nm23-H1 Gene Research Articles

Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy.

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.

Correlation of p16 and nm23-H1 expression levels with incidence and prognosis of soft tissue sarcoma.

The expression levels of p16 and nm23-H1 genes in soft tissue sarcoma (STS) were evaluated to investigate correlation of the expression levels with the incidence and prognosis of STS. Tumor tissues and para-carcinoma normal tissues were collected from 64 STS patients. The messenger ribonucleic acid (mRNA) expression levels of p16 and nm23-H1 in the tissues were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein expression levels of p16 and nm23-H1 in tissues were detected using immunohistochemistry. Spearman's correlation analysis was used for the correlation between expression levels of p16 and nm23-H1 in STS tissues and the correlation between p16 and nm23-H1 mRNA and protein expression. Moreover, the correlation of p16 and nm23-H1 expression levels in tumor tissues with pathological parameters and prognosis of STS patients were analyzed combined with clinical data. Results of RT-qPCR showed that mRNA expression levels of p16 and nm23-H1 in tumor tissues of STS patients were significantly lower than those in para-carcinoma normal tissues (P<0.01). Results of immunohistochemistry showed that the positive expression rates of p16 and nm23-H1 in tumor tissues of STS patients (43.75 and 39.06% respectively) were significantly lower than those in para-carcinoma normal tissues (85.93 and 89.06% respectively). The expression of p16 and nm23-H1 mRNA was positively correlated with protein expression levels. There was a positive correlation between the expression levels of p16 and nm23-H1 in tumor tissues of STS patients. The negative expression of p16 in tumor tissues of STS patients correlated with tumor size, tumor metastasis and clinical staging, and the negative expression of nm23-H1 correlated with tumor metastasis and clinical staging. The overall 5-year survival rate of patients was 54.68%, and the prognosis of patients with positive expression levels of p16 and nm23-H1 was better. Univariate survival analyses revealed that p16 and nm23-H1 were influencing factors of the overall survival rate of STS patients. p16 and nm23-H1 expression in STS is low, and their expression levels are closely related to the pathological parameters and prognosis of STS patients, so they can serve as reference indexes for prognosis estimation of STS.

Role of nm23H1 in predicting metastases in prostatic carcinoma

Non-metastatic nm23H1 gene is thought to play a critical role in cell proliferation. Studies of nm23H1 have been done in many other malignancies. But none of these studies took up nm23H1 gene as predictor in the metastases of prostatic carcinoma. To study the expression of nm23H1 in prostatic lesion and to correlate nm23H1 expression with presence of metastases, tumour stage, tumour grade and with PSA level serum. Tertiary hospital based retrospective and prospective study done in a period of one year from thirty patients having prostatic lesion confirmed by biopsy. Immunohistochemistry for nm23H1 was performed on unstained coated sections of prostatic lesions to study the relation with prostatic lesion and their correlation with age, PSA level, tumour stage, grading. Clinical data was collected from medical records. SPSS Version 15 analysis software was used. The value were presented in number(%) and Mean ± SD. Majority of patients belong to age group 61 to 70yrs.Gleason score >7 were seen in 55% of patients of adenocarcinoma with and without metastasis. The difference in PSA levels between BPH and adenocarcinoma was significant (P < 0.001). IHC expression for nm23H1 gene showed positive findings in all the cases (P = 1). PSA values >20ng/ml showed maximum % mean expression (98.64%) as compared to PSA levels <10 ng/ml (96.91%). IHC expression of nm23H1 is not an effective tool to distinguish among the cases of BPH, adenocarcinoma of prostate with and without metastasis. Hence nm23H1 gene does not behave like an antimetastatic gene in prostatic lesions.

Molecular prognostic factors in early-stage cervical adenocarcinoma.

Within the past years the proportion of cervical adenocarcinomas has increased, however, there is a shortage of data regarding immunohistochemical and molecular features and their prognostic relevance in early-stage cervical adenocarcinoma (esCAC). Aim of the present study was to evaluate molecular prognostic factors in esCAC patients treated with primary surgery. Analyses of surgical specimens in 59 patients with esCAC were performed on fixed paraffin-embedded sections of tumour tissue. Tumour tissue sections were routinely stained with hematoxylin and eosin followed by microscopic examination. Immunohistochemical analyses (IHC) were performed on paraffin-embedded section. Flow cytometry (FCM) analysis of paraffin-embedded tumor tissue was performed using flow cytometer FACSCalibur equipped with argon laser. DNA histogram analysis was performed with ModFit application. Treatment effectiveness was evaluated using overall 5-year survival. Survival probability was estimated using the Kaplan-Meier method. Overall survival rate estimated using Kaplan-Meier method was 74.6%. Among the IHC and FCM features univariate analysis showed statistical significance of nm23-H1 gene expression and total S-phase fraction ≤ 11.9% (S-TOT). In multi- variate analysis LVSI and parametrial involvement had significant, negative impact on survival (HR = 8.04, p < 0.003 and HR = 4.03, p < 0.017, respectively). However, none of the tested IHC and FCM features had any influence on overall 5-year survival.

Impact of laparoscopy on the biological behavior and gene expression of endometrial adenocarcinoma cells

The current study investigated the effect of laparoscopy on the biological behavior and gene expression of endometrial adenocarcinoma cells. Totally, 40 patients with stage I endometrial adenocarcinoma and 20 patients with benign uterine diseases were enrolled in this study. For patients with endometrial adenocarcinoma, laparoscopy was performed in 20 cases and laparotomy was carried out in the other 20 cases. Total laparoscopic hysterectomy was performed in patients with benign diseases. Cell apoptotic rate and the gene expression of N-myc, Fas, metastasis-associated protein 1 (MTA1), and nm23-H1 were determined in the normal and cancerous endometrial tissues both preoperatively and postoperatively. For endometrial adenocarcinoma cells, laparoscopy, instead of laparotomy, promoted the apoptosis of endometrial adenocarcinoma cells, down-regulated the expression of apoptosis suppressor gene N-myc and metastasis-promoting gene MTA1, up-regulated the expression of apoptosis-promoting gene Fas and metastasis suppressor gene nm23-H1. However, laparoscopy did not affect the apoptotic rate and gene expression in normal endometrial cells. Laparoscopy may be used as a safe and effective intervention for endometrial cancer.

Transfection of tumor metastasis suppressor gene nm23-H1 up-regulates the activity of glycogen synthase kinase -3β in lung cancer cell line L9981

Objective To investigate the influence of tumor metastasis suppressor gene nm23-H1 on the activity of glycogen synthase kinase -3β (GSK-3β) in lung cancer cell line L9981. Methods The levels of GSK-3β expression in cytoplasm and nucleus were determined with anti-GSK-3β antibody in lung cancer cell line control group (cell line with nm23-H1 gene deletion), nm23-H1 transfection group (cell line with nm23-H1 transfected) and blank transfection group (cell line with vector transfected) by Western blotting. The activity of GSK-3β among those three groups was detected by immunoprecipitation and analyzed by a radioactive isotope scintillation counter before and after treatment with 20 mmol/L LiCl. Results (1) The expression indensity of GSK-3β in cytoplasm and nucleus was (6 639±503) and (4 481±446) A in nm23-H1 transfection group, (645±352) and (726±68) A in blank transfection group, and (763±267) and (683±49) A in control group, respectively. There was very significance in GSK-3β expressive indensity of both cytoplasm and nucleus among nm23-H1 transfection group, blank transfection group and control group (P=0.025). There was significant difference between nm23-H1 transfection group and blank transfection group or control group (P=0.017), but no significant difference was observed between blank transfection group and control group (P=0.094). (2) The GSK-3β activity in cytoplasm and nucleus was (29 371±2 492) and (9 647±859) counts per minute in nm23-H1 transfection group, (11 241±1 495) and (1 492±176) counts per minute in blank transfection group, and (14 271±1 137) and (1 853±113) counts per minute in control group, respectively. There was very significant difference in GSK-3β activity of both cytoplasm and nucleus among nm23-H1 transfection group, blank transfection group and control group (P=0.032). The GSK-3β activity in nm23-H1 transfection group was significantly higher than that in blank transfection group and control group (P=0.027), but no significant difference was observed between the blank transfection group and control group (P=0.131). (3) After treatment with 20 mmol/L LiCl, the expression indensity of GSK-3β in cytoplasm and nucleus was (5 037±427) and (823±350) A in nm23-H1 transfection group, (2 174±126) and (248±12) A in blank transfection group, and (2 273±128) and (253±14) A in control group. No significant difference in GSK-3β expression indensity existed before and after treatment with LiCl in nm23-H1 transfection group (P=0.168). However, the GSK-3β expression indensity in cytoplasm and nucleus before treatment was remarkably higher than that after treatment in both blank transfection group and control group (P=0.012, 0.015). (4) After treatment with 20 mmol/L LiCl, the GSK-3β activity in cytoplasm and nucleus was (12 837±1 042) and (748±215) counts per minute in nm23-H1 transfection group, (4 739±401) and (947±126) counts per minute in control group, and (4 638±401) and (1 162±127) counts per minute in blank transfection group, respectively. The GSK-3β activity in cytoplasm and nucleus after treatment with LiCl was remarkably lower than that before treatment in nm23-H1 transfection group, blank transfection group and control group (P=0.006, 0.029, 0.010). Conclusion Transfection of nm23-H1 gene can significantly up-regulate the expression level and activity of GSK-3β in lung cancer cell line. The inhibitory effects of nm23-H1 gene on the signal transduction of Wnt pathway might be carried out through up regulating GSK-3β expression and activity in lung cancer cell line. Key words: Lung cancer cell lines; nm23-H1 gene; Wnt signal pathway; Glycogen synthase kinase -3β

NM 23-H1 gene expression of a migration suppressor in gastric lymphoma.

39 Background: We previously describedthat the NDPK-A(nucleoside diphosphate kinase A) contributes variably to the migration of cancer cell. To investigate the role of the NDPK-A in gastric lymphoma, we evaluated the expression of NDPK-A in two different cancer cell lines of the same gastric lymphoma patient. Methods: The primary cell line of gastric lymphoma, RF-1, was cultured from the primary gastric lymphoma site, and the other cell line RF-48 was metastasized to the peritoneum of the same patient after 6 months. We have generated stable gastric lymphoma transfectants, pIRES-NDPKA-GFP-neo, that constitutively over-express transfected and vector form of NDPK-A, in addition to a reporter, green fluorescent protein(GFP). The transfected gastric lymphoma cell migration assay was performed 24-well modified chemotaxis chambers with FluroBloc membrane. Results: In regard to the migration rate of the gastric lymphoma cell lines, RF-1 vector was 75.8±7.5%, RF-1 transfected was 54.4±12.3%, RF-48 vector was 84.7±15.4%, RF-48 transfected type was 62.3±9.2%, RF-48 migrated better than RF-1, and the vector type migrated better than the transfected type. NDPK-A was presented more in RF-1 than RF-48, and presented more in the transfected type than the vector type, and concerning the migration rate, RF-48 migrated better than RF-1, and the vector type migrated better than the transfected type. Conclusions: Concerning NM 23-H1 gene encodes the NDPK-A in gastric lymphomas, more abundant was NDPK-A, poorer was migration, and thus it played a role of the suppressor of migration. Our analysis of pooled data found that NDPK-A acts as a migration suppressor in gastric lymphoma.

Nm23H1 mediates tumor invasion in esophageal squamous cell carcinoma by regulation of CLDN1 through the AKT signaling.

Esophageal cancer is a lethal malignancy worldwide. Previously, low expression of metastasis suppressor Nm23H1 and tight junction (TJ) protein claudin-1 (CLDN1) have been known to correlate with poor prognosis in esophageal squamous cell carcinoma (ESCC). However, the molecular interaction between them has not been clarified. In the present study, we first examined the expression of Nm23H1 and CLDN1 in 74 surgical ESCC samples by immunohistochemistry (IHC) to verify their clinicopathologic significance. The biologic effects of Nm23H1 gene silencing or overexpression in ESCC cell lines were then studied by migration and invasion studies, and its regulation on CLDN1 expression was also investigated by western blot analysis. Moreover, the expression of Nm23H1 and CLDN1 at the same invasion front of ESCC tumors was verified by immunofluorescence. The results showed a significantly positive correlation between the expression of Nm23H1 and CLDN1 (γ=0.296, P=0.011) in surgical specimens, especially for the 34 tumors with lymph-node metastasis (γ=0.455, P=0.007). In ESCC cell lines, silencing of Nm23H1 expression markedly enhanced cell invasiveness, accompanied by increased Akt phosphorylation and decreased CLDN1 expression. Conversely, Nm23H1-expressed transfectants exhibited reduced invasiveness, decreased Akt phosphorylation and correspondingly increased CLDN1 expression. Regain of CLDN1 expression in ESCC cells significantly suppressed invasiveness, but did not influence the Akt phosphorylation. Moreover, treating Nm23H1-depleted cells with the AKT inhibitor MK2206 recovered CLDN1 expression, and diminished the invasiveness of ESCC cells. Finally, decreased expressions of both CLDN1 and E-cadherin were observed at the invasive front of the Nm23H1-negative tumors. Overall, our current study documented that reduced Nm23H1 expression activates the AKT signaling pathway, results in diminished CLDN1 expression and potentiates invasiveness of ESCC cells. Enhancement of Nm23H1 expression, inhibition of the AKT signaling pathway, or combined, might be a potential treatment strategy in selective ESCC patients.

Mutation of the nm23-H1 gene has a non-dominant role in colorectal adenocarcinoma.

Nm23-H1 is a metastasis suppressor gene, which is has a reduced expression in patients with digestive system cancer. However, the mechanistic basis for the genetic instability remains unknown. To study the expression of the nm23-H1 gene in patients with colorectal cancer, polymerase chain reaction-single strand conformation polymorphism was used to analyze any point mutation, and immunohistochemistry was used to detect the expression of nm23-H1. Results revealed that all 63 specimens of Chinese human colorectal cancer tissues exhibit no point mutation. Among those 63 specimens, 19 (30%) exhibited positive immunostaining for the nm23-H1 protein and 44 (70%) exhibited negative immunostaining. These observations suggested that the protein and gene expression levels of nm23-H1 are reduced in colorectal cancer compared with the adjacent normal tissues, and the point mutation in the nm23-H1 gene is not the dominant cause of metastatic colorectal cancer.

The milk-derived hexapeptide PGPIPN inhibits the invasion and migration of human ovarian cancer cells by regulating the expression of MTA1 and NM23H1 genes.

Some bioactive peptides derived from natural resources or synthesized by rational design have been proved to have very good anticancer effect. We studied the inhibition of PGPIPN, a hexapeptide derived from bovine β-casein, on the invasion and metastasis of human ovarian cancer cells in vitro and its molecular mechanism. The human ovarian cancer cells studied include the cell line SKOV3 as well as the primary ovarian cancer cells from ovarian tumor tissues of 37 patients at initial debulking surgery, diagnosed as serous ovarian adenocarcinoma. We showed that PGPIPN inhibited the invasion of ovarian cancer cells with Transwell chamber assay, the migration of ovarian cancer cells with cell scratch assay and colony formation of ovarian cancer cells. The expression (mRNAs and proteins) of genes relevant to invasion and metastasis, MTA1, and NM23H1 were analyzed by real-time PCR and western blotting. PGPIPN repressed the expression of MTA1, and promoted NM23H1. The effects of PGPIPN were dose-dependent. Thus, our study suggests that PGPIPN is a potential therapeutic agent for adjuvant therapy of human malignant ovarian tumors.

Clinical implication of voltage-dependent anion channel 1 in uterine cervical cancer and its action on cervical cancer cells.

Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry were performed to investigate the influence of human nonmetastatic clone 23 type 1 (nm23-H1), a metastasis-associated gene on proteomic alterations in cancer cells of the uterine cervix. It was validated by RT-PCR and Western blot analysis. The expression of voltage-dependent anion channel 1 (VDAC1) was increased in nm23-H1 gene silenced SiHa or CaSki cervical cancer cells. The clinical implication was shown that cervical cancer tissues with positive VDAC1 immunoreactivity exhibited deep stromal invasion (>10 mm in depth) and large tumor size (> 4 cm in diameter). Cervical cancer patients with positive VDAC1 immunoreactivity displayed higher recurrence and poorer overall survival than those with negative VDAC1. Silencing of VDAC1 reduced cell proliferation and migratory ability. Mitochondrial membrane potential was decreased and reactive oxygen species generation was increased in the VDAC1 gene-silenced cervical cancer cells. Cell cycle progression and autophagy were not changed in VDAC1 silencing cells. The cytotoxicity of cisplatin was significantly enhanced by knockdown of cellular VDAC1 and the compounds that interfere with hexokinase binding to VDAC. Therapeutic strategies may be offered using VDAC1 as a target to reduce cell growth and migration, enhance the synergistic therapeutic efficacy of cisplatin and reduce cisplatin dose-limiting toxicity.

Transcriptional factor FOXO3 negatively regulates the expression of nm23-H1 in non-small cell lung cancer.

Nm23-H1 was the first metastasis suppressor discovered in most tumor models and reduction or loss of nm23-H1 expression correlates with tumor progression and metastasis in non-small-cell lung cancer. Despite extensive studies, the regulatory mechanism of nm23-H1 expression is far from elucidated. The transcriptional factor forkhead box (FOX)O3 has been reported to be involved in multiple regulatory signaling pathways in the biological behavior of tumors. Therefore, we aimed to study the relationship between FOXO3 activity and nm23-H1 expression. Real time reverse transcriptase-polymerase chain reaction and Western blotting assays were employed to determine nm23-H1 messenger ribonucleic acid and protein expression after being transformed by different FOXO3 plasmid in A549 cells. A dual luciferase reporter system and chromatin immunoprecipitation assay, were used to determine the promoter activity of the nm23-H1 gene and to detect the binding of FOXO3 into the nm23-H1 promoter, respectively. We found that activated FOXO3 decreased nm23-H1 expression and dominant negative FOXO3 increased nm23-H1 expression. Modulation of FOXO3 activity with FOXO3 pathway inhibitors altered nm23-H1 promoter activity. Although there is a putative binding site of FOXO3 in the nm23-H1 promoter, FOXO3 regulated nm23-H1 expression in an indirect manner. We demonstrated that the transcriptional factor FOXO3 decreased the expression levels of the tumor suppressor gene nm23-H1 in the non-small-cell lung cancer A549 cell line and that the level of expression of nm23-H1 was controlled by FOXO3 in an indirect manner. This finding provided an insight into the upstream regulation of nm23-H1 and may provide promising targets for inhibition of the metastasis process.

The nucleoside diphosphate kinase-A as a migration suppressor in gastric lymphoma.

87 Background: The nm23-H1 gene encodes the NDPK-A (nucleoside diphosphate kinase A) that contributes variably to the migration of cancer cell. To investigate the role of the NDPK-A in gastric lymphoma, we examined the expression of NDPK-A in two different cancer cell lines of the same gastric lymphoma patient. Methods: The primary cell line of gastric lymphoma, RF-1, was cultured from the primary gastric lymphoma site, and the other cell line RF-48 was metastasized to the peritoneum of the same patient after 6 months. We have generated stable gastric lymphoma transfectants, pIRES-NDPKA-GFP-neo, that constitutively over-express transfected and vector form of NDPK-A, in addition to a reporter, green fluorescent protein(GFP). The transfected gastric lymphoma cell migration assay was performed 24-well modified chemotaxis chambers with FluroBloc membrane. Results: In regard to the migration rate of the gastric lymphoma cell lines, RF-1 vector was 75.8±7.5%, RF-1 transfected was 54.4±12.3%, RF-48 vector was 84.7±15.4%, RF-48 transfected type was 62.3±9.2%, RF-48 migrated better than RF-1, and the vector type migrated better than the transfected type. NDPK-A was presented more in RF-1 than RF-48, and presented more in the transfected type than the vector type, and concerning the migration rate, RF-48 migrated better than RF-1, and the vector type migrated better than the transfected type. Conclusions: In regard to the migration rate of the gastric lymphoma cell lines, RF-1 vector was 75.8±7.5%, RF-1 transfected was 54.4±12.3%, RF-48 vector was 84.7±15.4%, RF-48 transfected type was 62.3±9.2%, RF-48 migrated better than RF-1, and the vector type migrated better than the transfected type. NDPK-A was presented more in RF-1 than RF-48, and presented more in the transfected type than the vector type, and concerning the migration rate, RF-48 migrated better than RF-1, and the vector type migrated better than the transfected type.

Suppression of alpha-tocopherol ether-linked acetic acid in VEGF-induced angiogenesis and the possible mechanisms in human umbilical vein endothelial cells

Alpha-tocopherol ether-linked acetic acid (α-TEA) has been reported to exhibit both anti-tumor and anti-metastatic activities in cell culture and animal studies. However, it is unclear whether α-TEA possesses anti-angiogenic effects. In this study, we investigated the effect of α-TEA on vascular endothelial growth factor (VEGF)-induced angiogenesis and matrix metalloproteinase (MMP) expression both in vitro and ex vivo. We found that the α-TEA inhibited tube formation, invasion, and migration in human umbilical vein endothelial cells (HUVECs) and that such actions were accompanied by reduced expression of MMP-2. α-TEA also inhibited ex vivo angiogenesis, as indicated by chicken egg chorioallantoic membrane assay. We further showed that α-TEA attenuated protein expression of VEGF receptor-2 (VEGFR-2)-mediated p38 mitogen-activated protein kinase (p38), phosphorylated p38, and focal adhesion kinase (FAK). Moreover, α-TEA (30μM) significantly up-regulated protein expression of tissue inhibitors of MMP (TIMP)-2 (by 138%) and the metastasis suppressor gene nm23-H1 (by 54%). These results demonstrate that the anti-angiogenic effect of α-TEA both in vitro and ex vivo and its possible mechanistic action appears to involve the inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways and through up-regulation of TIMP-2 and nm23-H1 expression.

Associations of nm23H1, VEGF-C, and VEGF-3 receptor in human prostate cancer.

We studied the expression of the non-metastatic clone 23 type 1 (nm23H1) gene, vascular endothelial growth factor (VEGF)-C, and its receptor VEGFR-3 using an in situ hybridization technique and immunohistochemical analyses with prostate cancer tissues and adjacent benign tissues of 52 human archival cases. The association between VEGF-C expression, microlymphatic count (MLC), and staining intensity for nm23H1 and VEGFR-3 was used to evaluate tumor metastasis and survival rate. MLC values were significantly higher in tumorous tissue than in non-cancerous tissue. VEGF-C mRNA, VEGFR-3, and nm23H1 were highly expressed in tumorous tissue. VEGFR-3 expression was greater in VEGF-C mRNA-positive tumors than in VEGF-C mRNA-negative tumors. The association of VEGFR-3 expression with VEGF-C mRNA and MLC suggested that the poor prognosis and tumor metastasis associated with VEGFR-3 expression may be due, in part, to its role in promoting angiogenesis. VEGF-C expression was significantly associated with tumor lymphangiogenesis, angiogenesis, and immune response as a potent multifunctional stimulating factor in prostate cancer. Expression of nm23H1 was significantly inversely correlated with lymph node metastasis. Furthermore, there was a strong negative correlation between the expression of nm23H1, VEGF-C mRNA, and MLC. These findings provide important information for prophylactic, diagnostic, and therapeutic strategies for prostate cancer.

Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector

背景与目的已有的研究证明nm23-H1基因是一个重要的肿瘤转移抑制基因，但其抑制肿瘤转移的生化机理尚不完全清楚。Nm23-H1基因结构和功能异常与肿瘤的侵袭转移有密切关系。我们前期已构建了nm23-H1的短发夹RNA（short hairpin RNA, shRNA）载体以及可抵抗此shRNA降解的nm23-H1的cDNA的表达载体，在此基础上我们欲应用基因定点突变技术构建具有不同酶活性并能抵抗此shRNA降解的nm23-H1cDNA真核表达载体，并通过恢复实验验证其表达，为进一步研究肿瘤抑制基因nm23-H1的分子机制提供理论基础和实验依据。方法以pcDNA3.1(+)-shRNA-resistant-nm23-H1质粒为突变模板，应用重叠延伸PCR方法引入nm23-H1基因四个单点突变和一个联合位点突变，并将突变基因片段克隆到真核表达载体pcDNA3.1Hygro(+)。将突变质粒转染人肺腺癌细胞株A549/nm23-H1-shRNA（稳定沉默nm23-H1基因），利用Western blot技术验证不同突变体nm23-H1蛋白的表达。结果成功构建了shRNA抵抗的nm23-H1S44A、nm23-H1P96S、nm23-H1H118F、nm23-H1S120G、nm23-H1P96S-S120G五个突变型真核表达载体，经DNA序列分析突变的碱基序列与实验设计完全一致，经Western blot验证nm23-H1蛋白表达正常。结论成功构建了五个具有不同突变位点的shRNA抵抗的nm23-H1基因真核表达载体，并且突变蛋白质nm23-H1表达正常，同时也表明重叠延伸PCR技术是一种高效、便捷、经济的DNA定点突变方法。

Inhibition of nm23-H1 gene expression in chronic myelogenous leukemia cells

For solid tumors of a malignant origin, the expression of the nm23-H1 gene is a positive prognostic factor. However, for chronic myeloid leukemia (CML), the prognostic role of nm23-H1 gene expression is unknown. The present study investigated the impact of nm23-H1 gene expression on the proliferation and migration of the CML K562 cell line to elucidate the association between nm23-H1 gene expression and CML cell survival. An RNAi lipo-recombinant plasmid of the nm23-H1 gene (pGCsi-nm23-H1) was constructed and transfected into the K562 cells. RT-PCR and western blotting were used to detect nm23-H1 mRNA and protein expression, respectively. The anchorage-independent growth ability of the transfected cells was observed in soft agar culture and the ability of the K562 cells to migrate was determined using a Transwell assay. Following the successful construction and transfection of the pGCsi-nm23-H1 plasmid into the K562 cells, nm23-H1 mRNA and protein expression levels were significantly lower compared with the control group. The stably-transfected pGCsi-nm23-H1 K562 cells exhibited a markedly increased ability to form colonies and the number and sizes of the colonies were significantly increased compared with those of the control. In vitro, the cells migrated through a Matrigel-coated membrane during incubation for 20 h. The Transwell assay revealed that the quantitative number of pGCsi-nm23-H1 K562 cells that migrated into the lower compartment of the invasion chamber was markedly increased compared with the control. In conclusion, nm23-H1 gene expression may inhibit K562 cell proliferation and migration. nm23-H1 may be a cancer suppressor gene and play a significant role in inhibiting the survival of CML cells.

P-0012 - The Nm23-H1 Gene Acts as a Migration Suppressor in Gastric Lymphoma

P-0012 - The Nm23-H1 Gene Acts as a Migration Suppressor in Gastric Lymphoma

Anti-tumor activity and its mechanism of lycopene

Lycopene is an important natural carotenoid,and is widely found in human tissues and blood,with the prevention of many types of cancer,anti-aging effect.It can inhibit the proliferation of cancer cells of oral malignancy,and increasing of gap-junction communication between cells to prevent oral cancer,and also can inhibit the development of colorectal cancer by reducing the activity of matrix metalloproteinase.It plays the role of anti-metastasis through enhancing the expression of anti-metastatic gene nm23-H1. Key words: Neoplasms; Lycopene

Expression of nm23H1 and MMP2 in laryngeal carcinoma and its role in aggressiveness of the tumour and node metastasis

EnAbstract Introduction Squamous cell carcinomas of the head and neck are known for their aggressive growth and propensity to metastasize. The invasion is facilitated by matrix metalloproteinases (MMPs). The overexpression of many MMPs is positively associated with tumour metastasis. The nm23H1 gene has been implicated as a suppressor gene and reduced expression of its gene product has been observed in patients with positive lymph node metastasis. Objective The aim of this study was to examine the degree of expression of both MMP2 and nm23H1 proteins in 24 patients with primary laryngeal carcinoma using immunohistochemical technique and also to correlate the results with the clinical, radiological and histopathological data, in order to evaluate their role in predicting the local spread and lymph node metastasis. Patients and methods This study included 24 patients with primary laryngeal carcinoma involving various regions of the larynx and was carried out at the Department of Otolaryngology, Ain Shams University Hospitals. The laryngeal and nodal specimens were examined histopathologically and immunohistochemical analysis was carried out for the nm23H1 and MMP2 proteins. Results There was a significant correlation between MMP2 expression and the site of the tumour, as a drastic reduction in MMP2 expression was mainly associated with glottic carcinoma (P&lt;0.05). The tumour stages were associated with an increase in MMP2 expression, but this was not statistically significant (P=0.07). However, the presence of lymph node metastasis was significantly related to the overexpression of MMP2 (P&lt;0.05). As regards nm23H1, a statistically significant correlation was found between nm23H1 expression and the tumour stage (P&lt;0.05). Moreover, the presence of lymph node metastasis was significantly correlated to the loss of nm23H1 expression and vice versa (P&lt;0.05). However, the correlation between the expression levels of MMP2 and nm23H1 was found to be statistically insignificant (P&gt;0.05). Conclusion The increased expression of MMP2 was related to the occurrence of nodal metastasis of the tumour and also to the tumour sites characterized by frequent metastasis (supraglottic and transglottic). In contrast, nm23H1 expression was inversely related to the advancement of the tumour stage and the nodal metastatic state.