MicroRNA-421 confers paclitaxel resistance by binding to the KEAP1 3'UTR and predicts poor survival in non-small cell lung cancer.

MicroRNAs regulate post-transcriptional gene expression and play important roles in multiple cellular processes. In this study, we found that miR-421 suppresses kelch-like ECH-associated protein 1(KEAP1) expression by targeting its 3'-untranslated region (3'UTR). A Q-PCR assay demonstrated that miR-421 is overexpressed in non-small cell lung cancer (NSCLC), especially in A549 cells. Consistently, the level of miR-421 was higher in clinical blood samples from lung cancer patients than in those from normal healthy donors, suggesting that miR-421 is an important lung cancer biomarker. Interestingly, overexpression of miR-421 reduced the level of KEAP1 expression, which further promoted lung cancer cell migration and invasion, as well as inhibited cell apoptosis both in vivo and in vitro. Furthermore, knockdown of miR-421 expression with an antisense morpholino oligonucleotide (AMO) increased ROS levels and treatment sensitivity to paclitaxel in vitro and in vivo, indicating that high miR-421 expression may at least partly account for paclitaxel tolerance in lung cancer patients. To find the upstream regulator of miR-421, one of the candidates, ß-catenin, was knocked out via the CRISPR/Cas9 method in A549 cells. Our data showed that inhibiting ß-catenin reduced miR-421 levels in A549 cells. In addition, ß-catenin upregulation enhanced miR-421 expression, indicating that ß-catenin regulates the expression of miR-421 in lung cancer. Taken together, our findings reveal the critical role of miR-421 in paclitaxel drug resistance and its upstream and downstream regulatory mechanisms. Therefore, miR-421 may serve as a potential molecular therapeutic target in lung cancer, and AMOs may be a potential treatment strategy.

Identification of mitoxantrone as a new inhibitor of ROS1 fusion protein in non-small cell lung cancer cells.

Mitoxantrone, an FDA-approved drug for multiple sclerosis and hormone refractory prostate cancer, is identified as a potent inhibitor of ROS1 fusion protein by in silico screening in non-small cell lung cancer cells. Mitoxantrone can suppress the phosphorylation of ROS1 and subsequently inhibit its downstream signaling pathway and thus induce cell apoptosis.

Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor.

BACKGROUND: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southern China. Although there are conventional therapies, the side effects and toxicities are not always tolerable for patients. Recently, the tumoricidal effect of ginsenosides on different cancer cells has been studied. This study aims to investigate the anti-cancer effect of ginsenosides on NPC cells and their underlying mechanism. METHODS: The cytotoxicity of ginsenosides on NPC cell line HK-1 was measured by MTT assay. Apoptosis was detected by propidium iodide staining followed by flow cytometry. A xenograft tumor model was established by injecting nude mice with HK-1 cells. The activation of caspases and apoptosis-inducing factor (AIF) were evaluated by Western blot analysis. Nuclear translocation of AIF was also studied by immunofluorescence staining. Mitochondrial membrane potential was measured by JC-1 dye using flow cytometry. RESULTS: Four ginsenosides, 20 (S)-Rh2, compound K (CK), panaxadiol (PD) and protopanaxadiol (PPD), induced apoptotic cell death in HK-1 cells in a concentration-dependent manner. CK inhibited HK-1 xenograft tumor growth most extensively and depleted mitochondrial membrane potential depolarization and induced translocation of AIF from cytoplasm to nucleus in HK-1 cells. In addition, depletion of AIF by siRNA abolished CK-induced HK-1 cell death. CONCLUSION: Ginsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo.

14-3-3gamma is upregulated by in vitro ischemia and binds to protein kinase Raf in primary cultures of astrocytes.

The 14-3-3 protein family comprises critical regulatory molecules involved in signaling during cell division, proliferation, and apoptosis. Despite extensive study, the functions of the 14-3-3 proteins in brain remain unclear. 14-3-3gamma, a subtype of the 14-3-3 family of proteins, was thought to be brain- and neuron-specific. Using RNA arbitrarily primed PCR, we identified an upregulated cDNA fragment of the 14-3-3gamma gene in primary cultures of astrocytes. Using Northern blot analysis, we confirmed this fragment was brain-specific. In cultures of astrocytes, 14-3-3gamma genes and proteins were differentially expressed at different ages and the proteins were distributed only in the cytoplasm. These results indicated that 14-3-3gamma was not neuron-specific but also expressed in astrocytes. The function of this protein in brain is unclear. Northern and Western blot analyses demonstrated that 14-3-3gamma mRNA and protein were upregulated in cultured astrocytes in an anaerobic chamber-induced ischemia model. The induction of 14-3-3gamma proteins was neither suppressed by an MAP kinase inhibitor (U0126) nor a PI-3 kinase inhibitor (LY294002). These data indicated that induction of 14-3-3gamma might not involve PI-3 and MAP kinase-dependent pathways. Using coimmunoprecipitation, we demonstrated that endogenous 14-3-3gamma bound to c-Raf-1 and p-Raf 259. As Raf is one of the critical serine/threonine kinases controlling cell growth, differentiation, and death, the binding of 14-3-3gamma to Raf indicates the critical role of this protein in ischemia-induced apoptosis and the changes in signal transduction in astrocytes in culture.