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

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 cancer [29].

*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 [32, 33].

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

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

Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy 159

other Asian medicines for centuries due to its safety and tolerance [34, 35]. Curcumin has a unique conjugated structure including two methylated phenols linked by the enol form of a heptadiene-3,5-diketone that gives the compound a bright yellow color (Figure 3) [7, 35].

It has been demonstrated that anthocyanin-rich extracts from berries and grapes, and several pure anthocyanins and anthocyanidins, exhibit pro-apoptotic effects in multiple cell types such as colon [23, 36], breast [37, 38], prostate [39, 40], and leukemia cancer cells [10, 41]. They induce apoptosis through both intrinsic (mitochondrial) and extrinsic (Fas) pathways. In the intrinsic pathway, the treatment of cancer cells with anthocyanin results in destabilization of the mitochondrial membrane, cytochrome *c* release and activation of caspase-9, and -3 as well as pro-apoptotic protein such as apoptosis inducing factor [10, 37, 40]. In the extrinsic pathway, anthocyanins modulate the expression of Fas and FasL (Fas ligand) in cancer cells, which result in the activation of caspase-8, then cleaves Bid to tBid, and ultimately stimulates cytochrome *c* release [41]. Of note, several lines of evidence have indicated that oxidative stress resulted from stimulation of ROS production and/or insufficient ROS elimination is implicated in anthocyanins-triggered apoptosis induction in cancer cells, although broad biological activities including antimutagenesis and anticarcinogenesis of anthocyanins are generally attributed to their antioxidant activity [2, 7, 10, 22-25]. Indeed, it has been demonstrated that the most common type of anthocyanins, cyanidin-3-rutinoside, induced apoptosis in a human myeloid leukemia cell line, HL-60, in a dose- and timedependent manner accompanied by accumulation of peroxides [10]. Cyanidin-3-rutinoside treatment resulted in ROS-dependent activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), which contributed to cell death by activating the mitochondrial pathway mediated by Bim, one of proapoptotic gene of Bcl-2 family [10]. More importantly, cyanidin-3-rutinoside treatment did not lead to increased ROS accumulation in normal human peripheral blood mononuclear cells (PBMNC) without inducing cytotoxic effects on these cells [10] as indicated by our preliminary data concerning treatment of HL-60 and PBMNC with anthocyanidin (unpublished data). These results suggest that cyanidin-3-rutinoside could be used in leukemia therapy with the advantages of being widely available and selective against tumors. More recently, delphinidin and cyanidin were reported to induce apoptosis in a human metastatic colorectal cancer cell line, LoVo and LoVo/ADR, a doxorubicin-resistant LoVo, but not in cells originating from a primary tumor site, *i.e.* Caco-2 [23]. Furthermore, LoVo/ADR was more sensitive to anthocyanins than LoVo cells [23]. It has been reported that the rate of lactate production is significantly higher in LoVo/ADR than in LoVo cells [42]. Therefore, the differences in changes of cellular energy metabolism associated with neoplastic transformation has been suggested to contribute the differential sensitivities to these anthocyanins [43]. Moreover, ROS accumulation, inhibition of glutathione reductase, and depletion of GSH were observed in the apoptosis triggered by anthocyanidins in these metastatic colorectal cancer cell lines [23]. These experimental re-

**2.2. Involvement of altered redox status in apoptosis induction triggered by** 

**polyphenolic compounds** 

*2.2.1. Anthocyans* 

**Figure 1.** Chemical structure of anthocyanidins

**Figure 2.** Chemical structure of luteolin

**Figure 3. Chemical structures of naturally occurring curcumin.** The scheme shows the diketone and keto-enol forms of curcumin. Curcumin exists as an equilibrium mixture of two tautomeric forms in solution. The enol structure of curcumin, which is stabilized by intramolecular H-bonding, is the most energetically stabilized and favored form.

other Asian medicines for centuries due to its safety and tolerance [34, 35]. Curcumin has a unique conjugated structure including two methylated phenols linked by the enol form of a heptadiene-3,5-diketone that gives the compound a bright yellow color (Figure 3) [7, 35].
