CCT020312 exerts anti-prostate cancer effect by inducing G1 cell cycle arrest, apoptosis and autophagy through activation of PERK/eIF2α/ATF4/CHOP signaling.

PERK/eIF2α/ATF4/CHOP signaling pathway is one of three major branches of unfolded protein response (UPR) and has been implicated in tumor progression. CCT020312 is a selective PERK activator and may have a potential anti-tumor effect. Here we investigated the anti-prostate cancer effect and its underlying mechanism of CCT020312. Our results showed that CCT020312 inhibited prostate cancer cell viability by inducing cell cycle arrest, apoptosis and autophagy through activation of PERK/eIF2α/ATF4/CHOP signaling. CCT020312 treatment caused cell cycle arrest at G1 phase and increased the levels of cleaved-Caspase3, cleaved-PARP and Bax in prostate cancer C4-2 and LNCaP cells. Moreover, CCT020312 increased LC3II/I, Atg12-Atg5 and Beclin1 levels and induced autophagosome formation. Furthermore, knockdown of CHOP reversed CCT020312-induced cell viability decrease, apoptosis and autophagy. Bafilomycin A1 reversed CCT020312-induced cell viability decrease but had no effect on CCT020312-induced CHOP activation in C4-2 and LNCaP cells. In vivo, CCT020312 suppressed tumor growth in C4-2 cells-derived xenograft mouse model, activated PERK pathway, and induced autophagy and apoptosis. Our study illustrates that CCT020312 exerts an anti-tumor effect in prostate cancer via activating the PERK pathway, thus indicating that CCT020312 may be a potential drug for prostate cancer.

Targeting Neuregulin 1 (NRG1): A Novel Biomarker for Non-Small-Cell Lung Cancer.

The aim of this study was to identify the roles of neuregulin 1 (NRG1) during the tumor progression in non-small-cell lung cancer (NSCLC). NSCLC patients with lung squamous cell carcinoma and lung adenocarci-noma were enrolled in this study. The expression of NRG1, vascular endothelial growth factor (VEGF) and surviving in clinical specimens was examined using immunohistochemistry analysis. The cytokine production in plasma was evaluated by ELISA. The levels of NRG1-associated molecules were determined using western blotting. The proliferation and apoptosis of cells with NRG1 knockdown were accessed by CCK-8 assay and flow cytometry. Upregulation of NRG1 as well as tumor-associated angiogenesis markers VEGF and survivin was detected in tissue and serum samples of NSCLC patients compared with the control. Furthermore, positive correlation with NSCLC levels and VEGF/survivin was also found in NSCLC specimens. In addition, upregulation of NRG1, VEGF and survivin was associated with poor overall survival in NSCLC patients. Moreover, enhanced production of NRG1 was detected in serum samples from NSCLC patients compared with healthy donors, and ROC analysis revealed the importance of NRG1 levels on distinguishing NSCLC samples and the controls. These findings suggested the novel diagnostic value of NRG1 in NSCLC. Additionally, upregulated protein levels of NRG1 and its target genes were also found in tissues samples of NSCLC patients compared with normal controls. These data indicated that NRG1 was a promising marker NSCLC, and it could be involved in tumor progression by targeting its downstream target including ErbB-Akt axis. Furthermore, the growth of lung cancer cells was suppressed by the knockdown of NRG1. Our findings could provide guidance for more accurate diagnosis for NSCLC, and future therapeutic approaches might be developed by better understanding of NRG-1-modulated molecular mechanisms during the tumor development in NSCLC.

Construction of an artificial intercellular communication network using the nitric oxide signaling elements in mammalian cells.

To increase the functionality of synthetic genetic circuits for programming cell populations and coordinating behavior across a population, we developed and analyzed an artificial cell-to-cell communication system in mammalian cells using nitric oxide signaling elements by integrating nitric oxide synthesis with the c-fos promoter, whose transcription activity could be triggered by the nitric oxide pathway. In the system, engineered 'sender' cells synthesized the intercellular messenger nitric oxide, which diffused into the environment and activated the c-fos promoter, and subsequently, green fluorescence protein (GFP) reporter expression in nearby engineered 'receiver' cells. Next, the sender module was integrated into the receivers under positive-feedback regulation, resulting in population density-dependent GFP expression in a quorum-sensing pattern. This artificial cell-to-cell communication system in mammalian cells could serve as a versatile tool for regulated gene expression and as building blocks for complex artificial gene regulatory networks for applications in gene therapy, tissue engineering, and biotechnology.

[Construction and identification of synchronous nitric oxide-amplified gene circuits controlled by radiation].

BACKGROUND: It has been proven that the positive feedback gene circuits can increase the expression level of interested genes, and the synchronization of genetic circuits can further enhance the efficacy of gene therapy. In order to obtain an enhanced and prolonged gene expression in target cells, a radiation controlled positive feedback genetic circuit is constructed via linking the c-fos promoter with the inducible nitric oxide synthase (iNOS) cDNA, which can be synchronized by nitric oxide (NO) intercellular messenger. Ultimately, the efficacy of radiogenetic therapy for cancer will be improved. METHODS: Using the gene recombination techniques, the vector pfos-iNOS/green fluorescent protein (GFP) was generated by cloning the radiation-responsive c-fos promoter into the plasmid vector pIRES2-EGFP to replace the primary CMV promoter, and then inserting human iNOS cDNA downstream of c-fos promoter in the vector pIRES2-EGFP. The constructed plasmids were then downloaded into A549 cells with lipofectamine. With exposure of various doses of ionizing radiation, outputs of GFP and iNOS in the treated cells were observed and analyzed. RESULTS: The interested plasmid was successfully constructed, proved by restriction enzyme digestion analysis. The outputs of GFP and iNOS in the transfected cells were markedly increased compared with the control cells after radiation, the peak level was seen in 16 hours after radiation. CONCLUSIONS: A positive feedback genetic circuit is successfully developed, composed by c-fos promoter and iNOS cDNA, which can be synchronized by secreting the intercellular messenger NO. This genetic circuit will be utilized in further study.