Donghao Shang

TGFBI-promoted Adhesion, Migration and Invasion of Human Renal Cell Carcinoma Depends on Inactivation of von Hippel-Lindau Tumor Suppressor

OBJECTIVE To investigate the role of transforming growth factor-β-induced (TGFBI) in metastasis of renal cell carcinoma (RCC) and the associations between TGFBI expression and von Hippel-Lindau (VHL) status. METHODS In null type VHL cells stably transfected with the VHL vector, the expression of VHL in cells with wild type VHL was decreased by siRNA. We investigated the effects of hypoxia-inducible transcription factor (HIF) on TGFBI in RCC cells by decreasing the expression levels of HIF-1α and HIF-2α through siRNA. The secretion of transforming growth factor-β1 (TGF-β1) in RCC cells with different VHL status was analyzed by enzyme-linked immunosorbent assay. The role of TGFBI in metastasis and the effect of VHL activation on TGFBI-induced adhesion, migration, and invasion in RCC cells were examined using matrigel, chemotaxis, and the transwell system, respectively. RESULTS Our results suggested that TGF-β1 and TGFBI might be targets of VHL, and the suppression of TGFBI by VHL is not by way of the HIF-1α or HIF-2α pathway. The expression of TGFBI was significantly enhanced by TGF-β1 in VHL-inactive RCC cells compared with VHL-active cells. In addition, these results indicate that TGFBI participated in the adhesion, migration, and invasion of RCC cells, which are dependent on the inactivation of VHL. CONCLUSION The results of the present study suggest that TGFBI-promoted metastasis of RCC cells depends on inactivation of the VHL tumor suppressor and that TGFBI could be a therapeutic target against RCC in the future.

Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway.

Background Renal cell carcinoma (RCC) is one of the most common kidney cancers and is highly resistant to chemotherapy. Accumulating evidence suggests that interleukin-22 (IL-22) may mediate host defense against varietal pathogens as a proinflammatory and anti-inflammatory cytokine. The purpose of this study is to assess the inhibitory effects of IL-22 on human RCC cell line A498 and to investigate the possible mechanisms underlying the anti-tumor effects of this cytokine. Methodology A498 cells, a RCC cell line, were used to assess the inhibitory growth effects of IL-22 using the MTT assay and flow cytometric analysis in vitro. BALB/C nude mice bearing A498 cell xenografts were used to examine the antitumor efficacy of IL-22 in vivo. Western blotting assay was performed to detect the regulation of the intracellular signaling pathway of IL-22. Principal Findings We found that IL-22 suppressed the growth of A498 cells in a dose-dependent manner and inhibited the growth of A498 xenografts. We also observed that IL-22 produced a dose-dependent inhibitory effect on A498 cells that involved the induction of G2/M cell cycle arrest without cell apoptosis. Moreover, we showed that the phosphorylation of STAT1 was increased and the phosphorylation of ERK1/2 was attenuated in A498 cells exposed to IL-22. The growth inhibition of A498 cells was partially revised after IL-22 treatment as the expression of STAT1 was knocked down. And inflammatory cytokines, interferon-α and tumor necrosis factor-α (TNF-α) were barely involved in the suppression of A498 cell xenografts treated with IL-22. Conclusions IL-22 dose-dependently suppresses RCC cell line A498 cells in vitro and induces growth inhibition of A498 cell-bearing mouse xenografts. These results suggest that the anti-RCC effects of IL-22 are at least partially mediated through regulation of STAT1 signaling pathways and G2/M cell cycle arrest, rather than by inducing apoptosis and inflammatory cytokines.

Interferon-α Induces G1 Cell-Cycle Arrest in Renal Cell Carcinoma Cells Via Activation of Jak-Stat Signaling

The purpose of this study was to clarify the mechanism of IFN-α resistance in RCC. The effects of IFN-α on induction of apoptosis and cell-cycle arrest were analyzed by flow cytometric analysis. Jak-Stat pathway components induced by IFN-α was evaluated using Western blotting. The results suggested that IFN-α caused growth inhibition of RCC cell lines via arrest in the G1 phase without inducing apoptosis. The resistance of RCC to IFN-α was associated with the low expression of Stat1. This study indicated that the Jak-Stat pathway should be considered a primary target for improving the response of RCC to IFN-α treatment.

Gene expression profiling of the synergy of 5-aza-2 ′ -deoxycytidine and paclitaxel against renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is one of the most common kidney cancers and is highly resistant to chemotherapy. We previously demonstrated that 5-aza-2′-deoxycytidine (DAC) could significantly increase the susceptibility of renal cell carcinoma (RCC) cells to paclitaxel (PTX) treatment in vitro, and showed the synergy of DAC and PTX against RCC. The purpose of this study is to investigated the gene transcriptional alteration and investigate possible molecular mechanism and pathways implicated in the synergy of DAC and PTX against RCC.MethodscDNA microarray was performed and coupled with real-time PCR to identify critical genes in the synergistic mechanism of both agents against RCC cells. Various patterns of gene expression were observed by cluster analysis. IPA software was used to analyze possible biological pathways and to explore the inter-relationships between interesting network genes.ResultsWe found that lymphoid enhancer-binding factor 1 (LEF1), transforming growth factor β-induced (TGFBI), C-X-C motif ligand 5 (CXCL5) and myelocytomatosis viral related oncogene (c-myc) may play a pivotal role in the synergy of DAC and PTX. The PI3K/Akt pathway and other pathways associated with cyclins, DNA replication and cell cycle/mitotic regulation were also associated with the synergy of DAC and PTX against RCC.ConclusionThe activation of PI3K/Akt-LEF1/β-catenin pathway could be suppressed synergistically by two agents and that PI3K/Akt-LEF1/β-catenin pathway is participated in the synergy of two agents.

Synergistic roles of p53 and HIF1α in human renal cell carcinoma-cell apoptosis responding to the inhibition of mTOR and MDM2 signaling pathways

Introduction mTOR and MDM2 signaling pathways are frequently deregulated in cancer development, and inhibition of mTOR or MDM2 independently enhances carcinoma-cell apoptosis. However, responses to mTOR and MDM2 antagonists in renal cell carcinoma (RCC) remain unknown. Materials and methods A498 cells treated with MDM2 antagonist MI-319 and/or mTOR inhibitor rapamycin were employed in the present study. Cell apoptosis and Western blot analysis were performed. Results and conclusion We found that the MDM2 inhibitor MI-319 induced RCC cell apoptosis mainly dependent on p53 overexpression, while the mTOR antagonist rapamycin promoted RCC cell apoptosis primarily through upregulation of HIF1α expression. Importantly, strong synergistic effects of MI-319 and rapamycin combinations at relatively low concentrations on RCC cell apoptosis were observed. Depletion of p53 or HIF1α impaired both antagonist-elicited apoptoses to differential extents, corresponding to their expression changes responding to chemical treatments, and double knockdown of p53 and HIF1α remarkably hindered MI-319- or rapamycin-induced apoptosis, suggesting that both p53 and HIF1α are involved in MDM2 or mTOR antagonist-induced apoptosis. Collectively, we propose that concurrent activation of p53 and HIF1α may effectively result in cancer-cell apoptosis, and that combined MDM2 antagonists and mTOR inhibitors may be useful in RCC therapy.

Protein tyrosine phosphatase ζ enhances proliferation by increasing β-catenin nuclear expression in VHL-inactive human renal cell carcinoma cells

ObjectiveWe investigated the role of protein tyrosine phosphatase ζ (Ptprz1) in human renal cell carcinoma (RCC) cells’ proliferation and associations between Ptprz1 expression and von Hippel-Lindau (VHL) activation.MethodsA normal human renal cell line and four human RCC cell lines were used in this study. VHL or Ptprz1 expression in RCC cells was increased by transfection with a VHL or Ptprz1 vector. VHL or Ptprz1 expression was decreased in these cells by siRNA using Lipofectamine 2000. Cells’ proliferative activity was assessed by WST-1 assay.ResultsOur results suggested that Ptprz1 was a target of VHL, and a loss of VHL activation increased Ptprz1 expression in RCC cells. Ptprz1 enhanced β-catenin protein expressions in the nuclear fractions of RCC cells and participated in regulating proliferation by activating β-catenin and its downstream genes. In addition, a loss of VHL activity may enhance the proliferative activity of RCC cells by increasing Ptprz1 expression.ConclusionPtprz1-enhanced RCC cells’ proliferation depends on VHL inactivation, and the Ptprz1/β-catenin pathway may be a potential target for treating RCC with inactive VHL.

Profiling of mRNA and long non-coding RNA of urothelial cancer in recipients after renal transplantation

The molecular mechanism and signal transduction pathways involved in urothelial cancer (UC) after renal transplantation (RTx) remain unknown. In this study, we investigated the profiling of messenger RNA (mRNA) and long non-coding RNA (lncRNA) in RTx recipients with UC. The mRNA and lncRNA of six pairs of UC and corresponding normal urothelial tissues in RTx recipients were profiled using Arraystar Human lncRNA Microarray V3.0, which is designed for the global profiling of 26,109 coding transcripts and 30,586 lncRNAs. Quantitative real-time PCR (qRT-PCR) was used to validate the differentially expressed mRNAs and lncRNAs. Molecular function classification and biological process classification for the differentially expressed mRNAs were analyzed with Gene Ontology. The key pathways that were associated with UC after RTx were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Compared to normal urothelial tissues, 1597 mRNAs were upregulated and 1032 mRNAs were downregulated in UC; 2107 lncRNAs were upregulated and 1794 lncRNAs were downregulated (greater than twofold). Further qRT-PCR analysis of mRNA and lncRNA expression showed well consistency with the data of microarray analysis. The expression of matrix metalloprotease (MMP)-3, MMP-10, MMP-12, and MMP-13 was significantly increased, while the expression of CD36 was decreased in UC after RTx. Co-expression analysis of lncRNAs and their nearby coding genes showed that lncRNAs may play critical roles in regulating nearby genes in the carcinogenesis of UC. Our results also suggest that peroxisome proliferator-activated receptor (PPAR) signaling may be involved in UC after RTx. Moreover, several cytokines and their receptors were also significantly upregulated in UC after RTx, suggesting that cytokines might be modulated and participated in the carcinogenesis of UC after RTx. We analyzed the potential molecular mechanism and pathways involved in the UC of RTx recipients. Our results revealed that several key regulatory pathways and lncRNAs play critical roles in the carcinogenesis of UC, and suggest that UC in RTx recipients may be more likely to invade and metastasis. However, the detailed functional analysis of these mechanisms should be further performed in the future.

Interferon‑α enhances the susceptibility of renal cell carcinoma to rapamycin by suppressing mTOR activity

The aim of the present study was to investigate the antiproliferative effects of interferon (IFN)-α and rapamycin (RPM) on renal cell carcinoma (RCC) cells and examine the synergistic growth suppression conferred by IFN-α and RPM. The effects of IFN-α and/or RPM on RCC cells were determined using a WST-1 assay and the synergy of IFN-α and RPM against three RCC cell lines was analyzed with isobolographic analysis. The expression of mammalian target of rapamycin (mTOR) was downregulated by RNAi, and the expression and phosphorylation of proteins in the mTOR pathway following treatment with IFN-α and/or RPM was examined by western blot analysis. The observations indicated that IFN-α significantly increased the susceptibility of RCC cells to RPM and the synergistic effect of IFN-α and RPM against RCC cells was confirmed in all three RCC cell lines. The mTOR pathway was shown to be associated with the synergistic effect of IFN-α and RPM against RCC. IFN-α and RPM alone decreased the phosphorylation of mTOR, p70 S6 kinase, S6 and 4E binding protein 1, and IFN-α significantly enhanced the RPM-induced suppression of the mTOR pathway. However, in RCC cells with low mTOR activity, the synergy of IFN-α and RPM was eliminated. Therefore, the results of the present study indicate that the mTOR pathway plays an important role in the synergistic effect of IFN-α and RPM against RCC cells. Thus, mTOR may serve as an effective therapeutic target in the treatment of advanced RCC.

Expression and Proliferation-Promoting Role of Lymphoid Enhancer-Binding Factor 1 in Human Clear Cell Renal Carcinoma

Lymphoid enhancer-binding factor 1 (LEF1) has been regarded as an important gene for carcinogenesis in many malignancies, however, the role of LEF1 in the progression of human renal cell carcinoma (RCC) has not been well studied. In this study, we investigated the expression of LEF1 in human RCC and the effect on proliferative ability of RCC cells. RCC samples from 138 patients who underwent radical nephrectomy were used in this study, the expression of LEF1 protein was determined by immunohistochemistry and Western blot, mRNA expression was analyzed by RT-PCR and real-time PCR. To investigate the effect of LEF1 on the proliferation of RCC cells, a LEF1 vector was transfected into RCC cells and LEF1 expression was also decreased by using siRNA. Proliferative ability of RCC cells was examined by WST-1 assay and a xenograft study with BALB/C nude mice. Our results indicated that LEF1 expression was significantly increased in stage III, IV and grade 3 RCC than in normal kidney, however, decreased LEF1 expression was found in low-stage and grade RCC compared to that in normal kidney, the expression of LEF1 was correlated to tumor stages, histologic grade, and tumor sizes in RCC. The effect of LEF1 on the proliferation in RCC was also analyzed, our results suggested that RCC cells expressing high levels of LEF1 had significantly increased proliferative ability compared to control cell lines, in contrast, RCC cells with a low LEF1 expression had lower proliferative ability. Moreover, LEF1 promoted proliferation of RCC cells depending on suppressing G2/M cell-cycle arrest. Our study demonstrated that the expression of LEF1 is associated with the progression of RCC and that LEF1 maybe involved in the development of RCC, these suggested LEF1 play a key role and might serve as a therapeutic target in treating advanced RCC.