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Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells

Abstract

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in 40–50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

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Abbreviations

Ad:

adenovirus

APC:

allophycocyanine

AS:

antisense

CAR:

Coxsackievirus and adenovirus receptor

ECL:

enhanced chemiluminescence

ERK:

extracellular signal-regulated kinase

FACS:

fluorescent-activated cell sorting

FITC:

fluorescein isothiocyanate

gapdh :

glyceraldehyde-3-phosphate dehydrogenase

GFP:

green fluorescent protein

IFN-β:

beta (fibroblast) interferon

IL:

interleukin

MAPK:

mitogen-activated protein kinase

mda-7:

melanoma differentiation associated gene-7

MEK:

MAPK/ERK kinase

MEZ:

mezerein

m.o.i.:

multiplicity of infection

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

pfu:

plaque-forming units

PI:

propidium iodide

PKC:

protein kinase C

scAb:

single-chain antibody

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Acknowledgements

The present study was supported in part by NIH Grants R01 CA097318 and R01 CA098712, the Samuel Waxman Cancer Research Foundation, the Chernow Endowment and a sponsored research award from Introgen Therapeutics Inc. PBF is the Michael and Stella Chernow Urological Cancer Research Scientist and a SWCRF Investigator.

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Lebedeva, I., Su, ZZ., Emdad, L. et al. Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells. Oncogene 26, 733–744 (2007). https://doi.org/10.1038/sj.onc.1209813

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