Inhibition of colon cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA of the Ki-ras gene

Sakakura, Chouhei; Hagiwara, Akeo; Tsujimoto, H.; Ozaki, Kouichi; Sakakibara, Tateki; Oyama, Takayuki; Ogaki, Masaharu; Imanishi, Takeshi; Yamazaki, Junpei; Takahashi, Tetsuyuki

doi:10.1097/00001813-199508000-00008

Cited by 16 publications

(5 citation statements)

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“…With respect to the target sites in mRNA, the AUG translational initiation codon is preferentially chosen as a target, and the growth-inhibitory effects of the anti-sense oligonucleotides complementary to the initiation codon region of the K-ras oncogene have been reported (Liu et al, 1993;Kobayashi, 1995;Sakakura et al, 1995). However, in attempting K-ras mutation-specific inhibition of cell growth, we have to use the mutated codon 12 as a target site of anti-sense oligonucleotides.…”

Section: Discussionmentioning

confidence: 99%

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

et al. 1999

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About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched antisense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells. Int.

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Section: Discussionmentioning

confidence: 99%

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

et al. 1999

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“…AS K-ras PS ODN, CACAAGTTTATATTCAGT, were synthesized that were complementary to wt K-ras nucleotides 196-213 (adjacent to the start codon). Based on previous studies (26), MM K-ras PS ODN, CACTTGTAAATAT-TCAGT, and scrambled (SC) K-ras PS ODN, ACTAGC-TATACTAGCTAT, to the same region (nucleotides 196-213), were synthesized.…”

Section: Methodsmentioning

confidence: 99%

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells

Lebedeva

Gopalkrishnan

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

143

210

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Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.

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“…Moreover, the downregulation of Ki-ras by MPEG ODN 20 promoted a growth inhibition of 40%, as one expects from a gene involved in the transmission of mitotic signals. 47,53,54 We found that the MPEG moiety slightly hinders the binding of the conjugate to the Ki-ras pur/pyr motif. In fact, triplex formation between MPEG ODN 20 and Ki-ras pur/pyr duplex is characterized by a K d of 1.5 mM at 371C, which is about one order of magnitude higher than that observed with ODN 20 (0.2 mM at 371C).…”

Section: Discussionmentioning

confidence: 88%

Antiproliferative activity of a triplex-forming oligonucleotide recognizing a Ki-ras polypurine/polypyrimidine motif correlates with protein binding

et al. 2004

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The Ki-ras gene is frequently mutated and/or overexpressed in human cancer. Since it is suspected to play a key role in the pathogenesis of many tumors, there is interest to search for strategies aiming at the specific inhibition of this oncogene. In this paper, we investigated the capacity of a 20 mer G-rich oligonucleotide (ODN20) conjugated to high molecular weight monomethoxy polyethylene glycol (MPEG) to inhibit the expression of the Ki-ras gene and the proliferation of pancreatic cancer cells. The conjugate, MPEG ODN20, was designed to form a triplex with a critical pur/pyr sequence located in the promoter of the Ki-ras gene. To make the conjugate resistant to endogenous and exogenous nucleases, five phosphorothioate linkages were introduced in its backbone. Confocal microscopy and FACS experiments showed that MPEG ODN20 had a higher capacity to penetrate the cell membranes and accumulate in the nucleus of Panc-1 cells than ODN20. Incubation of Panc-1 cells with MPEG ODN20 reduced specifically the levels of Ki-ras mRNA and RAS protein p21RAS. A single-dose administration of MPEG ODN20 was sufficient to inhibit cell proliferation by about 50% compared with control. By contrast, the antiproliferative activity of the unconjugated ODN20 analog was found to be not significant. Band-shift and footprinting experiments showed that MPEG ODN20 formed a weak triplex (Kd approximately 1.5 microM at 37 degrees C, 50 mM Tris-acetate, pH 7.4, 10 mM NaCl, 10 mM MgCl2, 5 mM spermidine) with the Ki-ras pyr/pur motif, suggesting that its bioactivity can hardly be mediated by a triplex-based mechanism. Here, we provide evidence that, in vitro, ODN20 and MPEG ODN20 competitively inhibit the binding to the Ki-ras pur/pyr motif of a nuclear protein, suggesting that the activity of MPEG ODN20 occurs with an aptameric mechanism. The biological implications of this study are discussed.

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Inhibition of colon cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA of the Ki-ras gene

Cited by 16 publications

References 0 publications

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells

Antiproliferative activity of a triplex-forming oligonucleotide recognizing a Ki-ras polypurine/polypyrimidine motif correlates with protein binding

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