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Abstract

Objective:

The study explored the chemoprophylactic potential of roflumilast against 1,2-dimethylhydrazine (DMH) actuated preneoplastic colon damage in albino Wistar rats.

Methods:

Animals were arbitrarily divided into five groups of six animals each. DMH was used to induce preneoplastic colon damage (20 mg/kg/7 days, subcutaneously, for 42 days). Roflumilast was administered subcutaneously at two doses (1 and 5 mg/kg/day, from day 28 to 42). At the end of the study, the animals were recorded for the electrocardiographic changes and heart rate variability (HRV) paradigms on 42nd day, using PowerLab system. Blood samples were collected from all the animals to measure hydrogen sulfide (H₂S) and nitric acid. The colon tissue was dissected out and analyzed for inflammatory markers, biochemical parameters including, superoxide dismutase, thiobarbituric acid reactive substances, catalase, and glutathione reductase and histopathology.

Results:

DMH caused derangement of HRV factors, abnormal antioxidant markers, and elevated levels of inflammatory markers. H₂S and nitric oxide levels upsurge in DMH-treated rats and promoted preneoplastic damage. Histopathologically, loss of crypts, goblet cells, and distorted lamina propria were observed in toxic group. Treatment with roflumilast was able to curtail down oxidative stress and inflammatory markers and stabilitate the hemodynamic derangements as well as was able to restore the normal architecture of colonic mucosa.

Conclusion:

The findings from the present study conclude that treatment with roflumilast positively modulates the preneoplastic colon damage.

Keywords

Colon cancer DMH roflumilast phosphodiesterase-4 inhibitors

Introduction

The link between chronic inflammation and tumor progression is well established, long-standing inflammation secondary to chronic infection such as inflammatory bowel disease, Crohn’s disease, and ulcerative colitis predisposes to colon cancer.¹ The treatment options for colon cancer remain limited and prognosis remains poor, henceforth there is urge of novel therapeutic targets for treatment of colon cancer. The phosphodiesterases (PDEs) are a group of enzymes that are coded by 21 genes in humans and comprise a superfamily of 11 PDEs based on structural similarity, which function to break down the phosphodiester bond in the 2° messenger cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate.² Interestingly, phosphodiesterase-4 inhibitors (PDE4is) are implemented in the treatment of various cancer pathologies including B-cell malignancies, lung cancer, pancreatic cancer, and malignant melanomas by impairing growth and chemotaxis as well as increasing apoptosis.³ Evidence suggests the involvement of PDE4 in early stage of colon cancer and inhibition of PDE4 has been found to trigger luminal apoptosis in colon cancer crypt model.⁴

Recently, increased level of cAMP has been related to inhibition of cellular proliferation in colon cancer.⁵ The level of cAMP is regulated by equilibrium between venture of two categories of enzymes, the adenylyl cyclase and PDE, cAMP-generating and cAMP-degrading enzymes, respectively. Triggered by extracellularly explicated signals, the G-protein coupled receptor binds with diverse inflammatory mediators such as histamine, leukotrienes, prostaglandins, chemokines and results in activation of cAMP which further interacts with protein kinase A (PKA) and Epac, that is, exchange protein activated by cAMP to elicit downstream signaling.⁶ PKA further phosphorylates the cAMP response element-binding protein (CREB) and activating transcription factor (ATF-1), along with inhibition of inflammatory promoters like nuclear factor-kappa beta (NF-κβ). Such combined inhibitory effects on CREB, ATF-1, and NF-κβ result in downregulated messenger RNA expression of cytokines (TNF-α, IL-6, and IL-18) as well as an upsurge in anti-inflammatory activity.⁷ Henceforth, elevating the level of cAMP by inhibition of PDE can be effective strategy to combat preneoplastic colon damage.

Roflumilast is an isoenzyme-selective PDE4i. Roflumilast gets converted to its active metabolite roflumilast N-oxide in vivo which increases the level of cAMP by inhibiting PDE4.

Roflumilast being a selective PDE4i overcomes the drawbacks of nonselective PDE4i that produce a number of side effects, including nausea, vomiting, diarrhea, and weight loss that limit their clinical efficacy.

One of the serious problems in treating colon cancer is chemoresistance to cytotoxic chemoprophylactic agents so there is demand for novel therapeutic drugs. Henceforth, in this study, effort has been made to prevent colon cancer by inhibiting PDE4-activated inflammatory pathway of cancer by roflumilast.

Methods

Drugs and chemicals

Roflumilast (Rofmil tablet, manufactured by Intas Pharmaceuticals Ltd, Bagheykhola, Sikkim, India), leucovorin injection IP (Leutero, manufactured by Hetero Pharmaceuticals Ltd, Baddi, Himachal Pradesh, India), and 5-fluorouracil (Dabur Pharmaceuticals Ltd, Lucknow, Uttar Pradesh, India) were procured from the market. 1,2-Dimethylhydrazine (DMH) was purchased from ACROS Organics, Brand—Thermo Fisher Scientific, Fair Lawn, New Jersey, USA. Other reagents and chemicals were of analytical grade and purchased from Hi-media Labs (Mumbai, Maharashtra, India); else otherwise mentioned in the text.

Experimental animals

Male albino Wistar rats were obtained from central animal house facility. The animals were then sheltered in cages under controlled environment (23°C, 12 h light/dark cycle), with access to a standard pellet diet and drinking water ad libitum. The experiments were carried out according to the CPCSEA guidelines, Department of Animal Welfare, Government of India (Animal approval number: IAEC/SHUATS/17MBM14).

Experimental protocol

Experimental animals were randomly divided into five sets of six animals each (n = 6). Two doses of roflumilast were selected in present study.⁸ Group 1—control (received 1 mM ethylenediaminetetraacetic acid [EDTA] prepared in normal saline at the dose of 2 ml/kg/day, subcutaneously [s.c.] for 42 days); group 2—standard (DMH, 20 mg/kg/7 days, s.c. for 42 days + 5-fluorouracil, 25 mg/kg, and leucovorin, 25 mg/kg, both intraperitoneally [i.p.] on 33rd, 35th, 39th, and 42nd days of study); group 3—roflumilast low dose (DMH, 20 mg/kg/7 days, s.c. for 42 days + roflumilast, 1 mg/kg/day, orally [p.o.] from day 28 to 42); group 4—roflumilast high dose (DMH, 20 mg/kg/7 days, s.c. for 42 days + roflumilast, 5 mg/kg/day, p.o. from day 28 to 42); and group 5— toxic (DMH, 20 mg/kg/7 days, s.c. for 42 days).

The animals were recorded for the electrocardiographic (ECG) changes and heart rate variability (HRV) paradigms on 42nd day, using the protocol specified in the forthcoming section. After the completion of individual group as per the specified protocol, the blood aliquots were acquired under chloroform anesthesia through retro-orbital plexus in centrifugation tubes. The blood aliquots were incubated and centrifuged at 10,000 r/min for 15 min to collect serum. The serum samples were kept at −20°C till further use. Afterward, animals were euthanized under ether anesthesia. The colon tissue was dissected and removed by locking both ends with a surgical suture (to prevent drainage of the content). The colon contents and the tissues were further evaluated against various prototypic changes and samples were stored at −20°C till further use.

Weight variation

Percentage variation in weight among all the sets was determined by the following formula

%Weight variation= [final weight (g) - initial weight (g) /final weight (g) ×100]

Hemodynamic changes

The experimental animals were anesthetized using ketamine HCl (50 mg/kg, intramuscularly [i.m.]) and diazepam (5 mg/kg, i.m.) in cocktail and thereby mounted on a wax tray. The hemodynamic changes were taped using PowerLab system; electrodes for recording were placed on the skin of ventral and dorsal thorax to tape the ECG signals. The electrodes were coupled to bio-amplifier (ML-136) and PowerLab channel (ML-826) to convert analog to digital signals (AD Instruments, Australia). The ECG signals were documented on the hard disk and interpreted offline using Lab Chart Pro-8 (AD Instruments).

ECG and HRV analysis was performed on various sections of continuous ECG signals taped in the foregoing section. First of all, the raw data signals were interpreted manually to assure that all the R waves were identified accurately. Thereafter, heart rate (HR) was calculated by plotting the number of waves per unit time. On similar grounds, time and frequency domain paradigms of HRV were calculated using the Lab Chart Pro-8).^9,10

Assessment of pH and total acidity

Following the treatment, animals were euthanized by cervical dislocation following which the colon tissue was collected. The content of the colon tissue was collected and pH of the solution was measured using pH meter (Labman Scientific Instrument, Chennai, India, LMPH-10).¹¹

Total acidity was evaluated using the procedure described previously and expressed as mEq.¹²

Antioxidant markers

The colon tissues were accurately weighed (10% w/v) and homogenized in 0.15 M KCl and centrifuged at 15,000 r/min for 10 min at 4°C in a cooling centrifuge. The supernatants were collected and analyzed for biochemical parameters including, superoxide dismutase (SOD), thiobarbituric acid reactive substances (TBARS), catalase, and glutathione reductase (GRx) using the protocols established at our lab.^13,14

Cyclooxygenase (COX) estimation

Twenty microliter of serum and 160 µl Tris buffer (50 mM) was incubated for 5 min. A 10 µl of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) reagent and arachidonic acid (AA) solution were added and read at 630 nm using multiplate reader (ALERE Microplate Reader, Haryana, India, AM-2100) at 0 and 1 min interval. AA solution was prepared by mixing 50 µl of the 40 mM AA with 50 µl of 0.1 N potassium hydroxide using vortexing and subsequently 900 µl of double-distilled water. TMPD stock solution was prepared by dissolving 0.3 mg in 1 ml of distilled water and subsequent 1:10 dilution was prepared for the assay.^15,16

Lipoxygenase (LOX) estimation

Twenty-five microliter of AA solution was added to the 475 µl supernatant (as prepared for cyclooxygenase (COX) assay) and incubated for 6 min. A 500 µl of ferrithiocyanate reagent was added and read at 480 nm using ultraviolet (UV) spectrophotometer (Cary 60, Agilent Technologies International Private Limited, Santa Clara, California, USA, FeSO₄ in 0.2 M HCl) and reagent 2 (3% NH₄SCN in methanolic solution) in 1:1 ratio.^17,18

Estimation of plasma hydrogen sulfide

The hydrogen sulfide (H₂S) estimation was performed using the method of Ang et al.¹⁹ with slight modifications. Briefly, 150 µl of plasma was added to 500 µl of premixed zinc acetate (1% w/v) solution and the volume was made up to 1.5 ml with milli-Q water. This mixture was further mixed with 532 µl of dye (266 µl of 20 mM N, N-dimethyl-p-phenylenediamine [NNDP] in 7.2 M HCl and 266 µl of 30 mM FeCl₃ in 1.2 M HCl). The mixture was incubated at 37°C for 10 min. Five hundred microliter of trichloroacetic acid (10% w/v) was added to the above mixture and centrifuged for 5 min at 12,000 r/min. Supernatant was taken and absorbance was read spectrophotometrically at 670 nm (Carry 60, Agilent Technologies).²⁰

Assay for nitric acid—Griess method

Generation of nitric acid (NO) in the plasma sample was arbitrated by measuring nitrite accumulation using Griess reagent (1% sulphanilamide, 0.1% N-(1-napthyl)-ethylene diamine dihydrochloride in 5% H₃PO₄). Equal quantity (500 μl) of plasma and Griess reagent were mixed and incubated at 37°C for 30 min. The test mixture was subsequently read on a UV–visible spectrophotometer (Cary 60, Agilent Technologies) at 540 nm using appropriate blank. Sodium nitrite was used to prepare a standard curve.²¹

Histopathology

Colon tissues were evaluated histopathologically by hematoxylin and eosin (H&E) staining. The tissues were fixed in paraformaldehyde for overnight succeeded by 70% isopropanol overnight. Tissues were further exposed to increasing concentration of isopropanol (70%, 90%, and 100%), superseded by dehydration with 100% xylene. The tissues were embedded in paraffin wax and blocks were prepared. Five millimeter sections were prepared using microtome followed by staining with H&E. The sections were visualized and photographed at 40× using digital biological microscope (N120, BR-Biochem Life Sciences, New Delhi, India).^22,23

Statistical analysis

All data were presented as mean ± standard deviation and analyzed by one way analysis of variance followed by Bonferroni’s multiple comparison tests for the possible significance identification between the various groups *p < 0.05, **p < 0.001, and ***p < 0.01 were considered statistically significant. Statistical analysis was carried out using GraphPad Instat (3.10) (San Diego, California, USA).

Results

Effect of roflumilast on hemodynamic changes

HRV is a biomarker of the autonomic nervous system (ANS) and it provides a measure of ANS through sympathetic and parasympathetic nervous system. HRV is a useful noninvasive tool to evaluate the prognosis of cancer patients. The results show that DMH administration demonstrated distortion in the ECG profile characterized by shortening of RR interval, increase in the heartbeat along with prolongation of QRS, and decreased P wave amplitude in comparison to control (Figure 1).

Figure 1.

Effect of roflumilast treatment on ECG recording. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for autonomic dysfunction relevant in the context of pathophysiology of colon cancer. Groups were differentiated as (1) control (1 mM EDTA + saline, 2 ml/kg, s.c.); (2) standard (DMH, 20 mg/kg, s.c. + 5-FU, 25 mg/kg, i.p. + LU, 25 mg/kg, i.p.); (3) roflumilast low dose (DMH, 20 mg/kg, s.c + roflumilast, 1 mg/kg, p.o.); (4) roflumilast high dose (DMH, 20 mg/kg, s.c. + roflumilast, 5 mg/kg, p.o.); and (5) toxic (DMH, 20 mg/kg, s.c.). For presented ECG recordings, in the box plots, the boxes denote interquartile ranges, horizontal lines inside the box denote the median, and bottom and top boundaries of boxes are 25th and 75th percentiles, respectively. Lower and upper whiskers are 5th and 95th percentiles, respectively. ECG: electrocardiographic; DMH: 1,2-dimethylhydrazine.

Aberration in ECG profile was recorded for the time domain (average RR, median RR, standard deviation of RR intervals [SDRR], and coefficient of variation of RR intervals [CVRR]) and frequency domain (low frequency [LF], high frequency [HF], and LF/HF) parameters after DMH treatment. Roflumilast treatment restored the HRV paradigms toward normal with more profound effects by roflumilast low dose (Figure 2).

Figure 2.

Effect of roflumilast treatment on HRV recording. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for autonomic dysfunction relevant in the context of pathophysiology of colon cancer. Groups were differentiated as (1) control (1 mM EDTA + saline, 2 ml/kg, s.c.); (2) standard (DMH, 20 mg/kg, s.c. + 5-FU, 25 mg/kg, i.p. + LU, 25 mg/kg, i.p); (3) roflumilast low dose (DMH, 20 mg/kg, s.c + roflumilast, 1 mg/kg, p.o.); (4) roflumilast high dose (DMH, 20 mg/kg, s.c. + roflumilast, 5 mg/kg, p.o.); and (5) toxic (DMH, 20 mg/kg, s.c.). For presented HRV recordings, in the box plots, the boxes denote interquartile ranges, horizontal lines inside the box denote the median, and bottom and top boundaries of boxes are 25th and 75th percentiles, respectively. Lower and upper whiskers are 5th and 95th percentiles, respectively. HRV: heart rate variability; DMH: 1,2-dimethylhydrazine.

Effect of roflumilast on pH, total acidity, and percentage weight variation

The gastric pH showed a significant decrease in the toxic group (6.62 ± 0.26) as compared to control (7.12 ± 0.11). Roflumilast at low dose (7.09 ± 0.05) resulted in significant increase in pH but no significant variation in pH was observed in standard (6.84 ± 0.08) and roflumilast high dose (6.74 ± 0.06) groups as compared to toxic group (6.62 ± 0.26).

On the same note, there was a significant upsurge in total acidity value in the toxic group (70.00 ± 15.63) as compared to control (24.45 ± 5.44), standard (38.89 ± 7.79), roflumilast high dose (44.45 ± 8.07), and roflumilast low dose (36.67 ± 6.99) groups.

Variation in weight was found to be insignificant among toxic (20.35 ± 6.62), standard (24.06 ± 10.88), control (22.12 ± 7.73), roflumilast low dose (17.87 ± 5.19), and roflumilast high dose (9.97 ± 6.82) groups (Table 1).

Table 1.

Effect of roflumilast on weight variation, pH, and total acidity against DMH-induced preneoplastic colon damage.^a

Groups	Variation in weight (%)	pH	Total acidity (mEq/l)
1. Control 1 mM EDTA + saline (2 ml/kg, s.c.)	22.12 ± 7.73	7.12 ± 0.11^b	24.45 ± 5.44^b
2. Standard 5-FU + LU (25 mg/kg each i.p.) + DMH (20 mg/kg, s.c.)	24.06 ± 10.88	6.84 ± 0.08	38.89 ± 7.79^b
3. Roflumilast low dose Roflumilast (1 mg/kg, p.o.) + DMH (20 mg/kg, s.c.)	17.87 ± 5.19	7.09 ± 0.05^b	36.67 ± 6.99^b
4. Roflumilast high dose Roflumilast (5 mg/kg, p.o.) + DMH (20 mg/kg, s.c.)	9.97 ± 6.82	6.74 ± 0.06	44.45 ± 8.07^b
5. Toxic DMH (20 mg/kg, s.c.)	20.35 ± 6.62	6.62 ± 0.26	70 ± 15.63

DMH: 1,2-dimethylhydrazine; ANOVA: analysis of variance; SD: standard deviation.

^a Values are represented as mean ± SD (n = 6). Groups were compared on the basis of the one-way ANOVA followed by the Bonferroni test. All groups were compared to the toxic group.

^b p < 0.001.

Effect of roflumilast on oxidative stress markers

Oxidative stress is an imbalance between reactive oxygen species (ROS) production and the counteractive ability of antioxidants. DMH administration in albino Wistar rats resulted in significant increase in malondialdehyde (MDA) level (0.66 ± 0.01 nmol of MDA/µg of protein) as compared to control (0.43 ± 0.02 nmol of MDA/µg of protein), whereas treatment resulted in significant decrease in MDA level with most significant decrease in roflumilast low dose (0.48 ± 0.02 nmol of MDA/µg of protein) followed by roflumilast high dose (0.57 ± 0.03 nmol of MDA/µg of protein) and standard (0.54 ± 0.05 nmol of MDA/µg of protein) as compared to toxic group (Table 2).

Table 2.

Consequence of roflumilast administration on oxidative stress markers against DMH-induced colon damage.^a

Groups	TBARS × 10⁻² (nmol of MDA/µg of protein)	GRx (mg %)	Protein carbonyl (nmol/ml)	SOD (units of SOD/mg of protein)	Catalase (nM of H₂O₂/min/mg of protein)
1. Control 1 mM EDTA + saline (2 ml/kg, s.c.)	0.43 ± 0.02^b	0.50 ± 0.03^b	292.12 ± 35.17	0.017 ± 0.003^c	0.11 ± 0.03^b
2. Standard 5-FU + LU (25 mg/kg each i.p.) +D MH (20 mg/kg, s.c.)	0.54 ± 0.05^b	0.47 ± 0.03^b	427.6 ± 36.26^b	0.013 ± 0.006^b	0.07 ± 0.008^b
3. Roflumilast low dose Roflumilast (1 mg/kg, p.o.) + DMH (20 mg/kg, s.c.)	0.48 ± 0.02^b	0.31 ± 0.003^b	174.02 ± 10.02^b	0.016 ± 0.004^c	0.08 ± 0.013^b
4. Roflumilast high dose Roflumilast (5 mg/kg, p.o.) + DMH (20 mg/kg, s.c.)	0.57 ± 0.03^b	0.39 ± 0.01^b	216.15 ± 5.1^b	0.027 ± 0.006	0.18 ± 0.015
5. Toxic DMH (20 mg/kg, s.c.)	0.66 ± 0.01	0.73 ± 0.002	474.34 ± 13.32	0.033 ± 0.011	0.20 ± 0.005

DMH: 1,2-dimethylhydrazine; TBARS: thiobarbituric acid reactive substances; MDA: malondialdehyde; GRx: glutathione reductase; SOD: superoxide dismutase; ANOVA: analysis of variance; SD: standard deviation.

^a Values are represented as mean ± SD (n = 6). Groups were compared on the basis of the one-way ANOVA followed by the Bonferroni test. All groups were compared to the toxic group.

^b p < 0.001.

^c p < 0.01.

The GRx level was upregulated in the toxic group (0.73 ± 0.002) as compared to control (0.50 ± 0.03) whereas decreased significantly in standard drug (0.47 ± 0.03) followed by roflumilast high dose (0.39 ± 0.01) and roflumilast low dose (0.31 ± 0.003) in comparison to toxic group (Table 2).

As far as the level of protein carbonyl is concerned it was found to be significantly high in the toxic group (474.34 ± 13.32) as compared to control (292.12 ± 35.17). Comparing the treatment and standard group with the toxic and standard group was found effective and there was significant decrease with most prominent decrease in roflumilast low dose (174.02 ± 10.02).

Similarly, SOD was significantly upregulated in the toxic group (0.033 ± 0.011 units of SOD/mg of protein) as compared to control group (0.017 ± 0.003) but achieved normal values in standard (0.013 ± 0.006) and roflumilast low dose (0.016 ± 0.004) groups as compared to toxic group (Table 2).

The same trend was observed with catalase enzyme as it was found to be upregulated in the toxic group (0.20 ± 0.005 nM of H₂O₂/min/mg of protein) and roflumilast high dose (0.18 ± 0.015 nM of H₂O₂/min/mg of protein) as compared to control (0.11 ± 0.03 nM of H₂O₂/min/mg of protein) but normalized in standard (0.07 ± 0.008) followed by roflumilast low dose (0.08 ± 0.013 nM of H₂O₂/min/mg of protein) as compared to toxic group (Table 2).

Effect of roflumilast on inflammatory markers

Chronic inflammation is characterized by infiltration with macrophages, lymphocytes, and plasma cells; the secretion of COX and prostaglandins which leads to tissue destruction due to continuous production of ROS and nitrogen species. All these processes may lead to changes in cells and form cancer. For the estimation of these inflammatory markers, COX and lipoxygenase (LOX) activity was checked in the serum samples of the animals. The enzymatic COX and LOX activity was upregulated in the toxic group which was reverted back by roflumilast treatment especially with a low dose of roflumilast (Figure 3).

Figure 3.

Effect of roflumilast on COX, LOX, H₂S, and NO against DMH-induced colon carcinogenesis. Values are represented as mean ± SD (n = 6). Comparisons were made on the basis of the one-way ANOVA followed by the Bonferroni test. All groups were compared to the toxic group (**p < 0.01; ***p < 0.001): (1) control (1 mM EDTA + saline, 2 ml/kg, s.c.); (2) standard (DMH, 20 mg/kg, s.c. + 5-FU, 25 mg/kg, i.p. + LU, 25 mg/kg, i.p.); (3) roflumilast low dose (DMH, 20 mg/kg, s.c. + roflumilast, 1 mg/kg, p.o.); (4) roflumilast high dose (DMH, 20 mg/kg, s.c. + roflumilast, 5 mg/kg, p.o.); and (5) toxic (DMH, 20 mg/kg, s.c.). COX: cyclooxygenase; LOX: lipoxygenase; H₂S: hydrogen sulfide; NO: nitric acid; DMH: 1,2-dimethylhydrazine; ANOVA: analysis of variance; SD: standard deviation.

Effect of roflumilast on gaseous mediators of inflammation such as H₂S and NO

NO and H₂S are gaseous transmitters and responsible for proliferation and cell death. The gaseous mediators of inflammation such as H₂S and NO were found to be significantly elevated (p < 0.001) in the toxic group which was further restored by roflumilast treatment (Figure 3).

Effect of roflumilast on histopathological studies

Histopathologically control group showed normal crypts, goblet cells along with lamina propria. Loss of crypts, goblet cells, and distorted lamina propria was found in the toxic group and to some extent in roflumilast high dose (5 mg/kg); however, roflumilast low dose (1 mg/kg) helped to restore the normal architecture of colonic mucosa (Figure 4).

Figure 4.

Histopathological evaluation of the colonic tissue with H&E staining: (1) control (1 mM EDTA + saline, 2 ml/kg, s.c.); (2) standard (DMH, 20 mg/kg, s.c. + 5-FU, 25 mg/kg, i.p. + LU, 25 mg/kg, i.p.); (3) roflumilast low dose (DMH, 20 mg/kg, s.c. + roflumilast, 1 mg/kg, p.o.); (4) roflumilast high dose (DMH, 20 mg/kg, s.c. + roflumilast, 5 mg/kg, p.o.); and (5) toxic (DMH, 20 mg/kg, s.c.). H&E: hematoxylin and eosin; DMH: 1,2-dimethylhydrazine.

Discussion

The present study was carried out to assess the effect of roflumilast on DMH instigated preneoplastic colon damage in albino Wistar rats. Plethora of studies suggest poor survival prognosis in cancer patients with worsening of their ECG and HRV parameters. Autonomic dysfunction is a prominent marker of cancer. Deteriorated ECG and diminished HRV, being a strongest marker of autonomic dysfunction can be associated with the development of advanced cancer.²⁴ HRV also provides an insight into both the branches of ANS, sympathetic activity (LF band) and parasympathetic activity (HF band) and their ratio (LF/HF) represents the so called “Sympathovagal balance.”^25,26 A decrease in LF/HF signifies stress and imbalance between the two branches of ANS. As ratified by the results roflumilast treatment restored sympathovagal balance (LF/HF). The RR interval represents the time gap between two heartbeats. RR interval found to be decreased in toxic group signifying increased HR. As validated by the result, treatment with roflumilast restored normal HR.

Cancer cells derive their energy from anaerobic glycolysis pathway, making the microenvironment around the cancer cells much more acidic (due to formation of lactic acid) and a subsequent decrease in pH.²⁷ Henceforth, abrupt rise in total acidity values could be directly correlated with cancer cell proliferation. Results observed in the present study corroborates with the findings of Rawat et al.²⁸ as animals treated with DMH results in decreased pH and increased total acidity. The observed rise in total acidity and fall in pH in the toxic group was significantly restored by roflumilast treatment.²⁹

Plethora of research over the past few decades demonstrates that ROS plays a pivotal role in carcinogenesis as well as cancer cell survival. It is also well established that cancer cells thrive under oxidative stress. In addition, oxidative DNA damage by ROS generated by partial reduction of O₂ like $O_{2}^{2 -}$ anion, H₂O₂, and OH⁻ ion contribute to genetic mutations which result in carcinogenesis.³⁰ In comparison to non-cancer tissue, ROS levels are inflated in, colon, pancreatic, breast, prostate, and other types of cancers. Rise in the level of ROS results in lipid peroxidation (rise in MDA level) as well as upregulation of SOD, catalase, and glutathione peroxidase as these three enzymes work in conjunction to scavenge ROS. TBARS and PC are the sensitive and reactive markers of membrane damage and are stable products of lipid and protein peroxidation.¹¹ Similar findings were also observed in this study as the toxic group treated with DMH there was an upsurge in the level of SOD, catalase, and glutathione peroxidase as well as increased level of TBARS and protein carbonyl that could be accredited to the high level of ROS.³¹ Treatment with roflumilast established normal levels of biochemical oxidative stress markers.

COX and LOX serve as inflammatory markers in various pathological conditions including cancer. An upsurge in their activity contributes to chronic inflammation due to the formation of prostaglandins (especially PGE2) and leukotrienes (especially LTB4). Inflammatory cells have overexpression of inflammatory genes including COX and LOX and evidence suggests that tumor infiltration by inflammatory cells is hallmark of colon cancer.³² COX, in particular COX-2, has been shown to be increased in colon cancer.⁹ In the same pattern, we also observed increased levels of COX in the DMH-treated animals and roflumilast helped to downregulate the increased levels of COX. The increase in the level of LOX can also be correlated with colon cancer, similarly upregulated LOX expression was found in toxic group which was significantly restored by roflumilast treatment.

Role of gaseous mediators in colo-rectal cancer is still unclear but evidence suggests that elevated levels of these two gases serve as inflammatory mediators. In this regard, overexpression of cystathionine-β-synthase particularly in colon cancer tissue produces large amount of H₂S^33,34 as well as H₂S could also inhibit apoptosis^34,35; on the other hand, various in vitro and in vivo investigations suggest the possible involvement of NO in the advancement of tumor angiogenesis.³⁶ It has been demonstrated that NO induces endothelial cell growth and regulates the tumor blood flow. In addition, in a study it was seen that knocking down iNOS gene in APC^(Min/+) mice resulted in marked decrease in the number of intestinal polyps. Henceforth, it is clear that increased level of NO and H₂S promote colon carcinogenesis³⁷ and same way in this study also, high levels of NO and H₂S was found in toxic group which was reverted back to normal by roflumilast treatment.

Colorectal malignancies originate from budding of an adenomatous polyp which can be seen as well as differentiated masses of epithelial dysplasia and crypt cell proliferation. The histopathological picture revealed large number of serrated adenomas, distorted crypts, and lamina propria in the toxic group whereas the treatment with roflumilast nudged down epithelial dysplasia, crypt cell proliferation, and damage to lamina propria along with restoring the surface architecture of colonic mucosa.

Conclusion

The authors would like to conclude that treatment with roflumilast can positively modulate the preneoplastic colon damage by downregulating the inflammatory markers, antioxidant parameters, and stabilizing the histopathological parameters. All in all, our findings emphasize the potential role of roflumilast in the suppression of colon carcinogenesis and opens future endeavors of research toward exploring the potential of roflumilast.

Footnotes

Author contributions

ASS and SR designed the experiments and collected the samples. SR performed the experiment and collected the data. ASS, SR, and MNA analyzed the data and wrote the manuscript. ASS and SR contributed equally to this work. All the authors contributed to modify the final version of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Deanship of Scientific Research, Prince Sattam Bin Abdulaziz University, Saudi Arabia (Research Project Number: 2019/03/10464).

ORCID iD

MN Ansari

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Roflumilast counteracts DMH-induced preneoplastic colon damage in albino Wistar rats

Abstract

Objective:

Methods:

Results:

Conclusion:

Keywords

Introduction

Methods

Drugs and chemicals

Experimental animals

Experimental protocol

Weight variation

Hemodynamic changes

Assessment of pH and total acidity

Antioxidant markers

Cyclooxygenase (COX) estimation

Lipoxygenase (LOX) estimation

Estimation of plasma hydrogen sulfide

Assay for nitric acid—Griess method

Histopathology

Statistical analysis

Results

Effect of roflumilast on hemodynamic changes

Effect of roflumilast on pH, total acidity, and percentage weight variation

Effect of roflumilast on oxidative stress markers

Effect of roflumilast on inflammatory markers

Effect of roflumilast on gaseous mediators of inflammation such as H2S and NO

Effect of roflumilast on histopathological studies

Discussion

Conclusion

Footnotes

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

References

Effect of roflumilast on gaseous mediators of inflammation such as H₂S and NO