Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Inhibition of dihydroorotate dehydrogenase by doxorubicin and brequinar sensitize cancer cells to TRAIL-induced apoptosis

ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy is not especially successful due to the fact that many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL on cancer cells, but the exact mechanisms of actions are poorly understood. In this study we performed high-throughput siRNA screening and genome-wide gene expression profiling on doxorubicin treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified to be a potential candidate. DHODH is rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin and silencing of DHODH sensitized U1690 cells to TRAIL. Inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis, and was accompanied by downregulation of cFLIPL and mitochondrial depolarization. However, the expressions of DR4 and 5 were not changed in response to brequinar treatment in contrast to doxorubicin. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initialized by both brequinar and doxorubicin. Furthermore, other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel drug, brequinar, to be potentially utilized in TRAIL combinatorial cancer therapy and highlighted for the first time the importance of DHODH and pyrimidine pathway in mediating TRAIL sensitization in cancer cells. Total RNA obtained from small cell lung cancer U1690 cells either treated with DMSO control or 1 M-BM-5M doxorubicin for 12 or 24h.

ORGANISM(S): Homo sapiens

SUBMITTER: Tao He

PROVIDER: E-GEOD-42531 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Senescent cells secrete a plethora of factors with potent paracrine signaling capacity. Strikingly, senescence, which acts as a defense against cell transformation, exerts pro-tumorigenic activities through its secretome by promoting numerous tumor-specific features, such as cellular proliferation, epithelial-mesenchymal transition and invasiveness. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the unique activity of activating cell death exclusively in tumor cells. Given that the senescence-associated secretome supports cell transformation, we asked whether factor(s) of this secretome would establish a program required for the acquisition of TRAIL sensitivity. We found that conditioned media from several types of senescent cells (CMS) efficiently sensitized pre-transformed cells to TRAIL, while the same was not observed with normal or immortalized cells. Dynamic transcription profiling analysis of CMS-exposed pre-transformed cells revealed paracrine autoregulatory loop of senescence-associated secretome factors and a dominant role of CMS-induced MYC. Sensitization to TRAIL coincided with MYC upregulation and massive changes in gene regulation. CMS-induced MYC silenced its target gene CFLAR, encoding the apoptosis inhibitor FLIPL, thus leading to the acquisition of TRAIL sensitivity. Altogether, our results reveal that senescent cell-secreted factors exert a TRAIL sensitizing effect on pre-transformed cells by modulating the expression of MYC and CFLAR. Notably, CMS dose-dependent sensitization to TRAIL was observed with TRAIL-insensitive cancer cells and confirmed in co-culture experiments. Dissection and characterization of TRAIL-sensitizing CMS factors and the associated signaling pathway(s) may provide a mechanistic insight in the acquisition of TRAIL sensitivity and lead to novel concepts for the apoptogenic therapy of pre-malignant and TRAIL-resistant tumors. Pre-transformed BJEL cells were incubated with CMS for 0, 1, 3, 6, 8, 16 or 24 h respectively. Total RNA has been extracted from each time point and used for gene expression analysis (Affymetrix Human Gene 1.0 ST Arrays).

Project description:Neuroblastoma is an embryonal tumor which originates from neural crest progenitor cells that fail to differentiate along their predefined route to sympathetic neurons or sympatho-adrenergic adrenal cells. It is the most common extracranial tumor of childhood and accounts for 15% of all childhood cancer deaths. Especially patients suffering from high grade or relapsed neuroblastoma have poor outcome in spite of aggressive treatment regimens including autologous stem cell transplantation. Those patients are in urgent need of additional effective therapies which demands the development of targeted approaches. Dihydroorotatedehydrogenase (DHODH) is the fourth enzyme of the pyrimidine synthesis pathway which oxidizes dihydroorotate to orotate. In recent past it became a potential drug target for cancer treatment because of its keyrole in processing essential pyrimidine nucleotides. On the basis of the existing data, functional inhibition of DHODH is considered to be promising therapeutic option for several tumor entities like advanced colorectal, breast or lung-cancers. Leflunomide is an established drug in treatment of the autoimmune diseases rheumatoid arthritis and multiple sclerosis. In the liver Leflunomide becomes converted to its active metabolite called Teriflunomide, which inhibits the activity of DHODH directly. In recent times Leflunomide is also used for therapy against the Cytomegalovirus and the BK virus. Also for Melanoma was shown recently a decreased growth rate due to Leflunomide treatment in a zebrafish and a mouse model. As Melanoma is a malignant tumor of the skin, which derives also from neural crest progenitor cells, a coherent investigation of effictivity of Leflunomide in neuroblastoma celllines showed first promising results. The aim of our study was to reanalyse the effectivity of Leflunomide in Neuroblastoma and to shed further light in its biological mode of action. Three+B52 biological samples of the human neuroblastoma cell line IMR32 were treated with DMSO or Teriflunomide [118 ￂﾵM]

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Inhibition of dihydroorotate dehydrogenase by doxorubicin and brequinar sensitize cancer cells to TRAIL-induced apoptosis

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