Ameliorative effect of fluvoxamine against colon carcinogenesis via COX-2 blockade with oxidative and metabolic stress reduction at the cellular, molecular and metabolic levels

Highlights

• •Colorectal carcinoma (CRC) is third most frequent malignancy in the world.
• •We investigated the anti-CRC potential of fluvoxamine (FLX) using HT-29 cells (IC₅₀ ∼x223C10 µg/ml).
• •FLX suppress tumour, via blockade of COX-2 and IL6 in western blot and qRT-PCR analysis.
• •Histopathology and ACF count reveal the restoration of ruptured architecture of colon tissue after FLX treatment.
• •NMR based metabolomics revealed restoration of altered metabolites during carcinoma to normal levels.

Abstract

Fluvoxamine's (FLX's) anticancer potential was investigated in pre-clinical research utilizing a DMH-induced colorectal cancer (CRC) rat model. qRT-PCR and immunoblotting validated the mechanistic investigation. The CRC condition was induced in response to COX-2 and IL-6, however, following FLX therapy, the condition returned to normal. FLX's anti-CRC potential may be attributable to COX-2 inhibition since this molecular activity was more apparent for COX-2 than IL-6. FLX repaired the altered metabolites linked to CRC rats, according to ¹H-NMR analysis. FLX was shown to be similar to 5-FU in terms of tumor protection, which may be useful in future medication development.

1. Introduction

The third most frequent malignancy is colorectal carcinoma (CRC) which occurs in both women and men, and second-ranked for cancer-related death in the world in 2015 [1]. It has commonly occurred in old age, as well as the young generation in India due to changes in lifestyles and food habits [2], [3], [4]. CRC is treated with 5-fluorouracil (5-FU) for many years along with a few recent newer agents i.e. cetuximab and bevacizumab [4]. Synthetic agents have been shown to have moderate benefits due to high costs and drug resistance [5]. Hence, the development of a new molecule with a molecular pathway mechanism for CRC treatment is the first priority to researchers. Therefore, it is necessary to explore newer anti-CRC agents from the existing marketed drug molecule to prolong the survival of the patient.

Recent research suggested that 5-hydroxytriptamine (5-HT) promotes CRC via regulation of β-catenin/matrix metalloprotinase-7 (MMP7) pathway [6]. 5-HT reuptake inhibitor, fluoxetine had antineoplastic potential against in vitro colon cancer cells and also in the in vivo colonic carcinogen mouse model [7]. Therefore, we questioned whether fluvoxamine (FLX) has a similar effect to fluoxetine as both are structurally similar. Another literature pointed that FLX inhibited human glioblastoma invasion by disrupting acting polymerization [8]. Before performing an in vivo study, we investigated the anti-CRC potential of FLX using a human colon cancer cell line (HT-29 cells) and demonstrated its strong antiproliferative potential against HT-29 cells (50% inhibitory concentration, IC₅₀ ∼x223C10 µg/ml) (Supplementary Data Sheet, Fig. S1). Inspired by the aforementioned finding, we speculated that FLX might be an effective agent for the treatment of CRC.

The previous investigation further pointed out that oxidative stress-related cancer and injury have vital roles in CRC development and that the carcinogenic action of dimethylhydrazine (DMH) has a specific role in inflammation and oxidative stress in colorectal tissue [9,10]. The shielding effect of these compounds was investigated using a variety of physiological, histopathological, oxidative stress, and enzyme-linked immunosorbent assay (ELISA) assays in an attempt to figure out how their anti-CRC properties work on a molecular level. Later, a serum metabolomics investigation using proton nuclear magnetic resonance (¹H-NMR) was conducted to distinguish the metabolic disturbances associated with CRC before and after FLX therapy.

2. Materials and methods

2.1. Materials

All other chemicals were procured from Himedia Ltd., Mumbai, India, and Sigma Aldrich Ltd., USA. Enzyme assay kits were procured from Transasia Biomedicals Pvt. Ltd., Baddi, India. Interleukins were commercially availed from Sigma-Aldrich, USA. Cyclooxygenase-2 (COX-2) ELISA kit was acquired from Bioseps Technology Co., LTD, China. All of the solvents and chemicals used in the experiment were analytical grade with a purity of 99 percent, and distilled water was utilized throughout the experiment. All the antibodies were procured from Thermo Fisher Scientific, Waltham, MA, USA.

2.2. Experimental protocol

Albino Wistar rats (male, 100 to 120 g) were used for this experiment and protocol was approved previously by Institutional Animal Ethical Committee (Ref no. 1648/PO/Re/S/12/CPCSEA/48R2). Before the experiment, the animals were maintained for a week. Temperatures of 25  °C ± 5  °C and a 12-hour light/dark cycle were maintained (normal laboratory settings), with unrestricted access to a commercial pellet diet and water ad libitum.

All animals were arbitrarily separated into 5 groups of 8 animals each (n = 8). The groups were then divided as follows: group I [normal control, (NC)]: 0.25% carboxy methylcellulose (CMC, 2 mL/kg), group II [carcinogen control, (CC)]: DMH (40 mg/kg, subcutaneously, once in a week for 4 weeks), group III [positive control, (PC)]: DMH+5-FU (10 mg/kg, intraperitoneally, for 15 days after the induction of CRC), group IV [treatment 1, (T1)]: DMH+FLX (15 mg/kg, orally, for 15 days after the induction of CRC), group V [treatment 2, (T2)]: DMH+FLX (30 mg/kg, orally, for 15 days after the induction of CRC) [11], [12], [13], [14], [15]. The method for inducing CRC was adapted from previously published research [11], [12], [13], [14], in which albino Wistar rats were given a 40 mg/kg subcutaneous injection of DMH once a week for four weeks. All rats in groups II to VII were given DMH after a week of acclimating to their new environment. The dosage of FLX used in this research was previously published [15]. Animals were killed by cervical decapitation at the conclusion of the experiment, and their colons were promptly dissected, washed in ice-cold saline, and kept at -20  °C for future research. For additional examination, the serum was collected, processed, and stored.

2.3. Estimation of various physiological parameters and biochemical parameters in CRC tissue, enzyme levels, and lipid profiles in serum

Various physiological measures, such as body weight changes, tumor incidence number, tumor volume, pH of colonic content, and overall acidity, were assessed in accordance with prior research [5]. Using a commercially available kit, the levels of enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) in serum were also tested. The biochemical parameters such as catalase (CAT) [16], protein carbonyl (ProC) [10], superoxide dismutase (SOD) [9], glutathione (GSH) [17] and thiobarbituric acid reactive substances (TBARS) [9,16,17] were estimated in CRC tissue.

The lipid profile kit acquired from Agape Diagnostic Ltd., Kerala, India was utilized for the determination of triglycerides (TG), high-density lipoprotein (HDL), and total cholesterol (TC). Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) were estimated by employing the Friedewald's formula [18,19].

LDL (mg/dL) = TC – HDL – (TG/5)

VLDL (mg/dL) = TC – HDL – LDL

2.4. Estimation of inflammatory mediators in colon tissue

ELISA was performed for interleukins (IL-2, IL-6, IL-10, and IL-1β) and COX-2 as per the protocols provided by the manufacturer [18,19].

2.5. Molecular biology studies of CRC tissue

Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting analyses of colonic tissue were further performed in this experiment. 10 mg of CRC tissue was placed in Eppendorf tubes, total messenger RNA (mRNA) was isolated employing the TriaZol reagent, and the amount of mRNA contained in each sample was determined employing the NanoDrop instrument at 260 nm/280 nm. Furthermore, complementary deoxyribose nucleic acid (cDNA) was synthesized employing the GeneSure first-strand cDNA synthesis kit as per the manufacturer's procedure (Genetix Biotech Asia Pvt. Ltd., New Delhi, India). Finally, employing the Sybr® green PCR master mix, qRT-PCR was conducted in the Agilent Stratagene Mx3000P series (Applied Biosystems, Foster City, USA). The housekeeping control β-actin was used to normalize the mRNA. For all drugs, Ct levels were standardized to untreated control samples (ΔCt = Ct_{gene of interest} – Ct_{housekeeping gene}). The relative changes in the degree of expression of a single gene were estimated based on 2^−ΔΔCt (ΔΔCt = ΔCt_test– ΔCt_control) [15]. The primer sequences were as follows: IL-6, 5ʹ-GCCCTTCAGGAACAGCTATGA-3ʹ (forward) 5ʹ-TGTCAACAACATCAGTCCCAAGA-3ʹ (reverse) [20]; COX-2, 5ʹATCAGAACCGCATTGCCTCT-3ʹ (forward), 5ʹ-GCCAGCA ATCTGTCTGGTGA-3ʹ (reverse) [21]; and β-actin, 5ʹ-AAGTCCCTCACCCTCCCAAAAG-3ʹ (forward) 5ʹ-AAGCAATGCTGTCACCTTCCC-3ʹ (reverse) [22].

Immunoblotting was used to assess the expressions of IL-6, COX-2, and β-actin. To lyse the cells, they were lysed in RIPA buffer, centrifuged at 10,000 rpm for 15 min at 4  °C, and total proteins were calculated using Bradford reagent. A 12 percent sodium dodecyl sulfate (SDS)-polyacrylamide gel was used for electrophoresis, and the gel was immediately transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skimmed milk containing phosphate-buffered saline (PBS) with 0.1% tween-20 (PBS-T) for 3 h at 4 °C and probed with primary antibodies diluted in PBS-T: IL-6, COX-2, and β-actin mouse monoclonal antibody (1:500 dilution of each) overnight at 4 °C. 3 times with tris-buffered saline, the membranes were washed containing tween-20 (TBS-T), the next day before being incubated for three hours at room temperature with anti-rabbit secondary antibodies coupled to horse-radish peroxidase at a 1:3000 dilution. The membrane was developed using enhanced chemiluminescence ECL (PierceTM ECL Western Blotting Substrate) and pictures were taken using Chemidoc after the film was washed three times with TBST (Clinx Scientific Instruments, China) [9,10,23].

2.6. Histopathological studies, aberrant crypt foci (ACF), and digital image analysis

The procedures adopted for histopathology were described previously [9]. For counting ACF, a total colorectal portion was stained with methylene blue to coat the mucosal surface and observed under a microscope (40X) [24,25]. Microscopic data analysis using Image J standard program software (NIH), six regions were selected and analyzed individually. A surface plot experiment was subsequently used to identify the square plot. Finally, by selectively retaining stained zones of hematoxylin and eosin, a 3D image reconstruction and software-based analysis dataset of constructs representing score were generated using Image J (NIH) programme (H&E) and ACF images and pixel vs. intensity determination by the 3D interactive surface plot and log-histogram analysis [26,27].

2.7. ¹H-NMR based serum metabolic profiling

All the serum samples were prepared for NMR measurements using a previously published protocol [5,9,10,15,17]. The NMR spectra were recorded at 298 K with a proton frequency of 800.21 MHz on a Bruker BiospinAvance-III 800 MHz NMR spectrometer (equipped with Cryoprobe). In all, 400 mL of serum were transferred to a 5 mm NMR sealed tube, with a second sealed capillary tube containing a known concentration of TSP added for chemical shift reference and locking. For each serum sample, the standard Bruker's pulse programme library sequence (CPMGPR1D) was used to obtain transverse relaxation-edited CPMG (Carr–Purcell–Meiboom–Gill) NMR spectra, with pre-saturation of the water peak obtained by irradiating it continuously throughout the recycling delay (RD) of 5 sec.

By comparing chemical shifts and splitting patterns available with 800 MHz database library of NMR suite of commercial software CHENOMXv8.1 software (Chenomx Inc., Edmonton, Canada), the peaks in the 1D-¹H-CPMG NMR spectra were identified and assigned to various metabolites. Data from the HMDB (Human Metabolome Database) [28] and BMRB (Biological Magnetic Resonance Data Bank) [29], as well as previously reported NMR spectra of metabolites, were used to assign the remaining peaks in the CPMG ¹H-NMR spectra.

The 1D-¹H-CPMG NMR spectra features were further subjected to multivariate data (MVD) analysisusing the Metaboanalyst web-based tools server (version 3.0; publicly available web server for academic use:https://www.metaboanalyst.ca/) [30,31]. Prior to data processing, TopSpin3.0 was used to manually phase and baseline-correct all of the 1D ¹H CPMGNMR spectra (Bruker NMR data Processing Software). The CPMG NMR data were automatically binned for (8.5-0.5) ppm rangeand normalized with respect to total spectral intensity using the AMIX software programme (Version 3.8.7, Bruker, BioSpin). The resulted data matrix containing binned data from the CPMG spectra were finally used for multivariate statistical data analysis.

Using principal component analysis (PCA, an unsupervised MVD analysis method), the outliers were first detected. To reveal class separations and identify the metabolites that contributed significantly to group differentiation, supervised partial least squares discriminate analysis (PLSDA) with orthogonal signal correction (referred here as OPLS-DA) was employed. The cross-validation of OPLS-DA modelwas assessed using the R² and Q² and the metabolites of discriminatory potential were subsequently identified based onvariable significance on projection scores (i.e. VIPs). The discriminatory metabolites exhibiting statistical significance estimated using the univariate statistical analysis based on Mann-Whitney test with a p-value <0.05. The box plot representations for key metabolic features are used to highlight the variation (i.e. the up and down regulation) in the quantities of altered metabolites of discriminatory ability (as decided based on with VIP score values around 1.

2.8. Statistical data analysis

Statistical analysis was performed using GraphPad Prism 5.0 (San Diago, CA, USA). All results were expressed as mean ± standard deviation (SD). The data were analyzed by one-way ANOVA (analysis of variances) followed by Bonferroni multiple comparison test. For all estimations, statistical significance differences were considered with respect to CC group (***p < 0.001, **p < 0.01, *p < 0.05).

3. Results

3.1. Estimation of physiological parameters and biochemical parameters in the colon and various enzyme levels and lipid profiles in serum

In different physiological circumstances, the effects of FLX and 5-FU were compared. Body weight disparities were more apparent in DMH-treated rats (CC group), but returned to normal following treatment (Fig. 1A). Tumor volume and incidence no. were ∼x223C3.5 times higher in the CC group but both treatments successfully normalized the impact (Fig. 1B and 1C). Similar trends were seen in colonic content pH and total acidity, with CC having a lower pH and a higher total acidity than NC (Fig. 1D and 1E). The impacts of FLX and 5-FU on these parameters were similar to the previous parameters. Most significantly, the impact of FLX at 30 mg/kg is comparable to that of the commercially available chemotherapeutic 5-FU.

Fig. 1.

Effects of fluvoxamine (FLX) on (A) Body weight variation, (B) Tumor volume, (C)Tumor incidence no., (D) Total acidity in colon, (E) pH and (F) enzyme levels in plasma. Data are represented as mean ± SD (n = 8). Statistically significant differences were observed between carcinogen control and test groups [one way-ANOVA followed by Bonferroni multiple comparison test (***p < 0.001, **p < 0.01, *p < 0.05)].The studied groups are:(NC: Normal Control, CC: Carcinogen Control, PC: Positive Control, T1: FLX 15 mg/kg, T2: FLX 30 mg/kg).

Similarly, serum AST, ALT, and LDH were also measured, and levels were recorded in various groups (Fig. 1F). Enzyme levels were increased ∼x223C3.0 folds (p < 0.01) in CC than NC control but treatment with test compounds restored the levels to normalcy.

Lipid profile parameters (TC, TG, HDL, LDL, and VLDL) were assessed in comparable studies to determine the protective effect of FLX at different dosages. In DMH-treated rats (except HDL), TC, TG, VLDL, and LDL were significantly higher than in NC rats (except HDL) and then returned to normal following treatment (Fig. 2A). When comparing CC (31 mg/dL) to NC (60 mg/dL), the HDL level in CC (31 mg/dL) was lower. After oral treatment of FLX at a dosage of 30 mg/kg, which was similar to 5-FU, HDL levels in the blood returned to normal.

Fig. 2.

Effects of fluvoxamine (FLX) after oral administration of 15 mg/kg and 30 mg/kg for 15 days in carcinogen control rats (A) Lipid profile and (B) Anti-proliferative biomarkers COX-2, IL-2, IL-6, IL-10, IL-1β in colon tissue. Data are represented as mean ± SD (n = 8). Statistically significant differences were observed between carcinogen control and test groups [one way-ANOVA followed by Bonferroni Multiple Comparison Test (***p < 0.001, **p < 0.01, *p < 0.05)].The studied groups are: (NC: Normal Control, CC: Carcinogen Control, PC: Positive Control, T1: FLX 15 mg/kg, T2: FLX 30 mg/kg).

We looked at oxidative stress markers during CRC and the effect of FLX on oxidative stress and cancer development once again. The amounts of the enzymes SOD and CAT were decreased by 3.0 times in CC compared to NC (Table 1), however, 5-FU and FLX treatments successfully augmented its level to normalcy. A similar observation was found in the case of reduced GSH concentration as well. MDA and ProC formations were substantially greater in CRC mice, however, following 5-FU and FLX treatments, these formations were dramatically decreased (Table 1).

Table 1.

Study of anti-oxidative parameters in DMH-exposed carcinogenesis in rats. (NC: Normal Control, CC: Carcinogen Control, PC: Positive Control, T1: fluvoxamine (15 mg/kg) and T2: fluvoxamine (30 mg/kg).

Sr.No.	Parameters	NC	CC	PC	T1	T2
1.	SOD (U/μg of protein)	3.28 ± 0.04	1.25 ± 0.01	2.25 ±0.01***	2.02 ± 0.12***	2.93± 0.01***
2.	CAT (nM of H2O2/min/μg of protein)	3.93 ± 0.51	1.03 ± 0.30	2.74 ± 0.96***	2.29± 0.73 **	3.58± 0.34***
3.	ProC (μM/μg of protein)	0.10 ± 0.02	0.19 ± 0.02	0.12 ± 0.04**	0.14 ± 0.03	0.11± 0.07**
4.	TBARS (nM of MDA/mg of protein)	19.81± 2.16	35.59 ± 3.50	23.52 ± 2.50***	27.44 ± 3.85***	21.56± 2.19***
5.	GSH (mM/mg of Protein)	6.25±0.42	2.75±1.55	5.44±0.56***	4.66±1.13**	5.91±0.90***

Data represented here as mean±SD (n=8). Statistically significant differences were observed between CC and test groups [one way ANOVA followed by Bonferroni multiple comparison test (***p<0.001, **p<0.01 and *p<0.05)]

3.2. Effect of FLX on interleukins and COX-2 levels

To further elucidate the effect of FLX over inflammatory events, we measured various inflammatory mediators and COX-2 by ELISA in CRC tissue. There was a significant increase in IL-6 and COX-2 concentrations in DMH-treated rats than other mediators (Fig. 2B). Treatment with FLX (30 mg/kg) and 5-FU suppressed the concentration of these mediators to normalcy. This action was more prominent for COX-2 than any other mediator(s).

3.3. Molecular biology studies of CRC tissue

qRT-PCR analysis was performed to measure the gene expression levels of IL-6 and COX-2. dimethylhydrazine (DMH) treated CC group demonstrated an upstream regulation of both IL-6 and COX-2 genes as compared to NC. However, FLX and 5-FU treatments significantly normalized (p < 0.001) the expressions to normalcy. FLX (30 mg/kg) dosage effectiveness was found to be similar to that of commercially available chemotherapeutics, 5-FU (Fig. 3A). A similar observation was also noticed in immunoblotting assays where protein expressions of both COX-2 proteins upregulated in CC rats, followed by a decrease in expression after treatments (Fig. 3B and 3C). IL-6 protein expression had a less prominent effect in this experiment. The images of full blots are represented in Supplementary Data Sheet, Fig. S2-S4.

Fig. 3.

(A) Gene expression levels of COX-2 and IL-6 in colon tissue. (B) Protein expression levels of COX-2 and IL-6 in colon tissue after treatment with FLX (determined by quantitative western blot analysis). (C) Relative band density. Data are represented as mean ± SD (n = 3). Statistically significant differences were observed between carcinogen control and test groups [one way-ANOVA followed by Bonferroni multiple comparison test (***p < 0.001, **p < 0.01, *p < 0.05)]. The studied groups are: (NC: Normal Control, CC: Carcinogen Control, PC: Positive Control, T1: FLX 15 mg/kg, T2: FLX 30 mg/kg).

3.4. H&E and ACF analyses

Histopathological changes were further observed in colonic tissues the analysis was linear before and after treatments using H&E stains. Tissue analysis exhibited crypts (Cr), tumor stroma (TS), and goblet cell adenoma (GA), more vacuolated and damaged cells in DMH treated group (CC) than NC. The histological damage in the colon tissue was reversed with FLX therapy (30 mg/kg) (Fig. 4). Using Image J (NIH) software, a collection of constructs indicating score was created using 3D image reconstruction and software-based analysis, further documented by thresh holding stained zones of H&E images, followed by pixel vs. intensity determination by the log-histogram analysis and 3D interactive surface plot. Image J analysis further accentuated the results obtained from H&E stains (Fig. 4).

Fig. 4.

The colonic H&E analysis and digital image analysis in DMH- induced CC rats (Scale bar 50 µm). Temporal vacuole were prominent in DMH group which was absent after 5-FU, FLX 15 and 30 mg/kg administration.(Cr- Crypts, NE- Normal epithelium, CL- colon lumen, GA- Goblet cell adinoma, Ly- lymphatics, lined by epithelium TS- Tumor stroma). 3D image reconstruction and software-based analysis dataset of constructs representing score was done by using Image J (NIH) software by thresh holding of stained zones of H&E images followed by pixel vs intensity determination by the 3D interactive surface plot and log-histogram analysis.The studied groups are:(A: Normal Control, B: Carcinogen Control, C: Positive Control, D: FLX 15 mg/kg, E: FLX 30 mg/kg).

ACF count using methylene blue stains further represented the presence of more ACF nodules with the larger size in the CC group, indicating CRC condition (Fig. 5). Nodules size became decreased after treatments with 5-FU and FLX. Image J software analysis supported the previous results with bigger nodules formation in CRC rats and reduction in size after treatments.

Fig. 5.

DMH induced colonic ACF multiplicity along with tumor multiplicity count and digital image analysis in rats. 3D image reconstruction and software-based analysis dataset of constructs representing score was done by using Image J (NIH) software by thresh holding of stained zones of methylene blue images followed by pixel vs intensity determination by the 3D interactive surface plot and log-histogram analysis.The studied groups are:(A: Normal Control, B: Carcinogen Control, C: Positive Control, D: FLX 15 mg/kg, E: FLX 30 mg/kg). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.5. ¹H-NMR-based metabolomics to access the biochemical impact after FLX treatment

SIMCA-P + 11.0 (Umetrics, Umea, Sweden) was used to further analyze the data acquired from NMR studies, with orthogonal partial least-squares-discriminant analysis (OPLS-DA) and principal components analysis (PCA) being used to determine metabolic changes before and after treatments. PCA analysis was used to determine the performance of the analytical quality system and potential outliers. On the other hand, OPLS-DA (Fig. 7) was used to complete the summary and differentiate the variables among each group.Q² and R² were used to optimize the model (Fig. 7). We observed well separation between groups obtained from score plots (s-plots) through 1D-¹H-CPMG-NMR spectra (generated using both form OPLS-DA and s-plots, Fig. 6). The metabolites were selected based on significant thresholds of variable impact on projection (VIP) values (more than 1.0) produced by OPLS-DA models. Meanwhile, the statistics were analyzed using a two-tailed Student's t-test with a 0.05 significance level. Log2 fold change (FC) was applied to indicate how these particularly different metabolites varied among the groups. Finally, the database of all metabolites (from OPLS-DA) was presented into MetaboloAnalyst 3.0 to generate heat maps and multivariate statistics. When the p-value is less than 0.05, the area under the curve (AUC, form box, and whisker plots, Fig. 8) under the receiver operating characteristic curves (ROC) was used to determine the efficacy of putative biomarkers (Fig. 8).

Fig. 7.

Combined and Pairwise OPLS-DA analysis:(A)The 2D OPLS-DA analysis of 1D ¹H CPMG NMR spectra score plot derived from combined analysis comprising of all the groups: Carcinogen control (CC),Treatment low dose (T1) & Treatment high dose (T2), Positive control (PC), Normal Control (NC). (B) The potential discriminatorymetabolite entities identified from VIP scores derived from PLS-DA modelling of complete data matrix and resulted VIP scores for top 30 metabolite entities are shown in increasing order of VIP score values to highlight their discriminatory potential.

Fig. 6.

Stack plot of representative 1D ¹H CPMG NMR spectra of rat serum obtained from different groups. Carcinogen control (CC),Treatment low dose (T1) & Treatment high dose (T2), Positive control (PC), Normal Control (NC) groups.

Fig. 8.

Metabolic effects of fluvoxamine (FLX) treatment: The box-cum-whisker plots are showing relative variations in quantitative profiles of serum metabolites relevant in the context of the pathophysiology of colonic cancer. In the box plots, the boxes denote interquartile ranges, horizontal line 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. Where: Carcinogen control (CC), Treatment low dose (T1) & Treatment high dose (T2), Positive control (PC), Normal Control (NC) groups.

Both individual and pair-wise data analysis from OPLS-DA showed R² = 0.88, Q² = 0.72, indicating the metabolic separation between CRC and treatment groups Fig. 7, Fig. 8. show the differences between the NC and DMH treated groups, as well as variable importance on projection (VIP) score, and p-value. Box-cum-whisker plots revealed that there were significantly elevated concentrations of LDL/VLDL, formate, lactate, creatinine, glycine, trans-aconitate and decreased levels of glucose, o-acetyl choline, N-acetyl glutamate (NAG), lysine, leucine, 3-hydroxy butyrate, glutamate, tyrosine in DMH treated rats (Fig. 8). All these metabolites became almost normalized after treatments with 5-FU and FLX (30 mg/kg).

4. Discussion

Recent literature enlightened that CRC is the third common cancer-related death worldwide [1,32]. Marketed chemotherapeutics have severe adverse effects and tumor resistance, because of this reason, the clinical outcome of CRC treatment remains limited [5,33,34]. Thus, the major concern of our investigation is to search for new marketed cancer therapeutics that could have greater effectiveness and prominent selectivity against CRC treatment. During searching, we found 5-HT reuptake inhibitor, FLX an antidepressant, used for compulsive obsessive disorder, had antineoplastic potential against in vitro colon cancer cells and in the in vivo colonic carcinogen mouse model [7].

In the present study, in vitro HT-29 cell line experiment revealed that FLX had IC₅₀ of (∼x223C10 µg/ml) which was the primary indication of the anti-CRC potential of FLX. This observation was further strengthened by in vivo investigation of the anti-neoplastic potential of FLX using DMH-treated rats. CRC condition in DMH treated rats characterized by decreased body weight and increased tumor incidence number & tumor volume, alteration in pH, and increase in total acidity of colonic content further signified cancerous condition [9,10,14,17]. The treatments with 5-FU and FLX restored these altered physiological parameters to normal, signifying protective action by the treatments. Furthermore, various enzymes such as ALT, AST, and LDH enzymes are found in serum during liver metastases of CRC [35]. Treatment with FLX considerably reduced these enzyme levels nearer to normal as compared to the CRC group, demonstrating a decline of liver metastases in CRC. In addition, the formation of colorectal polyps increased during elevated serum TG and TG cholesterol in serum [36,37]. In continuation with the previous observation, colorectal damage during carcinogenesis was observed through increased TG and TC decreased HDL concentrations in CC rat sera compared with NC. These lipid profile parameters normalized after treatments that indicated protective action of FLX and 5-FU and the colorectal polyps formation may be decreased.

Moreover, a previous report suggested that the oxidation process was increased in cancerous conditions [38]. We observe the reactive oxygen species (ROS) releases from all types of cancerous cells and counterbalance through the antioxidant activity of the cellular system [39]. This unbalanced condition triggers cancerous conditions due to excessive cellular proliferation [40]. Altogether, an antioxidant defense mechanism occurs in our body when the antioxidant enzymes i.e. SOD and CAT scavenge free radicals and GSH engulfs ROS [41]. In general, the enzyme CAT catalyzes the conversion of H₂O₂ to oxygen and water, thereby protecting reactive oxygen species, while SOD neutralizes superoxide free radicals [42]. Cellular proliferation during oxidative stress was observed through lower concentrations of SOD, CAT, and GSH enzymes in DMH rats as compared to NC rats. FLX produced protective action via regularization of these parameters nearer to normal. Furthermore, proteins and lipids are oxidized during oxidative stress and characterized through MDA and ProC formations in higher amounts [43]. Similar trends were found out in CC rats, again signified aerobic condition of colorectal tissue during the cancerous condition. Defensive action was again documented after FLX and 5-FU treatments through reduced production of MDA and ProC in treated rats. Therefore, we say that FLX could prevent tumors with noticeable antioxidant effects.

Except for the antioxidant mechanism, we wanted to figure out whether FLX had any effects against CRC-specific pro-inflammatory biomarkers. Further, a recent investigation confirmed that pro-inflammatory biomarkers like interleukins (IL-2, IL-6, IL-10, and IL-1β) and COX-2 are generated at colon cancer sites [44], [45], [46]. The expression of the COX-2 enzyme at the tumerogenic site results in the synthesis of prostaglandins-E2 (PGE2) which inhibits apoptosis and promotes tumor angiogenesis, tumor cell growth [47], [48], [49]. Consequently, ELISA assays demonstrated that all inflammatory cytokines expressed a higher amount in CRC sites of DMH rats. All the concentrations moved to normalcy after treatments, indicating that FLX could reduce the concentrations at CRC sites. This observation was further supported by molecular biology experiments. qRT-PCR studies represented the higher expression of IL-6 genes and COX-2 genes at CRC tissue. The mRNA expression of both genes turned to normal after treatments, clearly proving that FLX could inhibit the expression of these genes at molecular levels. The expression of COX-2 was more prominent than IL-6. A similar observation was found in immunoblotting assays where overexpressed COX-2 genes were further downregulated after treatments with FLX and 5-FU. It is obvious from the results that the anti-neoplastic property of FLX against CRC may be due to COX-2 inhibition.

After evaluating the plausible mechanism of the anti-CRC potential of FLX, we tried to find out the morphological changes that occurred before and after treatments. We observed vacuolated and damaged cells, Cr, TS, and GA in DMH-treated rats, signifying inflammatory conditions during CRC [15]. This inflammatory mediated abnormal architecture was significantly vanished in treated FLX and 5-FU groups, indicating the protective ability of the compounds against CRC. Furthermore, higher ACF nodules (necrotic tissue) were found in CRC rats, again normalized after FLX treatment. ACF is the precursors of CRC progression [50] and more ACF nodules indicated CRC condition which recovered after FLX administration.

Later, NMR-based metabolomics with multivariate data analysis was further performed to know about metabolic alterations during CRC condition and after treatments over these altered metabolites. Metaboanalyst software along with HMDB and BMRB database, followed by OPLS-DA were employed to make the metabolic alterations among groups [5,9,10,14,17,[28], [29], [30], [31]]. During CRC condition, the directly involved biochemical cycles were tricarboxylic acid (TCA) cycle, glycolysis, glucogenesis, phosphatidylinositol, and choline pathways. Box and whisker plots from OPLS-DA analysis through NMR experiment differentiated perturbation in serum metabolites and restoration after treatments. Previously published literature revealed CRC condition primarily well documented through elevation of lactic acid and reduction of glucose concentrations in serum [9,10]. This action is similar to the War-burg effect where more cancerous tissues consumed glucose for energy production and released lactic acid as a byproduct [51,52]. The noticeable observation was recorded after FLX and 5-FU treatments i.e., to normalization of two metabolites and promisable effect against CRC progression of FLX (particularly higher dose).

In addition, elevated concentrations of lactate in DMH rats indicated an augmentation in the TCA cycle which may lead to gluconeogenesis pathway activation. Lactate alters the pH of extracellular fluid to acidic at cancerous sites and this acidic pH triggers cell invasion and metastasis [53,54]. Furthermore, glutamate and NAG are the predecessors of GSH synthesis, GSH is a well-known natural antioxidant and free radical scavenger during cellular oxidative damage [55,56]. Hence, we found a reduced concentration of glutamate in CRC rats, demonstrating oxidative stress condition at tumorigenic sites which is similar to previous findings [55]. The condition further recovered after FLX treatment perhaps due to the antioxidant property of FLX. Besides these, o-acetyl choline is responsible for cell membrane formation and integrity [55,56]. The reduced o-acetyl choline concentration in the tumerogenic sites of CRC rats further indicated o-acetyl choline consumed more by CRC tissue i.e. more cancerous cell formation. In addition, LDL/VLDL is the precursor of cholesterol biosynthesis where cholesterol is utilized for cell membrane production [37]. CRC condition again characterized by elevated level of LDL/VLDL in rat DMH treated rat serum in our experiment. Both FLX and 5-FU normalized LDL/VLDL concentrations in serum, further protective ability metabolites were documented in our studies.

Several amino acids have been associated with cancer cell homeostasis and regeneration. The data obtained from NMR-based metabolomics were correlated with previously published data where creatinine and glycine were remarkably enhanced during carcinoma conditions [57]. Higher utilization of amino acids implicated higher utilization of proteins for cellular proliferation and integrity. Restoration of amino acids after treatments further demonstrated the anti-neoplastic property of FLX. The metabolites i.e. formate, trans-aconitate, leucine, lysine, 3-hydroxy butyrate (3-HB), tyrosine had the least effects in our experiment.

In the present study, the anti-CRC potential of FLX was linked with physiological, biochemical, molecular mechanistic, and metabolic parameters. The anticancer potential of FLX was confirmed by both in vitro HT-29 cells and in vivo DMH rat model. The molecular insights of FLX may be due to the inhibition of overexpressed COX-2 enzymes. Furthermore, metabolic studies could provide a clear idea of metabolic perturbations before and after treatments which can give evidence of cellular functioning. All evidence demonstrated that FLX could serve anti-CRC drugs in the near future that complied specifically with the hypotheses put forward in the recent reports [58,59].

5. Conclusion

The present work substantiated that FLX normalized various oxidative stress parameters, demonstrating its potency to tackle pro-oxidative conditions during CRC. qRT-PCR and immunoblotting assays strongly supported the potential of FLX to inhibit COX-2 enzymes. Lastly, NMR-based serum metabolic profiling further ameliorated that FLX has the ability to normalize perturbated metabolites which changed in DMH treated rats, representing anti-CRC potency of FLX for preventing endogenous metabolic disorders. Finally, we concluded that FLX may be an effective molecule against colon cancer, which would be beneficial for future drug design perspectives.

Declaration of Competing Interest

The authors declare that they have no conflict of interests.