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

#### *2.2.1. Anthocyans*

158 Apoptosis and Medicine

**Figure 1.** Chemical structure of anthocyanidins

**Figure 2.** Chemical structure of luteolin

energetically stabilized and favored form.

**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 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-

sults are in good agreement with previous reports on a possible role of flavonoids, as well as other phytochemicals, in modulating the glutathione (GSH) antioxidant activity, including regulation of intracellular GSH levels through targeting its synthesis, induction of multiple resistant protein 1 mediated GSH efflux, or inhibition of glutathione peroxidase enzyme activity observed in hematopoietic malignant and solid cancer cells in vitro and in vivo [19, 24, 44]. Furthermore, an in vitro study showed that anthocyanidins inhibit glutathione reductase (GR) in an oxygen-dependent manner, presumably via the effect of superoxide [45]. Therefore, these results suggest that anthocyanidins may be used as sensitizing agents through modulating intracellular redox status in various cancer therapy.

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

Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy 161

effects of Vitex [22]. Together, our results suggest that a crosstalk between intrinsic and extrinsic pathway via Bid activation as a result of oxidative stress plays a critical role in Vitex-induced apoptosis in KATO-III cells. We also demonstrated that apoptosis induction was observed in Vitex-treated COLO 201, concomitantly with a significant increase in heme oxygenase-1 (HO-1) gene expression as observed in KATO-III cells [53]. On the other hand, unlike KATO-III, apoptosis induction was not abrogated in the presence of antioxidants, such as NAC [53]. We further demonstrated that after treatment with Vitex, the upregulation of ER stress-related genes, such as glucose-regulated protein 78 (GRP78) and C/EBPhomologous protein (CHOP) along with the activation of caspase-9 and -3 were observed in COLO 201 [21]. However, an inhibitor for JNK significantly suppressed the apoptosis induction associated with caspase-3 activation [21]. These results thus suggest that the activation of JNK, and caspase-9 and -3 resulted from ER stress contributed to the apoptosis induction in Vitex-treated COLO 201 cells. Taken together, it seems that either oxidative stressdependent or ER stress-dependent apoptosis would be triggered in cancer cells treated with Vitex depending on different cell types. Most importantly, our in vivo experimental data revealed that the administration of Vitex significantly suppressed tumor growth in COLO 201 xenograft mice, although more studies must be conducted to understand detailed in vivo pharmacological characterization of Vitex treatment [21]. In addition, as shown in Figure 4, we recently demonstrated a significant dose-dependent cytocidal effect in both a welldifferentiated hepatocellular carcinoma (HCC) cell line, HepG2 and an undifferentiated HCC cell line, HLE, although the levels of cytotoxic activities of Vitex varied between two cells. Similarly, a significant dose-dependent cytocidal effect was also observed in both cells when treated with as high as 20 μg/ml luteolin, one of major constituents of Vitex. However, there was a trend to increase cell proliferation in both cells when treated with a relatively lower concentration of luteolin. On the other hand, 5-FU induced a dose-dependent cytocidal effect on HLE, but not in HepG2. These results indicated that while the cytocidal effect of 5-FU was more selective against undifferentiated hepatocellular carcinoma HLE, Vitex and luteolin exhibited significant cytocidal effects on both well-differentiated and undifferentiated hepatocellular carcinoma cells, suggesting a possible broad usefulness of these compounds to hepatocellular carcinoma therapy. Of note, Vitex has been used to treat patients with various obstetric and gynecological disorders in Europe [30, 31]. Moreover, it is interesting to note that Vitexins, which are isolated from the seed of Chinese herb *Vitex Negundo* and bear a basic flavonoid structure, show cytotoxic and antitumor effects against breast, prostate and ovarian cancer cells through apoptosis induction via an intrinsic pathway based on in vitro and in vivo xenograft tumor models [54]. Therefore, our results provide a new insight into the clinical use of Vitex for colon cancer and hepatocellular carcino-

Curcumin has emerged worldwide as a potent therapeutic substance for treating diverse human diseases including various types of cancer, such as leukemia, colon cancer and pancreatic cancer [7, 25, 35, 55, 56]. Although the precise mode of action of this compound is

ma besides those cancers mentioned above.

*2.2.3. Curcumin* 

Intriguingly, a good correlation has been found between anthocyanin chemical structure and chemoprotective activity. Indeed, several lines of evidence have shown that the number of hydroxyl groups on the B-ring of anthocyanidins is associated with the potency of prooxidative [45-47], apoptotic induction [48], anti-transformation [49], as well as antioxidative activities [1, 8]. For instance, delphinidin and cyanidin that possess *ortho*dihydroxyphenyl structure on the B-ring, showed stronger apoptotic induction in human leukemia cells [48] and inhibitory effect on 12-*O*-tetradecanoylphorbol-13-acetate (TPA) induced cell transformation [49]. Furthermore, a similar trend of structure-activity relationship was also observed in the suppression of transcription factors closely associated with intracellular redox status, such as activator protein-1 (AP-1), nuclear factor-κB (NFκB), and CCAAT/enhancer-binding protein (C/EBPδ) [20, 49, 50]. Structure-activity studies also suggested that the potency as inhibitors of epidermal growth factor receptor (EGFR), a target of an expanding class of anticancer therapies [51], might be positively correlated with the presence of hydroxyl functions in positions 3' and 5' of ring B of the anthocyanidinos molecule, and inversely with the presence of methoxy groups in these positions. All of these findings provide important molecular basis for the antitumor properties of anthocyanidins.
