The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis.

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Identification of a novel cell-penetrating peptide targeting human glioblastoma cell lines as a cancer-homing transporter.

Cell-penetrating peptides (CPPs) as a novel biomedical delivery system have been highly anticipated, since they can translocate across biological membranes and are capable of transporting their cargo inside live cells with minimal invasiveness. However, non-selective internalization in various cell types remains a challenge in the clinical application of CPPs, especially in cancer treatment. In this study, we attempted to identify novel cancer-homing CPPs to target glioblastoma multiforme (GBM), which is often refractory and resistant to treatment. We screened for CPPs showing affinity for the human GBM cell line, U87MG, from an mRNA display random peptide library. One of the candidate peptides which amino-acid sequence was obtained from the screening showed selective cell-penetrating activity in U87MG cells. Conjugation of the p16(INK4a) functional peptide to the GBM-selective CPP induced cellular apoptosis and reduced phosphorylated retinoblastoma protein levels. This indicates that the CPP was capable of delivering a therapeutic molecule into U87MG cells inducing apoptosis. These results suggest that the novel CPP identified in this study permeates with high affinity into GBM cells, revealing it to be a promising imaging and therapeutic tool in the treatment of glioblastoma.

COPA and SLC4A4 are required for cellular entry of arginine-rich peptides.

Cell-penetrating peptides (CPPs) have gained attention as promising tools to enable the delivery of various molecules in a non-invasive manner. Among the CPPs, TAT and poly-arginine have been extensively utilized in numerous studies for the delivery of functional proteins, peptides, and macromolecules to analyze cellular signaling. However, the molecular mechanisms of cellular entry remain largely unknown. Here, we applied siRNA library screening to identify the regulatory genes for the cellular entry of poly-arginine peptide based on microscopic observation of the entry of fluorescent peptides in siRNA-treated cells. In this screening, we identified the cell membrane gene SLC4A4 and the trafficking regulator gene COPA, which also plays an important role in early endosome maturation. These results demonstrated that cellular entry of poly-arginine requires at least two different steps, probably binding on the cell surface and endosomal entry. The identification of genes for cellular entry of poly-arginine provides insights into its mechanisms and should further aid in the development of highly efficient cell-penetrating peptides.

Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems.

Cell-penetrating peptides have gained attention owing to their promise in noninvasive delivery systems. Among the identified cell-penetrating peptides, the TAT peptide has been preferentially used for transduction into cells of diverse origins. However, this activity is nonselective between neoplastic and non-neoplastic cells. Here we describe artificial cell-penetrating peptides that are selectively and efficiently incorporated into human tumour cells, according to their lineage. Ten representative tumour lineage-homing cell-penetrating peptides were obtained by screening of a random peptide library constructed using messenger RNA display technology, and some of the isolates were further modified by amino-acid substitution. Their advantageous tumour cell-targeting ability is corroborated in an in vivo mouse model for imaging and growth suppression of metastatic xenoplant tumours. These cell-penetrating peptides are potentially useful for the efficient targeting of human neoplasms in a tumour origin-dependent manner, and provide a framework for the development of peptide-based anti-tumour technologies.

Effects of hippuristanol, an inhibitor of eIF4A, on adult T-cell leukemia.

We evaluated the anti-adult T-cell leukemia (ATL) effects of hippuristanol, an eukaryotic translation initiation inhibitor from the coral Isis hippuris. Hippuristanol inhibited proliferation of HTLV-1-infected T-cell lines and ATL cells, but not normal peripheral blood mononuclear cells. It induced cell cycle arrest during G1 phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and induced apoptosis by reducing the expression of Bcl-x(L), c-IAP2, XIAP and c-FLIP. The induced apoptosis was associated with activation of caspase-3, -8 and -9. Hippuristanol also suppressed IkappaBalpha phosphorylation and depleted IKKalpha, IKKgamma, JunB and JunD, resulting in inactivation of NF-kappaB and AP-1. It also suppressed carbonic anhydrase type II expression. In addition to its in vitro effects, hippuristanol suppressed tumor growth in mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV-1-infected T cells. These preclinical data suggest that hippuristanol could be a potentially useful therapeutic agent for patients with ATL.