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| RESEARCH ARTICLE |
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| Year : 2023 | Volume
: 55
| Issue : 2 | Page : 97-107 |
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Raptinal ameliorates 1,2-dimethylhydrazine-induced colon cancer through p53/Bcl2/Bax/caspase-3-mediated apoptotic events in vitro and in vivo
Yongting Lan1, Yang Yang2, Abhijit Das3, Barshana Bhattacharya3, Souvik Roy3
1 Department of Gastroenterology, Zibo Central Hospital, Zibo, China
2 Department of Colorectal and Anal Surgery, Baoji People's Hospital, Baoji, China
3 Department of Pharmacy, NSHM Knowledge Campus Kolkata, Kolkata, West Bengal, India
| Date of Submission | 05-Mar-2022 |
| Date of Decision | 28-Apr-2023 |
| Date of Acceptance | 04-May-2023 |
| Date of Web Publication | 03-Jun-2023 |
Correspondence Address:
Souvik Roy
Department of Pharmaceutical Technology, NSHM Knowledge Campus, 124 B.L. Saha Road, Kolkata - 700 053, West Bengal
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijp.ijp_168_22
OBJECTIVES: Colon carcinoma stands as the most familiar malignancy throughout across the globe. Raptinal induce apoptosis through the alteration of cellular events. Thus, in the present investigation, the anticancer activity of raptinal counter to 1,2-dimethylhydrazine (DMH) persuaded colon carcinoma has been evaluated through both in vivo and in vitro systems.
MATERIALS AND METHODS: The pharmacophore analysis demonstrated the binding efficacy of raptinal with the apoptotic proteins. The chemotherapeutic activity of raptinal was examined through HT-29 human colorectal cancer (CRC) cell line as well as DMH persuaded CRC in the rat model. The cytotoxicity analysis, flow cytometry, and DAPI analysis have been carried out on HT-29 cell line through in vitro assessment. The colon carcinoma has been induced through DMH administration and subsequently Dextran sulfate sodium treatment in male Wistar rats. After 18 weeks of raptinal treatment, the colon tissues have been investigated for aberrant crypt foci (ACF) count, antioxidant status, histology, immunohistochemical assessment, and apoptotic analysis.
RESULTS: The raptinal therapy on HT-29 cells demonstrated a substantial % of early apoptosis followed by G0 and G1 phase arrest, which subsequently led to apoptosis. Furthermore, it inhibits ACF development with improved colonic abrasions and structural integrity of colonic mucosa with increased levels of antioxidants, proapoptotic biomarkers including p53, caspase-3, Bax and downstream effects of Bcl-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 mutation.
CONCLUSIONS: These findings indicate the raptinal effectively reduces colon cancer by inducing apoptosis through p53/Bcl2/Bax/caspase-3 pathway and suppressing IL-6, TNF-mediated chronic inflammation in the colon cancer microenvironment.
Keywords: Apoptosis, cell proliferation, chemotherapeutics, colon carcinogenesis, raptinal
How to cite this article:
Lan Y, Yang Y, Das A, Bhattacharya B, Roy S. Raptinal ameliorates 1,2-dimethylhydrazine-induced colon cancer through p53/Bcl2/Bax/caspase-3-mediated apoptotic events in vitro and in vivo. Indian J Pharmacol 2023;55:97-107 |
How to cite this URL:
Lan Y, Yang Y, Das A, Bhattacharya B, Roy S. Raptinal ameliorates 1,2-dimethylhydrazine-induced colon cancer through p53/Bcl2/Bax/caspase-3-mediated apoptotic events in vitro and in vivo. Indian J Pharmacol [serial online] 2023 [cited 2023 Oct 4];55:97-107. Available from: https://www.ijp-online.com/text.asp?2023/55/2/97/378026 |
| » Introduction | |  |
Colon carcinoma accounts for 10% of total carcinoma occurrences and 9.4% of carcinoma-related deaths in 2020.[1] Significantly higher prevalence rate and deaths in China are associated with the genetic factors and superior population.[2] Despite rigorous treatment strategies the prevalence of colorectal cancer (CRC) continues, but due to numerous side effects, the efficacy gets restricted. As a consequence, researchers are seeking for a novel chemotherapeutic approach to eradicate cancer cells without compromising the viability of healthy cells.
Deregulation of the apoptotic process is one of the most prominent factors that lead to cancer by reinforcing the survival of malignant cells. Therefore, most of the cancer treatments rely on the activation of the apoptotic pathway to prevent cancer progression.[3] The primary effector proteins for the intrinsic apoptotic pathway include p53, Bax, and Bcl2 which leads to mitochondrial outer membrane permeabilization (MOMP)[4] that triggers the cytochrome-C release and initiates intrinsic apoptosis.[5] Subsequently, the activation of caspase-3 leads to apoptosis.[6] Bax/Bak plays a crucial role in MOMP regulation in association with Bcl2 and condemns a cell to death. In addition, significant overexpression of Bcl2 has been observed in various malignancies whereas in the solid tumors p53 expression was predominantly found. Thus, targeting apoptotic proteins is considered an emerging strategy in the field of chemotherapy to induce apoptosis.[7]
Raptinal, a cell-permeable bifluorene-dicarbaldehyde compound (9H,9′H-9,9′-Bifluorene-9,9′-dicarbaldehyde), acts as a rapid activator of intrinsic apoptosis. Raptinal causes a complete release of cytochrome-C and activates caspase-3 within 1 h of exposure in various cell lines.[8] Raptinal-mediated cytochrome-C release in Bax/BAK/BOK-independent fashion, chemosensitizes the cancer cells that undergo functional or genetic loss of BAX/BAK genes.[9] The Raptinal-loaded silver nanoparticle (AgNPs) has been investigated against hepatocellular carcinoma. The experimental outcome showed that AgNPs increased the genetic expression of cytochrome-C and caspase-3.[10]
In colon cancer, aberrant crypt foci (ACF) have been reflected as utmost primeval malignancy abrasions with specific dysplastic features[11] and chronic inflammation. A comprehensively inflammatory cytokines have been created by the gut immune cells due to localized inflammation. Thus, the chronic inflammation accompanied by the epigenetic and genetic modification that facilitates the initiation of colon carcinoma. Among several inflammatory biomarkers, the interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) are mostly accountable to induce colon carcinoma.[12]
The pharmacophore analysis exhibited the effective pharmacological targets of raptinal to establish the chemotherapeutic mechanism against CRC. The 1,2-dimethylhydrazine (DMH) persuaded CRC model was assessed for the evaluation of chemotherapeutic activity of raptinal through examining Bcl2/p53/caspase-3/Bax and cytokine transduction ways correlated with apoptosis. The action of raptinal on HT-29 cell line was also considered to correlate with in vivo as well as in vitro assessment for demonstrating its anticancer efficacy.
| » Materials and Methods | | |
Chemicals
Entire chemicals have been an analytical reagent brand. Raptinal (97% pure by HPLC), Dextran sulfate sodium (DSS), 1,2 dimethylhydrazine (DMH), streptavidin peroxidase, biotinylated goat anti-rabbit immunoglobulin G, proteinase K and 3,3'-diaminobenzidine (DAB) been obtained by Sigma Aldrich Chemical Co.(St. Louis, MO, USA). Rabbit anti-rat Bcl-2, p53, caspase-3, Bax, IL-6, TNF-α, and Ki-67 have been purchased through BioLegend (San Diego, California, USA). The apoptotic kit has been procured by Takara Bio Inc (Kusatsu, Japan).
Pharmacophore analysis
Drug-likeness prediction and ADMET properties
Swiss ADME is used to evaluate the compounds' drug-likeness characteristics (Swiss Institute of Bioinformatics software). Based on physicochemical, pharmacokinetics, lipophilicity, and drug-likeness values, it provides a wide variety of chemoinformatic features of compounds. AdmetSAR is used to forecast the compounds' ADME and toxicity. The most excellent active molecules are selected experimentally applicable for molecular docking, taking into account the parameters of atoms depend on the human oral bioavailability, human intestinal absorption, plasma protein binding, Caco-2 permeability, blood–brain barrier diffusion, acute toxicity, LD50, carcinogenicity, and mutagenicity.[13]
Preparation of modeled proteins for ligands docking
The proteins (such as p53, Bcl2, Bax, and caspase-3) used were retrieved by using Protein Data Bank (PDB) and the PDB format collected. The proteins were cleaned and prepared for docking using PyMOL. In order to continue the ligand preparation process, this ligand molecule was imported into AutoDock Tools. Each produced receptor and ligand molecule were docked separately. AutoDock Vina was used to carry out the docking approach. The outcomes were presented according to binding affinity. Discovery Studio Biovia 2021 was used to examine the docking data.[14]
In vitro assessment
Cell culture preparation
HT-29, human colorectal carcinoma cell line have been procured by the American Type Culture Collection, United States, and incubated in DMEM growth media, 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells have been cultured at 37°C in 5% CO2 and 96% relative humidity.
Cell viability assessment
The cell viability assessment has been executed by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis. The HT-29 cells were seeded into 96 microtiter plate and incubated at 37°C in 5% CO2 for 24 h. Then the cells have been treated with several concentrations of raptinal and incubated for 24 h. After incubation, 0.5 mg/ml of MTT reagent in DMEM has been added to the cells and incubated for 3 h. Followed by MTT addition, DMSO has been poured into the solution to complete dissolve the crystal of formazan and absorbance has been quantified at 560 nm. The % of cell viability has been measured as per:
% Cell viability = 100-% of cytotoxicity
DAPI staining method
Using the method developed by Li et al., chromatin condensation and nuclear blebbing in cells were examined through DAPI staining method.[15]
Cell cycle analysis and apoptotic assay by flow cytometry
HT-29 cells have been suspended and their DNA has been labeled by propidium iodide. Fluorescence-activated cell sorting analysis has been used to determine how nuclear DNA was arranged during various cell cycle stages. The results obtained were analyzed by Modfit tools and the percentage of apoptotic cells was measured.[15]
In vivo assessment
Animals
To assess the CRC studies, male Wistar rats (70–120 g) of 6-week-old have been obtained from IICB Kolkata for chemotherapeutic analysis. All the experimental animals have been reserved in polypropylene cages along with the free supply of adequate food[16] and drinking water with 50%–58% relative humidity at 22°C ± 3°C and 12 h light/dark sequence. The experimental animals have been accustomed within 7 days previously being the start of the experimentation.
Experiments in vivo in rats
After the accustomed period, the experimental animals were dispersed haphazardly into five sets where per sets contained six rats. Wistar rats male, 6 weeks old (excluding group I) has been introduced DMH of 30 mg/kg in 0.9% saline through a single i. p. injection associated with 2% (w/v) DSS in portable water for 1 week preliminary 7 days later the injection.[17],[18] Subsequently, the experimental animals have been treated with raptinal by oral gavage route that was sustained for 5 months. We performed an acute oral toxicity study of raptinal and found the LD50 value was 650 mg/kg for Wistar rats (data not exposed). The experimentation animal sets have been divided as per:
Group I-vehicle control group, Group II-introduce chemical carcinogen (DMH + DSS) and designated as carcinogen control group, Group III-DMH induced carcinoma + administered 100 mg/kg raptinal, Group IV-DMH persuaded carcinoma + administered by 200 mg/kg raptinal, Group V-DMH persuaded carcinoma + administered by 400 mg/kg raptinal.
Aberrant crypt foci investigation and histopathology
Colon tissues at the distal end were carefully separated into two sections and then rinsed with 0.9 percent NaCl. The ACF evaluation portion was used in one, and the histology and immunohistochemical analysis segments were employed in the other. The tissues have been stained by 0.2% methylene blue solution in phosphate buffer saline (PBS)[19] as well as ACF multiplicity been estimated through light microscope (Olympus Corp., Tokyo, Japan).[20] The outcomes of each group have been denoted as the average of five distinct interpretations. Subsequently, the tissues have been embedded along with paraffin which was scotch into 5 μm width and retained onto the glass slides. Then, the colon tissues have been stained by hematoxylin and eosin (H and E) which was visualized by light microscope.[21]
Immunohistochemistry
The isolated colon tissue was embedded into the paraffin wax, incised within 5 μm width and mounted onto the poly-L-lysine-covered glass slides. Then, these tissue sections have been incubated by anti-mouse p53 (1:200), Bcl-2 (1:200), Bax (1:200), caspsase-3 (1:200), TNF-α (1:200), and IL-6 (1:200) antibodies at 4°C for 24 h. Then, the slides been washed through PBS and introduce HRP conjugated secondary antibody on the slides for half an hour. DAB was utilized for the color development on the tissue sections and counterstaining was carried out by using H&E solution. Then, the tissue section was visualized by a light microscope.
Cellular proliferation assay
The colon tissue section with 5 μm width has been fixed onto the poly-L-lysine-covered glass slides. Followed by the slides washed with PBS and added the anti-mouse Ki-67 antibody (1:500) on the tissue section at 4°C for over a night. Then, the section was once more introduced in HRP conjugated secondary antibody for half an hour at 22°C–25°C. DAB was used for color development on the tissue sections and counterstaining by H and E that have been visualized by a light microscope.
Apoptotic assessment through Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The colon tissues have been fixed onto the poly-L-lysine covered slides and introduced in proteinase K (20 μg/ml in PBS) for 10 min at 22°C–25°C, as a result of digestion of nonspecific proteins. Then the section had been introduced into terminal deoxynucleotidyl transferase (TdT) buffer solution (30 mM of Trizma base, pH 7.2, 1 mM of cobalt chloride, 140 mM sodium cacodylate) and also introduced in TdT solution comprising of TdT and dUTP at 37°C for 1 h 30 min. The responses of the tissue section have been hampered via treatment by 2% standard saline citrate for 10 min at 22°C–25°C. Then, these tissue sections have been incubated by the anti-digoxigenin peroxidase at 22°C–25°C for half an hour. DAB was used for the color development on the tissue sections and counterstaining was done with H and E staining after that the sections has been visualized by a light microscope.
The % of Ki-67-positive cells/total numberof cells has been determined as labeling index. The % of TUNEL-responsive cells/entire number of cells has been expressed as an apoptotic index (AI).
Antioxidant activity
Colon tissues have been homogenized (10% w/v) in 0.1M of PBS at pH 7.0 to evaluate antioxidant effects. Then, these homogenized tissues were centrifuged for 15 min, the aliquot has been collected and evaluated through enzymatic assessment.[22]
Superoxide dismutase
The superoxide dismutase (SOD) effect of homogenized breast tissue has been evaluated in accordance with the scheme elaborated by Awasthi et al. SOD assessment been uttered as units/min/mg protein.[23]
Catalase
In accordance with Sinha et al. homogenized breast tissue was utilized for the catalase assessment. Absorbance had been showed at 620 nm and the catalase assessment has been stated since μmol of H2O2 consumed/min/mg protein.[24]
Glutathione S-transferase
The assessment of Glutathione peroxidase (GPx) had been evaluated as per the scheme mentioned by Rotruck et al. GPx assessment has been explained since μmol of Glutathione S-transferase consumed/min/mg protein.[25]
Statistical analysis
The outcomes of the study have been revealed by way of mean ± standard error mean. The one-way analysis of variance through post hoc test (Tukey's test) has been accomplished to estimate the statistical importance via Developer: GraphPad Software, Inc. The variation had been hypothecated to be statistically significant with P < 0.05.
| » Results | | |
Pharmacophore analysis
Analysis of ADMET properties
The ADMET properties of raptinal are depicted in [Table 1]. As per the ADMET prediction raptinal is able to cross the blood–brain barrier and efficiently get absorbed by intestinal mucosa. It also showcased significant distribution properties in the subcellular localization through mitochondria. Raptinal is not carcinogenic and does not show AMES toxicity. | Table 1: Absorption, distribution, metabolism, excretion, and toxicity properties
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Molecular docking
According to the docking results the interaction of cancer-repressive gene p53 and the pro-apoptotic proteins Bax, caspase-3, and anti-apoptotic protein Bcl2 showed significant binding affinity [Table 2] and [Figure 1]. Among all those protein p53 and caspase-3 show the most promising result with docking scores of-7.6 and-7.2, respectively. | Figure 1: Molecular docking study of raptinal with (a) Bax (b) Bcl2 (c) p53 and (d) Caspase-3
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In vitro study
Raptinal prevents HT-29 cells from surviving
According to the cell viability investigation, raptinal was linked to the dose-dependent suppression of HT-29 cells [Figure 2]a. At doses of 5, 10, and 15 μM, respectively, the percentage of viability of HT-29 cells drop to 67.54%, 58.05%, and 34.63% after the treatment. | Figure 2: (a) Effects of raptinal on cell viability of HT-29 cells at 24 hours where *P < 0.05 as compared to 2.5 μm concentration. Similarly #P < 0.05 as compared to 5 μm concentration, $P < 0.05 as compared to 10 μm concentration and δP < 0.05 as compared to 15 μm concentration (b) DAPI stained HT-29 cells after 24 h of treatment with raptinal, white arrows represented nuclear fragmentations and white arrowhead represented membrane blebbing (c) detection of apoptosis in HT-29cells by Flow cytometry after treatment with raptinal (d) percentage of apoptotic cells versus concentration in HT-29 cells where *P < 0.05 as compared to control. Similarly #P < 0.05 as compared to 5 μm concentration, $P < 0.05 as compared to 10 μm concentration (e) percentage of apoptotic cells in early and late apoptosis stage in HT-29 cells. *P < 0.05 as compared to control. Similarly #P < 0.05 as compared to 5 μm concentration, $P < 0.05 as compared to 10 μm concentration (f) analysis of cell cycle phase distribution of HT-29 cells after the treatment with raptinal (g) quantitative of distribution of HT-29 cells in different phases of cell cycle. Data represent means ± SEM from three different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey's multiple comparison post hoc analysis. SEM = Standard error mean, ANOVA = Analysis of variance
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Raptinal causes chromatin condensation
Raptinal was shown to cause nuclear condensation in HT-29 colon cancer cells [Figure 2]b. At 24 h, the 15 μM raptinal dose in HT-29 cells showed the highest level of chromatin condensation, which denotes the highest incidence of apoptosis.
Flow cytometry
After being exposed to various raptinal concentrations for 24 h, the organization of HT-29 cells that had undergone apoptosis was shown in [Figure 2]c. The percentages of apoptotic cells on HT-29 cells after being treated with 5, 10, and 15 μM of raptinal are 15.93%, 39.16%, and 44.8% [Figure 2]d. Furthermore, after 24 h of treatment with raptinal, it was found that the number of cells in the early apoptotic stage had increased in the treated cells [Figure 2]e.
The flow cytometric data for cell cycle phase distribution has been depicted in [Figure 2]f. A substantially risen cellular proliferation at G0/G1 phase has been noticed in the raptinal-treated group. At Go/G1 phase the cell population was found to be 64.69%, 62.14% and 51.45% in 5 μM, 10 μM, and 15 μM raptinal-treated group, respectively. In addition, after treatment with the compound, an increase in cells at S-phase was seen on the HT-29 cell lines [Figure 2]g.
In vivo assessment
Aberrant crypt foci counting
The experimental animals in the vehicle control group [[Figure 3]a: A] did not have any detectable ACF in their colonic samples taken, while those in DMH-induced carcinogen control group [[Figure 3]a: B] developed ACF that could be identified as colon mucosal abrasions. These crypt foci could be identified from regular crypts by their distinctive elliptical figure, thick epithelial lining, dark stain, and perycriptal region. Raptinal medication dramatically decreased the ACF multiplicity in the group that received it [[Figure 3]a: C-E] (P < 0.05) [Table 3]. | Figure 3: (a) Effect of raptinal on ACF. Topographical view of colon mucosa after staining the tissue with methylene blue. ACF were observed and distinguished from the surrounding normal crypts by their increased size, distance from the lamina to basal cell surfaces and pericryptal zone at ×10. (A) normal control group (B) carcinogen control group (C) 100 mg/kg of raptinal-treated group (D) 200 mg/kg of raptinal-treated group (E) 400 mg/kg of raptinal-treated group. The arrows show the crypts. (b) Histological study of colon mucosa of rats at ×10 and ×40. (A) normal control group denoting M, Sm, Mm, CAC, Lamina propia, C, Ac and distinct Gc (B) carcinogen control groups showing DM, Hyp, Dys (C) 100 mg/kg raptinal-treated group depicting Dc, discontinuation of absorption cells denoted by arrowhead and loss of goblet cells (C), DM (D) 200 mg/kg raptinal-treated group depicting Hyp and neutrophil infiltration (Nu inf) (E) 400 mg/kg raptinal-treated group depicting Gc, newly formed C and no DM. (c) Effect of raptinal on in vivo antioxidant enzymes SOD and CAT, GST. *Significant difference as compared to carcinogen control group (P < 0.05). #Significant difference as compared to 100 mg/kg group (P < 0.05). $Significant difference as compared to 200 mg/kg group (P < 0.05). ACF = Aberrant crypt foci, CAC = Columnar absorptive cells, SOD = Superoxide dismutase, CAT = Catalase, GST = Glutathione, M = Mucosa, Sm = Submucosa, Mm = Muscularis mucosa, C = Crypts, Ac = Absorption cells, Gc = Goblet cells, DM = Depletion of mucin, Hyp = Hyperplasia, Dys = Dysplasia, Dc = Dilation of Column
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Histopathological analysis
The growth of noninvasive cancer involving abrasions on the mucosal layers was evident from the histological analysis of colorectal tissues of animals in the DMH-induced carcinogen control group [[Figure 3]b: B]. Both significant mucosal dysplasia and hyperplasia were present with the increase in the ACF multiplicity. The goblet and absorptive cells of the hyperplastic colon mucosa were mixed with some mucin loss. Comparing the ACF-bearing colon tissue to the normal colonic mucosa, the ACF-bearing colon tissue has a more pronounced and expanded lumen [[Figure 3]b: A]. In [[Figure 3]b: B] the dysplastic ACF exhibit mucosal layer expansion departing via mitosis, total mucin exhaustion, and goblet cell loss. Reduced occurrences of adenoma formation were seen in the raptinal treatment set since opposed to DMH persuaded carcinogen control group [[Figure 3]b: C-E]. There was no epithelial sluffing in the colonic tissue in 100 mg/kg and 200 mg/kg raptinal administered groups, although there was minimal hyperplasia in the epithelial region, as well as decreases in the ACF in lumen and no evidence of mucin deprivation [[Figure 3]b: C and D]. In the group of rats that received 400 mg/kg of raptinal, goblet cells have been visible in colonic tissue, as well as ACF production have been markedly reduced. Moreover, the colonic mucosa of the group administered 400 mg/kg of raptinal showed no signs of mucin depletion [[Figure 3]b: E].
Antioxidant study
Decreased intensity of Catalase (CAT), SOD, and glutathione has been discovered in the colonic tissue in the DMH control group. In contrast with the DMH control group, 400 mg/kg treatment group showed a significant increase in CAT, SOD, and glutathione levels with P < 0.05 [Figure 3]c.
Immunohistochemical analysis
The immunohistochemistry analysis to determine the expressions of the pro-inflammatory cytokines TNF-α and IL-6, tumor-suppressing protein p53, an anti-apoptotic protein Bcl-2, and the pro-apoptotic proteins Bax and caspase-3 [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, respectively. These proteins' expression in the vehicle control group was shown in [Figure 4]a: A and d: A. In contrast to the reduction in p53, Bax, and caspase-3 expression [[Figure 4]a: B, c: B and d: B] caused by the administration of DMH with DSS, a rise in the mutation of Bcl-2, TNF-α, and IL-6 were seen in mucosal area and goblet cells, as indicated by the white arrows in [Figure 4]b: B, e: B and f: B as compared with the vehicle control group (P < 0.05). White arrows in [Figure 4]a: C-E, c: C-E and d: C-E indicate a considerable superior the mutation of the proteins Bax, p53, and caspase-3 after therapy with raptinal, while the mutation of Bcl-2, TNF-α, and IL-6 has been significantly reduced [[Figure 4]b: C-E, e: C-E and f: C-E] as compared with DMH induced carcinogen control group (P < 0.05) [Table 4]. | Figure 4: (a) Immunohistochemical analysis of p53 expression in the colon tissues of rats at ×40. (A) Normal control group (B) carcinogen control group (C and D) 100 mg/kg and 200 mg/kg raptinal-treated animals (E) 400 mg/kg raptinal-treated animals showing significant expressions of p53, represented by white arrows (b) Immunohistochemical analysis of Bcl2 expression of rats at ×40. (A) normal control group (B) carcinogen control group showing significant expression of Bcl2 protein, represented by white arrows (C) 100 mg/kg treated animals (D and E) 200 and 400 mg/kg raptinal-treated animals (c) Immunohistochemical analysis of Bax expression in the colon tissues of rats at ×40. (A) normal control group (B) carcinogen control group (C and D) 100 mg/kg and 200 mg/kg raptinal-treated animals showing moderate expression of Bax (E) 400 mg/kg raptinal-treated animals showing significant expressions of Bax, represented by white arrows (d) Immunohistochemical analysis of caspase-3 expression in the colon tissues of rats at ×40. (A) normal control group (B) carcinogen control group (C and D) 100 mg/kg and 200 mg/kg raptinal-treated animals (E) 400 mg/kg raptinal-treated animals showing significant expressions of caspase-3, represented by white arrows (e) Immunohistochemical analysis of IL-6 expression in the colon tissues of rats at ×40. (A) normal control group (B) carcinogen control group showing significant expression of IL-6 as compared to normal control, represented by white arrows (C) 100 mg/kg treated animals (D and E) 200 and 400 mg/kg raptinal-treated animals (f) Immunohistochemical analysis of TNF-α expression in the colon tissues of rats at ×40. (A) normal control group (B) carcinogen control group showing significant expression of TNF-α as compared to normal control, represented by white arrows (C) 100 mg/kg treated animals depicting fewer expression of TNF-α as compared to carcinogen control group, represented by white arrows (D and E) 200 and 400 mg/kg raptinal-treated animals. IL = Interleukin, TNF-α = Tumor necrosis factor-α
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 | Table 4: Effect of raptinal on the expression of p53, Bcl2, Bax, caspase-3, interleukin-6, and tumor necrosis factor-α in colon tissues
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Raptinal causes suppression of Ki-67
Due to their distinct nuclear localization and ability to be measured by DAB-produced brown stains, the cells with Ki-67 labeling may be identified and discriminated. The percentage of cells having Ki-67 labeling as determined by the Labelling Index is shown in [Table 5] (LI). The manifestation of Ki-67 in vehicle control group is seen in [[Figure 5]a: A]. White arrowheads in the image above indicate that there were more Ki-67 marked cells in the lamina propria and mucosa in the carcinogen control groups [[Figure 5]a: B]. Animals treated with raptinal (100 mg/kg and 200 mg/kg) demonstrated a statistically decline (P < 0.05) in Ki-67 labeled cells [[Figure 5]a: C and D]. Comparing the 400 mg/kg raptinal administered group with the other treatment groups, the cell proliferation was dramatically reduced [[Figure 5]a: E]. | Figure 5: (a) Immunohistochemical analysis of Ki-67 expression at × 40. (A) normal control group (B) carcinogen control group showing strong expression of Ki-67, represented by white arrowheads (C and D) 100 and 200 mg/kg raptinal-treated groups (E) 400 mg/kg raptinal-treated groups (b) Immunohistochemistry of TUNEL apoptotic cells at ×40. (A) Normal control group (B) carcinogen control group (C and D) 100 and 200 mg/kg raptinal-treated groups (E) 400 mg/kg raptinal-treated group showing a significant increase in apoptosis, represented by white arrowheads. Approximately 700 cells were counted per field, 10 fields were examined per slide and 10 slides were examined per group. TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling
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Apoptotic assay through TUNEL method
There were TUNEL-positive apoptotic cells present when a brown stain was present covering the condensed chromatin of the apoptosis cells produced through DAB treatment. The animals in the vehicle control group showed normal levels of apoptotic events [[Figure 5]b: A] while, the DMH-induced carcinogen control group dramatically reduced the apoptotic events [[Figure 5]b: B]. A least 3-5 apoptotic cells have been found in around 700 cells in the carcinogen control group. White arrowheads in [[Figure 5]b: C-E] indicate that raptinal treatment significantly increased apoptotic events in the mucosal zone and goblet cells. On average, 10–15 cells have been seen in colonic tissues of 400 mg/kg raptinal administered group [[Figure 5]b: E]. In Table 5, the AI has been determined.
The ratio of cellular propagation to apoptotic events was exhibited through R value. The results of the TUNEL assay and cell proliferation assay showed a correlation between increased cellular propagation and significantly reduced the apoptotic events in the early stages of tumorigenesis. The increased proliferative activity in the carcinogen control group is indicated by its higher R value, while the increased apoptotic events in the treatment groups are indicated by its lower R value.
| » Discussion | | |
The pharmacophore analysis revealed the interaction of raptinal with Bcl-2 anti-apoptotic protein, p53 tumor repressive gene, Bax pro-apoptotic proteins, and caspase-3. The molecular docking of raptinal strongly suggest its ability to bind with the subsequent apoptotic proteins at their active site through a conventional hydrogen bond, van der Waals force, alkyl-alkyl interactions and brings about the initiation of apoptotic events in neoplastic cells.
Till now, the effects of raptinal in colorectal carcinoma and its promising chemotherapeutic action have not been demonstrated. Hence, the recent investigation focused on the evaluation of an anticancer activity of raptinal on HT-29 colorectal carcinoma cell line as well as DMH persuaded colorectal carcinoma in rat model. However, there are some confines in the investigation that additionally have to be highlighted which includes the evaluation of power assessment, investigation of numerous inflammatory biomarkers, that have to be examined for more attention of this molecule in clinical research.
MTT analysis on the HT-29 cell line, subsequently treatment by raptinal indicated its ability to reduce cellular propagation, thus inducing apoptotic events. The induction of apoptosis due to raptinal therapy has been analyzed by flow cytometry where a significantly increased % of early apoptosis was detected in HT-29 cell. Thus, the cell regulation halts in G0/G1 interphase which subsequently led to apoptotic events.
The histological assessment of colonic tissue demonstrated the existence of hyperplastic abrasions in mucosal region of DMH induced carcinogen control group. In addition, ACF count superior in hyperplastic abrasions and absolute mucosal dysplasia have been denoted in the DMH control group. The management of raptinal demonstrates a substantial reduce ACF counts which renovation of typical cell morphology along with significantly lower the hyperplastic abrasions and typical morphological features of goblet cells. This investigation notably depicts the chemotherapeutic effects of raptinal counter to DMH persuaded colorectal carcinoma in rat model.
The immunohistochemistry assessment of colon tissues represented the effects of Bcl2, p53, Bax, caspase-3, TNF-α/IL-6 to demonstrate anticancer activity of raptinal in DMH persuaded colorectal carcinoma. Raptinal triggers apoptotic events in the malignant cells via regulation of MOMP directly and by activating the proapoptotic proteins Bax and caspase-3 with subsequent lower mutation of Bcl-2 protein. Conversely, increased expression of Bcl2 was noted in malignant cells by the lower expression of apoptotic proteins. There is a bright connection with the chronic inflammation and cellular progression in the malignancy. The chronic inflammation can be generally facilitated through pro-inflammatory cytokines such as TNF-α and IL-6, that activated the epigenetic modification in promoter sites of cancer-reducing genes as well as cell cycle-controlling proteins. As a result of the deactivation of the cancer repressive gene, p53.[26] Together with, IL-6 causes the downstream effect of p53 proliferation concerned with inferior the apoptotic events that significantly result in malignant cell survival, growth and proliferation. Raptinal treatment significantly decreased the expression of inflammatory markers and thus a significant elevation of p53 level was also been exhibited in CRC cells as a comparison with the DMH-induced carcinogen control group.
In the carcinoma state, an inflammatory cytokine, TNF-α, acting as a crucial part which was frequently obtained in the tumor milieu.[27] In inflammation-related carcinoma, the participation of TNF-α was recognized that augments malignant cellular survival, growth, metastasis, and alteration of the immune systems.[28] In vivo assessment reported the higher mutation of TNF-α as well as IL-6 in the DMH control group since comparison with vehicle control group while the raptinal-treated group showed that the dramatically reduced the mutation of IL-6 and TNF-α for permitting us to conclude that the treatment with raptinal has an affirmative action on colorectal carcinoma.
The higher mutation of Ki-67 is designated by the marker of uncontrolled epithelial cellular progression and pre-cancerous state.[29] The current investigation revealed that the p53 is associated to control DNA imitation via the alteration of Ki-67 levels.[30] Therefore, carcinogen control group represented a significantly increases the number of Ki-67 marked cells and lower AI value that suggesting the abnormal cellular progression which been concomitant with ACF counts in colon mucosa. Raptinal-treated group has been reported that a remarkably lesser numeral of Ki-67 labeled cells which significantly higher the AI value. As a consequence, the outcomes of the study suggested that the treatment with raptinal increases p53 mutation thus obstructing the cellular propagation through the initiation of apoptosis in malignant cells that consequently lower the ACF counts and hyperplastic abrasions.
| » Conclusions | | |
At the end of the study, these findings indicate that raptinal effectively reduces colon cancer by inducing apoptosis via the Bcl-2/p53/caspase-3/Bax signaling ways as well as suppressing TNF-α and IL-6 induced chronic inflammation in the colorectal carcinoma microenvironment [Figure 6]. Furthermore, the in vitro and in vivo investigation revealed that the raptinal treatment may obstruct, repeal, or interrupt the development of colorectal carcinoma and additionally in future can provide a prospective competitor for colon carcinoma chemotherapy in clinical research. | Figure 6: Chemotherapeutic action of raptinal against colorectal carcinoma
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Acknowledgment
The authors are thankful to the Department of Pharmaceutical Technology, NSHM Knowledge Campus–Kolkata, for their continuous support throughout the experiment.
Financial support and sponsorship
Natural Science Foundation of Shandong Province (ZR2022MC174)
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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