Growth and neurite stimulating effects of the neonicotinoid pesticide clothianidin on human neuroblastoma SH-SY5Y cells.

Neonicotinoids are one of most widely used pesticides targeting nicotinic acetylcholine receptors (nAChRs) of insects. Recent epidemiological evidence revealed increasing amounts of neonicotinoids detected in human samples, raising the critical question of whether neonicotinoids affect human health. We investigated the effects of a neonicotinoid pesticide clothianidin (CTD) on human neuroblastoma SH-SY5Y cells as in vitro models of human neuronal cells. Cellular and functional effects of micromolar doses of CTD were evaluated by changes in cell growth, intracellular signaling activities and gene expression profiles. We examined further the effects of CTD on neuronal differentiation by measuring neurite outgrowth. Exposure to CTD (1-100 µM) significantly increased the number of cells within 24 h of culture. The nAChRs antagonists, mecamylamine and SR16584, inhibited this effect, suggesting human α3ß4 nAChRs could be targets of neonicotinoids. We observed a transient intracellular calcium influx and increased phosphorylation of extracellular signal-regulated kinase 1/2 shortly after exposure to CTD. Transcriptome analysis revealed that CTD down-regulated genes involved in neuronal function (e.g., formation of filopodia and calcium ion influx) and morphology (e.g., axon guidance signaling and cytoskeleton signaling); these changes were reflected by a finding of increased neurite length during neuronal differentiation. These findings provide novel insight into the potential risks of neonicotinoids to the human nervous system.

HIKESHI silencing can enhance mild hyperthermia sensitivity in human oral squamous cell carcinoma HSC­3 cells.

Hyperthermia (HT) is considered to be of value as a treatment modality in various cancers. However, the acquisition of thermotolerance in cancer cells due to the induction of heat shock proteins (HSPs) makes HT less effective. Recent findings have indicated that heat shock protein nuclear import factor hikeshi (HIKESHI), also referred to as C11orf73, acts as a nuclear import carrier of Hsp70 under heat stress conditions. The aim of the present study was to determine whether knockdown (KD) of HIKESHI by small interfering RNA (siRNA) can potentiate mild HT (MHT) sensitivity in human oral squamous cell carcinoma (OSCC) HSC­3 cells. The mRNA and protein expression of HIKESHI was found to be markedly suppressed in HSC­3 cells treated with siRNA for HIKESHI (siHIKE). Silencing HIKESHI significantly decreased the cell viability under MHT conditions (42˚C for 90 min). Immunocytochemical and western blot analyses clearly demonstrated that Hsp70 protein translocated from the cytoplasm to the nucleus under MHT conditions, and this translocation was significantly inhibited in cells treated with siHIKE. Treatment of the cells with MHT transiently increased the phosphorylation level of extracellular signal­regulated kinase (ERK)2. Furthermore, the phosphorylation was sustained in HIKESHI­KD cells under MHT conditions, and this sustained phosphorylation was abolished by pretreatment with U0126, an inhibitor of mitogen­activated protein kinase/ERK. In addition, U0126 significantly decreased the viability of cells treated with the combination of HIKESHI­KD and MHT. The data of the present study suggest that HIKESHI silencing enhanced the sensitivity of human OSCC HSC­3 cells to MHT.

Identification of genes and genetic networks associated with BAG3­dependent cell proliferation and cell survival in human cervical cancer HeLa cells.

Bcl­2­associated athanogene (BAG) 3, is a member of the BAG protein family and a known co­chaperone of heat shock protein (HSP) 70. BAG3 serves a role in regulating a variety of cellular functions, including cell growth, proliferation and cell death including apoptosis. BAG3 is a stress­inducible protein, however the constitutive expression level of BAG3 is increased in cancer cells compared with healthy cells. Recent proteomics technology combined with bioinformatics has revealed that BAG3 participates in an interactome with a number of proteins other than its typical partner HSP70. The functional types represented in the interactome included nucleic acid binding proteins and transcription factors, as well as chaperones, which indicated that overexpression of BAG3 may contribute to proliferation and cell survival through the alteration of gene transcription. While an increasing number of studies have addressed the function of BAG3 as a co­chaperone protein, BAG3­dependent alteration of gene transcription has not been studied extensively. The present study established two BAG3 knockout human cervical cancer HeLa cell clones and addressed the role of BAG3 in cell proliferation and survival through gene transcription, using DNA microarray­based transcriptome analysis and bioinformatics. The present study also identified two genetic networks associated with 'cellular growth and proliferation' and 'cell death and survival', which are dysregulated in the absence of BAG3, and may therefore be linked to BAG3 overexpression in cancer. These findings provide a molecular basis for understanding of BAG3­dependent cell proliferation and survival from the aspect of alteration of gene expression.