**Cytocidal Effects of Polyphenolic Compounds, Alone or in Combination with, Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy**

Bo Yuan, Masahiko Imai, Hidetomo Kikuchi, Shin Fukushima, Shingo Hazama, Takenori Akaike, Yuta Yoshino, Kunio Ohyama, Xiaomei Hu, Xiaohua Pei and Hiroo Toyoda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50218

**1. Introduction** 

154 Apoptosis and Medicine

134: 62-73.

Nature 457: 51-56.

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A growing body of clinical and experimental evidence has revealed a strong impact of drug resistance on clinical outcomes, especially in cancer therapy, since carcinogenesis is a multistep, multi-pathway and multi-focal process, which involves a series of epigenetic and genetic alterations [1-3]. In order to solve the serious issue facing clinical treatment, combination therapy is now widely advocated for clinical use and has been shown to have a beneficial effect on patient satisfaction [3, 4]. For instance, 5-fluorouracil (5-FU) and leucovorin with either irinotecan or oxaliplatin have been widely used for the treatment of patients with colorectal cancer [5, 6]. Furthermore, recently, various types of molecular target-based drugs, such as cetuximab and bevacizumab, are being used clinically. Although these continuous efforts to exploit potential combination therapies are ongoing, there is still a growing concern about treatment resistance, disease relapse and side effects of drugs clinically used. Of note, numerous components of edible plants, collectively termed phytochemicals that have beneficial effects for health, are increasingly being reported in the scientific literature and these compounds are now widely recognized as potential therapeutic compounds [1, 2, 4, 7, 8]. In fact, natural product derived substances, especially polyphenolic compounds with very little toxic effects on normal cells, have attracted great attention in the therapeutic arsenal in clinical oncology due to their chemopreventive, antitumoral, radiosensibilizing and chemosensibilizing activities against various types of aggressive and recurrent cancers [1, 8-10].

© 2012 Yuan et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Yuan et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Apoptosis, or programmed cell death, plays a key role in the development and growth regulation of normal cells, and is often dysregulated in cancer cells [11, 12]. It has been accepted that the aim of anticancer therapy is generally focused on apoptosis induction in premalignant and malignant cells, although other multiple molecular mechanisms such as modulation of carcinogen metabolism, anti-angiogenesis and induction of differentiation are also known to be implicated in its anticancer activity [4, 13]. So far, two principal signal pathways of apoptosis have been identified [11, 12]. The intrinsic mechanism of apoptosis involves a mitochondrial pathway. Apoptosis stimuli destruct mitochondrial membrane structure under the control of Bcl-2 (B-cell leukemia/lymphoma) family, resulting in the release of mitochondrial proteins including cytochrome *c*. Once cytochrome *c* is released it activates caspase-9 (initiator caspase) through the interaction with Apaf-1 (apoptotic protease activating factor-1) and dATP [14, 15], which ultimately leads to caspase-3 and -7 (effector caspases) activation [16]. On the other hand, the extrinsic pathway induced by death receptors, such as tumor necrosis factor receptor (TNFR) and Fas, is responsible for the activation of caspase-8 and caspase-10 (initiator caspase) accompanied by the activation of caspase-3 and -7 [16]. The effector caspases are the final mediators in the intrinsic and extrinsic pathways that cleave substrates and lead to cell death. Moreover, a third pathway involving endoplasmic reticulum (ER) stress and caspase-12 has been reported to be associated in apoptosis [4, 13]. A number of markers have been utilized to reveal apoptosis status including cell viability, cytochrome *c* release, caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation [17].

Cytocidal Effects of Polyphenolic Compounds, Alone or in Combination with,

Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy 157

associated with their antitumoral, chemopreventive and chemotherapeutic activities, have been shown to be implicated in alteration of redox status, since polyphenolic compounds are well known to possess both antioxidant and prooxidant activity [1, 2, 7, 8, 10, 20, 22-26]. In this chapter, we will highlight the recent advances on the cancer preventative activities of the polyphenolic compounds, including flavonoids such as anthocyanins, and *Vitex agnuscastus* fruit extract (Vitex) in which flavonoids are one of major components, as well as curcumin based on the most recent results from in vitro cell culture and in vivo animal model tumor systems. We will further summarize the detailed mechanisms underlying their cytocidal effects focusing on apoptosis induction. We will also provide detailed insight into potential future clinical application of these promising candidates endowed with potent antitumor activities, alone or in combination with other anticancer clinical drugs based on

**2. Cancer preventative activities of the polyphenolic compounds,** 

The most abundant flavonoid constituents of fruits and vegetables are anthocyans (i.e. anthocyanins (glycosides), and their aglycones, anthocyanidins) that confer bright red or blue coloration on berries and other fruits and vegetables [8, 20]. Anthocyanins are especially interesting with respect to other flavonoids because they occur in the diet at relatively high concentrations. The daily intake of anthocyanins in the US diet has been suggested to be 180-255 mg/day, in contrast, the daily intake of most other dietary flavonoids, including genistein, quercetin and apigenin, is estimated to be only 20-25 mg/day [27]. Anthocyanidins are a diphenylpropane-based polyphenolic ring structure, and are limited to a few structure variants including cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin (Figure 1), with a distribution in nature of 50%, 12%, 12%, 12%, 7%, and 7%, respectively, and they present almost exclusively as glycosides, anthocyanins [28]. Furthermore, epidemiological evidence has demonstrated that consumption of fruits and berries has been associated with decreased risk of developing

*Vitex agnus-castus* is a shrub of the Verbenaceae family and is found naturally in the Middle East and Southern Europe. Ripe fruit of *Vitex agnus-castus* has been used as a folk medicine for the treatment of various obstetric and gynecological disorders in Europe [30, 31]. Itokawa and colleagues have reported that Vitex (an extract from dried ripe *Vitex agnuscastus*) possesses cytocidal effects on P388, a mouse leukemia cells, suggesting its antitumor activity, and that flavonoids such as luteolin (Figure 2) are one of its major constituents

Curcumin, a hydrophobic polyphenol, also known as turmeric, is a major bioactive ingredient extracted from the rhizome of the plant *Curcuma longa* [34, 35]. Curcumin has been used as a dietary supplement as well as therapeutic agent in Chinese medicine and

**2.1. Resource and chemistry of anthocyanins, Vitex, and curcumin** 

preclinical and clinical trial results.

cancer [29].

[32, 33].

**anthocyanins, Vitex, and curcumin** 

Reactive oxygen species (ROS) have been widely believed to play a pivotal role in a wide variety of cellular functions, including cell proliferation and differentiation [3, 11]. Furthermore, oxidative stress, as a result of alterations of redox homeostasis due to an imbalance between ROS production and elimination, is known to be involved in many diseases such as hypoxic injury [11, 18]. Therefore, maintaining ROS homeostasis is crucial for normal cell growth and survival. Generally, cancer cells appear to generate more ROS than do normal cells due to its increased aerobic glycolysis. Furthermore, cancer cells exhibit increased ROS production and altered redox status. Recent studies suggest that these biochemical characteristics of cancer cells can be exploited for therapeutic benefits [3, 18]. Especially, tumors in advanced stage frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it is possible to preferentially eliminate these cells by pharmacological ROS insults. However, the upregulation of antioxidant capacity in adaptation to intrinsic oxidative stress in cancer cells can confer drug resistance [3, 18]. Thus, abrogation of such drugresistant mechanisms by redox modulation could have significant therapeutic implications [2, 3, 18, 19]. Indeed, it has been known that altered redox status is closely associated with apoptosis induction in various cancer cells [2, 3, 18, 19]. Collectively, manipulating ROS levels by redox modulation is a way to selectively kill cancer cells without causing significant toxicity to normal cells.

Polyphenolic compounds such as flavonoids and curcumin have been shown to induce apoptosis in various malignant cells including solid tumors and hematologic malignant cells [1, 2, 7-9, 20-22]. Interestingly, the mechanisms underlying the apoptosis induction, associated with their antitumoral, chemopreventive and chemotherapeutic activities, have been shown to be implicated in alteration of redox status, since polyphenolic compounds are well known to possess both antioxidant and prooxidant activity [1, 2, 7, 8, 10, 20, 22-26].

156 Apoptosis and Medicine

Apoptosis, or programmed cell death, plays a key role in the development and growth regulation of normal cells, and is often dysregulated in cancer cells [11, 12]. It has been accepted that the aim of anticancer therapy is generally focused on apoptosis induction in premalignant and malignant cells, although other multiple molecular mechanisms such as modulation of carcinogen metabolism, anti-angiogenesis and induction of differentiation are also known to be implicated in its anticancer activity [4, 13]. So far, two principal signal pathways of apoptosis have been identified [11, 12]. The intrinsic mechanism of apoptosis involves a mitochondrial pathway. Apoptosis stimuli destruct mitochondrial membrane structure under the control of Bcl-2 (B-cell leukemia/lymphoma) family, resulting in the release of mitochondrial proteins including cytochrome *c*. Once cytochrome *c* is released it activates caspase-9 (initiator caspase) through the interaction with Apaf-1 (apoptotic protease activating factor-1) and dATP [14, 15], which ultimately leads to caspase-3 and -7 (effector caspases) activation [16]. On the other hand, the extrinsic pathway induced by death receptors, such as tumor necrosis factor receptor (TNFR) and Fas, is responsible for the activation of caspase-8 and caspase-10 (initiator caspase) accompanied by the activation of caspase-3 and -7 [16]. The effector caspases are the final mediators in the intrinsic and extrinsic pathways that cleave substrates and lead to cell death. Moreover, a third pathway involving endoplasmic reticulum (ER) stress and caspase-12 has been reported to be associated in apoptosis [4, 13]. A number of markers have been utilized to reveal apoptosis status including cell viability, cytochrome *c* release, caspase-3 activation, poly (ADP-ribose)

Reactive oxygen species (ROS) have been widely believed to play a pivotal role in a wide variety of cellular functions, including cell proliferation and differentiation [3, 11]. Furthermore, oxidative stress, as a result of alterations of redox homeostasis due to an imbalance between ROS production and elimination, is known to be involved in many diseases such as hypoxic injury [11, 18]. Therefore, maintaining ROS homeostasis is crucial for normal cell growth and survival. Generally, cancer cells appear to generate more ROS than do normal cells due to its increased aerobic glycolysis. Furthermore, cancer cells exhibit increased ROS production and altered redox status. Recent studies suggest that these biochemical characteristics of cancer cells can be exploited for therapeutic benefits [3, 18]. Especially, tumors in advanced stage frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it is possible to preferentially eliminate these cells by pharmacological ROS insults. However, the upregulation of antioxidant capacity in adaptation to intrinsic oxidative stress in cancer cells can confer drug resistance [3, 18]. Thus, abrogation of such drugresistant mechanisms by redox modulation could have significant therapeutic implications [2, 3, 18, 19]. Indeed, it has been known that altered redox status is closely associated with apoptosis induction in various cancer cells [2, 3, 18, 19]. Collectively, manipulating ROS levels by redox modulation is a way to selectively kill cancer cells without causing signifi-

Polyphenolic compounds such as flavonoids and curcumin have been shown to induce apoptosis in various malignant cells including solid tumors and hematologic malignant cells [1, 2, 7-9, 20-22]. Interestingly, the mechanisms underlying the apoptosis induction,

polymerase (PARP) cleavage, and DNA fragmentation [17].

cant toxicity to normal cells.

In this chapter, we will highlight the recent advances on the cancer preventative activities of the polyphenolic compounds, including flavonoids such as anthocyanins, and *Vitex agnuscastus* fruit extract (Vitex) in which flavonoids are one of major components, as well as curcumin based on the most recent results from in vitro cell culture and in vivo animal model tumor systems. We will further summarize the detailed mechanisms underlying their cytocidal effects focusing on apoptosis induction. We will also provide detailed insight into potential future clinical application of these promising candidates endowed with potent antitumor activities, alone or in combination with other anticancer clinical drugs based on preclinical and clinical trial results.
