Ectopic expression of von Hippel-Lindau tumor suppressor induces apoptosis in 786-O renal cell carcinoma cells and regresses tumor growth of 786-O cells in nude mouse

The von Hippel-Lindau (VHL) is a known tumor suppressor that binds to alpha-subunits of hypoxia-inducible factors and induces ubiquitin-mediated degradation of the protein in an oxygen-dependent manner. VHL is also involved in the regulation of tumor angiogenesis, glycolysis, cell cycle regulation, and apoptosis. In the present study, we showed that ectopic expression of VHL induces apoptosis in renal cell carcinoma 786-O cells which contain only the mutant VHL, evidenced by TUNEL assay and DAPI staining. Furthermore, biochemical studies indicated that expression of VHL in 786-O cells results in both PARP and CPP32 cleavage, suggesting that VHL-induced apoptosis in 786-O cells is caspase dependent. Moreover, we also observed that apoptosis induced by ectopic VHL expression was associated with up-regulation of p27 as well as Bax, implicating the roles of these two proteins in VHL-induced apoptosis. The up-regulation of p27 and Bax by VHL was specific since we did not detect any changes in the level of other apoptotic factors including Fas and Bcl2 by the expression of VHL. We next examined the effect of VHL expression on the tumor growth of 786-O renal cell carcinoma cells in nude mouse. The results showed that injection of Ad.VHL adenovirus regresses the tumor growth of 786-O cells in nude mouse. The analysis by TUNEL assay as well as DAPI staining of 786-O tumors injected with Ad.VHL showed clear evidence of apoptosis. These results suggest that ectopic VHL expression induces apoptotic response in 786-O VHL mutant cells both in vitro and in vivo.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.     

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation

Background

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction.

Methodology/Principal Findings

Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing.

Conclusions/Significance

Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. these results suggest that Nutlin-3 might serve as a novel therapy for KS.Key words: Kaposi sarcoma (KS), nutlin-3, p53, cell cycle arrest, apoptosis, angiopoietin-2     

Differential gene expression in anticancer drug- and TRAIL-mediated apoptosis in renal cell carcinomas

Renal cell carcinomas (RCC) exhibit marked differences in susceptibility towards anticancer drug- and TRAIL-induced apoptosis. However, the underlying mechanisms determining apoptosis-sensitivity or -resistance are not well understood. The purpose of this study was to compare gene expression patterns induced by DNA-damage- and death receptor-induced apoptosis and to detect differentially expressed genes responsible for differences in apoptosis-susceptibility. Therefore, we performed a comparative cDNA-array analysis in an apoptosis-resistant and an apoptosis-sensitive RCC cell line. In the sensitive cell line an upregulation of multiple E2F1- and p53-inducible proapaptotic and cell-cycle regulating target genes by Topotecan as well as TRAIL was observed. Interestingly, several antiapoptotic NFκB-dependent target genes were also induced. In the resistant cell line, however, only a small number of E2F1-, p53- and NFκB-dependent target genes were differentially regulated. Conclusively, anticancer drug- as well as TRAIL-sensitivity go along with an upregulation of multiple proapoptotic genes. In contrast, the mechanisms of apoptosis-resistance are—at least in part—located upstream of gene induction and seem not to depend upon upregulation of de-novo-synthesized antiapoptotic genes. Conclusively, the proapoptotic stimuli are confronted with a cellular context which allows apoptosis to be conducted—in the sensitive cell line—or not—in the resistant cell line. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Sebastian Heikaus and Ercan Casliskan contributed equally to this work. This work was supported by the ’Deutsche Forschungsgemeinschaft (DFG).     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

TRAIL induces apoptosis and inflammatory gene expression in human endothelial cells

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes approximately 30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4',6'-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-kappaB, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL.     

Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.     

Protein kinase Cepsilon binds peripherin and induces its aggregation, which is accompanied by apoptosis of neuroblastoma cells

A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCepsilon induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCepsilon. PKCepsilon and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCepsilon. The interaction was specific for PKCepsilon since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCeta and -, which pulled down minute amounts. PKCepsilon interacts with vimentin through the same structures but does not induce its aggregation. When the PKCepsilon C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCepsilon through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCepsilon.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Tyrosine kinase inhibitor, methyl 2,5-dihydromethylcinnimate, induces PML nuclear body formation and apoptosis in tumor cells

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are the nuclear structure consisting of various proteins such as PML, SUMO-1, and p53. PML-NBs are implicated in the regulation of tumor suppression, antiviral responses, and apoptosis. In this study, we searched for bioactive metabolites that would promote the formation of PML-NBs in tumor cells. As a result, methyl 2,5-dihydromethylcinnimate (2,5-MeC), a tyrosine kinase inhibitor, enhanced expression and/or stability of PML proteins and induced PML-NB formation in p53 null H1299 cells established from non-small cell lung cancer (NSCLC) and wild-type p53-expressing U2OS cells derived from osteosarcoma. Furthermore, it enhanced apoptosis by exogenously expressed wild type p53 and the expression of p53-responsive genes, such as PUMA and p21, in H1299 cells. 2,5-MeC also activated endogenous p53 and induced apoptosis in U2OS cells. The results suggest that 2,5-MeC is likely to be a promising candidate drug for the clinical treatment of terminal cancer-expressing wild-type p53.     

HIV LTR-dependent expression of Bax selectively induces apoptosis in Tat-positive cells

HIV integrates into the host cell genome where it persists for the life of the cell. One approach to reducing viral burden is to selectively eliminate cells containing integrated provirus early following infection. We have used the HIV LTR promoter to selectively express transgenes in human cells positive for the HIV transactivator protein Tat. Transient transfection of Jurkat cells, or Jurkat cells stably expressing Tat (Jurkat-Tat), with a LTR construct containing luciferase reporter gene resulted in a 37-fold increase in gene expression when Tat was present. We have demonstrated that when pro-apoptotic Bax was used as the transgene, cytotoxicity was seen only in the Jurkat-Tat cells. Annexin-V staining indicated that Bax induced cell death by apoptosis. In mixed populations of Jurkat and Jurkat-Tat cells, the LTR-Bax construct was selectively cytotoxic to the Tat-positive cells. These results suggest that Bax under the control of the HIV LTR can be used to destroy cells harbouring HIV without affecting uninfected cells.     

Proline dehydrogenase (oxidase), a mitochondrial tumor suppressor, and autophagy under the hypoxia microenvironment

Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the pathway of proline catabolism, has been identified as a mitochondrial, metabolic tumor suppressor, which is downregulated in a variety of human tumors. However, our recent findings show that PRODH/POX is upregulated by hypoxia in vitro and in vivo. The combination of low glucose and hypoxia produces additive effects on PRODH/POX expression. Both hypoxia and glucose depletion enhance PRODH/POX expression through AMP-activated protein kinase (AMPK) activation to promote tumor cell survival. Nevertheless, the mechanisms underlying PRODH/POX prosurvival functions are different for hypoxia and low-glucose conditions. Glucose depletion with or without hypoxia elevates PRODH/POX and proline utilization to supply ATP for cellular energy needs. Interestingly, under hypoxia PRODH/POX induces protective autophagy by generating reactive oxygen species (ROS). AMPK is the main initiator of stress-triggered autophagy. Thus, PRODH/POX acts as a downstream effector of AMPK in the activation of autophagy under hypoxia. This regulation was confirmed to be independent of the mechanistic target of rapamycin (MTOR) pathway, a major downstream target of AMPK signaling.     

Silencing glypican-3 expression induces apoptosis in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common internal malignant tumors. Glypican-3 (GPC3) is involved in the biological and molecular events in the tumorigenesis of HCC. We used RNA interference to evaluate the molecular effects of GPC3 suppression at the translational level and demonstrated for the first time that GPC3 silencing results in a significant elevation of the Bax/Bcl-2 ratio, the release of cytochrome c from mitochondria and the activation of caspase-3. The results suggest that GPC3 regulates cell proliferation by enhancing the resistance to apoptosis through the dysfunction of the Bax/Bcl-2/cytochrome c/caspase-3 signaling pathway and therefore plays a critical role in the tumorigenesis of HCC. Thus, the knockdown of GPC3 should be further investigated as an attractive novel approach for the targeted gene therapy of HCC.     

Proline dehydrogenase (oxidase), a mitochondrial tumor suppressor,and autophagy under the hypoxia microenvironment

Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the pathway of proline catabolism, has been identified as a mitochondrial, metabolic tumor suppressor, which is downregulated in a variety of human tumors. However, our recent findings show that PRODH/POX is upregulated by hypoxia in vitro and in vivo. The combination of low glucose and hypoxia produces additive effects on PRODH/POX expression. Both hypoxia and glucose depletion enhance PRODH/POX expression through AMP-activated protein kinase (AMPK) activation to promote tumor cell survival. Nevertheless, the mechanisms underlying PRODH/POX prosurvival functions are different for hypoxia and low-glucose conditions. Glucose depletion with or without hypoxia elevates PRODH/POX and proline utilization to supply ATP for cellular energy needs. Interestingly, under hypoxia PRODH/POX induces protective autophagy by generating reactive oxygen species (ROS). AMPK is the main initiator of stress-triggered autophagy. Thus, PRODH/POX acts as a downstream effector of AMPK in the activation of autophagy under hypoxia. This regulation was confirmed to be independent of the mechanistic target of rapamycin (MTOR) pathway, a major downstream target of AMPK signaling.     

Dinitro-o-cresol induces apoptosis-like cell death but not alternative oxidase expression in soybean cells

In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.