Inducible nitric oxide synthase (iNOS) mediates the early increase of blood supply (EIBS) in colon carcinogenesis

Abstract

We have recently demonstrated that dramatic alteration in mucosal microvascular blood content termed early increase in blood supply (EIBS) is a hallmark of early colon carcinogenesis. In the current study, we elucidate the mechanism of EIBS by assessing iNOS/nitric oxide axis in the histologically normal colonic mucosa of rats treated with the colon-specific carcinogen, azoxymethane. We demonstrate that there was a strong temporal correlation between EIBS and iNOS expression/activity. Importantly, we also observed that short-term treatment with nitric oxide inhibitor abrogated EIBS. These data indicate that iNOS induction may have a critical role in augmenting the predysplastic mucosal blood supply and thereby fostering colon carcinogenesis.

Figure 1

iNOS induction occurs in a temporally consonant with EIBS (previously demonstrated to be 2 weeks post-AOM, prior to ACF development [8]). Fisher rats were euthanized 2 weeks after 2nd weekly injections of AOM (see methods section)in Panel A, iNOS expression (by RT-PCR) was upregulated in AOM treated colons (by ∼2.5 fold; p=0.02) compared to controls. Similarly as shown in Panel B, the protein expression (by IHC scoring intensity) was significantly upregulated in AOM rats (p< 0.05) compared to controls. However, at this stage no significant change was observed in the immunoreactivity of VEGF in AOM sample compared to controls.

Figure 2

iNOS induction/activity temporally correlates with the EIBS. To determine the associations of EIBS and iNOS induction during the carcinogenic progression animals were sacrificed serially at 4, 12 and 20 weeks post initiation (see text) * p<0.01 versus age-matched saline control. EIBS was measured by 4D-ELF, while iNOS and nitrotyrosine was quantified using immunohistochemistry.

Figure 2

iNOS induction/activity temporally correlates with the EIBS. To determine the associations of EIBS and iNOS induction during the carcinogenic progression animals were sacrificed serially at 4, 12 and 20 weeks post initiation (see text) * p<0.01 versus age-matched saline control. EIBS was measured by 4D-ELF, while iNOS and nitrotyrosine was quantified using immunohistochemistry.

Figure 3

Short term treatment with L-NAME mitigated EIBS in AOM-treated animals but had no effect on the colonic microvascular blood content in saline-treated rats (see text).