Introduction
Cancer disease is a serious health and social problem. Despite therapeutic advances, cancer is the second leading cause of morbidity and mortality worldwide (
https://www.who.int/health-topics/cancer#tab=tab_1). Although treating cancer with chemotherapy and radiotherapy is effective, it is associated with serious side effects, such as drug resistance or non-selectivity [
1]. These problems illustrate the need to develop new, more effective anticancer therapies and safer agents [
2]. Natural products or their direct derivatives play an important role in the discovery of new drugs for the treatment of cancer [
3]. The plant compounds have different inhibitory effects on cancer onset, development, progression, and metastasis [
4,
5]. Plants of the genus
Capsicum, belonging to the family
Solanaceae, are an important source of biologically active substances [
6]. We currently recognize 25 wild species and five domesticated species in the genus
Capsicum [
7],
Capsicum annuum,
Capsicum frutescens,
Capsicum chinense,
Capsicum baccatum, and
Capsicum pubescens [
8]. In addition to their use in gastronomy, peppers are also excellent producers of secondary metabolites, which have various pharmacological properties and contain cytotoxic compounds [
9].
A characteristic feature of wide varieties of peppers is their intense pungency caused by a group of bioactive phytochemicals, capsaicinoids, classified as alkaloids. They are vanilylamides derived from branched-chain C8-C11 (E) -monocline fatty acids and branched-chain or straight-chain saturated fatty acids [
10]. One of the capsaicinoids, capsaicin (48.6%) (CAP) is the most abundant compound in chili peppers, followed by 6,7-dihydrocapsaicin (36%) (DHK), nordihydrocapsaicin (7.4%), homodihydrocapsaicin (2%), and homocapsaicin (2%). CAP (trans-8-methyl-N-vanillyl-6-non enamide) is a crystalline, lipophilic, colorless, and odorless alkaloid soluble in fats, alcohols, and oils [
11,
12]. Many studies have shown that capsaicinoids have a wide range of biological and physiological effects. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes, where gene expression has identified candidate genes possibly involved in capsaicinoid biosynthesis [
10,
13]. CAP has analgesic [
14,
15] and anti-inflammatory effects [
16], decreases the prevalence of obesity [
17] and metabolic syndrome, improves gastrointestinal [
18] and cardiovascular symptoms [
19], and is characterized by antitumor activity [
20,
21]. The nutritional, and anti-obesity properties of different chili peppers was presented by Azlan et al. [
22]. Due to ability of CAP to mediate cell cycle arrest and induce cell apoptosis in in vitro experiments, it reduced the growth of human leukemia cells [
23], skin tumor cells [
24], prostate [
25,
26], bladder [
27], stomach [
28,
29], colon [
30], nasopharynx [
31], liver [
32], lung [
33], and breast cancer [
34]. Capsaicin can modify the function of many genes associated with the lifespan of cancer cells, initiating apoptosis, arresting cell growth, and suppressing angiogenesis and metastasis [
35,
36]. By inducing apoptosis in cancer cell lines, healthy cells remain intact [
37]. Several studies have shown that new combination therapies with various phytochemicals and chemopreventive drugs can induce increased antitumor activity through an additive or synergistic effect [
38]. Capsaicinoids potentiate the chemotherapeutic effect and relieve pain in cancer patients. CAP acts synergistically with other anticancer agents; thus, it can be used with other chemotherapeutic agents in cancer treatment [
39‐
41]. Colorectal cancer is one of the most commonly diagnosed diseases in the world [
42]. The incidence of this disease is closely related to the composition of the diet and the amount of vegetables consumed. Currently, preclinical studies testing the anticancer effects of CAP on colon cancer are lacking [
43‐
45].
DNA topoisomerases regulate conformational changes in DNA topology during normal cell growth, such as DNA replication, transcription, recombination, and DNA repair [
46]. They are also targets for several anticancer drugs [
47,
48]. Topoisomerase inhibitors interfere with human topoisomerases, or they can act as inhibitors without tumor cell toxicity [
49].
The aim of our work was to investigate the DNA-damaging/protective activities of the studied chili extracts. The DNA topology was studied with electrophoretic detection of topological changes induced in plasmid DNA. We hypothesized that an extract of CAP, DHK, and other varieties of chili peppers would influence both Topo I and II and exploit the ability to act on two distinct enzymatic targets, thereby maximizing the potential therapeutic effects. The biological activity of extracts was assessed using an MTT assay of the human colon cancer cell line HCT-116, potentially usable in cancer therapy and drug screening.
Materials and methods
Sample processing
All analyzed types of chili peppers: Habanero Red (HR), Habanero Maya Red (HMR), Trinidad Moruga Scorpion (TMS), Jalapeno (J), Serrano pepper (SP), Habanero Red Savina (HRS), Bhut Jolokia (BJ), Jamaica Rosso (JR) were grown and harvested at the Department of Food Hygiene, Technology and Safety of the University of Veterinary Medicine and Pharmacy in Košice. The cultivated peppers were dried in a laboratory oven with ventilation at 40 ± 5 °C. Before drying, the chili peppers were cut in half or quarters (depending on the size) in order to speed up the drying and to avoid undesired changes. The peppers were dried together with the placenta and seeds. After drying, they were stored in a closed glass container in a dry, dark place until analysis.
Completely dried fruits of various varieties of peppers were ground completely on an electric stainless steel mixer. From each pepper sample 0.2 g of ground pepper was weighed into 10 mL volumetric flasks and 2 ml of 96 (v/v) ethanol were added. The mixture was mixed on a Vortex homogenizer and placed in an ultrasonic bath for 5 minutes. After homogenization, the samples were macerated for 24 hrs in a dry and dark place under laboratory conditions. After the indicated time, the individual samples were filtered through filter paper into 10 mL volumetric flasks, washed with absolute ethanol and made up to 10 mL with the same solvent. The extracts were stored in sealed flasks at 5 °C in a refrigerator. Samples were filtered through a membrane syringe filter (Q-Max® RR Siringe Filters, Frisenette, 25 mm, 0.22 μm PVDF) prior to HPLC analysis. If CAP or DHK concentrations were outside the calibration range, the samples were diluted with absolute ethanol.
HPLC analysis
CAP and DHK standards were purchased from Sigma-Aldrich (USA), absolute ethanol from Emparta (Germany) and HPLC grade acetonitrile from Fisher (UK). The concentration of CAP and DHK in the extracts was determined using a Dionex UltiMate 3000 RS with a diode array detector (DAD) and a programmable Chromeleon Chromatography Data System, version 7.2 (Thermo Fisher Scientific, Germany). HPLC analysis was performed using a Polaris 5 column (C18-A 250 × 4.6 mm, 5 m, under isocratic conditions, at 40 °C and flow rate 1 mL.min−1. The sample was dosed using an autosampler and its volume was 10 L. The mixture of acetonitrile and water (70:30, v/v) was used as the mobile phase. CAP and DHK were measured with UV detector (DAD) at 282 nm. The quantification and HPLC method validation was based on the calibration curve fitting by linear regression analysis. Linear correlation between the peak area and the applied concentration was found in the concentration range 5–500 μg.mL−1, as confirmed by the correlation coefficient (0.99902 for CAP and 0.99932 for DHK). The x-axis in the graphical dependence represented the concentration of CAP or DHK and the y-axis was the peak area in the chromatographic record. The mean values for the regression equation were y = 0.027.x + 0.2049 for CAP and y = 0.0067x + 0.0057 for DHK.
Nuclease activity
A nuclease activity study was performed prior to the experiments, which confirmed that none of the samples were able to cleave plasmid DNA and that the ethanol content did not affect the plasmid. Nuclease activity of selected molecules were studied using isolated plasmid pUC19 (isolated by the alkaline lysis method in our laboratory). Mixture of pUC19 in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0, 2 μl) (Sigma-Aldrich), 10 mM Tris-HCl buffer (pH 7.4, 25 μL) (Sigma-Aldrich) and studied compounds (3 μL) in final concentration 1/10 of stock solution were incubated at 37 °C for 18 hrs. After incubation solution of bromophenol blue and xylene violet (3 μL) and samples were subjected on 1.0% v/v agarose (Sigma-Aldrich) gel. Electrophoresis ran 4 h at 35 V in 1xTAE (40 mM Tris, 20 mM acetic acid glacial (Centralchem), 1 mM EDTA) (Sigma-Aldrich) then it was stained with ethidium bromide for 15 min and destained in deionized water for 7 min. Electrophoretic record was photographed with electrophoretic system SYNGEN and processed with GeneSnap program.
Decatenation assay for topoisomerase II
Topoisomerase II (Topo II) decatenation assay was carried out according to the Inspiralis protocol using kinetoplast DNA (kDNA, 200 ng) in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) (Sigma-Aldrich) and appropriate amount of diluted human topoisomerase IIa (hTop IIa, 1.5 U) enzyme in dilution buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 50% v/v glycerol, 50 μg/mL albumin (Inspiralis). Assay was conducted in final concentration 1/10 of stock solutions (3 μL from stock solutions, in the case of CAP we used two stock solutions – 0.5 mg/mL and 1.0 mg/mL). Samples were prepared using a mixture of appropriate assay buffer (50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 100 μg/mL albumin, supplied as 10 × (Inspiralis)), 30 mM ATP (final concentration 1 mM) and deionized water in final volume 30 μL. Samples were incubated 30 min at 37 °C and after that reaction was stopped with STEB (30 μL, 40% v/v sucrose (Centralchem), 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5 g/dm3 bromophenol blue (Sigma-Aldrich)) and purified with chloroform:isoamylalkohol (Centralchem) (30 μL, 24:1) solution and subjected to the 1% v/v agarose gel in 1× TAE (40 mM Tris, 20 mM acetic acid glacial (Centralchem), 1 mM EDTA (Sigma-Aldrich)). Dilution of ethanol (% v/v) in samples for Topo I a Topo II was 3 μl ethanol/ 30 μl sample. Electrophoresis ran 4 hrs at 35 V and then agarose gel was stained with ethidium bromide solution and destained in water. Electrophoretic record was documented using UV light.
Relaxation assay for topoisomerase I
Impact of molecules on relaxation ability of topoisomerase I was studied on human topoisomerase I (hTopo I, Inspiralis) on plasmid pBR322 (Inspiralis). Mixture of plasmid (0.5 μg) in TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA), diluted hTopoI (0.5 U) in dilution buffer (10 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, 50% v/v glycerol, 50 μg/mL albumin (Inspiralis)) and studied compounds in final concentrations of 1/10 of stock solutions (3 μL from stock solution, in case of CAP were used two stock solutions – 0.5 mg/mL and 1.0 mg/mL) were incubated at 37 °C for 30 minutes in 1 × concentrated assay buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.25 mM EDTA, 5% glycerol, 50 μg/mL albumin, supplied as 10× stock (Inspiralis)) and deionized water in the final volume of 30 uL. After incubation reaction was stopped with STEB (30 μL, 40% v/v sucrose (Centralchem), 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5 g/dm3 bromophenol blue (Sigma-Aldrich)) and samples were purified with chloroform:isoamyl alcohol (Centralchem) (30 μL, 24:1) and upper layer of samples was subsequently subjected on 1% v/v agarose gel. Electrophoresis ran for 15 min at 20 V to allow subjected samples to penetrate the gel and then continued for 4 hrs at 35 V in 1 × TAE buffer (40 mM Tris, 20 mM acetic acid glacial (Centralchem), 1 mM EDTA (Sigma-Aldrich)). Then agarose gel was stained with ethidium bromide solution (15 min) and destained with deionized water (10 min). Electrophoretic record was visualized by UV light, photographed by SYNGEN system and processed in GeneSnap program.
Cell line
Human colon carcinoma cell line HCT116 (ATCC® CCL-247™) was cultured in RPMI medium supplemented by antibiotics (100 U/ml penicillin + 100 μg/mL penicilin-streptomycin) and 10% of FBS (fetal bovine serum) in the presence of 5% CO2 in a humidified atmosphere at 37 °C. If 5 × 106 cells were plated onto a 75 cm2 flask, the culture reaches 70-90% confluency in 2-3 days and was ready to split or harvest for experiments. To determine the linear range of each assay, six cell densities ranging from 50 to 10 000 cells/well were plated into sterile 96-well plates and incubated for 24, 48 or 72 hrs.
MTS assay
MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells. MTS was used for determining the number of viable cells in proliferation, cytotoxicity, or chemosensitivity. HCT116 cells were seeded at 5000 cells per well into 96-well microplates, after 24 hrs incubation treated with extracts of individual samples (extracts of peppers), CAP or DHK. After 24 hrs incubation of HCT116 cells, medium was removed and replaced with RPMI containing 10% fetal bovine serum (FBS) and extracts of dry extracts in ethanol peppers: HR, HMR, TMS, J, S, HRS, BJ, JR in dilution 100×, 500×,1000× and incubated at 37 °C and 5% CO2 for 24 hrs. Control group (cells HCT116) was not affected by extracts, CAP1 - CAP6 concentrations of CAP at 10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM. DHK1 - DHK6 concentrations of DHK at 10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM. The MTS assay was performed at 48 hrs and 72 hrs. After the 24 hrs exposure to the cells, 25 μL of CellTiter 96, AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA) was added to the cell culture medium, and incubated for 3 hrs at RT. The absorbance of wells at 490 nm was measured using a microplate reader. Results were expressed as means (±sd) of quadruplicate wells obtained by subtraction from cell-free equivalents, to eliminate A490 produced by the media alone. Effects of pepper extracts on HCT116 cells were analysed by MTS assay expressed as a fold of control [%] of absorbance generated in cell-mediated MTS assays to the control group.
Agilent × CELLigence real-time cell analysis
HCT116 cells (5 × 103 cells/well) were seeded in 96-well plates (RTCA E-Plates 96) on xCELLigence RTCA systems (Agilent). The cells were treated with chilli extracts 24 hrs after seeding. HCT116cells were cultured in the absence or presence of tested drugs at concentrations ranging from 100 μM to 100 nM. The cell adhesion and spread without the manipulation of the cells were continuously monitored in 60 min intervals over the course of a 120 hrs observation period using the ×CELLigence RTCA system.
Statistical analysis
Experiments under all conditions were performed in at least three independent measurements. Mean value and standard deviation were calculated using descriptive statistics. The data were analyzed by using the RTCA software Pro 1.2.1 (ACEA Bioscience). Statistical analysis was carried out by a non-parametric method, one-way ANOVA using SigmaPlot (Ver. 12.0). Differences were considered significant *p < 0.05; **p < 0.01; ***p < 0.001.
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