Motoki Takagi

Enhanced Growth Inhibition of Hepatic Multicellular Tumor Spheroids by Lactosylated Poly(ethylene glycol)‐siRNA Conjugate Formulated in PEGylated Polyplexes

PEGylated polyplexes (lac‐PEGylated polyplexes) composed of poly(L‐lysine) and lactosylated poly(ethylene glycol)‐small interfering RNA conjugate, which inhibits the RecQL1 gene product, were revealed to show an appreciable growth inhibition of multicellular HuH‐7 spheroids (human hepatocarcinoma cell lines) for up to 21 days (IC50=6 nM); this system used as an in vitro three‐dimensional (3D) model mimicking the in vivo biology of tumors. The PEGylated polyplexes thus prepared had a size of approximately 110 nm with clustered lactose moieties on their periphery as targeting ligands for the asialoglycoprotein‐receptor‐expressing HuH‐7 cells. In contrast, OligofectAMINE/siRNA (cationic lipoplex) was observed to have almost no growth‐inhibitory effect against HuH‐7 spheroids, even though the lipoplex showed a stronger growth‐inhibitory effect than the lac‐PEGylated polyplexes on conventional monolayer‐cultured HuH‐7 cells. The FITC‐tagged conjugate in the lac‐PEGylated polyplexes showed smooth penetration into the HuH‐7 spheroids compared with that in the lipoplexes, as observed by confocal fluorescence‐scanning microscopy. This indicates that the small size of approximately 100 nm and the reduced nonspecific interaction due to the nonionic and hydrophilic lactosylated PEG layer contributes to the smooth penetration of the PEGylated polyplexes into the spheroid interior, eventually facilitating their uptake into the cells composing the spheroids. Cellular apoptosis indicating programmed cell death was also observed in the HuH‐7 spheroids treated with the PEGylated polyplexes, revealing that the observed growth inhibition was indeed induced by the RNAi of the RecQL1 siRNA. These data suggest that the smart PEGylated polyplexes can indeed penetrate into the multiple cell layers of 3D tumor masses in vivo, exerting therapeutic effects through the RNAi.

Anticancer activity of RecQL1 helicase siRNA in mouse xenograft models.

Small interfering RNAs (siRNAs) are expected to have a medical application in human therapy as drugs with a high specificity for their molecular target mRNAs. RecQL1 DNA helicase in the human RecQ helicase family participates in DNA repair and recombination pathways in the cell cycle of replication. Silencing the RecQL1 expression by RecQL1‐siRNA induces mitotic death in vitro specifically in growing cancer cells. By contrast, the same RecQL1 silencing does not affect the growth of normal cells, emphasizing that RecQL1 helicase is an ideal molecular target for cancer therapy. In this study, we show that local and systemic administration of RecQL1‐siRNA mixed with polyethyleneimine polymer or cationic liposomes prevented cancer cell proliferation in vivo in mouse models of cancer without noticeable adverse effects. The results indicate that RecQL1‐siRNA in a complex with a cationic polymer is a very promising anticancer drug candidate, and that in particular, RecQL1‐siRNA formulated with a cationic liposome has an enormous potential to be used by intravenous injection for therapy specific for liver cancers, including metastasized cancers from the colon and pancreas. (Cancer Sci 2008; 99: 1227–1236)

Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of RecQL1 helicase

RecQL1 DNA helicase of the human RecQ helicase family participates in DNA repair and recombination pathways during cell‐cycle replication. When we examined the effect of RecQL1 suppression on cell growth, we found that RecQL1 silencing by small interference RNA efficiently prevented proliferation of a wide range of cancer cells by inducing mitotic catastrophe and mitotic cell death. In contrast, such mitotic cell death was not seen in the growing normal fibroblasts used as controls, even if RecQL1 expression was fully downregulated. Our results support the hypothesis that endogenous DNA damage that occurs during DNA replication and remains unrepaired in cancer cells due to RecQL1 silencing induces cancer cell‐specific mitotic catastrophe through a less‐strict checkpoint in cancer cells than in normal cells. We speculate that normal cells are exempt from such mitotic cell death, despite slow growth, because cell‐cycle progression is controlled strictly by a strong checkpoint system that detects DNA damage and arrests progression of the cell cycle until DNA damage is repaired completely. These results suggest that RecQL1 helicase is an excellent molecular target for cancer chemotherapy. (Cancer Sci 2008; 99: 71–80)