Targeting<i>MDR1</i>Gene: Synthesis and Cellular Study of Modified Daunomycin-Triplex-Forming Oligonucleotide Conjugates Able to Inhibit Gene Expression in Resistant Cell Lines

Stierlé, Vérène; Duca, Maria; Halby, Ludovic; Sénamaud-Beaufort, Catherine; Capobianco, Massimo L.; Laigle, Alain; Jollès, Béatrice; Arimondo, Paola B.

doi:10.1124/mol.107.042010

Cited by 13 publications

(12 citation statements)

References 42 publications

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“…However, the strategy has never been applied to an endogenous target of therapeutic interest. Other studies have demonstrated the cellular efficacy of TFOs conjugated to daunomycin, a Top2 inhibitor, without demonstrating a mechanism via a Top2‐mediated DNA cleavage (49–51). Thus, a clear demonstration of the mechanism of action in cells of the conjugates of TFOs attached to topoisomerase inhibitors was lacking.…”

Section: Discussionmentioning

confidence: 99%

Sequence‐specific targeting of IGF‐I and IGF‐IR genes by camptothecins

et al. 2010

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We and others have clearly demonstrated that a topoisomerase I (Top1) inhibitor, such as camptothecin (CPT), coupled to a triplex-forming oligonucleotide (TFO) through a suitable linker can be used to cause site-specific cleavage of the targeted DNA sequence in in vitro models. Here we evaluated whether these molecular tools induce sequence-specific DNA damage in a genome context. We targeted the insulin-like growth factor (IGF)-I axis and in particular promoter 1 of IGF-I and intron 2 of type 1 insulin-like growth factor receptor (IGF-IR) in cancer cells. The IGF axis molecules represent important targets for anticancer strategies, because of their central role in oncogenic maintenance and metastasis processes. We chemically attached 2 CPT derivatives to 2 TFOs. Both conjugates efficiently blocked gene expression in cells, reducing the quantity of mRNA transcribed by 70-80%, as measured by quantitative RT-PCR. We confirmed that the inhibitory mechanism of these TFO conjugates was mediated by Top1-induced cleavage through the use of RNA interference experiments and a camptothecin-resistant cell line. In addition, induction of phospho-H2AX foci supports the DNA-damaging activity of TFO-CPT conjugates at specific sites. The evaluated conjugates induce a specific DNA damage at the target gene mediated by Top1.

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Section: Discussionmentioning

confidence: 99%

Sequence‐specific targeting of IGF‐I and IGF‐IR genes by camptothecins

et al. 2010

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“…However, regulation of MDR-related gene expression is highly complex. For instance, such complexity is embodied in multiple transcription-regulatory elements in the 5' and 3' flanking sequences of the mdr-1 gene, and numerous protein factors involved in transcription-regulatory processes in a cell type-and stimulus-dependent manner [21] . The mechanism underlying the expression of MDR-related genes has not yet been fully understood.…”

Section: Discussionmentioning

confidence: 99%

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

Feng¹,

Wang²,

Pan³

et al. 2009

WJG

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AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 in multidrug resistant (MDR) hepatocellular carcinoma (HCC) cells. METHODS:MDR HCC cell lines, HepG2/adriamycin (ADM) and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein (P-gp) and multidrug resistant protein 1 (MRP1) expression levels. ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot. RESULTS:MTT assay showed that HepG2/ADM and SMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells (8.92% ± 0.22% vs 0.88% ± 0.05%, P < 0.001) and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells (7.37% ± 0.26% vs 1.74% ± 0.25%, P < 0.001). However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION: ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.

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“…Furthermore, DNM–TFO conjugates showed sequence-specific inhibition of the targeted gene, without however an effect of the anti-cancer activity of DNM. We recently demonstrated that conjugation of DNM to TFOs allowed to inhibit transcription of the target gene ( MDR 1) by a specific role of the DNM moiety (178). Furthermore, if these conjugates are employed in DNM-resistant cell lines, the uptake of DNM itself is permitted by the presence of the oligonucleotide, thus showing a mutual action of the partners of these conjugates, the DNM and the TFO.…”

Section: In Vitro Applications Of Tfos As Gene Modulatorsmentioning

confidence: 99%

The triple helix: 50 years later, the outcome

Duca

Vekhoff

Oussedik

et al. 2008

Nucleic Acids Research

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269

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Triplex-forming oligonucleotides constitute an interesting DNA sequence-specific tool that can be used to target cleaving or cross-linking agents, transcription factors or nucleases to a chosen site on the DNA. They are not only used as biotechnological tools but also to induce modifications on DNA with the aim to control gene expression, such as by site-directed mutagenesis or DNA recombination. Here, we report the state of art of the triplex-based anti-gene strategy 50 years after the discovery of such a structure, and we show the importance of the actual applications and the main challenges that we still have ahead of us.

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TargetingMDR1Gene: Synthesis and Cellular Study of Modified Daunomycin-Triplex-Forming Oligonucleotide Conjugates Able to Inhibit Gene Expression in Resistant Cell Lines

Cited by 13 publications

References 42 publications

Sequence‐specific targeting of IGF‐I and IGF‐IR genes by camptothecins

Sequence‐specific targeting of IGF‐I and IGF‐IR genes by camptothecins

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

The triple helix: 50 years later, the outcome

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