401-7
106. Yabu T, Tomimoto H, Taguchi Y, Yamaoka S, Igarashi Y, Okazaki T. Thalidomide-induced antiangiogenic action is mediated by ceramide through depletion of VEGF receptors, and is antagonized by sphingosine-1-phosphate. Blood. 2005; 106(1):125-134
107. Pitson SM, Moretti PA, Zebol JR, Lynn HE, Xia P, Vadas MA, Wattenberg BW. Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J. 2003; 22(20):5491-5500
108. Le Scolan E, Pchejetski D, Banno Y, Denis N, Mayeux P, Vainchenker W, Levade T, Moreau-Gachelin F. Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression. Blood. 2005; 106(5):1808-1816
109. 孙慧燕, 段海峰, 贾向旭, 李庆芳 , 刘玉和 , 吴祖泽 , 王立生. IL-6对多发性骨髓瘤细胞鞘氨醇激酶的激活作用.军事医学科学院院刊,2004;28:130-132
110. Cuvillier O, Levade T. Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria. Blood. 2001; 98(9):2828-2830
111. Greenhalgh CJ, Miller ME, Hilton DJ, Lund PK. Suppressors of cytokine signaling: Relevance to gastrointestinal function and disease. Gastroenterology. 2002;123(6) :2064- 2081
112. C Zhang, D Chaturvedi, L Jaggar, D Magnuson, JM Lee, TB Patel. Regulation of vascular smooth muscle cell proliferation and migration by human sprouty 2, Arterioscler. Thromb. Vasc. Biol. 2005; 25 (21) ;533–538.
113. N Hacohen, S Kramer, D Sutherland, Y Hiromi, MA Krasnow. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branchingof the Drosophila airways, Cell. 1998; 92 (13) 253–263.
114. T. Casci, J. Vinos, M. Freeman, Sprouty, an intracellular inhibitor of Ras signaling, Cell ;1999;96(5) ;655–665.
115. G. Minowada, L.A. Jarvis, C.L. Chi, A. Neubuser, X. Sun, N. Hacohen, Krasnow MA, Martin GR. Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed, Development , 1999;126 (20): 4465–4475.
116. Lo TL, Yusoff P, Fong CW, Guo K, McCaw BJ, Phillips WA, Yang H, Wong ES, Leong HF, Zeng Q, Putti TC, Guy GR.The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer. Cancer Res. 2004; 64(17): 6127–6136.
117. K Ozaki, R Kadomoto, K Asato, S Tanimura, N Itoh, M Kohno. ERK pathway positively regulates the expression of Sprouty genes, Biochem. Biophys. Res. Commun. 2001; 285 (34) 1084–1088.
118. Sasaki A, Taketomi T, Kato R, Saeki K, Nonami A, Sasaki M, Kuriyama M, Saito N, Shibuya M, Yoshimura A. Mammalian Sprouty4 suppresses Rasindependent ERK activation by binding to Raf1, Nat. Cell Biol. 2003; 5 (3) 427–432.
119. M.A. Impagnatiello, S. Weitzer, G. Gannon, A. Compagni, M. Cotton, G. Christofori, Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signalling in endothelial cells, J. Cell Biol. 2001;152 (17) 1087– 1098.
120. Lee CC, Putnam AJ, Miranti CK, Gustafson M, Wang LM, Vande Woude GF, Gao CF. Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis, Oncogene 2004; 23 (12)5193–5202.
121. Tsavachidou D, Coleman ML, Athanasiadis G. SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant. Cancer Res. 2004; 64: 5556- 5559
122. Sutterlüty H, Mayer CE, Setinek U, Attems J, Ovtcharov S, Mikula M, Mikulits W, Micksche M, Berger W.Down-regulation of Sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms. Mol Cancer Res. 2007; 5(5:509-20
123. M.A. Cabrita, F. J?ggi, S.P. Widjaja, G. Christofori, A functional interaction between sprouty proteins and caveolin-1, J. Biol. Chem. 2006; 281:29201-29212
124. 朱悦.王东煜.木村春彦。h-Spry2 基因在部分人类肿瘤中的表达和突变研究.中华实验外科杂志. 2002 :19(1) ;56-57
125. T.L. Lo, P. Yusoff, C.W. Fong, K. Guo, B.J. McCaw,W.A. Phillips, et al., The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer, Cancer Res. 2004;64(14) :6127–6136.
126. B. Kwabi-Addo, J. Wang, H. Erdem, A. Vaid, P. Castro, G. Ayala. The expression of Sprouty2, an inhibitor of fibroblast growth factor signal transduction, is decreased in human prostate cancer, Cancer Res. 2004 (21): 4728–4735.
127. McKie AB, Douglas DA, Olijslagers S, Graham J, Omar MM, Heer R, Gnanapragasam VJ, Robson CN, Leung HY. Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer, Oncogene.2005;24 (14) :2166–2174.
128. Fong CW, Chua MS, McKie AB, Ling SH, Mason V, Li R, Yusoff P, Lo TL, Leung HY, So SK, Guy GR. Sprouty 2, an inhibitor of mitogen- activated protein kinase signaling, is down-regulated in hepatocellular carcinoma. Cancer Res. 2006; 66(4): 2048-2058.
129. Miyoshi K, Wakioka T, Nishinakamura H, Kamio M, Yang L, Inoue M, Hasegawa M, Yonemitsu Y, Komiya S, Yoshimura A. The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization, Oncogene. 2004;23 (17) : 5567–5576.
130. Ogawa K, Nakanishi H, Takeshita F, Futakuchi M, Asamoto M, Imaida K, Tatematsu M, Shirai T. Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice. Int J Cancer. 2001;91(6):797-802
131.Yang JM, Peng ZH, Si SH, Liu WW, Luo YH, Ye ZY. KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97-H cells in vitro and in animal models. Liver Int. 2008; 28(1):132-9.
132. Si SH, Yang JM, Peng ZH, Luo YH, Zhou P. Influence of KAI1 gene on the growth and invasion of human hepatocellular carcinoma MHCC97-H cells.Zhonghua Gan Zang Bing Za Zhi. 2005; 13(8):615-6.
133. Mark D. Stenrlieht H. How matrix metalloproteniases regulate cell behaviour. Annu Rev Cell Dev Biol,2001; 17(2):463-469.
134. Brand K, Baker AH, Perez A, Possling A, Sacharjat M, Geheeb M, Arnold W. Treatment of colorectal liver metastases by adenoviral transfer of tissue inhibitor of metallopreteinases-2 into the liver tissue. Cancer Research, 2000; 60(20):5723-5730.
135. Zacchigna S, Zentilin L, Morini M, Dell'Eva R, Noonan DM, Albini A, Giacca M. AAV-mediated gene transfer of tissue inhibitor ofmetalloproteinases-1 inhibits vascular tumor growth and angiogenesis in vivo. Cancer Gene Ther, 2004; 11(1):73-80.
136. WM Liu, XA Zhang. KAI1/CD82, a tumor metastasis suppressor. Cancer Letters, 2006; 240 (2):183–194