Has Anyone so far Bridged the Gap between microRNA, Botanicals and Oropharyngeal Squamous Cell Carcinoma?

Background/Context: Current debate concerning properties of oropharyngeal squamous cell carcinoma (OSCC) is nothing new. More recent information can be found on functional aspects of microRNA (miRNA) in botanicals and mammals, suggesting that the aging process and cancer development are regulated by these molecules. Interestingly, some dietary supplements (especially botanicals) have been reported to have cancer chemopreventive and/or anti-cancer properties and are common ingredients in so-called "anti-aging" creams. Objective: The present study was therefore conducted to determine the state-of-the-art on botanicals, OSCC, and their relationship to miRNA. Methods/Design: PubMed and Google Scholar were interrogated for articles published in English between January 1, 2000 and December 31, 2010 that discuss botanicals and which could possibly be used in treating OSCC. Additional searches in Google, Google Scholar, PubMed and KoreaMed were conducted to detect possible associations between miRNAs and the aforementioned items. Results: The current body of literature suggests that botanicals might deliver some of their properties via miRNAs. No report was found however that makes a direct connection to OSCC. It therefore has not been determined whether there is an association between botanicals, OSCC, and miRNA molecules that could possibly have "anti-aging" as well as cancer chemopreventive and/or anti-cancer properties. Conclusions: Systematic studies are needed to determine whether it is miRNA that could dispense possible therapeutic properties of botanicals on OSCC.


Introduction
MicroRNAs (miRNAs) are small post-transcriptional regulators found in animals and plants ( Figure 1). Numerous miRNA sequences are evolutionarily conserved among plant species [1,2] as are their target sequences in the mRNAs [3]. This could possibly be due to the role of miRNAs in gene expression control. The average half-life of mammalian miRNAs has been calculated to be 119 hours which suggests that they are up to ten times more stable than mRNAs [4]. Among the differences between plant and animal miRNA biology are the processing, the position of target interaction and/or the degree of complementarity [5]. Health related functions may be mediated by miRNAs which regulate the expressome, metabolome or interactome [6] within plants (esp. botanicals) and/or molecules that modify the status of miRNAs within animals (esp. humans). Tanshinone IIA (Chemical Abstracts Service Registry Number, CAS no: 568-72-9) for instance, a constituent of the traditional Chinese medicine Dan shen (Salvia miltiorrhiza; National Center for Biotechnology Information, NCBI, Taxonomy Identifier: 226208) protects against particular variants of sudden cardiac death via repression of miRNA-1 which targets the inwardly rectifying potassium channel subunit Kir2.1 (Universal Protein Resource, UniProt, Identifier: Q64273) [7].
Oropharyngeal squamous cell carcinoma (OSCC, figure 2) has been defined as cancer of the anatomical regions that are covered by the classificators C00-C10 [8,9]. It is among the most common cancers worldwide, and continues to represent a public health problem [10]. Despite advances in therapy, cure rates and survival remain poor [11]. OSCC often appears to be preventable and is likely to be related to behavioral and lifestyle factors. Prevention of OSCC therefore remains the goal to reduce the incidence of this disease [12,13].
Anticancerous effects such as growth inhibition of OSCC cell lines can be mediated by miRNAs [14]. It is however noteworthy that some miRNAs can also promote cancer by supporting cell growth or metastasis [15], a phenomenon outside the scope of the present study. Figure 1: Graphic image representation of miRNA. Exemplarily selected results of single sequence RNA secondary structure prediction experiments for two miRNA species of Solanum lycopersicum, sly-miR169c [157] and sly-miR319 [160]. Two different computer programs, CONTRAfold (version: 2.02) [164] and RNAfold (Vienna RNA package version 2.0.0) [165,166], were used to allow for visual comparison of geometries.
[A] A secondary structure suggested for sly-miR169c based on conditional log-linear models (CLLM; CONTRAfold); [B] Coherence was found for the predicted minimum free energy structures and base pair probabilities for sly-miR169c (RNAfold); [C] A CLLM-based secondary structure suggested for sly-miR319 (CONTRAfold); [D] The secondary structure predicted for sly-miR319 based on minimum free energy structures and base pair probabilities (RNAfold) is not coherent, warranting further research The use of dietary supplements, especially botanicals, or parts of them such as extracts or seeds as medicines predates recorded history and may be seen to represent an antecedent to modern medicine [16]. Technological advancement has facilitated the delivery of breakthrough bioactive compounds of botanicals with potency more or less comparable to those of conventional drugs [17]. Also their low cost, (per) oral bioavailability, and assumed low-risk profile at pharmacologically relevant concentrations have tremendous appeal. As a result of this assumed safety a considerable number of women for example use herbal remedies during pregnancy [18]. Phytochemicals have been evaluated for cancer chemoprevention and anti-cancer activities, increasingly throughout the course of the past decade [19][20][21][22]. Interestingly, some of these botanicals are among the top 10 ingredients in 2010 "anti-aging" creams [23]. This and previous reports [24][25][26][27][28] are suggesting that cancer chemopreventive agents and/or anti-aging drugs could have something in common. What they have in common could be effects mediated by miRNA as these molecules play a role in controlling the aging process [29] and cancer development [30]. It may therefore be hypothesized that botanicals could execute the aforementioned cancer chemopreventive and/or anti-cancer effects via miRNAs. The present study was conducted to determine whether miRNAs are currently seen as a link between botanicals and issues related to solid malignant tumors as exemplified by OSCC.

Roots, rhizomes and barks of shrubs herbaceous and trees
Ashwaghanda shrub roots Ashwaghanda (Withania somnifera; NCBI Taxonomy ID: 126910) root extracts are commonly used in Indian traditional Ayurveda as a general tonic for overall health and remedy for a variety of ailments [60]. Withaferin-A (CAS no: 5119-48-2; 20 mg/kg bw), a steroidal lactone isolated from Withania somnifera, appeared to prevent alterations of both p53 (UniProt: Q00366) and Bcl-2 and decreases micronucleus formation in the bone marrow once administered to DMBA-painted hamsters [61,62]. Withaferin-A prevented tumor formation and helped maintain erythrocyte integrity during DMBA induction of OSCC [63].
Ginger (400 mg powder added to labarotory chow) might have a preventive effect on oropharyngeal carcinogenesis through induction of apoptosis and suppression of tumor growth and proliferation [72].

Portia tree barks
Portia tree or Thespesia populnea Soland ex Correa (NCBI Taxonomy ID: 3638; Family: Malvaceae; NCBI Taxonomy ID: 3629) is a large tropical tree found in coastal forests and India [73]. Ethanolic extract of Thespesia populnea bark (300 mg/kg bw) has been reported to execute cancer preventive effects in HBP contaminated with DMBA [74].

Fruits and their seeds Black raspberries
Black raspberries (Rubus occidentalis; NCBI Taxonomy ID: 75079) contain many compounds with both in vitro and in vivo preventive properties [75]. The lyophilized black raspberries (5% and 10%) had a preventive activity and inhibited the tumor formation in the oral cavity [76]. Black raspberries inhibitory effects on growth of dysplastic human cells from the oropharynx were attributed to certain components that target specific signaling pathways regulating the progression of cell cycle [77]. Ethanolic extract (10, 50, 100 µg/ ml, ddm) of freeze-dried black raspberries was a promising agent for prevention of oropharyngeal epithelial dysplasia [75]. Application of black raspberry bioadhesive gel (10% w/w gel or 0.5 g of 10% gel) containing anthocyanins (CAS no: 15067-77-7) represented a promising strategy for human OSCC chemoprevention by modulation of gene expression and reduction of cyclooxygenase-2 (UniProt: Q9NNY7) protein [78,79].

Tomato and its orange colored lycopene
Tomato is an edible fruit of the plant Solanum lycopersicum (NCBI Taxonomy ID: 4081) [86]. Combined administration of tomato (0.25 mg lycopene/ml; CAS no: 502-65-8;) and garlic (NCBI Taxonomy ID: 4682; 12.5 mg/ ml) inhibited the development of OSCC in the HBP by downregulation of Bcl-2 and upregulation of Bax (UniProt: Not Available), Bim, P53 and the caspases 3 and 8 [87]. They modulated xenobiotic-metabolizing enzymes mitigating the mutagenic and carcinogenic effects of DMBA [88]. Lycopene (a red carotene found in tomatoes; 2.5 mg/kg, bw) might exert its preventive effects by modulating lipid peroxidation and enhancing the activities of the enzymes in the glutathione redox cycle as it reduced glutathione (GSH; CAS no: 70-18-8), glutathione peroxidase (GPx; UniProt: P86215), glutathione S-transferase (GST; UniProt: P30116) and glutathione reductase (UniProt: Not Available) as biomarkers of cancer chemoprevention [89]. Lycopene (3 and 7 mol/L) inhibited proliferation and enhanced gap-junction communication of KB-1 tumor cells from the human oropharynx [90]. The observed effect of lycopene (4 or 8 mg/day) suggested that it can be effectively and safely used for the management of OLP [91]. Tomato paste containing weight lycopene (5 mg/kg bw reduced the incidence of OSCC in the HBP [92].

Plants used in Traditional Chinese Medicine
Levamisole (CAS no: 14769-73-4; ATC/DDD: P02CE01; antihelminthic drug) and/or Chinese medicinal herbs (root of Astragalus; NCBI Taxonomy ID: 20400; 12 g/day; fruit of Ligustrum, NCBI Taxonomy ID: 104487; 9 g/day; and fruit of Ziziphi jujuba; NCBI Taxonomy ID: 157914; 9 g/day) can modulate the level of the serum SCC associated antigen. For OLP patients, the combination therapy was superior to the single therapy of levamisole or of Chinese medicinal herbs [105]. Chinese herbal extracts of Drynaria bonii (NCBI Taxonomy ID: 272673), Angelica sinensis (NCBI Taxonomy ID: 165353) and Cornus officinalis Sieb (NCBI Taxonomy ID: 16906) were investigated for their antitumor potential on human OSCC cell lines (HSC-2 and NA) and the data demonstrated several unique antitumor properties of Drynaria bonii [106].
Herba erigerontis (NCBI Taxonomy ID: 124940) appeared to hamper the possible progression of OLP to tumor [110]. Also, Xian huayin (1.7 ml or 11.4 ml/kg bw/day) from China may exhibit a reversal effect on DMBA-induced premalignant lesions in the HBP [111]. The extract of the rhizome of Coptidium (NCBI Taxonomy ID: 568508, 13.5% w/w) induced cytochrome-c (UniProt: P99999) dependent apoptosis in immortalized and malignant human oropharyngeal keratinocytes via the mitochondrial signaling pathway [112].

Ferulic acid
Ferulic acid (hydroxycinnamic acid, CAS no: 1135-24-6), is found in the brans of grasses such as wheat (Triticum aestivum; NCBI Taxonomy ID: 4565), rice (Oryza sativa; NCBI Taxonomy ID: 4530), and oats (Avena; NCBI Taxonomy ID: 4496) [120]. It (40 mg/kg bw) has been said to reduce tumor incidence and size in DMBA-painted animals by exhibiting antilipidperoxidative effects as well as its ability to modulate the status of carcinogen detoxifying agents [121].

A Few Notes on Dosages
Additional searches were conducted to compare the dosages previously discussed in cell line and animal models with those suggested for humans. The following databases have been interrogated: About Herbs, a web site provided by the Memorial Sloan Kettering Cancer Center (MSKCC Integrative Medicine Service) [127], Herbs at a Glance, and the Complementary and Alternative Medicine (CAM) subset of PubMed both of which have been established by the National Center for Complementary and Alternative Medicine (NCCAM) [128].
This part of the study revealed that the current body of literature contains only few peer reviewed articles on effective clinical dosages in humans specified by good scientific practice experimental design. At least in parts this may be attributable to the fact that botanicals come in a variety of dosage forms such as teas, creams, gels, capsules or tablets, and/or that they can be formulated for various types of drug delivery supporting extended, sustained or other modes of drug release. Different dosages can therefore be readily extracted from the body of literature (table 1). Comparative studies are warranted to learn more about dose-response relationships in this context. Curcumin Curcumin: 0.1, 1.0 or 10.0 µM [66]; 1, 5, 10, 25, and 50 µM [67]; 0.1 µM to 1mM [68] Curcumin: 80 mg/kg bw [69] Turmeric: 1% [70] §Curcumin: 8 g/day [168]; 3.6 g/day [169]; 1-4 g/day [170] Black Raspberries Ethanolic extract: 10, 50 or 100 µg/ml [75] Lyophylized: 5% or 10% [76] Bioadhesive Gel: 1g of 5% gel [78] Bioadhesive

MicroRNA
Medicinal plants may exert the carcinogenic potential by modulating carcinogen detoxification, inhibiting lipid peroxidation, or by improving in vivo antioxidants defense mechanism [90]. Only a few of the described botanicals exhibit articles which describe a role of miRNAs in the corresponding plants. Interrogating PubMed on July 2 nd 2011 for entries containing for example the search terms "Salvia miltiorrhiza" and "miRNA" produced only a single hit, a publication mentioning the application of real-time reverse transcription polymerase chain reaction to determine the level of miRNA-1 [7]. Entering "Salvia miltiorrhiza" and "miRNA" yielded no findings.
In Camellia sinensis there are around 13 conserved miRNAs which belong to 9 miRNA families [129]. Computational homology search in expressed sequencing tags revealed four candidate miRNAs from four miRNA families together with their target [130].
Computational predictions of Vitis vinifera genome identified 5.778 putative miRNAs [131]. High-throughput sequencing in Vitis vinifera tagged 24 conserved miRNA families, 26 non-conserved miRNA families and 21 grapevine-specific miRNAs [132]. Several grape genes contain putative binding sites of miRNAs [133]. Differential miRNA patterns in tissue development and fruit maturation indicate a function of miRNAs in these processes [134].
In Solanum lycopersicum 78 highly conserved mature miRNAs were found by a miRNA detecting microarray [135] and miRNAs play a role in the symbiosis with arbuscular mycorrhizal [136] and draught response (miRNA-169c) [137]. The miRNAs-159 [138], 164 [139] and 319 [140] are involved in mechanisms like stamen regulatory network or leaf and flower development of Solanum lycopersicum.
In tomato plants the miRNA network is also associated with viral infection and is involved in: host defense response [141], altered host miRNA expression [142] and thereby miRNA mediated gene expression [143].
In safflower at least 236 miRNAs were identified which show different expression patterns in different tissues [144].
High throughput sequencing of wheat small RNAs revealed 58 miRNAs of 43 families from which 4 new miRNAs seem to be specific for monocots [145]. In the Triticeae 28 miRNA precursors were identified together with 337 target sequences in genes [146].
Approximately 40 potential miRNA genes were found in Oryza sativa [147]. Altered miRNA expression in rice was found as a result of heavy metal stress [148] and other biotic and abiotic stress factors [149]. Rice miRNAs control transcription factors which are involved in development and genes that regulate physiological processes [150].
Little is known about botanicals possibly inducing miRNAs in humans. Treatment of carcinoma cells with green tea polyphenons (polyphenon-60 or epigallocatechin gallate) leads to altered expression of miRNAs [151,152]. Epigallocatechin gallate changed miRNA expression in Hep G2 cells [153] and deregulated miRNA-21 and miRNA-330 in a mouse model of prostate carcinoma [154]. In a model of myocardial infarction miRNA-1 was downregulated by Tanshinone IIA a bioactive component of Salvia miltiorrhiza [7].
For the botanicals, ingredients, and properties that are not listed in this review, because there were no articles found which described the role of miRNAs in the plant or changes in human/animal miRNA expression upon treatment.