Mechanism of the Anticancer Effect of Phytochemicals

Preface

S. Zahra Bathaie

Fuyuhiko Tamanoi

In recent decades, the important role of phytochemicals in dietary and as a functional food as well as for therapeutic uses has attracted attention of a large number of scientists in different fields including molecular and cellular science, medical science, and food science. In this (Volume 37) and previous (Volume 36) volumes of The Enzymes, we attempted to compile studies on these topics and to discuss the mechanism of action of the phytochemicals in both cancer prevention and cancer treatment. Molecular mechanism of the anticancer effect of isoprenoids, polyphenols, and flavonoids was described in the previous volume. In the current volume (Volume 37), we continued and expanded the discussion to include some other families of compounds including quercetin, withanolides, dihydrochalcones, isothiocyanates, phytoestrogens, and sulfur-containing compounds. In Chapter 1, we summarized possible molecular mechanisms of anticancer compounds, especially phytochemicals and natural products. Detailed discussion on the mechanisms involving specific compounds can be found in other chapters. We hope that these discussions provide helpful guidelines for new researches on the mechanism of action of natural products. We are grateful to the authors for providing excellent and informative chapters in a timely fashion. We also thank Mary Ann Zimmerman and Helene Kabes of Elsevier for their guidance and encouragement during the preparation of this volume.

June 2015

Chapter One

How Phytochemicals Prevent Chemical Carcinogens and/or Suppress Tumor Growth?

S. Zahra Bathaie*,†,¹; Nasim Faridi*; Ahmad Nasimian*; Hamid Heidarzadeh*; Fuyuhiko Tamanoi†    * Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

† Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA

¹ Corresponding author: email address: bathai_z@modares.ac.ir, zbatha2000@yahoo.com

Abstract

Phytochemicals are a powerful group of chemicals that are derived from natural resource, especially with plants origin. They have shown to exhibit chemoprevention and chemotherapeutic effects not only in cell lines and in animal models of cancer but also some of them are in the clinical trial phase I and II. Despite numerous reports of these phytochemical effects on cancer, an overview of the mechanisms of their action and their effects on various cellular and molecular functions important in the inhibition of cancer progression has been lacking. In this review, we attempt to catalogue various studies to examine the effect of phytochemicals in cancer initiation, promotion, signaling, and epigenetic changes. Because of the numerous studies in these topics, we only pointed out to some examples in each section.

Keywords

Chemoprevention

Chemotherapeutic

Mechanism of action

Epigenetic

Enzyme inhibition

Signaling pathway

1 Introduction

Cancer is a growing health problem around the world; particularly with the steady increase in life expectancy, rising levels of urbanization and industrialization, increasing the fast food consumption, and the subsequent changes in environmental conditions, including the lifestyle and production of various pollutions.

On World Cancer Day 2014, a new global cancer report was compiled by UN Agency, the International Agency for Research on Cancer (IARC), showing that as a single entity, cancer is the biggest cause of mortality worldwide with an estimated 8.2 million deaths from cancer in 2012. Thus, this report suggests that cancer is now the world's biggest killer—with the number of cases set to explode in coming years. In fact, World Health Organization (WHO) indicates a 70% increase over next 20 years in worldwide cancer cases. Low- and middle-income countries are most at risk of cancer overwhelming their health systems and hindering economic growth, as they have the least resources and infrastructure to cope with the predicted levels of disease escalation. Restrictions on alcohol and sugar need to be considered, say WHO scientists as there now exists a real need to focus on cancer prevention by tackling smoking, obesity, and drinking. Compiled by IARC, The World Cancer Report series is recognized as an authoritative source of global perspective and information on cancer. The first volume appeared in 2003 and the second in 2008. The third volume in the series was released in 2014.

The concept of a magic bullet was popularized by Paul Ehrlich (March 14, 1854–August 20, 1915) a German physician and scientist, who worked in the fields of hematology, immunology, and chemotherapy. He defined a magic bullet as an ideal therapeutic agent that would be created and killed only the organism targeting a disease. He reasoned that if a compound could be made that selectively targeted a disease-causing organism, then it could be selectively delivered to that organism; this compound or magic bullet could only kill the target organism. This concept is now known as targeted therapy [1].

Since there appears to be no magic bullet to treat a diverse type of cancer, it has been apparent that cancer risks can be reduced by eliminating or at least minimizing the exposure to known carcinogens [2]. In 1981, Doll and Peto in a report based on the statistical and epidemiological data have announced that among all risk factors of cancer—tobacco, alcohol, occupation, and so on—about 35% (10–70%) of human cancer mortality is attributed to diet [3]. Although, it is a wide range variant, but it indicates the importance of diet as a risk factor of cancer. On the other hand, an inverse relationship between the risk of specific cancers and consumption of vegetables and fruits have been reported [2]. These indicate the importance of Phyto products in diet and in the life.

Phytochemicals (Phyto is from the Greek word meaning plant) are nonnutritive components in the plant-based diet that possess substantial anticarcinogenic and antimutagenic properties [2]. Phytochemicals have different roles in both cancer prevention and treatment. Despite remarkable progress in understanding the carcinogenic process and devising preventive/therapeutic effects of phytochemicals, the mechanisms of action of most phytochemicals have not yet been fully understood. Bioavailability, toxicity, pharmacodynamic, and pharmacokinetics of the plant components(s) should be investigated. Oral consumption of some phytochemicals results in lower plasma/serum concentration. The reasons for this include: low intestinal absorption, degradation by intestinal enzymes, and/or metabolization by phase I and/or II detoxifying enzymes. For example, crocin intestinal absorption is low and most of the orally consumed crocin appeared in the feces of rats [4]. In addition, it is degraded by the intestinal enzymes and after 2 h of oral administration of crocin, crocetin was detected in the serum of human subject [5]. Thus, oral administration of crocin may have low efficacy for therapeutic purposes, and it should be better that it is administered via injection [6].

In addition, adverse (or side) effects of phytochemicals should be considered. For example, there are several hundred published research articles and many review papers about the beneficial effects of resveratrol in various diseases, in both in vivo and in vitro studies [7–11]. Resveratrol is the most important stilbene related to cancer, and it is present in the foods like peanuts, pistachios, grapes, red and white wine, blueberries, cranberries, and even cocoa and dark chocolate. It possesses a natural antiproliferative activity, due to its role as a phytoalexin (plant antibiotic). It also increased the antitumor activity of several other drugs, such as rapamycin in breast cancer and gemcitabine in pancreatic cancer, both in vitro and in vivo [12]. Resveratrol affects all three stages of carcinogenesis, including: tumor initiation, promotion, and progression. It was found that it acts as an antioxidant and antimutagen, and induces phase II drug-metabolizing enzymes (anti-initiation activity). It also mediated anti-inflammatory effects and inhibited COX¹ and hydroperoxidase functions, as well as both COX-2 and MM-9² expression. It is a potent inhibitor of nuclear factor NF-κB activation in DMBA³-induced breast cancer in female Sprague-Dawley rats and other tumor types. Treatment of human breast cancer MCF-7 cells with resveratrol, in addition to the suppression of NF-κB activation, inhibited proliferation at S/G2/M phase (antipromotion activity). Extensive in vitro studies also revealed multiple intracellular targets of resveratrol, which in addition to inflammation, cell growth, and proliferation affect other targets like apoptosis, angiogenesis, invasion, and metastasis. Resveratrol induces human promyelocytic leukemia cell differentiation (antiprogression activity). It inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. Several other known targets of resveratrol are including: tumor suppressor p53 and Rb⁴; cell cycle regulators, cyclins, CDKs, p21WAF1, p27KIP and INK, and the checkpoint kinases ATM/ATR; transcription factors NF-κB, AP-1, c-Jun, and c-Fos; angiogenic and metastatic factors, VEGF, and matrix metalloprotease 2/9; and apoptosis and survival regulators, Bax, Bak, PUMA, Noxa, TRAIL, APAF, surviving, Akt, Bcl-2, and Bcl-xL. In some conditions, it also exerts the prooxidant activity and cause oxidative DNA damage that may lead to cell cycle arrest or apoptosis [13–15]. In contrast to the above-mentioned data, some papers also reported its adverse effects and show some hints about its application for chemoprevention or, even, its therapeutic effects in human subjects [16–21]. The renal toxicity of resveratrol in rat has been observed at the dose of 3000 mg/kg BW⁵ per day, but the dose of 300 was not toxic [19]. However, it has been reported that low concentration (5 mg/kg BW) of resveratrol promotes breast cancer in mice and has a role in metastasis. Resveratrol (50 mg/kg BW) induced tumor growth in both MDA-MB-231⁶ (ERα−, ERβ+) and MDA-MB-435 (ER− and highly aggressive) breast cancer cells. Investigation of the role of resveratrol in breast cancer metastasis indicated the lung metastasis in mice bearing MDA-MB-231 tumor, while metastasis of lung, liver, kidney, and bone from mice bearing MDA-MB-435 mammary tumors have been observed [18]. Resveratrol also affects the endocrine function and accelerates development of MNU⁷-induced mammary carcinomas of female rat [20]. Thus, resveratrol effect is dependent to both concentration and tumor type. Since impressive numbers of positive results were published, more attention on its safety should be considered for clinical usage of resveratrol.

In the present chapter, regardless of the phytochemical type, we focus on molecular mechanisms involved in the prevention or therapeutic activities of phytochemicals. Figure 1 summarized the most important aspect of molecular mechanism of phytochemicals action. Because of the considerable studies on the molecular mechanisms of many phytochemicals functions, and the extensive reviews presented by the experts in the volumes 36 and 37 of The Enzymes, we only presented here a few examples for each mechanism with the goal to provide a guidance to check for each phytochemical by researchers in the future.

Figure 1 Important aspects of the mechanisms of action of various phytochemicals.

2 Phytochemicals Application in Chemoprevention Strategies

While there is no magic bullet that can completely cure cancer, like many types of diseases, cancer might be prevented. To achieve this purpose, all the risk factors should be recognized completely and avoided. Without complete identification of risk factors, this type of prevention is difficult to implement. For primary prevention, there is a need for large lifestyle changes, but this is not easy to implement.

The population-based studies indicated the potential of some macronutrients (like fibers) and micronutrients (for example, vitamins and some trace elements) in vegetables and fruits to reduce the risk of cancer. While, some macronutrients like carbohydrates and lipids increase the risk of some diseases including cancer. The most exciting results have been obtained with antioxidant vitamins and their precursors, as well as the components which are found in dark, leafy green vegetables, and yellow/orange/red fruits and vegetables. NCI⁸ has produced a series of guidelines featuring each color of the rainbow of fruits and vegetables.

Different mechanisms involved in prevention with consideration to the more known phytochemicals that act through these mechanisms will be reviewed in the following sections.

2.1 Blocking Initiation/Reversing Promotion

The study of experimentally induced carcinogenesis in model animals indicated that tumor development consists of sequential separate steps: initiation, promotion, and progression. After the initial uptake or exposure to a carcinogen, the initiation step which is a rapid (may be 1–2 days) and irreversible process is beginning. This step involves a chain of extracellular and intracellular events. At first, the carcinogen should be distributed and transported to organs and tissues. Then, metabolic activation and detoxification can occur. It is also possible that the covalent interaction of reactive species with DNA results in genotoxic damages. In contrast, tumor promotion is relatively slow (> 10 years) and is known as a reversible process. In this step, actively proliferating preneoplastic cells accumulate. The last step, progression, is the neoplastic transformation and in some types of cancer, its duration is less than a year. It involves the growth of a tumor with the potential of invasion and metastasis [2].

A preventive strategy may block the initiation through different mechanisms. It may prevent the carcinogen from reaching the target sites, from undergoing metabolic activation or from interacting with the target cellular macromolecule (DNA, RNA, and proteins); i.e., preventing the DNA damage. It may also be accomplished through detoxification of carcinogen by phase II enzymes, scavenging free radicals by antioxidants, or through binding to DNA and DNA-adduct formation may prevent the attack of free radical to DNA. In addition to the mitochondrial source of reactive oxygen species,⁹ other important sources, both enzymatic and nonenzymatic, of the ROS production are shown in Fig. 2.

Figure 2 Various sources of reactive oxygen species in the body.

However, after initiation, a suppressing agent inhibits the malignant cell transformation in either the promotion or the progression steps. Chemopreventive phytochemicals can block initiation or reverse the promotion stage of multistep carcinogenesis or suppress proliferation of early preneoplastic lesions. They can also delay, interrupt, or terminate the progression of precancerous cells into malignant ones [2]. These mechanisms will be discussed later in this chapter.

I3C¹⁰ is a glucosinolate obtained from cruciferous vegetables. The preventive effect of I3C and its mechanism of action have been investigated using the mammoplasty-derived 184-B5 cells. Initiation of carcinogenesis was induced by chemical carcinogen BP¹¹ or with oncogene (HER) induction and the resulted cells were named as 184-B5/BP and 184-B5/HER, respectively. The results showed that treatment of 184-B5/BP, 184-B5/HER, and MDA-MB-231 cells with I3C resulted in a decrease in proliferation, a significant increase in the estradiol (E2) metabolite ratio and in cellular apoptosis, and inhibition of cell growth. It was concluded that the preventive effect of I3C on human mammary carcinogenesis possible is through regulation of cell cycle progression, increase the formation of antiproliferative E2 metabolites and induction of cellular apoptosis [22].

2.2 Activating Phase II Detoxifying Enzymes

Elimination of potential carcinogen from the body has been known as a highly effective strategy for reducing susceptibility to carcinogens. These mechanisms include: conjugation with endogenous ligands, chemical modification of reactive features of molecules that can damage DNA and other macromolecules, and the generation or increase of cellular antioxidants. This may happen through the conjugating enzymes and phase II drug-metabolizing (or detoxifying) enzymes/proteins.

The phase II enzyme induction system is an important component of the cellular stress response in which a diverse array of electrophilic and oxidative agents can be removed from the cell before they are able to damage biomacromolecules. The 5′-flanking regions of these genes contain a common cis-element, known as the antioxidant-responsive element.¹² Basic leucine zipper,¹³ and helix-loop-helix¹⁴ transcription factors (such as NRF2, JUN, FOS, FRA, MAF, and AH receptor) bind of these ARE sequences and regulate expression of some of the stress-response genes and induce phase II enzymes. The final result of these processes is the detoxification of carcinogens and protection against oxidative stress [23–25].

Antioxidants also exert their protective effects not only by scavenging ROS¹⁵ but also by inducing de novo expression of the aforementioned genes including phase II enzymes. Many xenobiotics can also activate stress-response genes in a manner similar to that achieved by antioxidants. These genes encode proteins/enzymes such as glutathione,¹⁶ catalase,¹⁷ superoxide dismutase,¹⁸ glutathione reductase,¹⁹ glutathione peroxidase,²⁰ gamma-glutamylcysteine synthetase,²¹ glutathione S-transferase,²² NAD(P)H:quinone oxidoreductase,²³ heme oxygenase-1,²⁴ and UDP-glucuronosyltransferase.²⁵ Other enzymes/proteins in this group include: epoxide hydrolase, dihydrodiol dehydrogenase, leukotriene B4 dehydrogenase, aflatoxin B1 dehydrogenase, and ferritin [23–27].

Several studies indicated the involvement of oxidative DNA damage and impaired antioxidant defense system in patients with various types of cancer [28–30]. For example, changes in the oxidant/antioxidant balance and DNA damage (8-hydroxy-deoxyguanosine²⁶ formation) in gastrointestinal cancer patients has been reported. In addition, significant increases in glutathione and decreases in both nitrite and nitrate, SOD, CAT activities, and antioxidant molecules in these patients lead to the suggestion of a mechanism involved in oxidative stress in gastrointestinal cancer [28]. Another study indicated the important roles of the antioxidant defense capacity and DNA repair system against oxidative damage as a known risk factor for pancreatic cancer [29].

The importance of dietary phytochemicals has been shown in various studies against oxidative stress. A number of phytochemicals have also been shown to induce expression of phase II enzymes via NRF2 [2]. Among them, the chemopreventive activity of four common phytochemicals present in cruciferous vegetables, the indoles: I3C, 3,3′-diindolylmethane²⁷; the isothiocyanates (ITCs): phenethyl isothiocyanate²⁸; and sulforaphane²⁹ has been investigated in HepG2-C8.³⁰ The cytotoxicity of the compounds and their mechanism of action through the potential activation of Nrf2-ARE-mediated transcriptional activation of phase II enzymes has been determined. The results indicated that the indoles like I3C or DIM alone could induce the expression of Nrf2-related genes. In addition, they can do the same in combination with the ITCs, SFN, or PEITC, which enhances their protective role against cancer [31]. The role of various phytochemicals in the regulation of UGT transcription has also been reviewed [32].

2.3 Prooxidant/Antioxidant Activities

The protective effect of antioxidants against reactive oxygen/nitrogen species (ROS/RNS) could be exerted through different ways. The endogenous antioxidants are classified into two essential groups, small molecules and enzymes. The antioxidant enzymes were explained in the last section.

The small molecule family of antioxidants is categorized into water-soluble (ascorbate, uric acid, glutathione, etc.) and lipid-soluble compounds (tocopherol, ubiquinol, carotenoids, etc.). According to the chemical structure, phytochemicals should belong to each of these classes.

Some phytochemicals have both antioxidant and prooxidant activities which may differ according to the concentration and/or other conditions. For example, exposure of cells to low or high concentrations of curcumin diminishes or enhances the ROS generation, respectively [33].

Another example is ascorbic acid, which has been known for the past several decades as an antioxidant and anticancer agent. Although it shows the toxic effect against cancerous cells, normal cells are relatively resistant to such cytotoxicity. It has been shown that ascorbic acid as a prooxidant leads to oxidative DNA breakage in lymphocytes and lymphocyte nuclei. The copper-dependent cellular redox status has been also suggested, which is an important element in the cytotoxic action of ascorbic acid against cancer cells [34].

Myricetin with both prooxidant and antioxidant activities in different conditions is the third example. Its antioxidant activity depends on both the ROS scavenging and iron ion chelation properties. In the presence of ascorbic acid, myricetin showed antioxidant properties, especially in complex with iron [35]. The dual property is very useful for medical application of the mentioned phytochemicals.

In a comparative study, the antioxidant and prooxidant activities of a series of phenolic compounds have been investigated. The results indicated most of the phenolic compounds have prooxidant activity at low concentrations. The antioxidant activity usually increases with an increase in the number of hydroxyl groups and a decrease in glycosylation [36].

Baicalin, a flavonoid obtained from Sho-saiko-to as a prooxidant showed the apoptotic effect on Jurkat cells³¹ [37].

2.4 Protection Against Radiation

In some studies, natural products derived from plant have been studied for their protective effect against various radiations [38,39]. Recently, a combination of caffeic acid, rosmarinic acid, and trans-cinnamic acid has been used for their protective effect against γ-radiation in human HaCaT³² cells by immunocytochemistry. The named compounds protect cells, with various degrees, against ROS production as a result of irradiation [40].

2.5 Alteration in Signaling Pathways

Many molecular alterations associated with carcinogenesis, in both promotion and progression steps, occur in the cell-signaling pathways, including the induction of cell cycle arrest and apoptosis or inhibition of signal-transduction pathways that regulate cell proliferation and differentiation. One of the central components of the intracellular-signaling network that maintains homeostasis is the family of MAPKs.³³ Numerous intracellular signal-transduction pathways converge with alteration in PKC,³⁴ PI3K,³⁵ and GSK,³⁶ which lead to abnormal COX-2,³⁷ AP-1,³⁸ NF-κB,³⁹ and c-myc expression. As these factors mediate pleiotropic effects of both external and internal stimuli in the cellular-signaling cascades, they are prime targets of diverse classes of chemopreventive phytochemicals [2,41,42].

The chemopreventive effect and mechanisms of curcumin have been well studied. Curcumin has been known as the inhibitor of NF-κB, which subsequently inhibits the proinflammatory pathways [43]. It also inhibits AP-1, some enzymes like COX-2 and MMPs, induces cell cycle arrest (cyclin D1), affects proliferation (EGFR and Akt), survival pathways (β-catenin and adhesion molecules), and TNF [44]. Although absorption of curcumin is limited through the intestinal tract, and its low systemic bioavailability decreased its adequate access in certain tissues, but active levels have been found in the gastrointestinal tract of animal and human