SummaryTumor necrosis factor alpha (TNF-α), 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-α by anti-TNF-α 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-α. The efficacies of various siRNA duplexes were tested against TNF-α mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-α by 55% (p<0.005) with a proportionate reduction in TNF-α 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 μg/kg body weight/day for two days) of liposome-encapsulated SSL3. The LPSinduced TNF-α secretion was inhibited by > 60% (p < 0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-α, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-α.

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.

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 19–21 base pairs 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 24 h, following which, TNF-α secretion was induced by exposure to LPS (0.1 ug/ml) for 2 h. 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 10 nM, 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 100 µg/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.

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. Earlier studies show that two daily doses (1.5 mg/kg body wt.) of an antisense oligonucleotide against rat TNF‐α mRNA, reduced lipopolysaccharide (LPS)‐induced liver injury by 60%. In this study, we introduce the new generation of gene‐silencing molecules, namely the short interfering RNA (siRNA), to reduce TNF‐α levels. Although siRNAs of 19‐21 base pairs are commonly used, it is reported that longer siRNAs may have much higher efficacies. In this study, we report the identification of a 27‐mer anti‐TNF‐α siRNA and compare its properties with a recently identified 21‐mer anti‐TNF‐α siRNA, SSL3. Primary cells of rat Kupffer cells were transfected with of three 27‐mer siRNA constructs (si27‐1, si27‐2 and si27‐3) for 24 h, following which, TNF‐α secretion was induced by exposure to LPS (0.1 ug/ml) for 2 h. TNF‐α released to the medium was measured by ELISA. Of the three si27 constructs two (si27‐2 and si27‐3) had profound inhibitory effect on TNF‐α secretion and the third had no effect at all. At 5 nM, si27‐3, almost totally (>97%) inhibited TNF‐α secretion compared to an 85% inhibition by the 21‐mer (SSL3); at 1 nM, si27‐3 was still inhibitory at 95% after 48 h whereas the inhibitory effect of SSL3 was lower at 75%. These data demonstrate the identification of a highly efficacious siRNA, which can be used in the treatment of TNF‐α mediated diseases (Supported by Grant AA 015081).

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