Phenomenon of atypical vascular effects of epinephrine and an increase of photodynamic response by nitroglycerin in rats with colon adenocarcinoma: adrenergic and nitrergic mechanisms and novel applied aspects

: Using an original model of stress-induced colon adenocarcinoma, we uncover atypical vasorelaxation effects of a mucosa injection of epinephrine assessed by laser speckle contrast imaging and a significant increase of fluorescent intensity of 5-ALA/PpIX from malignant colon tissues by a mucosa injection of nitroglycerine. We clearly demonstrate a high activity of adrenergic and nitrergic mechanisms underlying this phenomenon and discuss their application in improving of optical approaches for effective diagnosis of gastrointestinal cancer. pathological changes of colon tissues associated with chronic stress nitrosamines


Introduction
Chronic psychological stress and quality of life play a pivotal role in the tumor development, its progression and therapeutic outcome [1]. Gastrointestinal (GI) cancers, one of the most common cancers worldwide, are also influenced by stress to a great extent [2,3]. Activation of adrenergic system mediates most of stress effects. Indeed, the activation of beta2-adrenergic receptors (B2-ADRs) signaling pathway induced by catecholamine is considered as a key mechanism underlying stress responses [4,5].
In the past decade, it has become apparent that activation of adrenergic system under stress plays a key role in tumor initiation, progression and regulation of cancer vascular microenvironment [6][7][8]. The vascular B2-ADRs interacts with epinephrine, a stress hormone, and transduce signal to trimeric Gs proteins, which via adenylate cyclase stimulation, increase in cAMP, and downstream L-type calcium channel interaction, mediates vascular responses such as smooth muscle relaxation. The B2-ADRs and beta-arrestins as cofactors in adrenergic signaling pathway are the most involved in the carcinogenic processes [6,9,10]. The high expression of B2-ADRs promote cancer metastasis via generation of a proinflammatory environment, stimulation of angiogenesis and chemoattraction of tumour cells [12,13]. There are growing evidences showing the relationship between the stress-induced activation of adrenergic system and GI cancers [14]. However, the adrenergic mechanisms underlying invasion and metastasis of GI cancer remained poorly understood. On the one hand, the high production of endothelial nitric oxide (NO), as an important factor involved in B2-ADRs-related modulation of vascular tone, is considered a possible adrenergic mechanism responsible for initiation and progression of cancers [15]. On the other hand, concerning GI tumors, several studies in a contrary suggested that inducible NOS (iNOS) play the crucial role in development of GI cancer [16][17][18][19][20][21][22].
Thus, there are evidences about a key role of such factors as stress, catecholamines and NO in the development of GI cancer. However, there is no clear information, how these factors are related to each other and about stress-induced adrenergic and nitrergic mechanisms responsible for GI cancer initiation and progression.
To answer this question, here we studied the adrenergic and nitrergic mechanisms underlying GI tumor formation (colon adenocarcinoma) in rats induced by long term combination of two factors such as social stress (isolation) and nitrosamines diet.

Subject
Experiments were performed in mongrel male rats (250 to 300 g, n = 317) in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), protocols were approved by the Institutional Review Boards of the Saratov State University (Protocol 7, 07.02.2017). The rats were housed at 25 ± 2oC, 55% humidity, and 12:12 h light -dark cycle. Food and water were given ad libitum.

Modeling of stress-induced GI cancer
To induce GI cancer in rats, we used our original model of stress-induced highly heterogeneous adenocarcinoma, which develops spontaneously in different regions of GI tract, predominantly in the stomach and in the colon. To induce GI cancer, the rats underwent to a long term combination (during 9 months) of social stress (overpopulation) and daily diet including of toluidine (2 g/kg) in food and water with nitrites (2 g/l). In our previous work we studied formation of stress-induced gastric adenocarcinoma [23]. Here, we focused on the study of stress-induced colon cancer development (highly differentiated adenocarcinoma).
The experiments were performed using following groups: 1) the control group included rats, which lived in standard conditions; 2) the stress group included rats, which lived under chronic social stress (isolation) during 9 months; 3) the nitrosamine group included rats used daily nitrosamine diet during 9 months; 4) the stress + nitrosamines group included rats, which lived under chronic stress (isolation) in combination with nitrosamine diet during 9 months.

Tested substances
For the study of adrenergic mechanisms of development of stress-induced colon cancer we used: 1) pharmacological test including submucosal injection of epinephrine assessed by laser speckle contrast imaging (LSCI); 2) immunohistochemical (ICH) and quantitative analysis of expression of B2-ADR in GI tissues; 3) pharmacological test of activity of B2-ADR including submucosal injection of antagonist of BR-ADR (ICI 118551, 3-(isopropylamino)-1-[(7methyl-4-indanyl)oxy]butan-2-ol) and agonist of BR-ADR (isoproterenol) assessed by LSCI; 4) immunoassay of ARRB1 as co-factor of activation of BR-ADR, in the blood and in GI tissues.
For the analysis of activity of NO-ergic system we studied: 1) the level of NO in GI tissues by spectrofluorimetric assay; 2) pharmacological test of NOS activity including of submucosal injection of specific blocker of endothelial NOS (eNOS) -LN(5)-(1iminoethyl)ornithine, L-NIO, and specific blocker iNOS -L-NIL (N6-(iminoethyl)-Llysine, L-NIL assessed by LSCI; 5-ALA/PpIX-photodynamic effects before and after submucosal injection of nitroglycerin -donor of NO.

Laser speckle contrast imaging (LSCI) of colon microcirculation
Irradiation of He-Ne laser (Thorlabs HNL-210L, 21.5 mW, 632.8 nm) was guided by a single mode polarization maintained fiber (P3-630PM-FC-1, Thorlabs) to illuminate whole imaged area. Exposure time of a CMOS-sensor (Aca-2500-14gm, Basler) was set to 7 ms. F-number of a lens (21HC, Tamron) was adjusted until spatial spectrum of the speckle image fits entirely in a spectral band limited by the Nyquist criterion. Home-made software written in LabView was used for real-time processing of raw speckle frames. Recording rate was 40 frames per second. To speed up the calculation, a convolution approach was implemented to compute distribution of spatial speckle contrast using standard formula K = σ/<I>, where σ is a standard deviation and <I> is the mean value of speckle intensity fluctuations inside sliding window. Blood flow index was extracted from processed frames using histogram analysis algorithm [24].
The LSCI was performed on anesthetized rats with ketamine (Sigma Chemical Co, 40 mg/kg, i.v.), the abdomen was opened through a midline incision and the colon was gently exteriorized. To eliminate motion artifacts, the colon was stabilized by placing it on a Perspex stage. The tip of either probe was positioned at about 0.5-1 mm from, and perpendicular to, the surface of the colon mucosa. The duration of the recovering process was at least 1 hour after surgical preparation until mucosal blood flow was stabilized.

Histological, immuno -and immunohistochemical (IHC) assay of GI tissues
Mice were euthanized with an intraperitoneal injection of a lethal dose of ketamine. For histological analysis of GI tissues, the samples were fixed in 10% buffered neutral formalin. The formalin fixed specimens were embedded in paraffin, sectioned (4 µm) and stained with hematoxylin and eosin. The histological sections were evaluated by light microscopy using the digital image analysis system Mikrovizor medical µVizo-103 (LOMO, Russia).
The total area and intensity of IHC staining for B2-ADRs and ARRB1markers was carried out by microscopic method using a microscopic system Ariol SL50 (Genetix, UK) with photographic images and the subsequent processing of the photos in the program imageJ V1.43 (USA).
Beta-arrestin level was measured in GI tissues and in blood serum in rats by enzyme immunoassay (ARRB1, ELISA) according to a standard protocol.

Nitric oxide measurement in GI tissues
The NO content in GI tissue was measured spectrophotometrically (SF-2011, Moscow) using standard biochemical method [25].

Study of the photodynamic effects of a GI tumor
As a photosensitizer for photodynamic diagnostics (PDD) of GI tissues, we used 5aminolevulinic acid/protoporphyrin IX (5-ALA/PpIX, 20 mg/kg, per os, ALASENS, Niopik Inc., Moscow, Russia). PDD was performed 2 hours after the application of 5-ALA/PpIX. In all experiments, an excitation source at 405 nm was used to reveal the exogenous fluorescence signal, where is the Soret absorption band of PpIX. Excitation source used is based on high-power light emitting diode AFS-405 (Polironik Ltd., Moscow, Russia), with FWHM = 20 nm and output light power 25 mW. Spectral detection was carried out by a microspectrometer USB4000 (OceanOptics Inc., Dunedin, USA), with a spectral range of 350-1000 nm. Y-form optical fiber probe (6 + 1) was used to deliver excitation and emission light from the tissues investigated. From each tissue site -normal and abnormal colon mucosa were detected from 7 to 10 spectra which were averaged per tissue type and animal.

Statistical analysis
Differences from the initial level in the same group were evaluated by the Wilcoxon test. Intergroup differences were evaluated using the Mann-Whitney test and ANOVA-2 (post hoc analysis with the Duncan's rank test). Significance levels were set at p < 0.05 for all analyses.

Atypical vascular responses to epinephrine in rats with stress-induced colon cancer
To confirm the development of stress-induced colon cancer, as a first step, we studied the morphological changes in the colon tissues in the stress + nitrosamines group (n = 15) in comparison with the other three groups -the control/stress/nitrosamines (n = 10 in each group). Figure 1(a) illustrates the normal colon tissues in the intact rats. Figure 1(b) demonstrates example of peptic ulcers of colon tissues, which we observed in 8 of 10 rats from the stress group (11 small and 3 large ulcers). Figure 1(c) shows epithelial desquamation, which has been developed in all rats from the nitrosamine group. Figure 1(d) represents highly differentiated colon adenocarcinoma, which we detected in 12 of 15 rats from the group stress + nitrosamines. The animals no affected by stress or/and nitrosamine diet were no taken in the further analysis.
In the next step, we analyzed microcirculatory effects of epinephrine in all tested groups in the same rats. The mucosal epinephrine injection in the place of mucosal injuries induced expected reduction of microcirculation, which was similar between the control/stress/nitrosamines groups (Fig. 1(e)). On the contrary, rats with adenocarcinoma from the stress + nitrosamines group demonstrated atypical hyperperfusion after epinephrine treatment ( Fig. 1(f)). Indeed, the colon microcirculation was decreased from 11.1 ± 1.8 speckle contrast arbitrary units (a.u.) at baseline to 7.7 ± 1.3 a.u. after epinephrine injection (P<0.001, n = 15) in the control group, from 9.3 ± 1.1 a.u. to 5.8 ± 1.4 a.u. (P<0.001, n = 10) in the stress group, from 10.7 ± 1.9 a.u. to 4.2 ± 1.0 a.u. (P<0.001, n = 10) in the nitrosamines group, while the increase of colon microcirculation was observed after epinephrine injection in the stress + nitrosamine group (9.1 ± 1.2 vs 7.2 ± 1.5, P<0.001, n = 10).
Thus, the results of this series of experiment clearly demonstrate that colon cancer is characterized by a predominance of vasodilator effects of epinephrine that is not observed in the normal state and in pathological changes of colon tissues associated with chronic stress influences and nitrosamines diet.

Hyperactivity of beta2-adrenergic signaling system associated with colon adenocarcinoma in rats
Above, we showed epinephrine-induced hyperperfusion in the malignant colon tissues in rats reflecting vasorelaxation after pharmacological stimulation of vascular adreno-receptors. The epinephrine causes an endothelial relaxation via activation of vascular B2-ADRs [26]. The beta-arrestin-1 (ARRB1) is a membrane co-factor of activation of B2-ADRs playing an important role in adrenergic vascular responses to stress [5,27]. Considering these facts, we hypothesized the possible role of adrenergic vasorelaxant mechanisms in colon adenocarcinoma development in rats. Fig. 1. The morphological changes in the colon tissues induced by chronic stress, nitrosamine diet, stress + nitrosamine and LSCI monitoring of mesenteric microcirculatory response to mucosal epinephrine injection, associated with the development of colon cancer: A -the normal colon tissues (control group); B -the peptic ulcer (stress group); C -the epithelial desquamation (nitrosamine group); D -colon adenocarcinoma (stress + nitrosamine group): H&E x 246; E -Representative LSCI images of mesenteric microcirculatory during baseline recording (left) and after mucosal epinephrine injection (right) in healthy rats. Blue indicates areas of low flux and red -high flux; F -LSCI images of mesenteric microcirculatory in rats with colon adenocarcinoma, mean ± SEM, n = 15 in the stress + nitrosamine group and n = 10 in the control/stress/nitrosamine groups; * -P<0.05; ** -P<0.001. Figure 2(a) illustrates LSCI monitoring of microvascular responses to pharmacological modulation of B2-ADRs using ICI 118551 (antagonist of B2-ADRs) and isoproterenol (agonist of B2-ADRs). Our results clearly demonstrate that vascular sensitivity to blockade and stimulation of B2-ADRs was greater in rats from the stress + nitrosamine group compared with the control/stress/nitrosamine groups (n = 15 in each group). The rats from the stress + nitrosamine group also demonstrated higher B2-ADRs expression in the colon tissues and ARRB1 level in the blood and in the colon tissues than rats from the control/stress/nitrosamine groups (Fig. 2(b-h), n = 10 in each group). One could notice that the microvascular responses to pharmacological tests as well as expression of B2-ADRs in the colon tissues and the serum and tissue ARRB1 levels did not differ significantly between the control/stress/nitrosamine groups.
Thus, these series of experiments clearly uncover hyperactivity of beta2-adrenergic signaling system associated with colon adenocarcinoma in rats compared with the normal state and GI injuries induced by chronic stress and nitrosamine diet. Fig. 2. The effects of chronic stress, nitrosamine diet and stress + nitrosamine on the expression of B2-ADRs and ARRB1 (the colon tissues) and on the level of ARRB1 (in the blood and in the colon tissues) in rats: A -LSCI data of microcirculatory response to submucosal injection of ICI 118551 and isoproterenol (n = 15 in each group); Bimmunoassay of the level of ARRB1 both in the blood and in the colon tissues (n = 10 in each group); C -F -immunohistochemical analysis of B2-ADRs expression in the colon tissues (n = 10 in each group); G and H -quantitate study of B2-ADRs and ARRB1 expression, including analysis of intensity and area of fluorescent signal, respectively: * -p<0.05; ** -p<0.001; † -p<0.05 between groups.

Colon adenocarcinoma is characterized by the elevated activity of NO-ergic system in mucosa of the colon tissues
Here we analyzed activity of NO-ergic system in the mucosa of colon tissues as an important mechanism underlying B2-ADR vasorelaxation [28][29][30]. Using pharmacological tests, we obtained high microvascular response to L-NIL (blocker of iNOS) in rats with colon adenocarcinoma compared with the rats from the control/stress/nitrosamine groups, which did not differ between each other (Fig. 3(a), n = 15 in each group). There were no any changes in microvascular response to L-NIO (blocker of endothelial NOS -eNOS) between all tested groups (data not presented). Spectrophotometric analysis of NO production in the colon tissues demonstrated a higher mucosal NO level in rats with colon adenocarcinoma vs. the control/stress/nitrosamines groups (Fig. 3(b), n = 10 in each group). Fig. 3. The effects of chronic stress, nitrosamine diet and stress + nitrosamine on activity of NO-ergic system in GI tissues: A -LSCI data of microcirculatory response to submucosal injection of L-NIL (n = 15 in each group); B -the tissue level of NO in the control/ stress/ nitrosamine/ nitrosamine + stress groups (n = 10 in each group); C -fluorescence intensity values at 635 nm, corresponding to the emission of 5-ALA/PpIX for control group of animals (without exogenous 5-ALA application), and with applied 5-ALA, as a precursor of PpIX without and with submucosal injection of nitroglycerin in rats with colon adenocarcinoma (n = 8 in each group). * -p<0.05; ** -p<0.001 vs. the control group; † -p<0.05 between groups.
The mucosal NO levels were also high in the stress and in the nitrosamine groups vs the control group. However, these changes were a 1.3-fold and 1.5-fold greater in rats with colon adenocarcinoma vs. the stress and nitrosamine groups. Interesting note, that nitroglycerin (NO donor) significantly increased PDD signal of 5-ALA/PpIX in rats with colon adenocarcinoma (Fig. 3(c), n = 8 in each group). So, fluorescent signal from the injured mucosa was a 4.5-fold greater after submucosal injection of nitroglycerin vs. the single treatment with 5-ALA/PpIX.
Thus, mucosal injuries induced by stress, nitrosamines and stress + nitrosamines are characterized by a high production of mucosal NO that was more pronounced in rats with adenocarcinoma compared with the stress and nitrosamine groups. These changes in NO mucosal level in rats with colon adenocarcinoma was associated with a significant increase in microvascular sensitivity to pharmacological modulation of iNOS but not eNOS, as well as with significantly increased photodynamic diagnostics effectiveness by nitroglycerin.

Discussion
Here we demonstrate the development of rat colon adenocarcinoma induced by long combination of two factors, such as social stresses (isolation) and daily nitrosamines diet. Using this model, we uncover that stress-induced colon adenocarcinoma is associated with atypical vasorelaxation effects of epinephrine assessed by LSCI, as well as with the high expression of B3-ADRs and with the high level of ARRB1, both in the blood and in the colon tissues compared with the control, stress and nitrosamines groups. Our data are consistent with other results suggesting that the overexpression of B2-ADRs promotes of invasion and metastasis of GI cancers [31][32][33].
The uncovered atypical effects of epinephrine might be an important diagnostic approach in prognosis of catecholamine-induced immunotherapy resistance. So, the catecholamineinduced activation of B2-ADRs mediates resistance of GI tissues to immunotherapy by upregulating MUC4 expression [34]. Cross talk between Her2 and B2-ADRs activates a complex intracellular signaling network, which contributes to the drug resistance and failure of targeted therapeutics [4,35,36].
Our results clearly demonstrate that submucosal nitroglycerine injection significantly increased fluorescent intensity detected during photodynamic diagnostics procedure of malignant colon tissues using 5-ALA/PpIX that was also associated with the high mucosal NO level and elevated microvascular sensitivity to blockade of iNOS, but not eNOS.
Thus, our results indicate that the high level of NO associated with colon adenocarcinoma might be related to hyperactivity of iNOS. Among the NO synthase enzymes, the inducible isoform has the most diverse range, participating in numerous carcinogenic processes. Among human cancers, GI cancer is the one most closely associated with chronic inflammation. In the mid 1990's, many studies showed that iNOS promote GI tumor progression. So, Jenkins et al. demonstrated in experiments on nude mice that iNOS overexpression in a human COAD cell line increases the tumor growth [37]. Later, it has been shown that high expression of iNOS mRNA in GI tissues is associated with human colon adenoma and carcinoma [38,39]. Studies in the early 2000's showed the overexpression of iNOS in 50-60% of patients with colon cancer and that iNOS expression correlated with decreased survival and increased metastasis activity [40,41]. Further evidence of high NO levels in GI cancer comes from footprints such as nitrotyrosine formation and S-nitrosation of protein thiols. The abnormal S-nitrosation induced by the activation of iNOS modulates inflammation and GI cancer [22].
The increase in photodynamic response by nitroglycerin might be a novel pharmacological approach for improving of clinical value of photodynamic diagnosis (PDD) of GI cancer using 5-ALA/PpIX that has been reported in several publications [42][43][44]. 5-ALA itself is not a fluorophore, it is a precursor of fluorescence emitting protoporphyrin IX (PpIX), which accumulates specifically in tumor cells and emits fluorescence when the tumor is irradiated with excitation light on appropriate wavelength [45,46]. PpIX accumulation is observed in normal tissues, especially in the gastrointestinal mucosa, at 2-4 h after 5-ALA administration [47]. Thereafter, the accumulated PpIX in normal tissues is gradually metabolized, whereas PpIX accumulation in cancer cells is sustained for a while. After 6 h, the difference in PpIX concentrations between normal tissue and cancer tissue reaches its peak [46]. Thus, the timing of 5-ALA administration should be based on the estimated duration to fluorescence observation. However, the major limitation of the success of PDD is the relatively poor tumor selectivity after administration of the photosensitizer. The PDD of early-stage GI tumors continues to improve owing to technological advances in endoscopy, yet some tumors are still being missed. Thus, PDD itself is not sufficient to improve the detection rate of GI tumors and strongly needs to effective additional methods, which will increase diagnostic effectiveness of PDD [44,48].

Conclusions
Using an original model of stress-induced colon adenocarcinoma, we uncover atypical vasorelaxation effects of epinephrine in colon mucosa that is associated with the high expression of mesenteric B2-ADRs and with the high level of ARRB1 both in the blood and in the colon tissues in rats with colon cancer. Our results clearly demonstrate a high mucosal level of NO, elevated sensitivity of mesenteric vessels to blocked of iNOS and an increased photodynamic diagnostics effectiveness by nitroglycerin in rats with colon adenocarcinoma vs. the control, stress and nitrosamine groups. These data shed light on mechanisms underlying stress-induced colon cancer that is characterized by hyperactivity of adrenergic and NOergic systems in GI tissues.