Yasuomi Takagi

Selective Killing of Cancer Cells by Leaf Extract of Ashwagandha: Identification of a Tumor-Inhibitory Factor and the First Molecular Insights to Its Effect

Purpose: Ashwagandha is regarded as a wonder shrub of India and is commonly used in Ayurvedic medicine and health tonics that claim its variety of health-promoting effects. Surprisingly, these claims are not well supported by adequate studies, and the molecular mechanisms of its action remain largely unexplored to date. We undertook a study to identify and characterize the antitumor activity of the leaf extract of ashwagandha. Experimental Design: Selective tumor-inhibitory activity of the leaf extract (i-Extract) was identified by in vivo tumor formation assays in nude mice and by in vitro growth assays of normal and human transformed cells. To investigate the cellular targets of i-Extract, we adopted a gene silencing approach using a selected small hairpin RNA library and found that p53 is required for the killing activity of i-Extract. Results: By molecular analysis of p53 function in normal and a variety of tumor cells, we found that it is selectively activated in tumor cells, causing either their growth arrest or apoptosis. By fractionation, purification, and structural analysis of the i-Extract constituents, we have identified its p53-activating tumor-inhibiting factor as withanone. Conclusion: We provide the first molecular evidence that the leaf extract of ashwagandha selectively kills tumor cells and, thus, is a natural source for safe anticancer medicine.

Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection

Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Although it has been proposed that the relative thermodynamic stability at the 5′-ends of siRNAs plays a crucial role in siRNA strand selection, we demonstrate here that a character of the 2-nt 3′-overhang of siRNAs is the predominant determinant of which strand participates in the RNAi pathway. We show that siRNAs with a unilateral 2-nt 3′-overhang on the antisense strand are more effective than siRNAs with 3′-overhangs at both ends, due to preferential loading of the antisense strand into the RNA-induced silencing complex (RISC). Regardless of the relative thermodynamic stabilities at the ends of siRNAs, overhang-containing strands are predominantly selected as the guide strand; whereas, relative stability markedly influences opposite strand selection. Moreover, we show that sense strand modifications, such as deletions or DNA substitutions, of siRNAs with unilateral overhang on the antisense strand have no negative effect on the antisense strand selection, but may improve RNAi potency. Our findings provide useful guidelines for the design of potent siRNAs and contribute to understanding the crucial factors in determining strand selection in mammalian cells.

Cutting Edge: Lentiviral Short Hairpin RNA Silencing of PTEN in Human Mast Cells Reveals Constitutive Signals That Promote Cytokine Secretion and Cell Survival

Engagement of the FcεRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3′ position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34+ peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcεRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcεRI- responsiveness.

Differences among mechanisms of ribozyme-catalyzed reactions.

The catalytic properties of ribozymes depend on the sophisticated structures of the respective ribozyme-substrate complexes. Although it has been suggested that ribozyme-mediated cleavage of RNA occurs via a rather strictly defined mechanism, recent findings have clearly demonstrated the diversity of reaction mechanisms.

TEMPERATURE-DEPENDENT CHANGE IN THE RATE-DETERMINING STEP IN A REACTION CATALYZED BY A HAMMERHEAD RIBOZYME

To characterize the reaction catalyzed by a hammerhead ribozyme, the dependence on temperature of the reaction was examined. An Arrhenius plot revealed a transition that indicated a temperature‐dependent change in the activation energy at around 25°C. Thermodynamic parameters of the reaction were estimated at 10 and 35°C. The analyses led to the following conclusions. At 25–50°C. the chemical cleavage step (k cleav was the rate‐determining step, and the cleaved fragments dissociated from the ribozyme at a higher rate than the rate of the chemical reaction. When the temperature was below 25°C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate‐determining step. Above 50°C, the rate of the reaction decreased because, at such high temperatures, the formation of the Michaelis‐Menten complex (duplex formation) was hampered by thermal melting. A conformational change in the ribozyme‐substrate complex was not the rate‐determining step at any of the temperatures examined.

Similarity and Dissimilarity between Mg 2+ -Mediated and NH 4 + -Mediated Reactions Catalyzed by Hammerhead Ribozyme

In hammerhead ribozyme reactions, it was reported that a hammerhead ribozyme reaction proceeds in the presence of high concentrated NH4 ions. We performed an experiment on a solvent isotope effect of the ammonia-mediated hammerhead ribozyme reaction, resulting in the intrinsic isotope effect to be ∼2 (apparent isotope effect: ∼7.7; Figure 1). This result indicates the existence of transfer of a proton in the transition state of the ammonia-mediated ribozyme reaction. We previously reported that a Mg2+ ion might act as a Lewis acid catalyst at the leaving 5′-oxygen in the transition state of a hammerhead ribozyme reaction on the basis of the experiment of the solvent isotope effect.1 Thus, it strongly suggests the different catalysts between the magnesiummediated hammerhead ribozyme reaction and the ammonia-mediated.