key: cord-0030349-k7i5bq6c
authors: Molnar, Richard; Szabo, Laszlo; Tomesz, Andras; Deutsch, Arpad; Darago, Richard; Raposa, Bence L.; Ghodratollah, Nowrasteh; Varjas, Timea; Nemeth, Balazs; Orsos, Zsuzsanna; Pozsgai, Eva; Szentpeteri, Jozsef L.; Budan, Ferenc; Kiss, Istvan
title: The Chemopreventive Effects of Polyphenols and Coffee, Based upon a DMBA Mouse Model with microRNA and mTOR Gene Expression Biomarkers
date: 2022-04-12
journal: Cells
DOI: 10.3390/cells11081300
sha: 06dcee219f463255d6ea51fa3e77f2b3630d28ab
doc_id: 30349
cord_uid: k7i5bq6c

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.

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.

Therefore, novel chemopreventive strategies are warranted to enhance anticarcinogen mechanisms [5] [6] [7] [8] [9] [10] . In vitro studies and in vivo animal experiments suggest antimutagenic Thus, the mentioned molecular epidemiological biomarkers indicate DMBA exposure early and in a reliable manner [26, 32, 33, 35, 39, 40] . Furthermore, we also examined the gene expression level of the mammalian target of rapamycin (mTOR), which is involved in several cellular homeostasis mechanisms [36, 38] . More specifically, the liver, kidneys, and spleen parenchymal organs were studied because the DMBA treatment caused a significantly increased expression on relevant miR-134, miR-132, miR-124-1, miR-9-3, and mTOR in those organs for at least 24 h, based upon earlier research data [36] [37] [38] . Moreover, in earlier studies, the examined chemopreventive polyphenol extract, green tea extract, Chinese bayberry extract, and coffee extract ameliorated the DMBA, caused repetitive long interspersed element-1 (LINE-1) DNA hypomethylation [9] .

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.

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).

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. 

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] . 

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.

In the livers of animals, the consumption of the polyphenol extract significantly reduced the expressions of miR-9-3 (−41%; p < 0.05; SD = 11.1%), miR-124-1 (−68%; p < 0.001; SD = 10.1%), miR-132 (−62.9%; p < 0.001; SD = 9.2%), miR-134 (−77.9%; p < 0.001; SD = 5.6%), and mTORC1 (−49%; p < 0.001; SD = 8.4%) when compared to the positive DMBA control group ( Figure 1A) . We also observed a significant decrease in the expression of miR-9-3 (−38%; p < 0.05; SD = 12.1%), miR-124-1 (−59%; p < 0.05; SD = 9.8%), miR-132 (−62.4%; p < 0.001; SD = 8%), miR-134 (−60.4%; p < 0.001; SD = 8%), and mTORC1 (−39%; p < 0.001; SD = 8.6%) in the spleens of animals, compared to the positive DMBA control ( Figure 1B ). In the kidneys of animals, miR-9-3 (−59%; p < 0.001; SD = 7.8%), miR-124-1 (−62%; p < 0.05; SD = 13.1%), miR-134 (−81.4%; p < 0.001; SD = 3.7%), and mTORC1 (−59%; p < 0.001; SD = 6.3%) expressions were significantly lower in response to the polyphenol extract, compared to the positive DMBA control ( Figure 1C ), while the values for miR-132 (−27.1%; p = 0.051; SD = 13.7%) were not statistically significant.

The consumption of the green tea extract significantly reduced the expression of miR-9-3 (−33%; p < 0.05; SD = 12.9%), miR-124-1 (−69%; p < 0.001; SD = 7.4%), miR-132 (−45.4%; p < 0.05; SD = 10.2%), miR-134 (−59.2%; p < 0.001; SD = 8.9%), and mTORC1 (−57%; p < 0.001; SD = 6.7%) in the livers of animals, compared to the positive DMBA control ( Figure 2A ). The green tea extract resulted in a decrease in the expression of miR-9-3 (−56%; p < 0.001; SD = 8.5%), miR-124-1 (−62%; p < 0.001; SD = 11.3%), miR-132 (−61.1%; p < 0.001; SD = 9.1%), miR-134 (−47.6%; p < 0.05; SD = 11.2%), and mTORC1 (−58%; p < 0.001; SD = 5.1%) in the spleens, compared to the positive DMBA control ( Figure 2B ). In the kidneys, we also observed a significant decrease in the expression of miR-9-3 (−48%; p < 0.05; SD = 11.4%), miR-124-1 (−36%; p < 0.05; SD = 16.6%), miR-132 (−59.6%; p < 0.001; SD = 10.8%), miR-134 (−53.3%; p < 0.001; SD = 11.1%), and mTORC1 (−57%; p < 0.001; SD = 5.6%) in the group consuming the green tea extract, compared to the positive DMBA control ( Figure 2C ). 

The consumption of the green tea extract significantly reduced the expression of miR-9-3 (−33%; p < 0.05; SD = 12.9%), miR-124-1 (−69%; p < 0.001; SD = 7.4%), miR-132 (−45.4%; p < 0.05; SD = 10.2%), miR-134 (−59.2%; p < 0.001; SD = 8.9%), and mTORC1 (−57%; p < 0.001; SD = 6.7%) in the livers of animals, compared to the positive DMBA control ( Figure  2A ). The green tea extract resulted in a decrease in the expression of miR-9-3 (−56%; p < 0.001; SD = 8.5%), miR-124-1 (−62%; p < 0.001; SD = 11.3%), miR-132 (−61.1%; p < 0.001; SD = 9.1%), miR-134 (−47.6%; p < 0.05; SD = 11.2%), and mTORC1 (−58%; p < 0.001; SD = 5.1%) in the spleens, compared to the positive DMBA control ( Figure 2B ). In the kidneys, we also observed a significant decrease in the expression of miR-9-3 (−48%; p < 0.05; SD = 11.4%), miR-124-1 (−36%; p < 0.05; SD = 16.6%), miR-132 (−59.6%; p < 0.001; SD = 10.8%), miR-134 (−53.3%; p < 0.001; SD = 11.1%), and mTORC1 (−57%; p < 0.001; SD = 5.6%) in the group consuming the green tea extract, compared to the positive DMBA control ( Figure  2C ). 

In the liver, a statistically significant decrease was observed in miR-9-3 (−58%; p < 0.001; SD = 9.1%), miR-124-1 (−43%; p < 0.05; SD = 14.6%), miR-134 (−40.6%; p < 0.05; SD = 16.8%), and mTORC1 (−39%; p < 0.001; SD = 9.6%) in the Chinese bayberry extract group compared to the positive DMBA control, while the decrease in miR-132 (−19.1%; p = 0.14; SD = 14.9%) was not statistically significant ( Figure 3A ). There were statistically significant downward changes for miR-9-3 (−46%; p < 0.05; SD = 11.1%), miR-124-1 (−57%; p < 0.05; SD = 12.9%), miR-132 (−32.3%; p < 0.05; SD = 15.1%), miR-134 (−51.8%; p < 0.001; SD = 10.3%), and mTORC1 (−32%; p < 0.001; SD = 8.6%) in the spleens of the Chinese bayberry extract-consuming group, compared to the positive DMBA control ( Figure 3B ). In the kidneys, compared to the positive DMBA control, a statistically significant decrease could be observed for miR-9-3 (−40%; p < 0.05; SD = 13.2%), miR-124-1 (−51%; p < 0.05; SD = 14%), miR-132 (−57.9%; p < 0.001; SD = 10.5%), miR-134 (−28.8%; p < 0.05; SD = 12.8%), and mTORC1 (−22%; p < 0.05; SD = 11.9%) in the Chinese bayberry extract group ( Figure 3C ). 

In the liver, a statistically significant decrease was observed in miR-9-3 (−58%; p < 0.001; SD = 9.1%), miR-124-1 (−43%; p < 0.05; SD = 14.6%), miR-134 (−40.6%; p < 0.05; SD = 16.8%), and mTORC1 (−39%; p < 0.001; SD = 9.6%) in the Chinese bayberry extract group compared to the positive DMBA control, while the decrease in miR-132 (−19.1%; p = 0.14; SD = 14.9%) was not statistically significant ( Figure 3A ). There were statistically significant downward changes for miR-9-3 (−46%; p < 0.05; SD = 11.1%), miR-124-1 (−57%; p < 0.05; SD = 12.9%), miR-132 (−32.3%; p < 0.05; SD = 15.1%), miR-134 (−51.8%; p < 0.001; SD = 10.3%), and mTORC1 (−32%; p < 0.001; SD = 8.6%) in the spleens of the Chinese bayberry extractconsuming group, compared to the positive DMBA control ( Figure 3B ). In the kidneys, compared to the positive DMBA control, a statistically significant decrease could be observed for miR-9-3 (−40%; p < 0.05; SD = 13.2%), miR-124-1 (−51%; p < 0.05; SD = 14%), miR-132 (−57.9%; p < 0.001; SD = 10.5%), miR-134 (−28.8%; p < 0.05; SD = 12.8%), and mTORC1 (−22%; p < 0.05; SD = 11.9%) in the Chinese bayberry extract group ( Figure 3C ). 

In the livers, we observed a significant decrease in miR-9-3 (−37%; p < 0.05; SD = 19.8%) and mTORC1 (−37%; p < 0.05; SD = 14%) expressions in the group consuming the coffee extract, compared to the positive DMBA control, while the results for miR-124-1 (−21%; p = 0.21; SD = 23.6%), miR-132 (−16.7%; p = 0.24; SD = 19.4%), and miR-134 (−12.7%; p = 0.32; SD = 16.7%) were not statistically significant ( Figure 4A ). In the spleens, the expression of miR-9-3 (−46%; p < 0.05; SD = 10.7%), miR-134 (−38.9%; p < 0.05; SD = 12.7%), and mTORC1 (−20%; p < 0.05; SD = 8.9%) showed a statistically significant decrease in the coffee extract-consuming group, compared to the positive DMBA control, while the decrease in the expression of miR-124-1 (−15%; p = 0.37; SD = 22.9%) and the slight increase in the expression of miR-132 (13.1%; p = 0.40; SD = 23%) was not statistically significant ( Figure 4B ). In the kidneys, statistically significant decreases could be observed in miR-9-3 (−31%; p < 0.05; SD = 12.8%), miR-124-1 (−47%; p < 0.05; SD = 13.6%), miR-134 (−31.6%; p < 0.05; SD = 13.5%), and mTORC1 (−22%; p < 0.05; SD = 8.7%) in the coffee extract group, compared to the positive DMBA control, while the slight increase in miR-132 (22.1%; p = 0.18; SD = 25.4%) was not statistically significant ( Figure 4C ). 

In the livers, we observed a significant decrease in miR-9-3 (−37%; p < 0.05; SD = 19.8%) and mTORC1 (−37%; p < 0.05; SD = 14%) expressions in the group consuming the coffee extract, compared to the positive DMBA control, while the results for miR-124-1 (−21%; p = 0.21; SD = 23.6%), miR-132 (−16.7%; p = 0.24; SD = 19.4%), and miR-134 (−12.7%; p = 0.32; SD = 16.7%) were not statistically significant ( Figure 4A ). In the spleens, the expression of miR-9-3 (−46%; p < 0.05; SD = 10.7%), miR-134 (−38.9%; p < 0.05; SD = 12.7%), and mTORC1 (−20%; p < 0.05; SD = 8.9%) showed a statistically significant decrease in the coffee extract-consuming group, compared to the positive DMBA control, while the decrease in the expression of miR-124-1 (−15%; p = 0.37; SD = 22.9%) and the slight increase in the expression of miR-132 (13.1%; p = 0.40; SD = 23%) was not statistically significant ( Figure 4B ). In the kidneys, statistically significant decreases could be observed in miR-9-3 (−31%; p < 0.05; SD = 12.8%), miR-124-1 (−47%; p < 0.05; SD = 13.6%), miR-134 (−31.6%; p < 0.05; SD = 13.5%), and mTORC1 (−22%; p < 0.05; SD = 8.7%) in the coffee extract group, compared to the positive DMBA control, while the slight increase in miR-132 (22.1%; p = 0.18; SD = 25.4%) was not statistically significant ( Figure 4C ). Table 3 shows the summary of expression changes caused by feeding in the observed DMBA pretreated organs. Table 3 . Summary table of expression changes caused by feeding in the observed DMBA pretreated organs (* decreasing significantly p < 0.05; *** decreasing significantly p < 0.001; D decrease was not significant; O decrease was questionably; I increase was questionably).

miR-124-1 miR-132 miR-134 mTORC1 Polyphenol extract Liver * *** *** *** *** Spleen * * *** *** *** Kidneys *** * D *** *** Green tea Liver * *** * *** *** Spleen *** *** *** * *** Kidneys * * *** *** *** Chinese bayberry Table 3 shows the summary of expression changes caused by feeding in the observed DMBA pretreated organs. Table 3 . Summary table of expression changes caused by feeding in the observed DMBA pretreated organs (* decreasing significantly p < 0.05; *** decreasing significantly p < 0.001; D decrease was not significant; O decrease was questionably; I increase was questionably).

Polyphenol extract Liver * *** *** *** *** Spleen * * *** *** *** Kidneys *** * D *** *** Green tea Liver * *** * *** *** Spleen *** *** *** * *** Kidneys * * *** *** *** Chinese bayberry Liver *** * D * *** Spleen * * * *** *** Kidneys * * *** * * Coffee extract Liver

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.

According to in vitro studies, resveratrol, EGCG, and myricetin inhibit CYP 1A1 and 1A2 enzymes [48] [49] [50] , which activate DMBA [35] . If the DMBA activation is hindered, then the consequent HA-RAS and C-MYC oncogene overexpression is also blocked [32] .

Polyphenol structures are generally ROS-scavenging antioxidants that also exert antiinflammatory effects [51, 52] . Thus, molecular features of flavonoids [53] , chlorogenic acids, hydroxycinnamic acids, and the caffeine content of coffee [54] and melanoidins [17] exert antioxidant (ROS-quenching) effects, directly mitigating the ROS-induced cellular damage [55] [56] [57] [58] . Moreover, resveratrol [59] , myricetin [53, 58] , GTC [60] , and chlorogenic acid [61] induce the protective superoxide-dismutase (SOD) and glutathione-S-transferase (GST) enzymes. Furthermore, both resveratrol, as well as chlorogenic acids, decrease IL-1β, IL-6, and TNF-α expressions [62, 63] among others. Flavonoids thereby regulate the carcinogen and/or inflammatory effect-activated signal transduction pathways; for example, they inhibit protein tyrosine and focal adhesion kinases, as well as matrix metalloproteinases (MMPs) [57, 64] .

Resveratrol [62] and myricetin [65] up-regulate cAMP-response element-binding proteins (CREB) through the activated silent Information Regulator T1 (SIRT1)-dependent pathway [66, 67] resulting in a decrease in miR-134, miR-124, and mTOR expressions [68] . In contrast, the EGCG inhibited SIRT1, and both EGCG and resveratrol inhibited NF-κB activity as well [62, 63, 69] , while NF-κB generally decreases miR-124 [70] and miR-132 [71] . Theoretically, decreased NF-κB1 activity (in a seemingly mutually exclusive mechanism) increases the expression of the anticarcinogen miR-134 [72] . However, NF-κB, TNF-α, and IL-1β increase miR-9 expression, which downregulates NF-κB expression in a negative feedback loop [73] .

Moreover, in the coffee consuming group, the chlorogenic acids exert a kidney protective effect by inducing miR-134 [74] , which suppresses MMP-9 and MMP-7 [75] , ultimately decreasing cyclin D1 [76] , which is encoded by CCND1 and is in inverse correlation with miR-134 [74] . In addition, resveratrol [77] , EGCG [78] , and caffeine [79] induce phosphatase and tensin homolog (PTEN) gene activity, which decreases cyclin D1 cell cycle proteins [80] as well. Still, the stronger negative feedback regulation of miRs [36, 37, [81] [82] [83] prevail, ultimately decreasing miR-134 expression [36, 38, 81] .

Resveratrol [13, 63] and EGCG [84] inhibit antiapoptotic cascades by suppressing MAPK pathways [84] . This induces cell cycle arrest in the G0/G1 phase, which downregulates miR-132 and upregulates miR-9 [84] . In all examined groups, the presumably decreased cyclin D1 level led to the decrease in mTOR expression [85] . In addition, myricetin blocks phosphoinositide 3-kinase (PI3K) [86] , ultimately decreasing mTOR expression [87] .

MiR-132 inhibits the RAS p21 protein activator GTPase activating protein 1 (RASA1), which inhibits NRAS expression and HRAS activation [88] ; thus, miR-132 ultimately supports the MAPK cascade in this context. Therefore, the silencing of miR-132 expression could mediate the chemopreventive effect of resveratrol and EGCG. However, miR-9 blocks neurofibromin, which inhibits NRAS activation [88] . Thus, the induction of miR-9 seems to contradict the chemopreventive effect of resveratrol and EGCG in this context. The same is true for the pro-inflammatory cytokines' increased intercellular adhesion molecule-1 (ICAM1) expression [83] , which is blocked by resveratrol [89] . Despite that, ICAM1 positively modulates anti-inflammatory miR-124 expression [83] , which inhibits MAPK signal transduction [88] . The MAPK signaling pathway upregulates C-MYC, which activates AKT and cyclinD1 [90, 91] . Still, CREB downregulates miR-9 strongly in a negative feedback minicircuitry [92] , while miR-9 is also negatively correlated with NF-κB1 activity [93] , which decreases miR-124 [69] , corresponding to the results of this study.

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 ). 

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) .

These experimental agents possess similar chemopreventive molecular mechanisms, including ROS scavenging, as well as signal transduction modulating effects; namely, both DMBA induced inflammatory and proliferative pathways were inhibited, presumably through deactivating TNF, IL1, IL6, and NF-κB [29, 38, 46, 101] . According to the literature, chemopreventive agents presumably decrease all expressions of the examined miRs and mTOR [68, 70, 71, 93] by induced CREB and decreased NF-κB activities [62, 63, 65, 69] . However, miR-134 was expected to increase with decreased NF-κB activity [72, 74] and anti-inflammatory mir-124 should have been positively modulated by ICAM1 [83] , contradicting our results.

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] . 

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) .

These experimental agents possess similar chemopreventive molecular mechanisms, including ROS scavenging, as well as signal transduction modulating effects; namely, both DMBA induced inflammatory and proliferative pathways were inhibited, presumably through deactivating TNF, IL1, IL6, and NF-κB [29, 38, 46, 101] . According to the literature, chemopreventive agents presumably decrease all expressions of the examined miRs and mTOR [68, 70, 71, 93] by induced CREB and decreased NF-κB activities [62, 63, 65, 69] . However, miR-134 was expected to increase with decreased NF-κB activity [72, 74] and antiinflammatory mir-124 should have been positively modulated by ICAM1 [83] , contradicting our results.

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. 

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Results of a phase I pilot clinical trial examining the effect of plant-derived resveratrol and grape powder on Wnt pathway target gene expression in colonic mucosa and colon cancer

Green tea and liver cancer risk: A meta-analysis of prospective cohort studies in Asian populations

Japan Collaborative Cohort Study for Evaluation of Cancer. Nutrition and disease in the Japan Collaborative Cohort Study for Evaluation of Cancer (JACC). Asian Pac

Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae

Tea, coffee, and caffeine and early-onset basal cell carcinoma in a case-control study

(a)anthracene on onco/suppressor gene action in vivo: A short-term experiment

Formation and persistence of benzo(a)pyrene metabolite-DNA adducts

Exposure of coelomocytes from the earthworm Eisenia hortensis to Cu, Cd, and dimethylbenz[a]anthracene: An in vitro study examining reactive oxygen species production and immune response inhibition

Reactive oxygen species in tumor progression

Role of oxygen free radicals in cancer development

Inhibition of N-methyl-N-nitrosourea-and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations

Effect of E-2-(4 -methoxybenzylidene)-1-benzosuberone on the 7,12-dimethylbenz[alpha]anthraceneinduced onco/suppressor gene action in vivo. I: A 24-hour experiment

A study on CYP1A inhibitory action of E-2-(4 -methoxybenzylidene)-1-benzosuberone and some related chalcones and cyclic chalcone analogues

Early modification of c-myc, Ha-ras and p53 expressions by N-methyl-N-nitrosourea

Early modification of c-myc, Ha-ras and p53 expressions by chemical carcinogens (DMBA, MNU)

Effect of 7,12-Dimethylbenz(α)anthracene on the Expression of miR-330, miR-29a, miR-9-1, miR-9-3 and the mTORC1 Gene in CBA/Ca Mice

Changes in miR-124-1, miR-212, miR-132, miR-134, and miR-155 Expression Patterns after 7,12-Dimethylbenz(a)anthracene Treatment in CBA/Ca Mice

Kiss, I. In vivo effects of olive oil and trans-fatty acids on miR-134, miR-132, miR-124-1, miR-9-3 and mTORC1 gene expression in a DMBA-treated mouse model

MicroRNAs as lung cancer biomarkers and key players in lung carcinogenesis

DLK1-DIO3 genomic imprinted microRNA cluster at 14q32.2 defines a stemlike subtype of hepatocellular carcinoma associated with poor survival

MicroRNAs and their role in environmental chemical carcinogenesis

Role of microRNAs in translation regulation and cancer

KRAS induces lung tumorigenesis through microRNAs modulation

A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice

Early life stress enhances behavioral vulnerability to stress through the activation of REST4-mediated gene transcription in the medial prefrontal cortex of rodents

Oxidative stress, inflammation, and cancer: How are they linked? Free Radic

Regulation of glutathione synthesis

Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor

Inhibitory Effects of Eight Green Tea Catechins on Cytochrome P450 1A2, 2C9, 2D6, and 3A4 Activities

Human CYP1A1 inhibition by flavonoids

Phenol-rich fulvic acid as a water additive enhances growth, reduces stress, and stimulates the immune system of fish in aquaculture

Vitro Determination of Inhibitory Effects of Humic Substances Complexing Zn and Se on SARS-CoV-2 Virus

Dietary flavonoids: Bioavailability, metabolic effects, and safety

Coffea arabica instant coffee-Chemical view and immunomodulating properties

Free radical scavenging and antioxidant activity of plant flavonoids

Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic

The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer

Dietary ortho phenols that induce glutathione S-transferase and increase the resistance of cells to hydrogen peroxide are potential cancer chemopreventives that act by two mechanisms: The alleviation of oxidative stress and the detoxification of mutagenic xenobiotics

Antioxidant Properties of Resveratrol and its Protective Effects in Neurodegenerative Diseases

Potential health benefits of green tea (Camellia sinensis): A narrative review

Role of Chlorogenic Acids in Controlling Oxidative and Inflammatory Stress Conditions

Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase

Resveratrol alleviates lipopolysaccharide-induced inflammation in PC-12 cells and in rat model

The antitumor activities of flavonoids

Myricetin improves endurance capacity and mitochondrial density by activating SIRT1 and PGC-1α

Resveratrol improves learning and memory in normally aged mice through microRNA-CREB pathway

Resveratrol prevents cognitive deficits induced by chronic unpredictable mild stress: Sirt1/miR-134 signalling pathway regulates CREB/BDNF expression in hippocampus in vivo and in vitro

SIRT1 negatively regulates the mammalian target of rapamycin

Regulation of SIRT1 in cellular functions: Role of polyphenols

NF-κB-mediated miR-124 suppresses metastasis of non-small-cell lung cancer by targeting MYO10

NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation

NF-κB1, c-Rel, and ELK1 inhibit miR-134 expression leading to TAB1 upregulation in paclitaxel-resistant human ovarian cancer

Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals

Hsa-miR-134 suppresses non-small cell lung cancer (NSCLC) development through down-regulation of CCND1

Matrix metalloproteinases: Their functional role in lung cancer

Renoprotective mechanisms of chlorogenic acid in cisplatin-induced kidney injury

Chemoprevention by resveratrol: Molecular mechanisms and therapeutic potential

PI3K/AKT/mTOR signaling is involved in (-)-epigallocatechin-3-gallate-induced apoptosis of human pancreatic carcinoma cells

Caffeine activates tumor suppressor PTEN in sarcoma cells

PTEN induces cell cycle arrest by decreasing the level and nuclear localization of cyclin D1

The expression of circulating miR-504 in plasma is associated with EGFR mutation status in non-small-cell lung carcinoma patients

Identification of microRNA-124 in regulation of Hepatocellular carcinoma through BIRC3 and the NF-κB pathway

ICAM-1 regulates macrophage polarization by suppressing MCP-1 expression via miR-124 upregulation

Next-Generation Sequencing Reveals the Role of Epigallocatechin-3-Gallate in Regulating Putative Novel and Known microRNAs Which Target the MAPK Pathway in Non-Small-Cell Lung Cancer A549 Cells

mTOR-what does it do? Transpl. Proc

Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: Rationale and importance to inhibiting these pathways in human health

RAS-MAPK pathway epigenetic activation in cancer: miRNAs in action

Resveratrol attenuates ICAM-1 expression and monocyte adhesiveness to TNF-α-treated endothelial cells: Evidence for an anti-inflammatory cascade mediated by the miR-221/222/AMPK/p38/NF-κB pathway

MAPK signaling regulates c-MYC for melanoma cell adaptation to asparagine restriction

Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells

The CREB-miR-9 negative feedback minicircuitry coordinates the migration and proliferation of glioma cells

Resveratrol-Induced Changes in MicroRNA Expression in Primary Human Fibroblasts Harboring Carnitine-Palmitoyl Transferase-2 Gene Mutation, Leading to Fatty Acid Oxidation Deficiency

Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin. Effects on lipid peroxidation, hydroxyl radical generation and bleomycin-dependent damage to DNA

Photoperoxidation of unsaturated organic molecules. II. Autoperoxidation of aromatic hydrocarbons

Enzyme Inhibitors and Activators; Senturk, M

MicroRNA 132 promotes oxidative stress induced pyroptosis by targeting sirtuin 1 in myocardial ischaemia reperfusion injury

Acrylamide induces HepG2 cell proliferation through upregulation of miR-21 expression

Low doses of the carcinogen furan alter cell cycle and apoptosis gene expression in rat liver independent of DNA methylation

Reactive metabolites in the biotransformation of molecules containing a furan ring

NF-kappaB functions as a tumour promoter in inflammation-associated cancer

miR-134: A Human Cancer Suppressor?

The pharmacology of resveratrol in animals and humans

The note that they have no conflict of interest.