Abstract
Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2˙̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80–90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2–3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.
Highlights
Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation
We found that the mean mRNA expression ratios of NADPH oxidase 1 (NOX1) or its accessory genes in HT-29 cells were Ϸ15,000 (ϫ10Ϫ6) for NOX1; Ϸ20,000 (ϫ10Ϫ6) for NOXO1; and 1100 (ϫ10Ϫ6) for NOXA1 (Table 1); these experiments, in conjunction with our previous study [40], strongly suggest that among human tumor cells lines, NOX1 is most frequently expressed in those originating from the colon [31]
NOX1 mRNA expression did not change through 20 passages for clones 6A, G6, and SC; levels of NOX1 expression were significantly lower in clones 6A and G6 (Fig. 1A) than in parental HT-29 cells or cells expanded from the SC clone (p Ͻ 0.001 for either 6A or G6 compared with either parental HT-29 cells or cells from the SC clone)
Summary
HT-29 cells were utilized for these experiments because previous investigations had demonstrated that this cell line expressed high levels of NOX1 mRNA [27]. When stable HCT-116 clones expressing our NOX1 shRNA were compared with the parental line, vector control cells, or cells expressing our scrambled shRNA vector, we could demonstrate no effect of NOX1 shRNA expression on rates of cell growth (data not shown) or on the levels of protein-tyrosine or serine/threonine phosphatase activity in HCT-116 cells (Fig. 7, A and B). The mRNA expression levels of genes related to cell proliferation, angiogenesis, and invasion were measured by real-time RT-PCR in HT-29 parental cells and in clonal variants expressing a scrambled shRNA sequence (SC) or Nox shRNA (6A) during logarithmic growth in cell culture or from tumor xenografts established in immunoincompetent mice as described under “Experimental procedures.”. Genes in cell lines Down-regulated CCL14/15 c-MYB c-MYC CXCR4 FGFR3 VEGF-A HMOX1 NOX1
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