RNA interference is not only being used in cancer research as a means for detecting gene targets for small molecule drugs (See RNAi News, 11/07/03). Some researchers have been exploring ways to translate the technology directly into adjuvant therapies. According to research published in the Nov. 13 edition of Oncogene, scientists from the German Cancer Research Center (DKFZ) have found that siRNAs that knock down apoptosis-inhibiting genes may be able to enhance the effect of existing cancer treatments.
Karin Butz and DKFZ colleagues reported in the article that they targeted for knockdown the Livin/ML-IAP/KIAP member of the inhibitor of apoptosis protein (IAP) family. Ectopic expression of Livin, the researchers wrote, has been shown to block apoptosis induction by a variety of pro-apoptotic stimuli — in part by inhibiting caspase-3, a main effector caspase for the induction of apoptotic cell death — and has been found to be expressed in certain tumor cells but not in substantial amounts in normal tissue.
The researchers used the plasmid pSUPER vector system, which employs the polymerase-III H1-RNA gene promoter, to generate siRNAs against the Livin-1 and Livin-2 regions of the livin cDNA in HeLa cervical carcinoma cells. “After transfection,” they wrote, “both pSUPER-Livin-1 and pSUPER-Livin-2 siRNAS efficiently inhibited endogenous Livin protein expression. This was not observed for the transfection control.”
Additionally, expression of both Livin-1 and Livin-2 siRNAs led to a reduction of livin transcripts in HeLa cells.
Further analyses of pSUPER-Livin-2 — chosen because it regularly suppressed endogenous livin expression more strongly than its Livin-1 counterpart — showed that the siRNA-expressing vector increased caspase-3-like activities in HeLa cells, indicating that the down-regulation of Livin expression is associated with the release of caspase-3 from negative regulation by Livin, according to Butz et al. The researchers also found that pSUPER-Livin-2 also markedly increased the concentrations of the active form of caspase-3 following treatment of HeLa cells with the chemotherapeutic agent doxorubicin, UV irradiation, and tumor necrosis factor alpha, compared with cells receiving the control transfection.
TdT-mediated dUTP biotin nick-end labeling (TUNEL) analyses were performed to assess the cellular consequences of siRNA-mediated silencing of the livin gene, the researchers wrote. In the absence of a pro-apoptotic stimulus, the number of TUNEL-positive cells increased by about twofold following expression of pSUPER-Livin-2.
However, the pSUPER-Livin-2 apoptosis rate was strongly increased when stimulated with doxorubicin, UV irradiation, and TNF-alpha, demonstrating that “siRNA directed against livin can efficiently sensitize Livin-positive cancer cells toward pro-apoptotic stimuli,” the researchers stated. Additionally, colony formation assays showed that the “stable transfection of an expression vector generating siRNA against livin selectively inhibited the outgrowth of Livin-expressing tumor cells … with high efficiency. This suggests that a longer exposure to agents interfering with the livin function may suffice to induce apoptotic cell death, even in the absence of additional pro-apoptotic stimuli.”
“In view of a clinical perspective,” Butz and colleagues concluded, “a combination treatment of livin-targeting siRNAs with pro-apoptotic drugs, such as chemo- or radiotherapeutic agents, appears to be most promising.”
It may come as no surprise, however, that they cited delivery as a major hurdle to realizing this goal.
“Vector-borne expression of siRNAs … under therapeutic aspects, will have to meet the general challenges of gene therapy approaches, such as efficient delivery into the target cells or the circumvention of immune responses,” the researchers wrote. They also noted that the feasibility of using the hydrodynamic injection technique in primates is questionable.
“Alternative delivery strategies could be envisioned: translocating peptides have been developed, which efficiently transport plasmid DNA into living cells; other peptides were found to introduce compounds into all cell layers of intact human skin,” they wrote. “It remains to be seen whether the further development of these techniques may also provide a new route for the topical application of siRNA-encoding vectors in order to treat lesions of the skin and mucosa.”
Felix Hoppe-Seyler, one of Butz’s co-investigators, told RNAi News in an email that the researchers are planning to “use the siRNAs as molecular tools in order to elucidate the anti-apoptotic function of livin in detail.”
They also intend to test potential delivery systems for siRNAs with the overall aim that they may form a basis for a future topical application,” Hoppe-Seyler added. “This may be particularly relevant for livin, since this gene has been linked [to cancers including] malignant melanoma.”