Purpose: The RNA interference effect is an alternative to antisense DNA as an experimental method of downregulating 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.
1 There are at least three cxl-adrenoceptor subtypes, cxla, alb and ald, 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 ala. The total abundance of al-adrenoceptor mRNA in BPH samples was over six times that in non-BPH samples. This increase was mostly accounted for by ala, which was almost nine times as abundant in BPH samples as in non-BPH samples. The abundance of alb was almost the same between BPH and non-BPH samples, and the abundance of ald in BPH samples was about three times that in non-BPH samples. The ratio of the numbers of the subtype mRNAs, ala: alb: ald, 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 Lxladrenoceptor subtype mRNAs between BPH and non-BPH samples. atla and acld were clearly detected in the interstitium of the prostate, where axla was stained more intensely than Lxld, and the positive sites were primarily smooth muscle cells. In contrast, alb 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 Lxla subtype may be primarily responsible for the contraction of the prostate.
A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.
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.
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