RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells

The efficacy of doxorubicin in the treatment of cancer is limited by its side effects and by the onset of drug resistance. Reverting such resistance could allow the decrease of the dose necessary to eradicate the tumor, thus diminishing the toxicity of the drug. We transfected doxorubicin-sensitive (HT29) and doxorubicin-resistant (HT29-dx) human colon cancer cells with RhoA small interfering RNA. The subsequent decrease of RhoA protein was associated with the increased sensitivity to doxorubicin in HT29 cells and the complete reversion of doxorubicin resistance in HT29-dx cells. RhoA silencing increased the activation of the nuclear factor-κB pathway, inducing the transcription and the activity of nitric oxide synthase. This led to the tyrosine nitration of the multidrug resistance protein 3 transporter (MRP3) and contributed to a reduced doxorubicin efflux. Moreover, RhoA silencing decreased the ATPase activity of P-glycoprotein (Pgp) in HT29 and HT29-dx cells as a consequence of the reduced expression of Pgp. RhoA silencing, by acting as an upstream controller of both MRP3 nitration and Pgp expression, was effective to revert the toxicity and accumulation of doxorubicin in both HT29 and HT29-dx cells. Therefore, we suggest that inactivating RhoA has potential clinical applications and might in the future become part of a gene therapy protocol. (Mol Cancer Res 2008;6(10):1607–20)

[1]  A. Duncan,et al.  Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. , 1992, Science.

[2]  L. Goldstein.  MDR1 gene expression in solid tumours. , 1996, European journal of cancer.

[3]  B. Kaina,et al.  Rho GTPases: promising cellular targets for novel anticancer drugs. , 2006, Current cancer drug targets.

[4]  J. Cusack,et al.  Inducible chemoresistance to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothe cin (CPT-11) in colorectal cancer cells and a xenograft model is overcome by inhibition of nuclear factor-kappaB activation. , 2000, Cancer research.

[5]  J. Kushleika,et al.  RhoA regulation of NF-kappaB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells. , 2007, American journal of physiology. Cell physiology.

[6]  A. Shirahata,et al.  Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes , 2007, Free radical research.

[7]  Deborah A. Corey,et al.  Mechanisms of NOS2 regulation by Rho GTPase signaling in airway epithelial cells. , 2002, American journal of physiology. Lung cellular and molecular physiology.

[8]  S. Raguz,et al.  Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1 , 2004, Gene Therapy.

[9]  C. Riganti,et al.  Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux. , 2005, Cancer research.

[10]  Kyung Soo Park,et al.  Identification of a classic cytokine‐induced enhancer upstream in the human iNOS promoter , 2007, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  P. Opolon,et al.  Intravenous delivery of anti-RhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer. , 2006, Human gene therapy.

[12]  H. Lage,et al.  Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi) , 2003, FEBS letters.

[13]  P. Betta,et al.  Simian Virus 40 Infection Down-Regulates the Expression of Nitric Oxide Synthase in Human Mesothelial Cells , 2004, Cancer Research.

[14]  Shailendra Giri,et al.  Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha. , 2003, Free radical biology & medicine.

[15]  Bharat B. Aggarwal,et al.  Simvastatin Potentiates TNF-α-Induced Apoptosis through the Down-Regulation of NF-κB-Dependent Antiapoptotic Gene Products: Role of IκBα Kinase and TGF-β-Activated Kinase-11 , 2007, The Journal of Immunology.

[16]  S. Simon,et al.  Cell biological mechanisms of multidrug resistance in tumors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Gottesman,et al.  Multidrug resistance in cancer: role of ATP–dependent transporters , 2002, Nature Reviews Cancer.

[18]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[19]  Marianne Fillet,et al.  NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells , 2003, Oncogene.

[20]  S. Payne,et al.  Role of ABCC1 in export of sphingosine-1-phosphate from mast cells , 2006, Proceedings of the National Academy of Sciences.

[21]  M. Merville,et al.  Phosphorylation of NF-κB and IκB proteins: implications in cancer and inflammation , 2005 .

[22]  Hee-Sun Kim,et al.  Simvastatin prevents oxygen and glucose deprivation/reoxygenation‐induced death of cortical neurons by reducing the production and toxicity of 4‐hydroxy‐2E‐nonenal , 2006, Journal of neurochemistry.

[23]  S. Ghosh,et al.  Differential Phosphorylation of the Signal-responsive Domain of IκBα and IκBβ by IκB Kinases* , 2003, Journal of Biological Chemistry.

[24]  I. Pastan,et al.  Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Karin,et al.  The IKK/NF-κB activation pathway—a target for prevention and treatment of cancer , 2004 .

[26]  Ernest Beutler,et al.  Red Cell Metabolism: A Manual of Biochemical Methods , 1975 .

[27]  E. Beutler.  Red cell metabolism , 1971 .

[28]  J. Stamler,et al.  S-nitrosylation in health and disease. , 2003, Trends in molecular medicine.

[29]  J. Brugge,et al.  Activation of NF-κB following detachment delays apoptosis in intestinal epithelial cells , 2005, Oncogene.

[30]  G. Meinhardt,et al.  The HMG-CoA reductase inhibitor simvastatin overcomes cell adhesion-mediated drug resistance in multiple myeloma by geranylgeranylation of Rho protein and activation of Rho kinase. , 2004, Blood.

[31]  G. Bonadonna,et al.  Cardiac toxicity of daunorubicin. , 1969, Lancet.

[32]  Korey R. Johnson,et al.  Nuclear Factor-κB/IκB Signaling Pathway May Contribute to the Mediation of Paclitaxel-induced Apoptosis in Solid Tumor Cells , 2000 .

[33]  Wan-Wan Lin,et al.  The Role of Rho-Associated Kinase in Differential Regulation by Statins of Interleukin-1β- and Lipopolysaccharide-Mediated Nuclear Factor κB Activation and Inducible Nitric-Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells , 2006, Molecular Pharmacology.

[34]  P. Betta,et al.  Statins revert doxorubicin resistance via nitric oxide in malignant mesothelioma , 2006, International journal of cancer.

[35]  D. Rigal,et al.  Control of P-glycoprotein activity by membrane cholesterol amounts and their relation to multidrug resistance in human CEM leukemia cells. , 2005, Biochemistry.

[36]  M. Díaz-Guerra,et al.  Inhibition of IκB Kinase and IκB Phosphorylation by 15-Deoxy-Δ12,14-Prostaglandin J2 in Activated Murine Macrophages , 2000, Molecular and Cellular Biology.

[37]  A. Józkowicz,et al.  Anti-angiogenic and anti-inflammatory effects of statins: relevance to anti-cancer therapy. , 2005, Current cancer drug targets.

[38]  P. Opolon,et al.  Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[39]  S. Kaye,et al.  Drug resistance reversal--are we getting closer? , 2003, European journal of cancer.

[40]  H. Coley,et al.  Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein. , 2003, Cancer control : journal of the Moffitt Cancer Center.

[41]  H. Kalthoff,et al.  Inhibition of NF-κB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin , 2001, Oncogene.

[42]  P. Liu,et al.  RhoA‐mediated, tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin , 2006 .

[43]  W. Hait,et al.  Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells. , 2003, Cancer research.