The Chemopreventive Effects of Polyphenols and Coffee, Based upon a DMBA Mouse Model with microRNA and mTOR Gene Expression Biomarkers

Polyphenols are capable of decreasing cancer risk. We examined the chemopreventive effects of a green tea (Camellia sinensis) extract, polyphenol extract (a mixture of blackberry (Rubus fruticosus), blackcurrants (Ribes nigrum), and added resveratrol phytoalexin), Chinese bayberry (Myrica rubra) extract, and a coffee (Coffea arabica) extract on 7,12-dimethylbenz[a]anthracene (DMBA) carcinogen-increased miR-134, miR-132, miR-124-1, miR-9-3, and mTOR gene expressions in the liver, spleen, and kidneys of CBA/Ca mice. The elevation was quenched significantly in the organs, except for miR-132 in the liver of the Chinese bayberry extract-consuming group, and miR-132 in the kidneys of the polyphenol-fed group. In the coffee extract-consuming group, only miR-9-3 and mTOR decreased significantly in the liver; also, miR-134 decreased significantly in the spleen, and, additionally, miR-124-1 decreased significantly in the kidney. Our results are supported by literature data, particularly the DMBA generated ROS-induced inflammatory and proliferative signal transducers, such as TNF, IL1, IL6, and NF-κB; as well as oncogenes, namely RAS and MYC. The examined chemopreventive agents, besides the obvious antioxidant and anti-inflammatory effects, mainly blocked the mentioned DMBA-activated factors and the mitogen-activated protein kinase (MAPK) as well, and, at the same time, induced PTEN as well as SIRT tumor suppressor genes.


Introduction
Nowadays, the incidence and mortality of cancer in high-income countries (HIC) is decreasing [1,2], but in the low-and middle-income countries (LMIC), the trend-line is still supposed to increase slightly [1]. According to the WHO's assessment, 30-50% of cancer cases could have been prevented [3]. Indeed, the improving tendency in HIC is the result of successful primer prevention, early detection, and advanced therapies [1]. However, cancer is still, globally, the second leading cause of death (with approximately 9.6 million deaths in 2018) [4] and is also the greatest disease burden.
In this study, the preventive effects of several polyphenols, namely the green tea extract (catechin content of 80%), Chinese bayberry extract (myricetin content of 80%), polyphenol extract (with 4 g/100 mL added to resveratrol), and coffee extract were examined in a DMBA-treated mouse model to elucidate the effects of chemopreventive agents on the expression profile of the mentioned miRs and mTOR, in order to decide if their elevation caused by DMBA exposure can be mitigated or not.

Animal Treatment
The experimental setting in our study was similar to that described by Szabo et al. 2021 [9]. We utilized six groups of female CBA/Ca mice (n = 6) aged 12 weeks. Pre-feeding was not given to the untreated and DMBA-treated control groups; however, one group received 4 mg/day of the animal green tea (Camellia sinensis) excerpt (Xi'an Longze Biotechnology Co. Ltd., Xi'an, China); one group received 2.5 mg/day of the animal Chinese bayberry (Myrica Rubra) supplement (Xi'an Longze Biotechnology Co. Ltd., Xi'an, China); one group received 30 mg/day of the animal polyphenol extract (common grapevine (Vitis vinifera 'Cabernet Sauvignon') seed and peel, blackberry 'thorn free' (Rubus fruticosus 'Thornfree') seed and peel, and blackcurrants, plus an additional 4 g/100 mL of resveratrol, in particular, FruitCafe TM (Slimbios Ltd., Budapest, Hungary); and one group received a coffee (Coffea arabica) extract for two weeks (30 mg/day/animal, up to 150 mL) in addition to their regular feed. All other five classes of animals received 20 mg/bwkg DMBA intraperitoneally (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 0.1 mL of corn oil, with the exception of the untreated control group. Animals were put to death by cervical dislocation after 24 h of DMBA exposure, and their kidneys, liver, and spleen were extracted. Table 1 summarizes the specifics of the experimental setup, as well as the substances used.
Tomesz et al.'s 2020 publication [36] utilized the same experimental procedure. Animal experimentation standards and criteria were followed when housing mice. All the measures have been taken to avoid unnecessary pain. The experiment was carried out in accordance with current ethical rules (the Animal Welfare Committee of the University of Pécs issued the ethical license no. BA02/2000-79/2017).

Collective Isolation of RNA
A TRIZOL reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to isolate total cellular RNA, according to the manufacturer's guidelines. The quality of the RNA was determined using NanoDrop absorption photometry, and only RNA fractions with A > 2.0 at 260/280 nm were utilized in the RT-PCR (reverse transcription polymerase chain reaction) procedure.

Polymerase Chain Reaction in Reverse Transcription (RT-PCR)
On a LightCycler 480 qPCR system (Roche Diagnostics, Indianapolis, IN, USA), onestep PCR, containing a reverse transcription and a target amplification, was done in a 96-well plate using Kapa SYBR FAST One-step qPCR equipment (Kapa Biosystems, Wilmington, MA, USA).
The following temperatures of the program were used: After a 5-min incubation at 42 • C, a 3-min incubation at 95 • C, 45 cycles (95 • C for 5 s, 56 • C for 15 s, and 72 • C for 5 s) was executed, with a fluorescence reading taken at the finish of each cycle. To improve the specificity of the amplification, a melting curve analysis was done on each run (95 • C for 5 s, 65 • C for 60 s, and 97 • C). The following components were used in the reaction mixture: 10 µL of KAPA SYBR FASTqPCR Master Mix, 0.4 µL of KAPA RT Mix, 0.4 of dUTP, 0.4 µL of primers, and 5 µL of a miR template in a total amount of 20 µL of sterile double-distilled water. Table 2 summarizes the primer sequences (5 -3 ) of the mTORC1 gene, the studied miRs (miR-134, miR-132, miR-124-1, and miR-9-3), and the internal control (the mouse U6 gene). Integrated DNA Technologies (Integrated DNA Technologies Inc., Coralville, Iowa, USA) synthesized the primers, and the sequences were obtained from earlier publications [44,45].

Calculations and Statistical Analyses
The 2 −∆∆CT approach was used to determine and compare relative miR expression levels. The Kolmogorov-Smirnov test, Levene's test, and the T-probe were used to compare averages and test distributions and variances throughout the statistical study. For computations and analyses, the IBM SPSS 21 statistical program (International Business Machines Corporation, Armonk, NY, USA) was utilized. The statistical standard of significance was set at p < 0.05.

Discussion
DMBA induces cellular damage by releasing reactive oxygen species (ROS), which triggers the production of cytokines (such as TNF, IL1, IL6) and transcription factors (such as NF-kB) [29,38,46], as well as lowering the protective glutathione (GSH) level [29,38,46,47]. These consequences result in redundantly activated inflammatory and proliferative secondary signal transduction pathways that are self-induced.
Myricetin in liver cells as a pro-oxidant increases hydroxyl radicals (˙OH) if catalase (CAT) and SOD enzymes are blocked [94]. Aromatic hydrocarbons (such as DMBA) produce singlet molecular oxygen [95] that reacts with the histidine group of CAT and SOD, deteriorating these enzymes [96], leading to further increased˙OH levels, which induces protective miR-132 expression [97] in coherence with the results of this study.
Moreover, in the coffee consuming group, the traces of acrylamide and furan exert antagonistic effects against the examined chemopreventive agents; namely, acrylamide (≤100 µmol/L) in vitro, which significantly increases the proliferation of human HCC HepG2 cells and induces the EGFR/PI3K/AKT/cyclin D1 pathway, leading to decreased PTEN levels [98]. The epigenetic carcinogen furan also alters relevant cell cycles, as well as the apoptosis regulator gene expression in the rat's liver [99], and forms metabolites, which decreases GSH levels with chemical reactions [100].
The above-mentioned experimental materials and their decay products, substrates, enzymes, proteins, and signal transducer molecules orchestrate the observed expression patterns of miRs and mTORs, as well as influencing the cell proliferation ( Figure 5).

Conclusions
In all the examined organs in the green tea, myricetin, and flavonoid extract-treated groups, the DMBA elevated expression levels of miR-134, miR-132, miR-124-1, miR-9-3, and mTOR decreased significantly-except for miR-132 in the liver of the Chinese bayberry extract-consuming group, and miR-132 in the kidneys of the flavonoid fed group. However, in the coffee consuming group, only miR-9-3 and mTOR decreased significantly in the liver, miR-134 decreased in the spleen, and additionally, miR-124-1 decreased in the kidneys (Table 3).
Individual molecular features were indicated also; for example, the liver-specific prooxidant effect of myricetin increased only in the liverthe ROS sensitive miR-132 expression, in comparison with other studied organs [97]. In the coffee consuming group, the effects of beneficial flavonoids, chlorogenic acids, and melanoidins [17] were most likely partly antagonized by the carcinogen acrylamide and furan content of coffee [98,99].

Conclusions
In all the examined organs in the green tea, myricetin, and flavonoid extract-treated groups, the DMBA elevated expression levels of miR-134, miR-132, miR-124-1, miR-9-3, and mTOR decreased significantly-except for miR-132 in the liver of the Chinese bayberry extract-consuming group, and miR-132 in the kidneys of the flavonoid fed group. However, in the coffee consuming group, only miR-9-3 and mTOR decreased significantly in the liver, miR-134 decreased in the spleen, and additionally, miR-124-1 decreased in the kidneys (Table 3).
Individual molecular features were indicated also; for example, the liver-specific prooxidant effect of myricetin increased only in the liverthe ROS sensitive miR-132 expression, in comparison with other studied organs [97]. In the coffee consuming group, the effects of beneficial flavonoids, chlorogenic acids, and melanoidins [17] were most likely partly antagonized by the carcinogen acrylamide and furan content of coffee [98,99].
Moreover, the results could be deceptive, since in the late stages, malignant tumors mostly also downregulated anticarcinogen miRs, for example, miR-134 in invasive and metastatic HCC and RCC [102], or both miR-124 and miR-134 in glioblastoma, and miR-124 in squamous cell carcinoma [88]. However, miR-9 is upregulated in glioma [88]. Therefore, we can suppose that expression levels of mTOR and miRs are biomarkers, rather than relevant signal transductors, in this context [103].
In summary, the novel finding of this study is that the expression patterns of miR-9-3, miR-124-1, miR-132, miR-134, and mTOR, as molecular epidemiological biomarkers, indicated the early carcinogen effect of DMBA and the anticarcinogen effects of the polyphenol extract, green tea extract, Chinese bayberry extract, and coffee extract, which are chemopreventive agents against DMBA exposure, in accordance with the specific molecular features of the contained compounds. Our results contribute to the research of chemoprevention by assuming that the regular consumption of a diet abundant in polyphenols, as well as coffee, exerts anti-inflammatory and anti-cancer effects. These assumptions may form further investigations to improve our eating habits.