Knockdown of Ki-67 by dicer-substrate small interfering RNA sensitizes bladder cancer cells to curcumin-induced tumor inhibition.

Transitional cell carcinoma (TCC) of the urinary bladder is the most common cancer of the urinary tract. Most of the TCC cases are of the superficial type and are treated with transurethral resection (TUR). However, the recurrence rate is high and the current treatments have the drawback of inducing strong systemic toxicity or cause painful cystitis. Therefore, it would be of therapeutic value to develop novel concepts and identify novel drugs for the treatment of bladder cancer. Ki-67 is a large nucleolar phosphoprotein whose expression is tightly linked to cell proliferation, and curcumin, a phytochemical derived from the rhizome Curcuma longa, has been shown to possess powerful anticancer properties. In this study, we evaluated the combined efficacy of curcumin and a siRNA against Ki-67 mRNA (Ki-67-7) in rat (AY-27) and human (T-24) bladder cancer cells. The anticancer effects were assessed by the determination of cell viability, apoptosis and cell cycle analysis. Ki-67-7 (10 nM) and curcumin (10 µM), when treated independently, were moderately effective. However, in their combined presence, proliferation of bladder cancer cells was profoundly (>85%) inhibited; the rate of apoptosis in the combined presence of curcumin and Ki-67-7 (36%) was greater than that due to Ki-67-7 (14%) or curcumin (13%) alone. A similar synergy between curcumin and Ki-67-7 in inducing cell cycle arrest was also observed. Western blot analysis suggested that pretreatment with Ki-67-7 sensitized bladder cancer cells to curcumin-mediated apoptosis and cell cycle arrest by p53- and p21-independent mechanisms. These data suggest that a combination of anti-Ki-67 siRNA and curcumin could be a viable treatment against the proliferation of bladder cancer cells.

Dicer-substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro: in vivo targeting of Kupffer cells by siRNA-liposomes.

Tumor necrosis factor alpha (TNF-α) plays a major role in the pathogenesis of many inflammatory diseases. Neutralizing TNF-α by antibodies or antisense oligodeoxynucleotides, alleviate disease symptoms. In this study, we introduce the new generation of gene-silencing molecules, namely the small interfering RNAs (siRNAs) to reduce TNF-α. Although siRNAs of 19-21bp are commonly used, it is reported that longer siRNAs have much higher efficacies. Here, we report the identification of a 27-mer Dicer-substrate siRNA (DsiRNA) against TNF-α mRNA. Primary cells of rat Kupffer cells were transfected with five 27-mer siRNA constructs (si27-1, si27-2 si27-3, si27-4 and si27-5) for 24h, following which, TNF-α secretion was induced by exposure to LPS (0.1µg/ml) for 2h. TNF-α released to the medium was measured by ELISA. Of the five si27 constructs, si27-3 had the highest inhibitory effect on TNF-α secretion. At 10nM, si27-3 inhibited TNF-α secretion by 80% compared to a 60% inhibition by a 21-mer (SSL3). Following encapsulation in anionic liposomes, si27-3 at 100µg/kg body weight, on two successive days by intravenous administration, inhibited the secretion of TNF-α by 50%. These data demonstrate the identification of a highly efficacious siRNA formulation, which can be used in the treatment of TNF-α mediated diseases.

Inhibition of tumor necrosis factor alpha secretion in rat Kupffer cells by siRNA: in vivo efficacy of siRNA-liposomes.

Tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease. In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-alpha by anti-TNF-alpha antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-alpha. The efficacies of various siRNA duplexes were tested against TNF-alpha mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-alpha by 55% (p<0.005) with a proportionate reduction in TNF-alpha mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 microg/kg body weight/day for 2 days) of liposome-encapsulated SSL3. The LPS-induced TNF-alpha secretion was inhibited by >60% (p<0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-alpha, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-alpha.