**3. Potential future application of polyphenolic compounds, alone or in combination with, anticancer drugs**

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 accumulated in red blood cells during the consecutive administration of ATO to APL patients [66]. Furthermore, we have demonstrated for the first time that these arsenic metabolites also existed in cerebrospinal fluid [67], in which the concentrations of arsenic reached levels necessary for differentiation induction [68, 69]. We further investigated for the first time the arsenic speciation in plasma of bone marrow (BM), and demonstrated that speciation profiles of BM plasma were very similar to those of PB plasma, suggesting that speciation analysis of PB plasma could be predicative for BM speciation [70]. These findings on the pharmacokinetics of ATO in APL patients provide a new insight into clinical applications of ATO, and may contribute to better therapeutic protocols [4]. Recently, in order to understand the clinical side effects of arsenite, we also investigated the effects of arsenite on human-derived normal cells, since the clinical side effects of drugs are always found as a harmful and undesired effect on normal cells and/or tissues. Based on a study using a unique in vitro cell culture system comprising the primary culture chorion and amnion cells established in our laboratory [71-73], we demonstrated for the first time that transporter genes, such as aquaporin 9 and multidrug resistance associated protein 2 are involved in controlling intracellular arsenic accumulation in these primary cultured normal cells, which then contribute to differential sensitivity to arsenite cytotoxicity among these cells [74].

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

Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy 165

ATO. Indeed, quercetin and/or genistein are flavonoid with multiple biochemical effects such as downregulation of phosphoinositide 3-kinase/Akt signaling pathway and NF-κB transcription factor activity [79, 80], tyrosine kinase inhibition [81, 82]. Furthermore, both quercetin and genistein have been reported to selectively potentiate ATO-induced apoptosis via ROS generation resulted from intracellular GSH depletion, and activation of intrinsic and extrinsic apoptotic pathway in human leukemia cell lines such as HL-60, U937 and THP-1, but not in phytohemagglutinin-stimulated non-tumor peripheral blood lymphocytes [24, 83]. These results thus suggest that these flavonoids might be used to increase the clinical efficacy of ATO. Similar to flavonoids, subcytotoxic concentrations of curcumin also has been demonstrated to stimulate ROS production and potentiate apoptosis induction by ATO in leukemia cell lines via deregulation of Akt phosphorylation

With these considerations in mind, a lot of preclinical and clinical trials have been carried out. For instance, we recently reported for the first time that 5-FU in combination with Vitex achieved an enhanced cytocidal effect on COLO 201 cells, but lesser cytotoxic effect on human PBMNC [9]. It has also been demonstrated that delphinidin induces apoptosis and inhibited NF-κB signaling in prostate tumor cells in vitro and in a human prostate tumor xenograft in nude mice in vivo [39]. Furthermore, twenty-five colon cancer patients without receiving prior therapy and surgery consumed 60 g/day (20 g/3x/day) of black raspberry powder daily for 2–4 weeks. Biopsies of normal-appearing and tumor tissues were taken from these patients before and after berry treatment. Intake of berries reduced proliferation rates and increased apoptosis in colon tumors but not in normal-appearing crypts [8]. On the other hand, supplementation of anthocyanins in the diet of cancer patients receiving chemotherapy did not result in increased inhibition of tumor development when compared to chemotherapy alone [84]. These conflicting findings suggest that a large scale of clinical

A phase I clinical trial has shown that quercetin, another one of flavonoids, can be safely administered to patients with ovarian cancer or hepatoma by intravenous injection of bolus at a dose of 1400 mg/m2 [82]. Moreover, the evidence of antitumor activity was seen in the clinical trial based on sustained fall in serum CA 125 levels, which is proposed for the use as a surrogate marker of response [85]. Furthermore, similar to our previous report [9], CDF (a difluorinated analog of curcumin), alone or in combination with 5-FU and oxaliplatin, was more potent than curcumin alone in reducing the number of chemoresistant HCT-116 and HT-29 colon cancer cells expressing CD44 and CD166 stem cell-like markers [56]. Concomitantly, cell growth inhibition, apoptosis induction and disintegration of colonospheres in these colon cancer cells were also observed in the study [56]. Moreover, clinical trials have confirmed the safety and feasibility to use curcumin in combination therapy with current chemotherapeutic treatments [7]. More recent results from a phaseI/II study on 21 advanced pancreatic cancer patients with gemcitabine-based chemotherapy have indicated that the median overall survival time of the patients after a treatment with curcumin plus gemcitabine or gemcitabine/S-1 combination was 161 days and 1-year survival rate of 19% (95% confidence interval) [86]. Among eighteen evaluable patients, no

[25].

trial is needed.

The successful clinical efficacy of ATO in the treatment of APL patients has led to investigations on exploring potential treatment applications for other malignancies, including ATO-resistant hematopoietic cancer and solid tumors [75, 76]. In order to further extend our previous study and promote the clinical application of arsenite, we have been seeking to explore potential candidate agents, which are expected to not only potentiate the efficacy of ATO but also possibly reduce its dosages [4]. In this regard, using HL-60 cells which are reported to show resistance to arsenite, we found that delphinidin showed selective cytotoxic effects on the cells, but minimal effects on PBMNC, and sensitized the cells to arsenite, resulting in the enhancement of arsenite cytotoxicity (Yuan et al. manuscript in preparation). Therefore, our experimental data suggest that sensitization of HL-60 cells to arsenite achieved by the combination with delphinidin could benefit a reduced dosages of arsenite in clinical application, contributing to minimize side effect. The clinical trial planning is now underway.

Of note, it is well known that oxidative stress is involved in the mechanisms underlying the therapeutic efficacy of arsenite and plays a major role in the toxicity of arsenite [4, 13, 77]. In fact, in order to maximally exploit effective ROS-mediated cell death without causing significant toxicity to normal cells, redox-based drug combination strategies have been proposed [3, 11, 18]. Based on the strategies, ROS-generating reagents including natural products derived substances, especially phenolic compounds, have received much attention due to their cytocidal effects on tumor cells but little on normal cells. In this regard, like the cytocidal effects of cyanidin-3-rutinoside on leukemia cells [10], delphinidin 3-sambubioside has also been demonstrated to induce apoptosis in HL-60 through ROS-mediated mitochondria pathway [78], suggesting these anthocyanins are good candidates for ROS-generating reagents, thereby possibly potentiate the action of ATO. Indeed, quercetin and/or genistein are flavonoid with multiple biochemical effects such as downregulation of phosphoinositide 3-kinase/Akt signaling pathway and NF-κB transcription factor activity [79, 80], tyrosine kinase inhibition [81, 82]. Furthermore, both quercetin and genistein have been reported to selectively potentiate ATO-induced apoptosis via ROS generation resulted from intracellular GSH depletion, and activation of intrinsic and extrinsic apoptotic pathway in human leukemia cell lines such as HL-60, U937 and THP-1, but not in phytohemagglutinin-stimulated non-tumor peripheral blood lymphocytes [24, 83]. These results thus suggest that these flavonoids might be used to increase the clinical efficacy of ATO. Similar to flavonoids, subcytotoxic concentrations of curcumin also has been demonstrated to stimulate ROS production and potentiate apoptosis induction by ATO in leukemia cell lines via deregulation of Akt phosphorylation [25].

164 Apoptosis and Medicine

cells [74].

clinical trial planning is now underway.

accumulated in red blood cells during the consecutive administration of ATO to APL patients [66]. Furthermore, we have demonstrated for the first time that these arsenic metabolites also existed in cerebrospinal fluid [67], in which the concentrations of arsenic reached levels necessary for differentiation induction [68, 69]. We further investigated for the first time the arsenic speciation in plasma of bone marrow (BM), and demonstrated that speciation profiles of BM plasma were very similar to those of PB plasma, suggesting that speciation analysis of PB plasma could be predicative for BM speciation [70]. These findings on the pharmacokinetics of ATO in APL patients provide a new insight into clinical applications of ATO, and may contribute to better therapeutic protocols [4]. Recently, in order to understand the clinical side effects of arsenite, we also investigated the effects of arsenite on human-derived normal cells, since the clinical side effects of drugs are always found as a harmful and undesired effect on normal cells and/or tissues. Based on a study using a unique in vitro cell culture system comprising the primary culture chorion and amnion cells established in our laboratory [71-73], we demonstrated for the first time that transporter genes, such as aquaporin 9 and multidrug resistance associated protein 2 are involved in controlling intracellular arsenic accumulation in these primary cultured normal cells, which then contribute to differential sensitivity to arsenite cytotoxicity among these

The successful clinical efficacy of ATO in the treatment of APL patients has led to investigations on exploring potential treatment applications for other malignancies, including ATO-resistant hematopoietic cancer and solid tumors [75, 76]. In order to further extend our previous study and promote the clinical application of arsenite, we have been seeking to explore potential candidate agents, which are expected to not only potentiate the efficacy of ATO but also possibly reduce its dosages [4]. In this regard, using HL-60 cells which are reported to show resistance to arsenite, we found that delphinidin showed selective cytotoxic effects on the cells, but minimal effects on PBMNC, and sensitized the cells to arsenite, resulting in the enhancement of arsenite cytotoxicity (Yuan et al. manuscript in preparation). Therefore, our experimental data suggest that sensitization of HL-60 cells to arsenite achieved by the combination with delphinidin could benefit a reduced dosages of arsenite in clinical application, contributing to minimize side effect. The

Of note, it is well known that oxidative stress is involved in the mechanisms underlying the therapeutic efficacy of arsenite and plays a major role in the toxicity of arsenite [4, 13, 77]. In fact, in order to maximally exploit effective ROS-mediated cell death without causing significant toxicity to normal cells, redox-based drug combination strategies have been proposed [3, 11, 18]. Based on the strategies, ROS-generating reagents including natural products derived substances, especially phenolic compounds, have received much attention due to their cytocidal effects on tumor cells but little on normal cells. In this regard, like the cytocidal effects of cyanidin-3-rutinoside on leukemia cells [10], delphinidin 3-sambubioside has also been demonstrated to induce apoptosis in HL-60 through ROS-mediated mitochondria pathway [78], suggesting these anthocyanins are good candidates for ROS-generating reagents, thereby possibly potentiate the action of With these considerations in mind, a lot of preclinical and clinical trials have been carried out. For instance, we recently reported for the first time that 5-FU in combination with Vitex achieved an enhanced cytocidal effect on COLO 201 cells, but lesser cytotoxic effect on human PBMNC [9]. It has also been demonstrated that delphinidin induces apoptosis and inhibited NF-κB signaling in prostate tumor cells in vitro and in a human prostate tumor xenograft in nude mice in vivo [39]. Furthermore, twenty-five colon cancer patients without receiving prior therapy and surgery consumed 60 g/day (20 g/3x/day) of black raspberry powder daily for 2–4 weeks. Biopsies of normal-appearing and tumor tissues were taken from these patients before and after berry treatment. Intake of berries reduced proliferation rates and increased apoptosis in colon tumors but not in normal-appearing crypts [8]. On the other hand, supplementation of anthocyanins in the diet of cancer patients receiving chemotherapy did not result in increased inhibition of tumor development when compared to chemotherapy alone [84]. These conflicting findings suggest that a large scale of clinical trial is needed.

A phase I clinical trial has shown that quercetin, another one of flavonoids, can be safely administered to patients with ovarian cancer or hepatoma by intravenous injection of bolus at a dose of 1400 mg/m2 [82]. Moreover, the evidence of antitumor activity was seen in the clinical trial based on sustained fall in serum CA 125 levels, which is proposed for the use as a surrogate marker of response [85]. Furthermore, similar to our previous report [9], CDF (a difluorinated analog of curcumin), alone or in combination with 5-FU and oxaliplatin, was more potent than curcumin alone in reducing the number of chemoresistant HCT-116 and HT-29 colon cancer cells expressing CD44 and CD166 stem cell-like markers [56]. Concomitantly, cell growth inhibition, apoptosis induction and disintegration of colonospheres in these colon cancer cells were also observed in the study [56]. Moreover, clinical trials have confirmed the safety and feasibility to use curcumin in combination therapy with current chemotherapeutic treatments [7]. More recent results from a phaseI/II study on 21 advanced pancreatic cancer patients with gemcitabine-based chemotherapy have indicated that the median overall survival time of the patients after a treatment with curcumin plus gemcitabine or gemcitabine/S-1 combination was 161 days and 1-year survival rate of 19% (95% confidence interval) [86]. Among eighteen evaluable patients, no

patients experienced a partial or complete response and five patients (28%) demonstrated stable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) [86].

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

Anticancer Drugs Against Cancer Cells: Potential Future Application of the Combinatory Therapy 167

anthocyanidins, such as delphinidin, cyanidin and cyanidin-3-rutinoside, act as BCRP inhibitor, while some of them, such as malvidin, malvidin-3-galactoside and petunidin, exhibited bimodal activities serving as BCRP substrates and inhibitors at low concentrations and high concentrations, respectively [92]. These findings suggest that a variety of biological activities of anthocyanins and anthocyanidins may be attributed in part to their inhibitory effects on those drug transporters, paradoxically, may be abolished as a result of efflux through those transporters. These findings also raised a pharmacological and pharmaceutical concern about formulatability of the dietary constituent, and warn us against the casual use of herbs and/or other botanicals in cancer

Bo Yuan, Masahiko Imai, Hidetomo Kikuchi, Shin Fukushima, Shingo Hazama,

*Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy &* 

*National Therapeutic Center of Hematology of Traditional Chinese Medicine, XiYuan Hospital,* 

*The Third Affiliated Hospital of Beijing University of Traditional Chinese Medicine, Beijing,* 

This work was supported in part by grants from Japan China Medical Association to B.Y. This work was also supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology and by the Promotion and Mutual Aid Corporation for Private Schools of Japan. The authors thank Dr. Chieko Hirobe for encouraging suggestions and arranging sample supply, and Dr. Yamato Kikkawa for providing HCC cell lines for

[1] Fimognari C, Lenzi M, Hrelia P (2008) Chemoprevention of cancer by isothiocyanates and anthocyanins: Mechanisms of action and structure-activity relationship. Curr. Med.

[2] Reddy L, Odhav B, Bhoola KD (2003) Natural products for cancer prevention: A global

[3] Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ros-mediated mechanisms: A radical therapeutic approach? Nat. Rev. Drug Discov. 8:579-591.

Takenori Akaike, Yuta Yoshino, Kunio Ohyama and Hiroo Toyoda

*China Academy of Traditional Chinese Medicine, Beijing, P.R. China* 

patient care.

Xiaomei Hu

Xiaohua Pei

*P.R. China* 

this study.

**5. References** 

Chem. 15:440-447.

perspective. Pharmacol. Ther. 99:1-13.

**Acknowledgement** 

**Author details** 

*Life Sciences, Hachioji, Tokyo, Japan* 

Although many encouraging results of in vitro and in vivo studies suggest polyphenolic compounds as a promising candidate for cancer therapy, either alone or in combination with current anticancer drugs, the therapeutic applications of these compounds in humans are limited by their high metabolic instability as well as poor absorption and bioavailability [1, 7, 8, 20, 87]. In this regard, the selective delivery of nanotechnology-based formulations of these polyphenolic compounds to tumors, alone or in combination with other anticancer drugs, has been of great interest [7, 26, 88]. For instance, pegylated liposomal quercetin was shown to significantly improve its solubility and bioavailability and be a potential application in the treatment of tumor based on a study using CT26 (a mouse colorectal carcinoma cell line), LL/2 (Lewis lung cancer cell line) and H22 (a hepatoma cell line) xenograft mice [88]. Furthermore, diverse curcumin formulations have been developed with different nanotechnology consisting of its encapsulation or conjugation with nanoparticles, polymeric micelles or liposomes to improve its stability, bioavailability and specific and sustained delivery into cancer cells and, consequently, its anticarcinogenic effects [7]. In particular, the systemic administration of gemcitabine plus polymeric micelle-encapsulated curcumin formulation enhanced greater bioavailability in plasma and tissues as compared to that of free curcumin in xenograft models of human pancreatic cancer established in athymic mice [89]. In consequence, the combinatory administration efficiently block tumor growth and metastases in this animal model of pancreatic cancer. Furthermore, an inhibition of NF-κB and its targeted genes are implicated in the tumor growth inhibition [89]. Therefore, the use of nanotechnology-based formulations of polyphenolic compounds and their novel chemical analogs probably represents a potential alternative strategy of great clinical interest for overcoming the high metabolic instability and poor bioavailability of these compounds, which are among the principal factors limiting their therapeutic applications.
