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KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2- c ]quinolines

View Article: PubMed Central - PubMed

ABSTRACT

KRAS is one of the most frequently mutated oncogenes in human cancer, yet remaining undruggable. To explore a new therapeutic strategy, a library of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc) with a range of alkyldiamine side chains was designed to target DNA and RNA G-quadruplexes (G4) in the promoter and 5′-UTR mRNA of the KRAS gene. Biophysical experiments showed that di-substituted IQc compounds are potent and selective KRAS G4 stabilizers. They preferentially inhibit the proliferation of KRAS mutant cancer cell lines (0.22 < IC50 < 4.80 μM), down-regulate KRAS promoter activity in a luciferase reporter assay, and reduce both KRAS mRNA and p21KRAS steady-state levels in mutant KRAS colon cancer cell lines. Additionally, IQcs induce cancer cell death by apoptosis, explained in part by their capacity to repress KRAS expression. Overall, the results suggest that targeting mutant KRAS at the gene level with G4 binding small molecules is a promising anticancer strategy.

No MeSH data available.


Related in: MedlinePlus

KRAS promoter activity is decreased following exposure of HEK293T cells lines to equitoxic (IC50) concentrations of compounds TMPyP4, 2d, 3d and 3e.Cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four h later, cells were replated in 96 wells plates, at 5000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC50 equitoxic concentration of test compounds IQcs and TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. The results are expressed as the mean ± SEM fold-change compared to DMSO exposure, from three independent experiments. *p < 0.05 and § p < 0.01 from DMSO vehicle control.
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f5: KRAS promoter activity is decreased following exposure of HEK293T cells lines to equitoxic (IC50) concentrations of compounds TMPyP4, 2d, 3d and 3e.Cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four h later, cells were replated in 96 wells plates, at 5000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC50 equitoxic concentration of test compounds IQcs and TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. The results are expressed as the mean ± SEM fold-change compared to DMSO exposure, from three independent experiments. *p < 0.05 and § p < 0.01 from DMSO vehicle control.

Mentions: A first evaluation of the compounds' ability to down-regulate KRAS gene transcription by directly acting on the KRAS promoter was undertaken with a luciferase reporter assay. For this purpose two different size wild-type promoter constructs were used, containing the G4 sequence and cloned into the Firefly luciferase pGL3 Basic backbone (pGL-Ras0.5, PGL-Ras2.0). These and pGL3 Basic empty (Firefly Luciferase negative control/no promoter) were co-transfected into HEK293T cells together with Renilla luciferase pRL-TK (transfection efficiency normalization) as a G4 negative control. This late construct does not harbour G4-forming sequences and is insensitive to G4-related effects/regulation. In general, the data in Figure 5 clearly portraits a reduction of luciferase activity by ~25–50% when compared to Renilla basal levels induced by all G4 ligands. Moreover, the same level of promoter activity decrease is induced by compounds in cells transfected with plasmids of different sizes (500 and 2000 bp), suggesting that the target region of tested compounds is at most 500 bp upstream from the start of the coding region, thus coinciding with the region where the G4 sequence is located.


KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2- c ]quinolines
KRAS promoter activity is decreased following exposure of HEK293T cells lines to equitoxic (IC50) concentrations of compounds TMPyP4, 2d, 3d and 3e.Cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four h later, cells were replated in 96 wells plates, at 5000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC50 equitoxic concentration of test compounds IQcs and TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. The results are expressed as the mean ± SEM fold-change compared to DMSO exposure, from three independent experiments. *p < 0.05 and § p < 0.01 from DMSO vehicle control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5382548&req=5

f5: KRAS promoter activity is decreased following exposure of HEK293T cells lines to equitoxic (IC50) concentrations of compounds TMPyP4, 2d, 3d and 3e.Cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four h later, cells were replated in 96 wells plates, at 5000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC50 equitoxic concentration of test compounds IQcs and TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. The results are expressed as the mean ± SEM fold-change compared to DMSO exposure, from three independent experiments. *p < 0.05 and § p < 0.01 from DMSO vehicle control.
Mentions: A first evaluation of the compounds' ability to down-regulate KRAS gene transcription by directly acting on the KRAS promoter was undertaken with a luciferase reporter assay. For this purpose two different size wild-type promoter constructs were used, containing the G4 sequence and cloned into the Firefly luciferase pGL3 Basic backbone (pGL-Ras0.5, PGL-Ras2.0). These and pGL3 Basic empty (Firefly Luciferase negative control/no promoter) were co-transfected into HEK293T cells together with Renilla luciferase pRL-TK (transfection efficiency normalization) as a G4 negative control. This late construct does not harbour G4-forming sequences and is insensitive to G4-related effects/regulation. In general, the data in Figure 5 clearly portraits a reduction of luciferase activity by ~25–50% when compared to Renilla basal levels induced by all G4 ligands. Moreover, the same level of promoter activity decrease is induced by compounds in cells transfected with plasmids of different sizes (500 and 2000 bp), suggesting that the target region of tested compounds is at most 500 bp upstream from the start of the coding region, thus coinciding with the region where the G4 sequence is located.

View Article: PubMed Central - PubMed

ABSTRACT

KRAS is one of the most frequently mutated oncogenes in human cancer, yet remaining undruggable. To explore a new therapeutic strategy, a library of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc) with a range of alkyldiamine side chains was designed to target DNA and RNA G-quadruplexes (G4) in the promoter and 5&prime;-UTR mRNA of the KRAS gene. Biophysical experiments showed that di-substituted IQc compounds are potent and selective KRAS G4 stabilizers. They preferentially inhibit the proliferation of KRAS mutant cancer cell lines (0.22 &lt; IC50 &lt; 4.80 &mu;M), down-regulate KRAS promoter activity in a luciferase reporter assay, and reduce both KRAS mRNA and p21KRAS steady-state levels in mutant KRAS colon cancer cell lines. Additionally, IQcs induce cancer cell death by apoptosis, explained in part by their capacity to repress KRAS expression. Overall, the results suggest that targeting mutant KRAS at the gene level with G4 binding small molecules is a promising anticancer strategy.

No MeSH data available.


Related in: MedlinePlus