NDRG2 suppresses the proliferation of clear cell renal cell carcinoma cell A-498

Background

Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described.

Methods

The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot.

Results

The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell.

Conclusions

We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.     

Nicotinamide N-methyltransferase induces cellular invasion through activating matrix metalloproteinase-2 expression in clear cell renal cell carcinoma cells

Nicotinamide N-methyltransferase (NNMT) was recently identified as one clear cell renal cell carcinoma (ccRCC)-associated gene by analyzing full-length complementary DNA-enriched libraries of ccRCC tissues. The aim of this study is to investigate the potential role of NNMT in cellular invasion. A strong NNMT expression is accompanied with a high invasive activity in ccRCC cell lines, and small interfering RNA-mediated NNMT knockdown effectively suppressed the invasive capacity of ccRCC cells, whereas NNMT overexpression markedly enhanced that of human embryonic kidney 293 (HEK293) cells. A positive correlation between the expression of NNMT and matrix metallopeptidase (MMP)-2 was found in ccRCC cell lines and clinical tissues. The treatment of blocking antibody or inhibitor specific to MMP-2 significantly suppressed NNMT-dependent cellular invasion in HEK293 cells. Furthermore, SP-1-binding region of MMP-2 promoter was found to be essential in NNMT-induced MMP-2 expression. The specific inhibitors of PI3K/Akt signaling markedly decreased the binding of SP1 to MMP-2 promoter as shown by chromatin immunoprecipitation assay. We also demonstrated that PI3K/Akt pathway plays a role in NNMT-dependent cellular invasion and MMP-2 activation. Moreover, short hairpin RNA-mediated knockdown of NNMT expression efficiently inhibited the growth and metastasis of ccRCC cells in non-obese diabetic severe combined immunodeficiency mice. Taken together, the present study suggests that NNMT has a crucial role in cellular invasion via activating PI3K/Akt/SP1/MMP-2 pathway in ccRCC.     

Modulation of human renal cell carcinoma 786-0 MMP-2 and MMP-9 activity by inhibitors and inducers in vitro

A hallmark of renal cell carcinoma (RCC) invasion is its ability to degrade ECM by local production of gelatinase enzymes. Although many studies on RCC have demonstrated the importance of MMPs, very little information is currently known regarding the effect of inducers and inhibitors. We therefore investigated the effect of inducers and inhibitors on RCC 786-0 in vitro. Human RCC 786-0 (ATCC) was grown in RPMI medium supplemented with 10% FBS, penicillin, and streptomycin in 24-well tissue plates. At near confuence, the cells were washed with PBS; the serum-free medium was incubated with various inducers: phorbol ester (PMA), tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-β) and lipopolysaccharides (LPS). Cells were also incubated with inhibitors: EGCG, doxycycline, and a nutrient mixture with and without PMA; retinoic acid, dexamethasone, H-7; actinomcin D, or cyclohexamide. After 24 h, the medium was removed and analyzed for MMP-2 and MMP-9 by gelatinase zymography. RCC 786-0 secreted two bands, a major band corresponding to MMP-2 and a faint band corresponding to MMP-9. PMA and TNF-α, with increased concentration, increased MMP-9 secretion, while IL-1β and LPS did not significantly modify MMP-9 activity. MMP-2 secretion was not affected by any of the inducers. All the inhibitors tested without and with PMA showed a dose-dependent decrease in both MMP-2 and-9 expression. Further studies are in progress to confirm the role of MMP-9 on Matrigel invasion using PMA, cytokines, and LPS.     

肾透明细胞癌组织MMP-26和PDZK1表达临床意义的研究

目的:分析基质金属蛋白酶-26(MMP-26)和PDZK1在不同分化程度肾透明细胞癌(RCCC)中表达模式的测定,探讨其与RCCC的关系.方法:免疫组织化学(ABC法)和RT-PCR技术分别检测RCCC75例(A组)、肾脏良性病变的正常肾组织20例(B组)、RCCC新鲜组织30例(C组)和10例新鲜肾脏良性病变正常组织(D组)的MMP-26和PDZK1表达情况.结果:A组MMP-26表达率为82.7％ (62/75),B组为50.0％ (10/20),x2=9.183,P=0.002;A组PDZK1表达率为80.0％(60/75),B组为30.0％ (6/20),x2=18.631,P=0.000.MMP-26 mRNA在C、D组分别为0.830±0.126和0.600±0.035,t=2.931,P=0.009;PDZK1 mRNA在C和D组分别为0.950±0.055和0.570±0.043,t=4.494,P=0.000.C组MMP-26 mRNA在Ⅰ～Ⅱ级和Ⅲ～Ⅳ级表达分别为1.030±0.104和0.850±0.087,t=3.923,P=0.000;C组PDZK1 mRNA在Ⅰ～Ⅱ级和Ⅲ～Ⅳ表达分别为0.720±0.044和0.920±0.108,t=2.724,P=0.011.相关分析MMP-26与PDZK 1 mRNA在C组表达呈负相关,r=-0.737,P=0.000.结论:MMP-26在RCCC组织中高表达,且随其分级增高减低表达降低,PDZK1在RCCC组织中高表达,随其分级增高表达增高,MMP-26与PDZK1可能与肿瘤的发生发展有关,有望成为新的肿瘤治疗靶点.     

MMP-2和MMP-9表达与卵巢上皮性癌侵袭转移的关系

目的研究基质金属蛋白酶(MMP-2和MMP-9)在卵巢上皮性癌组织中的表达,探讨MMP-2、MMP-9表达与肿瘤的侵袭转移及预后的关系.方法用免疫组化S-P法对47例卵巢上皮性癌组织中MMP-2和MMP-9的表达情况进行检测.结果 MMP-2和MMP-9的表达在卵巢上皮性癌各组织类型之间差异无显著性,但在临床分期、初发、复发及细胞分级之间差异有显著性.MMP-2和MMP-9的表达与生存时间呈负相关.结论 MMP-2、MMP-9与卵巢上皮性癌的侵袭转移及预后有关.     

CIAPIN1 inhibits the growth and proliferation of clear cell renal cell carcinoma

Our previous studies indicated a direct correlation with loss of CIAPIN1 and carcinogenesis of tumor in human gastric cancer. Here we presented that the expression of CIAPIN1 was absent or significantly decreased in 102 cases of clear cell renal cell carcinoma (CCRCC) tissues (P < 0.05). Up-regulating CIAPIN1 by adenoviral vectors exhibited significant inhibition of CCRCC-derived cell growth in vitro and in vivo with G1 cell cycle arrest. Simultaneously, CIAPIN1-induced growth suppression was found partially to regulate various proteins, including inhibition of cyclinD1, cyclinE, cdk2, cdk4, p-Rb and VEGF, but up-regulation of p27^Kip1 and Rb.     

Prognostic significance of tenascin-C expression in clear cell renal cell carcinoma

Tenascin-C is an extracellular matrix protein that plays an important role in cell proliferation, migration and tumor invasion in various types of cancer. However, few reports exist on tenascin-C expression in renal cell carcinoma (RCC). This study aimed to assess the prognostic significance of tenascin-C in clear cell RCC. Using immunohistochemistry, 137 formalin-fixed, paraffin-embedded tissue sections obtained from patients with clear cell RCC were examined for tenascin-C expression. Tenascin-C expression was observed in 55 (40.1%) of the 137 clear cell RCC sections. Tumor cells displayed membranous and/or cytoplasmic staining for tenascin-C. Tenascin-C expression was more prominent near the pseudocapsule of the tumor and around the tumor vessels. Tenascin-C expression was significantly associated with a higher stage (P=0.0065) and higher nuclear grade (P=0.0001). However, there was no correlation between the tenascin-C expression and venous involvement. The cancer-specific survival rate in patients with a tenascin-C-positive primary tumor was significantly lower than that in those with a tenascin-C-negative primary tumor in univariate analysis (P=0.0017). However, tenascin-C expression did not exhibit a significant value for cancer-related death in the Cox regression analysis. In patients with stage 1-3 disease, the 5-year metastasis-free rate in patients with the tenascin-C-positive primary tumor was significantly lower than that in those with the tenascin-C-negative primary tumor (67.8 vs. 88.5%, respectively; P=0.0038). The Cox regression analysis showed that tenascin-C expression is a significant predictor of metastasis (P=0.0345). The tenascin-C expression was strongly related to the stage, nuclear grade and 5-year metastasis-free rate. Therefore, tenascin-C expression may be a possible marker for the metastatic potential of clear cell RCC.     

基质金属蛋白酶-9的表达与肾细胞癌血管生成的关系

背景与目的:基质金属蛋白酶-9(matrixmetalloproteinase-9,MMP-9)与肿瘤的侵袭、转移密切相关,近年发现它与肿瘤的血管生成也有关。本研究旨在探讨MMP-9的表达与肾细胞癌血管生成的关系及其临床意义。方法:应用免疫组化SP法检测78例肾细胞癌组织中MMP-9、血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)的表达及微血管密度(microvesseldensity,MVD)。结果:MMP-9的表达与肾细胞癌的病理分期和组织学分级有关(P<0.05);MMP-9表达阳性组的MVD值为110.46±50.16,明显高于阴性组的MVD值(84.77±44.52)(P<0.05),亦明显高于对照组的MVD值(74.03±36.06)(P<0.01);肾细胞癌组织中MMP-9的表达水平与VEGF的表达呈显著性正相关(r=0.4096,P<0.01)。结论:MMP-9与肾细胞癌的血管生成有关,检测MMP-9的表达可作为反映肾细胞癌侵袭和转移潜能的生物学指标。     

MiR-200c-3p inhibits cell migration and invasion of clear cell renal cell carcinoma via regulating SLC6A1

In this study, we investigated the mechanism of miR-200c-3p and SLC6A1 in regulating cell activity of clear cell renal cell carcinoma (CCRCC). The mRNA and miRNA expressions of tissue specimens were analyzed by CapitalBio Corporation (Beijing, China). The expression of SLC6A1 in CCRCC cells was examined through qRT-PCR and western blot. The migration and invasion ability of 786-O cells was testified by transwell assay after transfected. 786-O cell proliferation ability was detected by MTT assay. Dual luciferase reporter assay verified the association between SLC6A1 and miR-200c-3p. SLC6A1 was high expressed and miR-200c-3p was low expressed in CCRCC tissues and cells. Besides, lower SLC6A1 expression indicated longer survival time and higher survival rate. MiR-200c-3p could directly target at SLC6A1 and reduce its expression. MiR-200c-3p inhibited the proliferation, migration and invasion in 786-O cells by down-regulating SLC6A1 expression. The results suggested that the miR-200c-3p served as a suppressor for CCRCC via down-regulating SLC6A1.     

High expression of Ran GTPase is associated with local invasion and metastasis of human clear cell renal cell carcinoma

The Ran small GTPase (Ran) is involved in the regulation of nuclear transport, microtubule nucleation and dynamics, and spindle assembly. To address the question of whether Ran protein is associated with the progression of renal cell carcinoma (RCC), we compared by Western blotting the Ran protein levels in surgical RCC specimens from 180 consecutive Japanese patients with those in the corresponding nontumor tissue from the same patient. We also examined the Ran protein levels in tumors of different grades and stages. Ran proteins were more abundant in RCC tumor tissues than in nontumor tissues (p < 0.0001). High Ran expression was associated with higher grade, local invasion, and metastasis (p < 0.0001). Kaplan-Meier plots linked high Ran protein expression to a shorter overall survival in all cases (p < 0.0001) and a shorter disease-free survival in those without metastasis at radical or partial nephrectomy (M0; 131 cases, p < 0.0001). Ran protein expression was an independent factor influencing overall survival univariate analysis (p < 0.0001) and disease-free survival by multivariate analysis (p < 0.05). Our findings suggest that Ran is associated with the progression of RCC.     

Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor growth and invasion

Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock- or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.     

Microarray analysis of microRNA expression in renal clear cell carcinoma

Aim

To explore the microRNA (miRNA) expression in renal clear cell carcinoma (RCCC).

Methods

We compared the miRNA expression profiles in 11 pairs of RCCC and adjacent nontumorous tissue (NT) from 11 RCCC patients, using a mammalian miRNA microarray containing whole human mature and precursor miRNA sequences. To verify microarray results, Northern blotting was carried out on 5 randomly selected miRNAs.

Results

Totally 81 miRNAs were identified valid expression in RCCC samples, 48 of which specifically detected in RCCC samples, 17 of which detected downregulated in RCCC compared to NT sample, 2 upregulated and 14 without significant difference. MiRNAs in RCCC tissues exhibit an overall higher expression level than NT tissue. The chip results were confirmed by northern blot analysis.

Conclusion

Our study may help to clarify the molecular mechanisms involved in the pathogenesis of RCCC, and miRNAs potentially serve as a novel diagnostic biomarker of RCCC.     

Loss of expression of TIMP3 in clear cell renal cell carcinoma

AimsIn clear cell renal cell carcinoma (CCRCC), vascular endothelial growth factor (VEGF) represents the central positive mediator of tumour angiogenesis while VEGF receptor (VEGFR) is the primary target of anti-angiogenic therapies. TIMP3 is a physiological VEGFR-2 antagonist and thus could be considered as an anti-angiogenic factor. We therefore determined the status of this physiological inhibitor in CCRCC.Patients and methodsArchival tumour from 105 patients was studied. TIMP3 expression was analysed using immuno-histochemistry and real-time RT-PCR. Results were correlated with clinicopathological variables. To analyse the mechanisms of gene silencing involved, we performed Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA (MS-MLPA). At last, we evaluated the main upstream pathway described implicating TGFβRII, which induces TIMP3 expression.ResultsA down-expression of TIMP3, determined by immunohistochemistry, affected 100/105 renal cancers (95.2%). TIMP3 mRNA levels were significantly lower in high-grade tumours. Loss of heterozygosity of the TIMP3 gene was observed in 8 tumours (7.6%) and the 5′CpG island of the TIMP3 promoter was found to be methylated in 25 tumours (23.8%). A down-expression of TGFβRII was found in 85/105 CCRCCs (80.9%). A significant correlation was found between TIMP3 expression and TGFβRII expression.ConclusionsThis is the first demonstration that the loss of TIMP3 expression is observed in almost all CCRCCs. This loss of expression is a common molecular event in CCRCC. It may be an important initiation step for tumour development in a complex process implicating loss of heterozygosity on chromosome 22q, promoter hyper-methylation and inactivation of the TGFβRII pathway.     

Imbalance between MMP-2, 9 and TIMP-1 promote the invasion and metastasis of renal cell carcinoma via SKP2 signaling pathways

S-phase kinase-associated protein-2 (Skp2) is overexpressed in human cancers and acted as an oncogenic protein associated with poor prognosis by enhancing tumor metastasis. The present study has demonstrated that Skp2 overexpresses stable transfectants from 786-0 human renal cancer cells. We found that these stable transfectants exhibited increased migratory and invasive abilities. In addition, expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was upregulated and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was downregulated. In contrast, RNA interference-mediated knockdown Skp2 expression suppressed the ability of ACHN cells to migratory and invasive. Skp2 depletion increased P27 and decreased cyclin E activity, and then induced cell cycle arrest in the G0/G1 phase. Skp2 depletion also downregulated MMP-2 and MMP-9, while upregulated the TIMP-1 activity and expression. The results suggest that Skp2 signaling pathways promoted the ability to metastasize, by stimulating cell proliferation and increasing the ratio of MMP-2 and MMP-9/TIMP-1. So, in conclusion, we provide the first evidence that the imbalance of MMP/TIMP, including upregulation of MMP-2 and MMP-9 and downregulation of TIMP-1, is one of the mechanisms by which Skp2 promotes cell invasion.     

Fructose transporter GLUT5 expression in clear renal cell carcinoma

Renal cell carcinomas (RCC) can be subclassified for general purposes into clear cell, papillary cell, chromophobe cell carcinomas and oncocytomas. Other tumours such as collecting duct, medullary, mucinous tubular and spindle cell and associated with Xp 11.2 translocations/TFE 3 gene fusion, are much less common. There is also a residual group of unclassified cases. Previous studies have shown that RCC has high glycolytic rates, and expresses GLUT transporters, but no distinction has been made among the different subtypes of renal cell tumours and their grades of malignancy. In clear renal cell carcinoma (cRCC) glycogen levels increase, glycolysis is activated and gluconeogenesis is reduced. The clear cell subtype of RCC is characterized histologically by a distinctive pale, glassy cytoplasm and this appearance of cRCC is due to abnormalities in carbohydrate and lipid metabolism, and this abnormality results in glycogen and sterol storage. Several isoforms of glucose carriers (GLUTs) have been identified. We show here in a panel of 80 cRCC samples a significant correlation between isoform 5 (GLUT5) and many pathological parameters such as grade of differentiation, pelvis invasion and breaking capsule. GLUT5 expression also appears to associate more strongly with the clear cell RCC subtype. These data suggest a role for the GLUT5 isoform in fructose uptake that takes place in cRCC cells and which subsequently leads to the malignant RCC progression.     

Soybean saponin inhibits tumor cell metastasis by modulating expressions of MMP-2, MMP-9 and TIMP- 2

The antimetastatic properties of soybean saponin were investigated by evaluating matrix metalloproteinase (MMP-2 and MMP-9) production in HT-1080 cells. The mRNA expression levels of MMP-2 and MMP-9 were determined by RT-PCR analysis. The levels of secreted MMP-2, MMP-9 and tissue inhibitor of metalloproteinase-2 (TIMP-2) were determined by gelatin zymography and/or ELISA. The invasion of a Matrigel-coated membrane by human fibrosarcoma HT-1080 and HT-29 colon cancer cells was quantitatively assessed by counting the migrated cells. The treatment of HT-1080 cells with soybean saponin inhibited the mRNA expression of and reduced the amounts of secreted MMP-2 and MMP-9, whereas it increased the amount of secreted TIMP-2 dose-dependently. Soybean saponin significantly inhibited the invasion of HT-1080 cells through a Matrigel-coated membrane. The antimetastatic properties by soybean saponin were further confirmed by in vivo mice experiment via the tail vein injection of CT-26 colon cancer cells after feeding the mice the dietary soybean saponin. The incidence of metastatic tumor colonization of lungs of mice moderately decreased 2 weeks after the tail vein injection of CT-26 cells. Our current data support the notion that soybean saponin inhibits tumor cell metastasis by suppressing MMP-2 and MMP-9 productions, and stimulating TIMP-2 secretion, thereby suggesting that soybean saponin has a chemopreventive property against cancer metastasis.     

Impaired expression of the cell cycle regulator BTG2 is common in clear cell renal cell carcinoma

The prognosis of patients with renal cell carcinoma (RCC) is poor. A full understanding of the molecular genetics and signaling pathways involved in renal cancer development and in the metastatic process is of central importance for developing innovative and novel treatment options. In this study, BD Atlas Human Cancer 1.2 cDNA microarrays were used to identify genes involved in renal tumorigenesis. By analyzing gene expression patterns of four clear cell RCC (cRCC) cell lines and normal renal tissue, 25 genes were found differentially expressed. To determine the relevance of these genes, RNA in situ hybridization was performed on a tissue microarray generated from 61 snap-frozen primary renal cell carcinomas and 12 normal renal cortex biopsies. B-cell translocation gene 2 (BTG2), a negative cell cycle regulator, which was expressed in normal renal tissue but down-regulated in cRCC cell lines and primary cRCCs, was selected for additional experiments. Quantitative BTG2 mRNA expression analysis in 42 primary cRCCs and 18 normal renal cortex biopsies revealed up to 44-fold reduced expression in the tumor tissues. Decrease of BTG2 expression was not associated with tumor stage, grade, and survival. Cell culture experiments demonstrated that BTG2 expression was weakly inducible by the phorbolester 12-O-tetradecanoylphorbol-13-acetate in one of four cRCC cell lines. In contrast, increasing cell density led to elevated BTG2 mRNA expression in three of four cRCC cell lines. In both experiments, BTG2 mRNA levels did not reach values observed in normal renal tissue. These data suggest that down-regulation of BTG2 is an important step in renal cancer development.     

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel-Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.