Junichi Yano

Antitumor Activity of Small Interfering RNA/Cationic Liposome Complex in Mouse Models of Cancer

Purpose: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel cationic liposome, LIC-101. Experimental Design: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. Results: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. Conclusions: By combining siRNA with our cationic liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.

Quantification and distribution of α1‐adrenoceptor subtype mRNAs in human prostate: comparison of benign hypertrophied tissue and non‐hypertrophied tissue

1 There are at least three α 1‐adrenoceptor subtypes, α1a, α1b and α1d, in human tissues. Using an RNase protection assay, we have now determined the amount of each subtype mRNA in human prostatic tissue, for both benign prostatic hypertrophy (BPH) and non‐BPH. In all tissue samples examined, the predominant subtype mRNA was α1a. The total abundance of α1‐adrenoceptor mRNA in BPH samples was over six times that in non‐BPH samples. This increase was mostly accounted for by α1a, which was almost nine times as abundant in BPH samples as in non‐BPH samples. The abundance of α1b was almost the same between BPH and non‐BPH samples, and the abundance of α1d in BPH samples was about three times that in non‐BPH samples. The ratio of the numbers of the subtype mRNAs, α1a:α1b:α1d, was 85:1:14 in BPH samples and 63:6:31 in non‐BPH samples. 2 In situ hybridization studies showed no significant differences in the tissue localization of α1‐adrenoceptor subtype mRNAs between BPH and non‐BPH samples, α1a and α1d were clearly detected in the interstitium of the prostate, where α1a was stained more intensely than α1d, and the positive sites were primarily smooth muscle cells. In contrast, α1b staining was very faint. 3 This increase in mRNA abundance may be directly related to the contraction of prostatic tissue that leads to obstruction of the urinary tract in BPH patients. Specifically, our data suggest that increased expression of the α1a subtype may be primarily responsible for the contraction of the prostate.

Cloning, functional expression and tissue distribution of human α1C-adrenoceptor splice variants

We report the cloning and characterization of two isoforms of human α 1C‐adrenoceptor cDNA (α 1C‐2, α C‐3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (α 1C‐1) in their length and sequences of the C‐terminal domain. Tissue distribution of mRNAs showed that these variants co‐express with α 1C‐1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Quantification and distribution of α1‐adrenoceptor subtype mRNAs in human proximal urethra

We performed RNase protection assays and in situ hybridization to investigate the ratio of the three α1‐adrenoceptor subtype mRNAs, α1a, α1b and α1d, in human proximal urethra, and their localization in urethral cross‐sections. As revealed by the RNase protection assays, α1a was the predominant subtype mRNA in both male and female urethral samples. α1d mRNA was detected only in the female sample, and α1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, α1a : α1b : α1d, was 100 : 0 : 0 in the male urethra and 90 : 0 : 10 in the female urethra. In situ hybridization studies showed no significant differences in the cross‐sectional distribution of α1‐adrenoceptor subtype mRNAs between male and female urethras. Intense α1a staining was observed in the smooth muscle of the urethra, but α1b and α1d staining was much less intense. Of the three cloned α1 subtypes, α1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective α1 drugs, α1‐selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders.

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2′-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-μmol scale corresponds to 1 mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100 nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.

Tumor regression in mice by delivery of Bcl-2 small interfering RNA with pegylated cationic liposomes.

The pharmacokinetics and antitumor activity of pegylated small interfering RNA (siRNA)/cationic liposome complexes were studied after systemic administration to mice. We designed pegylated-lipid carriers for achieving increased plasma concentrations of RNA and hence improved accumulation of RNA in tumors by the enhanced permeability and retention effect. We compared the pharmacokinetics of siRNA complexed with liposomes incorporating pegylated lipids with longer (C-17 or C-18), shorter (C-12 to C-16), or unsaturated (C-18:1) acyl chains. When longer acyl chains were used, the plasma concentrations of siRNA obtained were dramatically higher than when shorter or unsaturated chains were used. This may be explained by the higher gel-to-liquid-crystalline phase-transition temperature (Tc) of lipids with longer acyl chains, which may form more rigid liposomes with reduced uptake by the liver. We tested a siRNA that is sequence specific for the antiapoptotic bcl-2 mRNA complexed with a pegylated liposome incorporating a C-18 lipid (PEG-LIC) by i.v. administration in a mouse model of human prostate cancer. Three-fold higher accumulation of RNA in the tumors was achieved when PEG-LIC rather than nonpegylated liposomes was used, and sequence-specific antitumor activity was observed. Our siRNA/PEG-LIC complex showed no side effects on repeated administration and the strength of its antitumor activity may be attributed to its high uptake by the tumors. Pegylation of liposomes improved the plasma retention, uptake by s.c. tumors, and antitumor activity of the encapsulated siRNA. PEG-LIC is a promising candidate for siRNA cancer therapy.

Signalling mechanisms in sphingosine 1‐phosphate‐promoted mesangial cell proliferation

Background: The bioactive sphingolipid sphingosine 1‐phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet‐derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P‐induced proliferation of mesangial cells is poorly known.

Therapeutic Effects of MicroRNA-582-5p and -3p on the Inhibition of Bladder Cancer Progression

Many reports have indicated that the abnormal expression of microRNAs (miRNAs) is associated with the progression of disease and have identified miRNAs as attractive targets for therapeutic intervention. However, the bifunctional mechanisms of miRNA guide and passenger strands in RNA interference (RNAi) therapy have not yet been clarified. Here, we show that miRNA (miR)-582-5p and -3p, which are strongly decreased in high-grade bladder cancer clinical samples, regulate tumor progression in vitro and in vivo. Significantly, the overexpression of miR-582-5p or -3p reduced the proliferation and invasion of UM-UC-3 human bladder cancer cells. Furthermore, transurethral injections of synthetic miR-582 molecule suppressed tumor growth and metastasis in an animal model of bladder cancer. Most interestingly, our study revealed that both strands of miR-582-5p and -3p suppressed the expression of the same set of target genes such as protein geranylgeranyltransferase type I beta subunit (PGGT1B), leucine-rich repeat kinase 2 (LRRK2) and DIX domain containing 1 (DIXDC1). Knockdown of these genes using small interfering RNA (siRNA) resulted in the inhibition of cell growth and invasiveness of UM-UC-3. These findings uncover the unique regulatory pathway involving tumor suppression by both strands of a single miRNA that is a potential therapeutic target in the treatment of invasive bladder cancer.

Quantification and distribution of α1‐adrenoceptor subtype mRNAs in human vas deferens: comparison with those of epididymal and pelvic portions

1 This study was intended to quantify the amounts of the α1‐adrenoceptor subtype mRNAs in human vas deferens, and demonstrate the receptor subtype responsible for the vas contraction. 2 The RNase protection assay showed that the mean total amount of α1a mRNA was 7.4±2.2 pg/5 μg of poly (A)+ RNA (97.0% of the total α1 mRNA) in the epididymal portion (E‐vas) and 4.9±0.8 pg/5 μg of poly (A)+ RNA (96.3% of the total) in the pelvic portion (P‐vas). The E‐vas showed a tendency to have a greater α1a mRNA abundance than the P‐vas (P=0.11). The α1b and α1d mRNAs were absent or of extremely low abundance. 3 By an in situ hybridization, the α1a and α1d mRNAs were recognized in the smooth muscle cells of the E‐vas and the P‐vas, and the distribution pattern the same in both tissues. The α1b mRNA positive site was scarcely detectable in both vas portions. 4 In a functional study, l‐phenylephrine produced concentration‐dependent contraction in the E‐vas (Emax=2.24±0.70 g; pD2=5.32±0.09) and the P‐vas (Emax=2.46±0.46 g; pD2=5.07±0.12). KMD‐3213, a novel α1A‐adrenoceptor‐selective antagonist, caused parallel rightward shifts of the concentration–response curves for l‐phenylephrine. Apparent pKB values were 9.90±0.16 for the E‐vas and 9.71±0.17 for the P‐vas. There was no significant difference in Emax, pD2 or pKB estimates between the two portions. 5 We have found that α1a mRNA is predominant in the human vas deferens, and confirmed that contraction of this organ is mediated by the α1A‐adrenoceptor.

Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents

Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2′-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5′-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5′-cap and a 3′-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications.