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Neuroblastoma is the most common extracranial solid childhood tumor. Despite the availability of advanced multimodal therapy, high-risk patients still have low survival rates. p21-activated kinase 4 (PAK4) has been shown to regulate many cellular processes in cancer cells, including migration, polarization and proliferation. However, the role of PAK4 in neuroblastoma remains unclear. In the present study, we demonstrated that PAK4 was overexpressed in neuroblastoma tissues and was correlated with tumor malignance and prognosis. To investigate the function of PAK4 in neuroblastoma, we used a small-molecule inhibitor that targets PAK4, that is, PF-3758309. Our results showed that PF-3758309 significantly induced cell cycle arrest at the G1 phase and apoptosis in neuroblastoma cell lines. Meanwhile, the inhibition of PAK4 by PF-3758309 increased the expression of CDKN1A, BAD and BAK1 and decreased the expression of Bcl-2 and Bax. In addition, we screened the target genes of PAK4 by PCR array and found that 23 genes were upregulated (including TP53I3, TBX3, EEF1A2, CDKN1A, IFNB1 and MAPK8IP2) and 20 genes were downregulated (including TNFSF8, Bcl2-A1, Bcl2L1, SOCS3, BIRC3 and NFKB1) after PAK4 inhibition by PF-3758309. Moreover, PAK4 was found to regulate the cell cycle and apoptosis via the ERK signaling pathway. In conclusion, the present study demonstrated, for the first time, the expression and function of PAK4 in neuroblastomas and the inhibitory effect of PF-3758309, which deserves further investigation as an alternative strategy for neuroblastoma treatment.

Decreased autophagy is accompanied by the development of a myeloproliferative state or acute myeloid leukemia (AML). AML cells are often sensitive to autophagy‑inducing stimuli, prompting the idea that targeting autophagy can be useful in AML cytotoxic therapy. AML NB4 cells overexpressing microtubule-associated protein 1 light chain 3-green fluorescent protein were screened with 69 inhibitors to analyze autophagy activity. AML cells were treated with the polo-like kinase 1 (PLK1) inhibitors RO3280 and BI2536 before autophagy analysis. Cleaved LC3 (LC3-II) and the phosphorylation of mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase, and Unc-51-like kinase 1 during autophagy was detected with western blotting. Autophagosomes were detected using transmission electron microscopy. Several inhibitors had promising autophagy inducer effects: BI2536, MLN0905, SK1-I, SBE13 HCL and RO3280. Moreover, these inhibitors all targeted PLK1. Autophagy activity was increased in the NB4 cells treated with RO3280 and BI2536. Inhibition of PLK1 expression in NB4, K562 and HL-60 leukemia cells with RNA interference increased LC3-II and autophagy activity. The phosphorylation of mTOR was reduced significantly in NB4 cells treated with RO3280 and BI2536, and was also reduced significantly when PLK1 expression was downregulated in the NB4, K562 and HL-60 cells. We demonstrate that PLK1 inhibition induces AML cell autophagy and that it results in mTOR dephosphorylation. These results may provide new insights into the molecular mechanism of PLK1 in regulating autophagy.

Background: Kawasaki disease (KD) is a self-limiting illness with acute systematic vascular inflammation. It causes pathological changes in mostly medium and small-sized arteries, especially the arteria coronaria, which adds the risk of developing coronary heart disease in adults. Materials and methods: We detected the miR-223-3p expression in 30 KD patients combined with 12 normal controls using miRNA microarrays and RT-PCR. A KD mouse model was constructed using Candida albicans water insoluble substance (CAWS). We also checked the miR-223-3p's expression using qRT-PCR. The Luciferase reporting system was implemented to validate the correlation between miR-223-3p and Interleukin-6 receptor subunit beta (IL-6ST). TNF-α was used to stimulate human coronary artery endothelial cells (HCAECs), and miR-223-3p activator or inhibitor and KD serum were used to treat HCAECs. A Western blotting automatic quantitative analysis protein imprinting system was used to test the expression of signal transducer and the activator of transcription 3 (STAT3), phosphorylated-signal transducer and the activator of transcription 3 (pSTAT3) and NF-κB p65. Results: Clinical trials found that miR-223-3p expressions were markedly different (more than 2-fold) between the acute KD group and the control group. E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) levels were also significantly higher (about 2-fold) in KD especially with coronary artery lesions. MiR-223-3p could alleviate vascular endothelial damage in KD mice, and IL-6 (Interleukin-6), E-selectin and ICAM-1 were simultaneously negative. The values of IL-6, E-selectin, and ICAM-1 mRNA expressions decreased, while the value of IL-6ST was increased between the agonist treated mice and KD mice. The RT-qPCR consequences displayed that miR-223-3p explored the highest expression on the third day in both the KD mice as well as the agonist group. MiR-223-3p can directly combine with IL-6ST 3' untranslatable regions (UTR) and held back the IL-6's expression. Overexpression of miR-223 down regulated IL6ST expression and decreased the expression of p-STAT3 and NF-κB p65, while the miR-223 inhibitor could reverse the above process. Conclusion: MiR-223-3p is an important regulatory factor of vascular endothelial damage in KD and could possibly become a potential target of KD treatment in the future.

Background Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Methods Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Results Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. Conclusions We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence. Electronic supplementary material The online version of this article (doi:10.1186/s12935-017-0405-y) contains supplementary material, which is available to authorized users.