Renal Cancer Cell Line 786-O Research Articles

IMN041 is an immunotherapeutic and an effective treatment in mouse xenograft models of pancreatic cancer, renal cancer and triple negative breast cancer

BackgroundiMN013 (5-aza-2',2'-difluorodeoxycytidine), a DNA methyl transferase inhibitor and ribonucleotide reductase inhibitor, and its prodrug iMN041 (3',5'-di-trimethylsilyl-2',2'-difluro-5- azadeoxycytidine), have been shown to be active in mouse xenograft models of hematogenous and solid tumors. In a xenograft of non-small cell lung cancer (NCI-H460), iMN041 treated mice demonstrated a marked inflammatory response upon analysis of tumor histology, which was hypothesized to be mediated by upregulation of natural killer (NK) cells. This study aimed to characterize the antitumor immune responses generated by iMN041 and test the efficacy iMN041 in solid tumors with poor prognosis.MethodsIn the Renca syngeneic mouse model, tumors were harvested following two doses of iMN041 or vehicle control, and analyzed by fluorescent-activated cell sorting for an antitumor immune response. iMN041 was also tested for tumor growth inhibition and animal survival for up to 42 days in xenograft models of pancreatic, renal, and triple negative breast cancer.ResultsTumors from mice implanted with the Renca cell line and treated with iMN041 demonstrated an increase in granzyme B in NK (p = 0.024) and NKT cells (p = 0.004), an increase in the ratios of CD8-T to regulatory T cells (Treg) (p = 0.0026) and CD4-T to Treg cells (p = 0.022) and a decrease in myeloid-derived suppressor cells (p = 0.040), compared to vehicle controls. A significant decrease in MAGE-A positive tumor cells in treated mice, concordant with a proportional decrease in all live tumor cells, suggests that these cells are one of the main targets of the activated immune system. Xenograft models of the triple negative breast cancer cell line DU4475, renal cancer cell lines 786-O and Caki-1, and pancreatic cancer cell lines CFPAC-1 and SW1990, demonstrated significantly lower tumor volumes, and, where there were a sufficient number of events, significantly improved survival in treated mice compared to vehicle control mice.ConclusionsIn mouse cancer models, iMN041 is an effective treatment for solid tumors mediated in part through a unique antitumor immune response.

SLC14A1 is a new biomarker in renal cancer.

Renal cancer is one of the common malignant tumors of the urinary tract, prone to distant metastasis and drug resistance, with a poor clinical prognosis. SLC14A1 belongs to the solute transporter family, which plays a role in urinary concentration and urea nitrogen recycling in the renal, and is closely associated with the development of a variety of tumors. Transcription data for renal clear cell carcinoma (KIRC) were obtained from the public databases Gene Expression Omnibus database (GEO) and The Cancer Genome Atlas (TCGA), and we investigated the differences in SLC14A1 expression in cancerous and normal tissues of renal cancer, its correlation with the clinicopathological features of renal cancer patients. Then, we verified the expression levels of SLC14A1 in renal cancer tissues and their Paracancerous tissues using RT-PCR, Western-blotting and immunohistochemistry. Finally, we used renal endothelial cell line HEK-293 and renal cancer cell lines 786-O and ACHN to explore the effects of SLC14A1 on the biological behaviors of renal cancer cell proliferation, invasion and metastasis using EDU, MTT proliferation assay, Transwell invasion assay and scratch healing assay. SLC14A1 was lowly expressed in renal cancer tissues and this was further validated by RT-PCR, Western blotting, and immunohistochemistry in our clinical samples. Analysis of KIRC single-cell data suggested that SLC14A1 was mainly expressed in endothelial cells. Survival analysis showed that low levels of SLC14A1 expression were associated with a better clinical prognosis. In biological behavioral studies, we found that upregulation of SLC14A1 expression levels inhibited the proliferation, invasion, and metastatic ability of renal cancer cells. SLC14A1 plays an important role in the progression of renal cancer and has the potential to become a new biomarker for renal cancer.

Diallyl Trisulfide Suppresses the Renal Cancer Stem-like Cell Properties via Nanog

Cancer stem-like cells (CSCs), which play an important role in tumor initiation and progression, have been identified in many cancers. Diallyl trisulfide (DATS) is an organosulfur compound extracted from garlic with anticancer activities. Nanog is a transcription factor responsible for maintaining the stemness of CSCs, but its role in the DATS-induced attenuation of renal CSC properties is unknown. In this study, renal CSCs were enriched from human renal cancer cell lines 786-O and ACHN cultured in a serum-free medium (SFM). The properties of CSCs were analyzed by evaluating the ability of the cells in sphere formation and measuring the expression of stem cell markers. We found that downregulation of Nanog inhibited renal CSC properties. DATS suppressed renal CSC activities by reducing tumorsphere formation, decreasing stem cell markers including Nanog, CD44, ALDH1A1, and Oct4, inhibiting cell proliferation and promoting apoptosis. We further revealed that overexpression of Nanog reversed the suppressive effects of DATS on renal CSCs. Taken together, our results demonstrated that DATS inhibited renal CSCs by suppressing Nanog. These novel findings suggested that, through Nanog targeting, DATS can potentially be used as an anti-tumor agent for renal cancer.

Ferroptosis is involved in the anti-tumor effect of lycorine in renal cell carcinoma cells.

Renal cell carcinoma (RCC) is a most common malignant tumor in the genitourinary system. Studies have shown that Lycorine has promising anticancer activities with minor side effects. However, the effect of lycorine on the proliferation of RCC cells and its underlying anti-tumor mechanism have not yet been fully elucidated. The human renal cancer cell lines 786-O, A498 and Caki-1 were cultured and treated with different concentrations of lycorine or ferrostatin-1, a ferroptosis inhibitor. Cell viability and colony formation assays were used to measure cell proliferation. The 5-, 12- and 15-HETE hydroxyeicosatetraenoic acid (HETE) and MDA levels, as well as the reduced to oxidized glutathione (GHS/GSSG) ratio, were analyzed. Western blot analysis was used to detect the expression of glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long chain family member 4 (ACSL4), which are key markers of ferroptosis. Transmission electron microscopy was used to observe the morphological features associated with ferroptosis. Lycorine was found to inhibit the proliferation of RCC cells. After lycorine treatment, the expression levels of GPX4 in RCC cells decreased, whereas those of ACSL4 increased. Lycorine induced the expression of 5-HETE, 12-HETE, 15-HETE and MDA in RCC cells, and reduced the GSH/GSSG ratio. In addition, ferrostatin-1 could prevent lycorine-induced ferroptosis in RCC cells.

Discovery of Oral Anticancer 1,2-Bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine Hybrids That Inhibit Angiogenesis and Induce DNA Cross-Links.

Designing hybrid molecules with dual functions is one approach to improve the therapeutic efficacy of combination treatment. We have previously conjugated phthalazine and bis(hydroxymethyl)pyrrole pharmacophores to form hybrids bearing antiangiogenesis and DNA interstrand cross-linking activities. To improve the bioavailability, we adopted a benzology approach to design and synthesize a new series of 1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazines. These new hybrids retained the dual functions and could be formulated into vehicles for intravenous and oral administration. Among them, we demonstrated that compound 19a with dimethylamine at the C6 position markedly suppressed the tumor growth of human small cell lung cancer cell line H526, squamous lung cancer cell line H520, and renal cancer cell line 786-O in nude mice, implying that compound 19a is a broad-spectrum anticancer agent. Our results implicated that the conjugation of antiangiogenic and DNA cross-linking is likely to be a helpful approach to improving the efficacy of combination therapy.

α-1,2-Mannosidase MAN1C1 Inhibits Proliferation and Invasion of Clear Cell Renal Cell Carcinoma.

Background: This study investigated the biological function of the gene MAN1C1 α-mannosidase in renal cell carcinoma. It has been reported that MAN1C1 is probably a potential tumor suppressor gene in Wilms. However, the role of MAN1C1 in human clear cell renal cell carcinoma (ccRCC) has not been reported.Methods: In this study, MAN1C1 gene over-expression was used to transfect human renal cancer cell lines 786-O and OS-RC-2 to study apoptosis and the underlying mechanisms which influence epithelial-mesenchymal transition.Results: MAN1C1 was down-regulated in ccRCC and related to the clinicopathological factors and prognosis of ccRCC. We revealed that over-expression MAN1C1 showed anti-tumor effect by inducing apoptosis, as determined by Cell Counting Kit-8 (CCK-8) assay, cell cycle analysis, and western blot analysis. What's more, MAN1C1 over-expression remarkably increased the ratio of Bax/Bcl-2 and inhibited epithelial-mesenchymal transition by increasing the expression of E-CA. In addition, the ratio of Bax/Bcl-2 and E-CA were also increased in MAN1C1 gene over-expression renal cancer cells compared with the control cells.Conclusion: We find that re-expression of silenced MAN1C1 in ccRCC cell lines inhibited cell viability, colony formation, induced apoptosis, suppressed cell invasion and migration. In conclusion, MAN1C1 is a novel functional tumor suppressor in renal carcinogenesis. This is the first time that the function of MAN1C1 gene has been verified in the renal tumor tissue so far.

CIP2A Promotes Proliferation, Invasion and Chemoresistance to Cisplatin in Renal Cell Carcinoma.

CIP2A is a well-known oncoprotein whose expression is elevated in multiple human solid tumor types. However, its role in renal cell carcinoma (RCC) development is poorly understood. Thus, in our present study, we used the renal cancer cell lines 786-O, A498 and CAKI-1 and the renal epithelial cell line HK-2 to clarify the function of CIP2A in RCC. We found that CIP2A expression is much higher in the RCC cells than in the normal renal epithelial cell. Lentivirus covered coding region CIP2A cDNA sequence and CIP2A siRNA were used to up and down regulate CIP2A expression in vitro. We found that overexpression of CIP2A promoted G1/S transition and cell proliferation. In addition, up-regulation of CIP2A significantly enhanced the invasion and migration capabilities of the cells. Furthermore, CIP2A promoted epithelial-mesenchymal transformation (EMT) and chemoresistance to cisplatin in RCC cells. Taken together, our findings demonstrate that CIP2A plays an important role in proliferation, invasion and chemoresistance to cisplatin in RCC cells. CIP2A may serve as an ideal molecular target for RCC therapeutics.

Biological characteristics of renal cancer cells after CTP-mediated cancer suppressor gene NPRL2 protein treatment.

Nitrogen permease regulator like-2 (NPRL2) has been proved to be a useful suppressor gene in treating many cancers containing renal cancer based on experiments. Transgenic technology which transfect exogenous NPRL2 gene into cancer cell was used in these experiments. However, this technology has defects, such as gene mutation and loss. Cytoplasmic transduction peptide (CTP) can be used to avoid these defects because it can directly mediate proteins to penetrate cell membrane and specifically locate in cytoplasm. In this article, CTP was used to directly mediate NPRL2 protein into the renal cancer cell line 786-O, then cell proliferation was detected by the CCK-8 method, cell cycle and apoptosis were detected by flow cytometry, cell invasion and migration ability were detected by the Transwell assay. Bcl-xl, Cyt-c and caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot for the analysis of the related mechanism. The result showed that CTP successfully mediated NPRL2 protein into renal cancer cells and the growth of cells was significantly inhibited. The mechanism may be NPRL2 down-regulating the expression of Bcl-xl which can up-regulate Cyt-c and further activate caspase-3, and then a cascade reaction is caused for cell apoptosis on the classic mitochondrial apoptosis pathway.

MiR-630 functions as a tumor oncogene in renal cell carcinoma.

IntroductionMicroRNAs (miRNAs) are non-coding RNAs that regulate multiple cell processes during cancer progression. Renal cell carcinoma (RCC) is a malignancy with a poor prognosis. In this study, we aimed to investigate the roles of miR-630 in RCC progression.Material and methodsExpression of miR-630 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in four renal cancer cell lines (786-O, ACHN, Caki-1, and Caki-2) and one normal human proximal tubule epithelial cell line (HK-2). Next, miR-630 inhibitor was used to inhibit miR-630 expression in 786-O cells. Finally, its effects on cell proliferation, apoptosis, migration, and invasion were evaluated.ResultsThe expression level of miR-630 was higher in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 than that in the normal renal cell line HK-2 (p < 0.05). Furthermore, a proliferation assay, apoptosis assay, migration assay and invasion assay were performed, and the results showed that down-regulation of miR-630 expression by miR-630 inhibitor significantly inhibited cell proliferation, migration, and invasion, which meanwhile induced cell apoptosis of the renal cancer cell line 786-O.ConclusionsThis is the first time that miR-630 expression has been shown to be associated with renal cancer progression, and down-regulation of miR-630 can inhibit tumor progression, which provides a potential therapeutic target for renal cancer treatment.

MiR-506 Is Down-Regulated in Clear Cell Renal Cell Carcinoma and Inhibits Cell Growth and Metastasis via Targeting FLOT1

BackgroundSome microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC).MethodsmiR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines.ResultsmiR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506.ConclusionsmiR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.

Methylation‑associated inactivation of LATS1 and its effect on demethylation or overexpression on YAP and cell biological function in human renal cell carcinoma.

Large tumor suppressor 1 (LATS1) gene is one of the key factors in Hippo signaling pathway. Inactivation of LATS1 by promoter methylation was found in colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), astrocytoma, breast cancer and it was proved to be a tumor suppressor. However, its role is unclear in renal cell carcinoma (RCC). In this study, the expression of LATS1 was determined by reverse transcription polymerase chain reaction (RT‑PCR) and immunohistochemistry in 30 pairs of RCC tissues and matched normal kidney tissues and RCC cells. We found that the expression of LATS1 was markedly reduced in RCC tissues and cells, in the RCC tissue in 46.7% (14/30), while in the normal kidney tissues in 76.7% (23/30), and was associated with pathological grade and clinical stage of RCC. We detected methylation status of LATS1 by bisulfite sequence-PCR (BSP) in renal cancer cell line 786-O which lowers expression of LATS1, and we found it hypermethy-lated (in 97.5%). In addition, pharmacological demethylation using 5-Aza-2'-deoxycytidine (5-Aza) restored the expression of LATS1 mRNA and protein in 786-O cells, both LATS1 demethylation and overexpression of LATS1 downregulated the expression of Yes-associated protein (YAP), inhibited cell proliferation, induced cell apoptosis and cell cycle G1 arrest in 786-O cells. Thus, this report for the first time demonstrates the inactivation of LATS1 by promoter methy-lation and it is a tumor suppressor in kidney cancer. LATS1 may serve as a biomarker for possible early diagnosis and as a potential therapeutic target for human RCC.

Up-Regulation of pVHL along with Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal Cancer Cells

The majority of renal cell carcinomas (RCCs) are characterized by loss of function of the tumor suppressor gene von Hippel Lindau (VHL), which acts as ubiquitin ligase for hypoxia-inducible factor-1α (HIF-1α). In the absence of VHL, HIF-1α protein becomes stabilized and contributes to tumorigenesis. Recent data demonstrate the antitumor efficacy of VHL promoter in RCC cells. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of VHL. NDRG2 is involved in proliferation and invasion of cancer cell, furthermore it is frequently down-regulated in renal cell carcinoma. Herein we evaluated the effect of NDRG2 overexpression on proliferation and invasion in human renal cancer cells. The human renal cancer cell line 786-O and A498 were infected with Ad-NDRG2 or Ad-LacZ. Overexpression of NDRG2 not only inhibited the growth of the cells, but also suppressed the invasion. Further study showed that the tumor suppressor gene VHL were up-regulated, whereas transcription factor HIF-1a and vascular endothelial growth factor (VEGF) were down-regulated in 786-O cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for a total of seven times significantly inhibited the growth and angiogenesis of xenografted 786-O tumors. In conclusion, these data indicate that NDRG2 may be involved in proliferation and invasion by impacting the expression of VHL and HIF-1α. NDRG2 may be an attractive therapeutic target for renal cell carcinoma.

Aberrant methylation and loss of CADM2 tumor suppressor expression is associated with human renal cell carcinoma tumor progression

Cell adhesion molecules (CADMs) comprise a protein family whose functions include maintenance of cell polarity and tumor suppression. In this report, we show that the CADM2 gene is repressed in human clear renal cell carcinoma by DNA promoter hypermethylation and/or loss of heterozygosity. Moreover, the loss of CADM2 expression is associated with a higher tumor pathology stage (p<0.05). The re-expression of CADM2 in the renal cancer cell line 786-O significantly suppressed tumor cell growth in vitro and in mouse xenografts by a G1 phase cell cycle arrest and the induction of apoptosis. Lentivirus-mediated CADM2 expression also significantly suppressed cancer cell anchorage-independent growth and invasion. Furthermore, the inhibition of endogenous CADM2 expression using siRNAs induced a tumorigenic phenotype in polarized non-tumorigenic MDCK cells. Thus, we conclude that CADM2 functions as a novel tumor suppressor and may serve as a potential therapeutic target for human renal cell carcinoma.

Biological characteristics of Rh123high stem-like cells in a side population of 786-O renal carcinoma cells

The aim of the present study was to investigate the differences in biological characteristics between the rhodamine 123 (Rh123)high and Rh123low subpopulations of the renal cancer cell line 786-O and to identify evidence for the existence of cancer stem cells in renal cell carcinoma (RCC) cells. In vitro cultured RCC 786-O cells were stained with Rh123, analyzed and sorted with flow cytometry. The differences in proliferative activity, long-term differentiation and radiation sensitivity between the two subpopulations were measured and the oncogenicity of each subpopulation was evaluated according to their neoplastic growth ability in soft agar and tumor-forming ability in NOD/SCID immunodeficient mice. There were two subpopulations in the cultured 786-O cells, Rh123high and Rh123low cells. Rh123low cells were the majority among 786-O renal carcinoma cells and barely formed solid tumors in NOD/SCID mice and colonies in soft agar. By contrast, the Rh123high cells were the minority, exhibited high proliferative activity, differentiation ability and resistance to radiation and showed high tumorigenesis potential and colony forming efficiency. The Rh123high cells had stem-like characteristics in cultured RCC 786-O cells in vitro.

Upregulated microRNA-16 as an oncogene in renal cell carcinoma.

MicroRNAs (miRs) are small, endogenous noncoding RNAs that serve a significant function in various biologic processes, including those involved in cancer. The present study aimed to determine the expression and function of miR-16 in renal cell carcinoma (RCC). Quantitative polymerase chain reaction was used to quantify the expression of miR-16 in 48 paired RCC tissues and adjacent normal tissues. The impact of miR-16 on cell proliferation, migration and apoptosis was analyzed by transfecting miR-16 mature molecules into the renal cancer cell lines 786-O and ACHN. The results indicated that miR-16 was significantly upregulated in RCC tissues (P<0.05). Downregulation of miR-16 resulted in reduced cell proliferation and migration and increased levels of apoptosis, while overexpression of miR-16 resulted in accelerated cellular proliferation and migration, suggesting that miR-16 may function as an oncogene in RCC. The present study demonstrated for the first time, to the best of our knowledge, that miR-16 is upregulated in RCC and acts as an oncogene by inducing cellular proliferation, migration and reducing apoptosis. Further study of miR-16 in RCC may clarify the molecular mechanisms of RCC carcinogenesis and aid in the development of novel biomarkers and therapeutic options.

Tanshinone IIA arrests cell cycle and induces apoptosis in 786-O human renal cell carcinoma cells

Tanshinone IIA (Tan IIA), isolated from the Chinese medicinal herb Danshen, has been reported to have anticancer effects in several tumor models, while its effects on renal cell carcinoma have not been studied. In the present study, we evaluated the effects of Tan IIA on growth inhibition and apoptosis in the renal cancer cell line 786-O and its mechanism of action. Results of the MTT assay indicated that the treatment of 786-O cells with Tan IIA resulted in a concentration-dependent decrease in cell viability. Flow cytometry analysis revealed that Tan IIA treatment caused apoptosis following cell cycle perturbation. Furthermore, we examined the expression of cell cycle and apoptosis-related proteins using immunoblotting, which indicated an upregulation of p53, p21, bax and caspase-3 in Tan IIA-treated cells compared with Tan IIA-untreated cells. These results suggest that the activation of p53 and the upregulation of its target genes, including p21 and bax, may be involved in the mitochondrial apoptosis induced by Tan IIA in 786-O cells.

Adiponectin receptor expression in human malignant tissues.

Adiponectin has been proposed to be a mediator of obesity-associated malignancies and to have direct antineoplastic effects acting via adiponectin receptors AdipoR1 and AdipoR2. We describe herein the expression of AdipoR1 and AdipoR2 in several cancers not previously studied. We used immunohistochemistry to assess expression of adiponectin receptors in archival specimens of renal cell carcinoma (n = 64), hepatocellular carcinoma (n = 123), melanoma (n = 20), cholangiocarcinoma (n = 20), transitional cell carcinoma of the bladder (n = 24), ovarian epithelial carcinoma (n = 63), cervical squamous cell carcinoma (n = 49), and adrenocortical carcinoma (n = 48). To compare expression in malignant versus nonmalignant tissues, we also studied AdipoR1 and AdipoR2 expression in pairs of renal cell carcinoma and adjacent healthy kidney tissue specimens by immunohistochemistry. We also studied mRNA expression in 45 specimens of renal cell carcinoma by real-time polymerase chain reaction. Finally, we utilized Western blotting to confirm the presence of adiponectin receptors and subsequently studied cell signaling pathways of adiponectin in the renal cancer cell line 786-O. Cancers associated with obesity were significantly more likely to express AdipoR1 than cancers not associated with obesity. Of the specimens of renal cell carcinoma, which is strongly associated with obesity, 93.8% expressed AdipoR1 compared to 44.9% of the specimens of cervical cell carcinoma, which is not associated with obesity (p < 0.001). There was no difference in the expression of adiponectin receptors or their mRNA between malignant and benign kidney tissue specimens. Overall, there were no correlations between expression of adiponectin receptors or their mRNA and tumor prognostic factors. Finally, Western blotting confirmed the presence of AdipoR1 in the renal cancer cell line 786-O, and adiponectin activates in vitro several signaling pathways in this cell line. In summary, we report for the first time expression of AdipoR1 and AdipoR2 in the above cancers and that AdipoR1 is more ubiquitously expressed in obesity-associated cancers.

Von Hippel-Lindau Gene Product Modulates TIS11B Expression in Renal Cell Carcinoma

TIS11B belongs to a group of RNA-binding proteins (including TIS11/tristetraprolin and TIS11D) that share characteristic tandem CCCH-type zinc-finger domains and can be rapidly induced by multiple stimuli. TIS11B has been shown to regulate vascular endothelial growth factor (VEGF) mRNA stability in adrenocorticotropic hormone-stimulated primary adrenocortical cells. TIS11B has also been documented as a negative regulator of VEGF during development, but nothing has yet been reported in the context of human cancers. The Von Hippel-Lindau (VHL) tumor suppressor protein regulates VEGF gene expression at both the transcriptional and post-transcriptional levels in normoxia. However, whether it can do so in hypoxia is still unclear. Here, we report a unique regulatory function of VHL in VEGF expression in hypoxia that is mediated through modulation of TIS11B protein levels in renal cancer cells. In normoxia, we detected increased expression of the microRNA hsa-miR-29b in the VHL-overexpressing renal cancer cell line 786-O. We also show that this increased expression of hsa-miR-29b decreased TIS11B protein expression by post-transcriptional regulation in normoxia. In contrast, in hypoxia, increased TIS11B expression paralleled an increased TIS11B mRNA stability in VHL-overexpressing 786-O cells. This VHL-mediated TIS11B up-regulation in hypoxia may be important for TIS11B-regulated gene expression: we observed a down-regulation of VEGF mRNA in hypoxia in VHL-overexpressing cells compared with parental 786-O cells, and this effect was reversible by silencing TIS11B expression.

ABT-737 Induces Expression of the Death Receptor 5 and Sensitizes Human Cancer Cells to TRAIL-induced Apoptosis

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-kappaB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-kappaB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.