Role of dietary phytochemicals in modulation of miRNA expression:Natural swords combating breast cancer

Banzeer Ahsan Abbasi, Javed Iqbal✉, Tariq Mahmood✉, Ali Talha Khalil, Barkat Ali, Sobia Kanwal, Sayed Afzal Shah, Riaz Ahmad

1Department of Plant Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan

2Nanosciences African Network (NANOAFNET), iThemba LABS-National Research Foundation Somerset West, Western Cape 7129, South Africa

3Department of Zoology, University of Gujrat, Sub Campus Rawalpindi 46000, Pakistan

4Department of Eastern Medicine and Surgery, Qarshi University, Lahore 56000, Pakistan

5UNESCO UNISA Africa Chair in Nanoscience and Nanotechnology, College of Graduate Studies, University of South Africa, Pretoria 0002, South Africa

6College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China

Keywords:Breast cancer miRNAs Dietary phytochemicals In vitro In vivo

ABSTRACT The National Cancer Institute had projected breast cancer (BC) as one of the topmost prevalent malignancies around the globe. In many cases, BC becomes resistant to chemotherapy, radiation and hormonal therapies. Traditional BC therapies are associated with adverse side effects, drug resistance and recurrence. Extensive research work has shown that these dietary phytochemicals (DPs) may exert therapeutic effects by regulating the miRNA expression. A large number of DPs have been researched as miRNA regulatory agents against BC and some other DPs have not yet been tested against BC. We have discussed the effects of curcumin, diallyl disulphide, 3,3′ diindolylmethane, ellagic acid, genistein,indole-3-carbinol, quercetin, resveratrol, and sulforaphane on regulation of expression of BC miRNAs in a wide range of in vitro and in vivo models. We have also shown some of the possible DPs (Oleanolic acid, capsaicin, benzyl isothiocyanate, epigallocatechin gallate,phenethyl isothiocyanate and ursolic acid) that have shown miRNA regulatory activities and have not yet been tested against BC miRNAs. Finally, current limitations, challenges, future perspectives of DPs and BC research are also critically discussed.

1. Introduction

Breast cancer (BC) is a serious global health concern in both developed and developing countries. It is one of the major causes of cancer associated death among women across the world accounting for 25% of all new cancer cases and 15% of all new cancer deaths[1,2]. BC is not only restricted to women, it also affects men[3], transgender individuals and people from all racial and ethnic backgrounds[4]. At present, large number of treatment methods are available for BC including surgical treatment, adjuvant chemotherapy, radiotherapy, hormonal therapy, targeted therapies together with immunotherapy, monoclonal antibody therapy and surgery [lumpectomy (breast-conserving surgical treatment)or mastectomy (surgical removal of breast tissues)][5]. But, the development of drug resistance and their side effects has weakened the potentials of these treatment strategies on cancer cells[6]. As a result, development of novel and potent drugs with no/less side effects is crucial to control the incidence of BC.

Recently, dietary phytochemicals (DPs) have appeared as chemopreventive and chemotherapeutic agents for BC because they have no/less side effects and low toxicity compared to synthetic drugs.Additionally, they are inexpensive and readily available. Growing evidences indicates that these phytochemicals could be used as a chemo-preventive and chemo-therapeutic agent in wide range of cancer including BC[7,8]. Emerging evidences suggested that miRNAs have played important role in the initiation, promotion and progression of various types of cancers including BC. They control the expression level of different genes and proteins related to cell growth, metastasis, proliferation and apoptosis[9,10]. Due to their significant roles in cancer initiation and proliferation, targeting miRNAs has been considered as an effective treatment option for cancer. Recent evidences indicate that DPs may inhibit BC progression through the modulation of miRNA expression[11,12].

A large number of DPs such as sulforaphane, ellagic acid (EA),genistein, curcumin, indole-3-carbinol (I3C), resveratrol (RV), diallyl disulphide (DADS), 3,3’-diindolylmethane (DIM), and quercetin have been tested as potent agents for regulating miRNAs against BC and a large number of DPs are still under clinical trials for their potential role against miRNAs regulation in BC. This review article focuses on some potential DPs which has shown promising results while targeting miRNAs in BC.

2. miRNAs involved in BC regulation

miRNAs are playing a vital role in regulating BC, and it is clear from large number of evidences that disturbance in the regulation of these miRNAs are mainly involved in the initiation promotion and progression of BC. An extensive research work has been carried out to identify large number of these dysregulated miRNAs in BC.These dysregulated miRNAs play a significant role in the regulation of a wide range of different molecular processes such as invasion,proliferation, metastasis, caspase mediated cell death, self-renewal,and epithelial to mesenchymal transition. miRNAs can perform dual functions as oncogenes (biological accelerators) and tumor suppressor genes (biological breaks). Onco-miRNAs expression is up-regulated (Figure 1); whereas, tumor-suppressor miRNAs expression (Figure 2) is downregulated in BC. Some important miRNAs along with their molecular mechanism of actions are given in Table 1 and 2.

Table 1 Different kinds of onco-miRNAs along with their molecular mechanism of actions involved in initiation, promotion and progression of breast cancer.

Table 2 Different kinds of tumor suppressor miRNAs along with their molecular mechanism of actions involved in suppression of breast cancer.

Figure 1. Effect of some dietary phytochemicals against breast cancer while up-regulating expression of large number of miRNAs in order to inhibit breast cancer.

Figure 2. Effect of some dietary phytochemicals against breast cancer while down-regulating expression of large number of miRNAs in order to cope up with breast cancer.

Table 3 In vitro and in vivo effects of dietary phytochemicals against breast cancer while up-regulating and down-regulating the expression of different miRNAs.

3. Dietary phytochemicals as potential miRNA regulatory agents

DPs are found in large number of dietary supplements such as vegetables, fruits, grains, beans, and other plants. Some DPs have been tested against BC miRNAs (Table 3). Here we discussed the effects of some DPs on BC miRNAs expression. They are chemically diverse and can regulate the miRNA expression through different strategies (Figure 3).

Figure 3. Effects of dietary phytochemicals on miRNA processing and expression.

3.1. I3C

I3C are strong phytochemicals widely distributed in the cruciferous vegetables such as kohlrabi, cauliflower (Brassica oleraceavar.botrytis), broccoli (Brassica oleraceavar.italica), horseradish(Armoracia rusticana), cabbage (Brassica oleraceavar.capitata),collard greens (Brassica oleraceavar.acephala), mustard (Brassicaspp.), brussels sprouts (Brassica oleraceavar.gemmifera), kale(Brassica oleraceavar.acephala), rutabaga (Brassica napobrassica),turnips (Brassica rapavar.rapa), bok choy (Brassica rapavar.chinensis), wasabi (Wasabia japonica), Chinese cabbage (Brassica rapavar.chinensis), arugula (Eruca sativa), radish (Raphanus sativus)and watercress (Nasturtium officinale)[58-60].

I3C has been shown to modulate miRNA expression in BC cells.I3C up-regulate miR-34a expression level in MCF-7 BC cells as a result of miR-34a expression, and I3C treatment increases the expression ofp21,p53, andp53ser15genes, eventually inducing apoptosis and inhibiting cell growth[48]. Hence, I3C may inhibit BC progression by modulating miRNA expression. These I3C may need further research and investigations to unfold their biological potentials.

3.2. EA

EA is an anti-BC flavonoid polyphenol present in grapes (Vitisvinifera), cloudberry (Rubus chamaemorus), wolfberry (Lycium barbarum), pomegranates (Punica granatum), strawberries (Fragariaspp.), raspberries (Rubusspp.), blackberries (Rubusspp.), pecans(Caryaspp.) and walnuts (Juglansspp.)[61].

Female ACI rats were treated with estrogen to induce mammary tumorigenesis and the efficacy of EA on the miRNAs expression was observed. EA treatment up-regulated the synthesis of miR-182,miR-375, miR-183, miR-34c, miR-196c, and miR-429, and downregulated the expression of miR122, miR-127, miR-335, miR-205,and miR-206 in tumors cells. EA also reduced the expression of their targetsERa(miR-206),cyclin D1(miR-206),cyclin G1(miR-182, -122),Bcl-w(miR-122),Bcl-2(miR-122), and increased the expression of their targetsRASD1(miR-182),FoxO3a(miR-182),FoxO1(miR-182, -183), and thereby caused the inhibition of tumor growth[52]. This finding indicates that EA has the capability of inhibiting BC progression by regulating miRNA expression; thereby,this phytochemical can be a potent anti-BC agent and needs further studies.

3.3. Pomegranate polyphenols

Pomegranate (Punica granatumL.) has been consumed for different medicinal purposes and is defined as “nature’s power fruit”. Pomegranate extract has been shown to inhibit cell survival and inflammation by modulating the expression of miRNA in BT-474 and MDA-MB-231 BC cells. It decreased miR-155 expression,contributing to inhibition ofpAkt, pPI3K, andAktexpression,and eventually caused the induction of phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 (SHIP-1) expression in BT-474 and MDA-MB-231 cells. Moreover, it was also noticed that pomegranate extract down-regulated miR-27a expression, resulting in transcriptional repressorZBTB10upregulation, and subsequently caused the downregulation of Sp (specificity protein)-1, -3, -4,VEGF, VEGF receptor-1, survivin, and nuclear factor kappa-lightchain-enhancer of activated B cells (NF-jB) p65 expression in cancer cells. Thus, pomegranate extract inhibited cell survival and inflammation[51]. Furthermore, pomegranate extract also regulated miRNA expression in BT-474 xenograftsin vivostudy. Pomegranate extract reduced miR-155 expression, inducedSHIP-1expression,and inhibitedpAktand pPI3K expression. Moreover, it decreased miR-27a expression, increasedZBTB10expression, and decreased Sp1, Sp3, Sp4 expression as well as VEGF, survivin, andNFκBp65 expression[51]. Therefore, the potential impact of pomegranate extract against BC miRNAs may be warranted for future exploration.

3.4. Sulforaphane

Sulforaphane, an isothiocyanate, is found in higher concentration in cauliflower (Brassica oleraceavar.botrytis), Chinese cabbage(Brassica rapavar.chinensis), brussels sprouts (Brassica oleraceavar.gemmifera), arugula (Eruca sativa), radish (Raphanus sativus),broccoli (Brassica oleraceavar.italica), kohlrabi (Brassica oleraceaGongylodes Group), turnips (Brassica rapavar.rapa), bok choy(Brassica rapavar.chinensis), horseradish (Armoracia rusticana),mustard (Brassicaspp.), rutabaga (Brassica napobrassica), collard greens (Brassica oleraceavar.acephala) and watercress (Nasturtium officinale)[61]. Sulforaphane has been researched to regulate miRNA expression in BC cells. Sulforaphane treatment was found to increase the expression level of exosomal miR-140 and decrease the expression of exosomal miR-29a and miR-21 in CD49f+/CD24 and ALDH1+MCF10DCIS stem-like cells. Moreover,sulforaphane decreased cancer stem cell markerALDH1expression and mammosphere formation in these cells. These results indicated that sulforaphane may inhibit cancer stem-like cells by regulating the expression level of miRNA[50]. Furthermore, researchers found that sulforaphane treatment up-regulated miR-140 expression in MCF10DCIS cellsin vitro. Moreover, sulforaphane treatment also increased the expression of miR-140 and decreasedALDH1andSOX9expression in CD44+/CD24 and ALDH1+ MCF10DCIS cellsin vivo. These results suggested that sulforaphane targetsSOX9andALDH1by regulating the expression of miR-140 and eventually suppress BC stem-like cells[50]. These findings would gain further attention for the investigations of sulforaphane as a chemoprevention and chemo-therapeutic agent for BC.

3.5. DADS

DADS is an organosulphur compound present in garlic[7,62]. It has been researched that DADS has shown to modulate miRNA expression in BC cells. Investigators reported that DADS treatment up-regulated the expression level of miR-34a in MDA-MB-231 cells. By up-regulating miR-34a expression, DADS treatment down-regulated sarcoma (SRC,a proto-oncogene) protein levels,and caused the suppression of Ras-GTP, leading to inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation.These effects indicate that DADS inhibits BC cell proliferation and invasion via miR-34a mediatedSRC/Ras/ERKinhibition[57]. DADS plus miR-34a treatment reduced tumor volume (as compare to control) in rats implanted with MDA-MB-231 BC cells, indicating miR-34a increases antitumor effect of DADS[57]. Hence, DADS could be a promising anticancer phytochemical against BC by targeting miRNAs and needs further research work in future.

3.6. DIM

DIM is widely distributed in cruciferous vegetables including brussels sprouts (Brassica oleraceavar.gemmifera), cauliflower(Brassica oleraceavar.botrytis), kale, broccoli (Brassica oleraceavar.italica), kohlrabi (Brassica oleraceaGongylodes Group), watercress(Nasturtium officinale), cabbage (Brassica rapavar.chinensis),arugula (Eruca sativa), daikon (Raphanus sativus), bok choy (Brassica rapavar.chinensis), turnips (Brassica rapavar.rapa), collard greens(Brassica oleraceavar.acephala), mustard greens (Brassica juncea)and radishes (Raphanus sativus)[63,58].

DIM has been shown to regulate miRNA expression. It has been researched that DIM increased the expression level of miR-212/132 cluster and down-regulated the expression ofSOX4in T47D and MDA-MB-231 BC cellsin vitroandin vivoin an aryl hydrocarbon receptor-dependent fashion. This study indicates that DIM inhibits BC metastasis via miR-212/132 mediatedSOX4downregulation[11].Study also reported that treatment of BC SKBR-3 and MDAMB-468 cells with DIM up-regulated miR-200a and miR-200b expression and down-regulated oncogenic forkhead box M1(FoxM1) andpAktexpression. This in turn resulted in the arrest of cell growth[53]. These results indicated that DIM can be a potent BC miRNA regulatory agent and need further research studies.

3.7. Curcumin

Curcumin is a natural polyphenolic compound with potent anti-BC potential. And high concentration of curcumin was found in the rhizomes of turmeric[64]. It has been proven from different scientific evidences that curcumin can regulate BC miRNAs expression.Curcumin has been shown to alter bisphenolA-induced upregulation of miR-19a and miR-19b and dysregulation of tumor suppressor-PTEN, phospho serine/threonine-specific protein kinase (p-Akt),p-MDM2,p53, and proliferating cell nuclear antigen (PCNA) in BC MCF-7 cells, which lead to suppression of cell proliferation[54]. It has also been researched that curcumin inhibit metastasis in BC MDA-MB-231 cells. This effect was related with down-regulation of pro-inflammatory c-x-c motif chemokine ligand (CXCL)-1 and -2 cytokines expression through up-regulation of miR-181b expression[65]. Curcumin alone or in combination with emodin upregulated miR-34a expression in MDA-MB-231 and MDA-MB-435 BC cellsviadownregulating the expression of anti-apoptotic gene B-cell lymphoma 2 (Bcl-2) and oncogeneBMI-1, which finally suppressed cell proliferation and invasion in the surrounding cells[55].Curcumin also increased the expression of miR-15a and miR-16,translated into a decrease expression ofBcl-2and finally induced caspase mediated cell death in MCF-7 cells[66]. These scientific studies have shown that curcumin has the potential to inhibit BC progression via modulating miRNA expression; therefore, curcumin can be a potential anti-BC agent and needs further research.

3.8. Genistein

Genistein is an isoflavone widely distributed in lupine (Lupinussp.),kudzu (Puerariasp.), fava beans (Vicia faba), soybeans (Glycine max)and psoralea (Psoralea corylifolia)[67]. It has also been researched and proven that genistein regulated miRNA expression in BC cells.Genistein down-regulated miR-155 expression and altered miR-155 targetsFoxo3,PTEN, and casein kinase 1a (CK1a), b-catenin andp27expression in MDA MB-435 and Hs578t cells. As a consequence,genistein inhibited cancer cell survival and proliferation, and induced caspase mediated cell death[56]. This study indicates that genistein can be useful for targeting BC miRNAs. Further investigations are needed.

3.9. Quercetin

Quercetin is a flavonoid found in onions (Alliumspp.), apples(Malusspp.), red wine, tea (Camellia sinensis), lemon (Citrusspp.),tomato (Solanum lycopersicum), honey, broccoli (Brassica oleraceavar.italica), kale, and beans[68,69]. Quercetin exhibited effect on BC cell growth by regulating miRNA expression. Quercetin increased the expression level of pro-apoptotic-Bax(Bcl-2associated X),caspase-3, and decreased oncogenic-EGFRexpression in MCF-7 and MDA-MB-231 cells via increasing the expression level of miR-146a expression. Quercetin induced apoptosis throughBaxand caspase-3 activation by up-regulating miR-146a expression and inhibited invasion throughEGFRdownregulation[70]. Quercetin also up-regulated the expression of miR-146a in xenografted cells and reduced tumor volume in mice[70]. Based on this study, quercetin could be a promising anti-BC agent and needs further investigations.

3.10. RV

RV is a polyphenol found in grapes (Vitis vinifera), berries, tomato(Solanum lycopersicum), peanuts (Arachisspp.), and red wine[61]. RV down-regulated the expression of different miRNAs such as, miR-125b-5p, miR-214-3p, miR-512-5p and miR-542-3p in MCF-7 BC cells and induces apoptosis. Moreover, it also down-regulated the expression of miRNAs as for example miR-32-5p, miR-134, miR-200c-3p and miR-542-3p in MDA-MB-231 BC cells resulting in BC arrest. Furthermore, RV also increased the expression of apoptosisrelated miR-409-3p and miR-122-5p in MCF-7 and MDA-MB-231BC cell lines respectively[12]. Moreover, RV down-regulated the expression of 18 miRNAs including miR-125b-1-3p and miR-93-5p in MCF-7 cells as well as 9 miRNAs such as miR-20a-5p and 125b-1-3p in MDA-MB-231 cells. In both cell lines, RV decreasedBcl-2,X-linked inhibitor of apoptosis protein (XIAP), cyclin-dependent kinase (CDK)-2, 4 and 6 protein expression and up-regulated caspase-8 and 9 protein expression. These results indicated RV induces apoptosis in BC cellsviaregulating miRNA expression[12].According to a research study performed by Qinet al., RV also increased the expression of miR-21, miR-129, miR-204, and miR-489 and down-regulated the expression of DNA methyltransferase 3b (DNMT3b) inin vivorat tumors[71]. Additionally, RV increased the expression of miR-663 and miR-744 by down-regulating eukaryotic translation elongation factor 1A2 (eEEF1A2) expression in MCF-7 cells resulting in the inhibition of invasion and cell proliferation[72].Study revealed that RV treatment increases tumor suppressive miR-16, miR-141, miR-143 and miR-200c expression in MDAMB-231-luc-D3H2LN, MCF-7, and MCF-7-ADR cells. In addition,miR-26a, miR-34a, miR-125a-3p, miR-126, miR-128, miR-185,miR-193b, miR-195, miR-196a, miR-335, miR-340, and miR-497 expression and argonaute2 (Ago2) expression were also up-regulated in RV treated MDA-MB-231-luc-D3H2LN cells. This study indicates that RV increasesAgo2activity by upregulating tumor suppressive miRNAs, leading to suppression of tumor growth[73].RV also suppressed tumor formation in mice injected with MB-231-luc-D3H2LN cells[73]. Clearly, RV has the potential to inhibit BC progression by regulating miRNA expression. All these studies suggested that RV needs further research to develop more anti-BC treatment agents.

4. Current challenges and emerging alternatives

Although DPs have been recognized as BC miRNA regulatory weapons, in this literature review much more attention is given to cope up with the following limitations.

(1)Low bioavailability and poor potency of DPs are some of the limitations associated with theirin vivouse. However, these problems can be resolved by developing semi-synthetic analogs of them. For example, synthetic curcumin analog 5-bis (4-hydroxy-3-methoxybenzyli dene)-N-methyl-4-piperidone has shown higher bioavailability than curcumin in mice[74] and RV analog 4,4′-dihydroxy-trans-stilbene inhibited BC cell proliferation and invasion with higher efficiency than RV[75].

The development of nanoparticle encapsulated phytochemical formulations is another solution for these bioavailability and potency limitations[2]. For example, nano-encapsulated curcumin and quercetin have shown higher bioavailability than free compounds in rats and nano-encapsulated quercetin has improvedin vivoanti-BC effects compared to the free forms[61].

Another study found that nanoparticles encapsulated with both quercetin and doxorubicin significantly suppressed doxorubicinresistant MCF-7 cellsin vivo[76]. Combinations of two or more DPs may also be beneficial for bioavailability and greater potency. For example, combination of genistein and capsaicin exhibited higher anti-inflammatory and anticarcinogenic effects in MCF-7 and tissuetype plasminogen activator-induced rat mammary tumor cells than either agent alone[77].

(2)Regulation of different signaling pathways by DPs may result in some undesirable changes. For instance, the antiangiogenic characteristic of RV is not only associated with pathological angiogenesis but it also disturbs the physiological angiogenesis inside the cells[78].

(3)It is largely unknown whether advantageous effects of DPs will be seen in humans since many DP potentials have been examined only in pre-clinical trials. Based on the above evidences, it is crystal clear that a much better understanding of the efficacy of DPs in BC prevention is needed. Future research work should emphasis on:(a) Perfect characterization of these DPs, (b) Better elucidation and explanation of the molecular mechanisms actions of these DPs, (c)Confirmation of their efficacy byin vivostudies using proper animal models of BC, (d) Demonstration of their effectiveness in clinical trials and (e) Demonstration of their safety.

5. Concluding remarks

BC is a serious concern and miRNAs are dysregulated in BC playing critical roles in regulating different stages of carcinogenesis such as tumor initiation, promotion, progression and chemoresistance. Therefore, miRNAs are gaining more attraction and are novel targets for BC treatment. Different research studies have suggested that DPs can potentially modulate the expression level of different miRNAs involved in cancer. In the present review,we have discussed some DPs that have shown promising role for targeting BC miRNAs and we have also suggested some potential DPs that can be tested against BC miRNAs for possible molecular mechanism of actions. RV, sulforaphane, genistein, curcumin,DADS, DIM, EA, I3C and quercetin exhibit promising anti-BC results inhibiting BC progression through regulating miRNA expression and it has shown efficacy to regulate miRNAs in bothin vitroandin vivostudies. Among these different DPs, RV has been tested against more miRNAs than the rest of the phytochemicals.Besides, there are some other DPs (benzyl isothiocyanate, capsaicin,EGCG, oleanolic acid, phenethyl isothiocyanate, ursolic acid,etc.,)which have anti-BC potentials but yet they have not been tested and researched against miRNAs involved in BC. Importantly,further studies of these potential bioactive compounds might lead to development of strategies for BC control. Recently, different miRNAs along with their molecular targets have been identified in BC and have been recognized as therapeutic targets as well. To the best of our knowledge and after careful literature survey, DPs have been tested against only a limited number of BC miRNAs and a large number of miRNAs still need extensive research work in order to unfold their molecular mechanism of action.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

All authors listed have made substantial, direct and intellectual contribution to the work. Banzeer Ahsan Abbasi and Javed Iqbal summarized the literature, wrote the manuscript and drew the figures.Barkat Ali, Sayed Afzal Shah, Sobia Kanwal Ali Talha Khalil and Riaz Ahmad revised the manuscript. TM helped in interpretation by reviewing several draft of the manuscript.