Ruthenium oligonucleotides, targeting HPV16 E6 oncogene, inhibit the growth of cervical cancer cells under illumination by a mechanism involving p53

Abstract

High-risk Human Papillomaviruses (HPV) has been found to be associated with carcinomas of the cervix, penis, vulva/vagina, anus, mouth and oro-pharynx. As the main tumorigenic effects of the HPV have been attributed to the expression of E6 and E7 genes, different gene therapy approaches have been directed to block their expression such as antisense oligonucleotides (ASO), ribozymes and small interfering RNAs. In order to develop a gene-specific therapy for HPV-related cancers, we investigated a potential therapeutic strategy of gene silencing activated under illumination. Our aim according to this antisense therapy consisted in regulating the HPV16 E6 oncogene by using an E6-ASO derivatized with a polyazaaromatic ruthenium (Ru II) complex (E6-Ru-ASO) able, under visible illumination, to crosslink irreversibly the targeted sequence. We examined the effects of E6-Ru-ASO on the expression of E6 and on the cell growth of cervical cancer cells. We demonstrated using HPV16 þ SiHa cervical cancer cells that E6-Ru-ASO induces after illumination, a reactivation of p53, the most important target of E6, as well as the inhibition of cell proliferation with a selective repression of E6 at the protein level. These results suggest that E6-Ru ASOs, activated under illumination and specifically targeting E6, are capable of inhibiting HPV16 þ cervical cancer cell proliferation.

Highly DNA‐Photoreactive Ruthenium 1,4,5,8‐Tetraazaphenanthrene Complex Conjugated to the TAT Peptide: Efficient Vectorization inside HeLa Cells without Phototoxicity – The Importance of Cellular Distribution

“…Indeed, Ru-TAP complexes are very efficient for inducing a PET with the guanine bases of DNA, as shown in several previous studies. [4,28,29] The present results show that the TAT peptide transports the Ru complex mainly into vesicles and into the cytoplasm; therefore, the Ru-TAP complex cannot reach its DNA or RNA targets. In contrast, the Ru-phen complex also transported into the vesicles by the TAT peptide can behave as an excellent singlet-oxygen photosensitizer, which is not the case for the Ru-TAP complex.…”

“…[28,29] Experimental Section Synthesis: Conjugates 1 and 2 (Figure 1, b) were synthesized as described previously. [32,33] Briefly, a modified phenanthroline ligand bearing an aminooxy group was coordinated to the desired bischelated precursor {either [Ru(TAP) 2 Cl 2 ] or [Ru(phen) 2 Cl 2 ]}.…”

“…[23,27] We have demonstrated that DNA-photoreactive Ru-TAP complexes conjugated to specific antisense oligonucleotides and internalized into living cells by particular synthetic polymers specifically inhibit the expression of targeted genes. [28,29] This specific photosilencing has demonstrated the efficiency of Ru-TAP complexes under irradiation inside living cells.…”

Ru–TAP complexes and DNA: from photo-induced electron transfer to gene photo-silencing in living cells

“…The E6 oncogene is thus an ideal target for targeted gene-silencing therapy in HPV + cervical cancer. HPV + SiHa cancer cells containing the gene E6 have thus been treated with photoreactive Ru-ODNs to test whether they behave as good Ruantisense oligonucleotides (Ru-ASOs) using commercial oligofectamine as the transfecting agent [77]. The Ru complex tethered to the 3 end of the ODN corresponds to [Ru(TAP) 2 phen] 2+ and the ODN sequence has been chosen so as to correspond to a sequence that is complementary to a 21-base-pair sequence of E6 and could thus behave as an ASO (sequence (a) of figure 19).…”

Vaginal gene therapy

“…In mouse tumor models, after intratumor administration, E7 mRNA expression was reduced to 45-50%. In a recent work, Reschner et al [112] investigated a potential therapeutic strategy of gen silencing activated under illumination. Their approach consisted in regulating the HPV 16 E6 oncogene by using an E6 antisense oligonucleotides (E6-ASO) derivatized with a polyazaariomtic ruthenium complex (E6-Ru-ASO) able, under visible illumination, to crosslink irreversibly the targeted sequence.…”