*2.2.2. Vitex*

We have been interesting the effects of naturally derived flavonoids on the growth of various types of cancer cells. Of those, we have demonstrated that Vitex exhibits cytotoxic activities against various types of solid tumor cells, such as KATO-III (a human gastric signet ring carcinoma cell line), COLO 201 (a human colon adenocarcinoma cell line), MCF-7 (a human breast carcinoma cell line) [52]. More interestingly, no apparent cytotoxicity was observed in non-tumor cells, such as human uterine cervical canal fibroblast (HCF) and human embryo fibroblast (HE-21) when treated with concentrations showing significant cytotoxicity in tumor cells, suggesting a selective cytotoxic activity against tumor cells [52]. We further demonstrated that Vitex induced apoptosis accompanied by an accumulation of intracellular ROS along with the decrease in the levels of intracellular GSH in KATO-III cells [22]. At the same time, our experimental data demonstrated a decrease in the amount of Bcl-2, Bcl-xL and Bid proteins; an increase in Bad protein; activation of caspase-8, -9 and -3; a leakage of cytochrome *c* from mitochondria in the cells [22]. Furthermore, the addition of an antioxidant, N-acetyl-L-cysteine (NAC), or exogenous GSH significantly abrogated the 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 carcinoma besides those cancers mentioned above.

#### *2.2.3. Curcumin*

160 Apoptosis and Medicine

properties of anthocyanidins.

*2.2.2. Vitex* 

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

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

We have been interesting the effects of naturally derived flavonoids on the growth of various types of cancer cells. Of those, we have demonstrated that Vitex exhibits cytotoxic activities against various types of solid tumor cells, such as KATO-III (a human gastric signet ring carcinoma cell line), COLO 201 (a human colon adenocarcinoma cell line), MCF-7 (a human breast carcinoma cell line) [52]. More interestingly, no apparent cytotoxicity was observed in non-tumor cells, such as human uterine cervical canal fibroblast (HCF) and human embryo fibroblast (HE-21) when treated with concentrations showing significant cytotoxicity in tumor cells, suggesting a selective cytotoxic activity against tumor cells [52]. We further demonstrated that Vitex induced apoptosis accompanied by an accumulation of intracellular ROS along with the decrease in the levels of intracellular GSH in KATO-III cells [22]. At the same time, our experimental data demonstrated a decrease in the amount of Bcl-2, Bcl-xL and Bid proteins; an increase in Bad protein; activation of caspase-8, -9 and -3; a leakage of cytochrome *c* from mitochondria in the cells [22]. Furthermore, the addition of an antioxidant, N-acetyl-L-cysteine (NAC), or exogenous GSH significantly abrogated the

through modulating intracellular redox status in various cancer therapy.

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

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

Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy 163

expression [59]. However, the antioxidants, NAC, L-ascorbic acid, alpha-tocopherol, catalase and superoxide dismutase, all effectively prevented curcumin-induced apoptosis, suggesting that curcumin-mediated apoptosis was closely related to the increase in intracellular ROS [59]. Besides hematopoietic cancer cells, curcumin-mediated apoptosis in human breast epithelial cells (H-ras MCF10A) involved generation of ROS as well as downregulation of Bcl-2 and up-regulation of Bax, suggesting redox signaling as a mechanism responsible for curcumin-induced apoptosis in these cells [60]. Syng-Ai et al. also demonstrated that curcumin-induced apoptosis in human breast tumor cell lines (MCF-7, MDAMB) and HepG2 cells is also mediated through the generation of ROS, and that depletion of glutathione by buthionine sulfoximine (BSO) promoted the increased generation of ROS, thereby further sensitizing the cells to curcumin [61]. Interestingly, curcumin had no cytocidal effect on normal rat hepatocytes, because of no superoxide generation [61]. These observations suggest that curcumin with broad biological actions could be developed into an effective chemopreventive and chemotherapeutic agent based on its ability to modulate intracellular redox status. However, the use of curcumin as a therapeutic agent has met with considerable skepticism, since as much as 75% of curcumin is excreted in the feces [62] and also undergoes repaid inactivation by glucuronidation [63], similar to metabolisms of flavonoids [2]. Recently, in order to increase its metabolic stability, numerous approaches have been undertaken, such as generating the fluoro-analog of curcumin termed Diflourinated-Curcumin (referred to as CDF) that exhibits increased metabolic stability [64, 65]. Furthermore, the CDF has been found to exhibit superior growth inhibitory properties against cancer cells to the parental compound curcumin [56, 64, 65].

**3. Potential future application of polyphenolic compounds, alone or in** 

As mentioned in the previous section, the deregulation and sustained activation of multiple tumorigenic pathways are typically implicated in cancer development and progression with locally advanced and aggressive nature. Consequently, the use of therapeutic agents acting on different deregulated gene products, alone or in combination therapy, may represent a potentially better strategy than the targeting on one specific oncogenic product to overcome treatment resistance and prevent cancer development and disease recurrence [2-4, 7]. So far, one of the most successful models for combinatory cancer therapies is all-*trans* retinoic acid (ATRA)/arsenic trioxide (ATO, arsenite) combination as a synergistic therapy for acute promyelocytic leukemia (APL) patients, in which ATRA synergizes ATO activity to provide a superior efficacy of combination therapy in patient through promoting the effects of ATO on several signaling pathway, such as apoptosis induction, differentiation as well as the degradation of PML-RARα [a fusion gene between promyelocytic leukemia (PML) gene and retinoic acid receptor (RAR) α], a causative gene for APL [4, 13]. In order to understand the mode of action of ATO and provide an effective treatment protocol for individual APL patients, we recently conducted studies on the pharmacokinetics of ATO in APL patients using biological samples such as peripheral blood (PB) and cerebrospinal fluid, and demonstrated that not only inorganic arsenic but also methylated arsenic metabolites

**combination with, anticancer drugs** 

**Figure 4. Cytocidal effects of Vitex, luteolin and 5-FU on HepG2 and HLE cells.** Hepatocellular carcinoma (HCC) cell lines, HepG2 (well-differentiated) and HLE (undifferentiated) were kindly provided by Dr. Yamato Kikkawa (Laboratory of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan). After treatment with Vitex (final concentrations: 1, 10, 50 and 100 μg/ml), luteolin (final concentrations: 0.1, 1, 10 and 20 μg/ml) or 5-FU (1, 10 and 100 μM) for 48 h, cell viability was determined by XTT dye-reduction assay according to the method described previously [53]. Significant differences between treatment group and control (\*: *p*<0.05; §: p<0.01; †: *p*<0.001)

not yet elucidated, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis through multiple signaling pathways, including intrinsic and extrinsic pathways as well as ER stress pathway [7, 57]. It has been suggested that curcumin-induced apoptosis is associated with ROS production and/or oxidative stress in cancer cells, in spite of its normal antioxidant capacity [7, 57]. Indeed, it has been demonstrated that curcumin can generate ROS as a prooxidant in the presence of copper in HL-60 cells, resulting in DNA damages and apoptotic cell death [58]. Furthermore, the prooxidant action of curcumin may be related to the conjugated β-diketone structure of this compound [58]. Kuo et al. also demonstrated that curcumin induced a dose- and timedependent apoptotic cell death in the same cells, concomitant with a decrease of Bcl-2

**Figure 4. Cytocidal effects of Vitex, luteolin and 5-FU on HepG2 and HLE cells.** Hepatocellular carcinoma (HCC) cell lines, HepG2 (well-differentiated) and HLE (undifferentiated) were kindly provided by Dr. Yamato Kikkawa (Laboratory of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan). After treatment with Vitex (final concentrations: 1, 10, 50 and 100 μg/ml), luteolin (final concentrations: 0.1, 1, 10 and 20 μg/ml) or 5-FU (1, 10 and 100 μM) for 48 h, cell viability was determined by XTT dye-reduction assay according to the method described previously [53]. Significant differences between treatment group and control (\*: *p*<0.05; §: p<0.01; †: *p*<0.001)

not yet elucidated, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis through multiple signaling pathways, including intrinsic and extrinsic pathways as well as ER stress pathway [7, 57]. It has been suggested that curcumin-induced apoptosis is associated with ROS production and/or oxidative stress in cancer cells, in spite of its normal antioxidant capacity [7, 57]. Indeed, it has been demonstrated that curcumin can generate ROS as a prooxidant in the presence of copper in HL-60 cells, resulting in DNA damages and apoptotic cell death [58]. Furthermore, the prooxidant action of curcumin may be related to the conjugated β-diketone structure of this compound [58]. Kuo et al. also demonstrated that curcumin induced a dose- and timedependent apoptotic cell death in the same cells, concomitant with a decrease of Bcl-2 expression [59]. However, the antioxidants, NAC, L-ascorbic acid, alpha-tocopherol, catalase and superoxide dismutase, all effectively prevented curcumin-induced apoptosis, suggesting that curcumin-mediated apoptosis was closely related to the increase in intracellular ROS [59]. Besides hematopoietic cancer cells, curcumin-mediated apoptosis in human breast epithelial cells (H-ras MCF10A) involved generation of ROS as well as downregulation of Bcl-2 and up-regulation of Bax, suggesting redox signaling as a mechanism responsible for curcumin-induced apoptosis in these cells [60]. Syng-Ai et al. also demonstrated that curcumin-induced apoptosis in human breast tumor cell lines (MCF-7, MDAMB) and HepG2 cells is also mediated through the generation of ROS, and that depletion of glutathione by buthionine sulfoximine (BSO) promoted the increased generation of ROS, thereby further sensitizing the cells to curcumin [61]. Interestingly, curcumin had no cytocidal effect on normal rat hepatocytes, because of no superoxide generation [61]. These observations suggest that curcumin with broad biological actions could be developed into an effective chemopreventive and chemotherapeutic agent based on its ability to modulate intracellular redox status. However, the use of curcumin as a therapeutic agent has met with considerable skepticism, since as much as 75% of curcumin is excreted in the feces [62] and also undergoes repaid inactivation by glucuronidation [63], similar to metabolisms of flavonoids [2]. Recently, in order to increase its metabolic stability, numerous approaches have been undertaken, such as generating the fluoro-analog of curcumin termed Diflourinated-Curcumin (referred to as CDF) that exhibits increased metabolic stability [64, 65]. Furthermore, the CDF has been found to exhibit superior growth inhibitory properties against cancer cells to the parental compound curcumin [56, 64, 65].
