Flavonoids from Achyrocline satureioides: promising biomolecules for anticancer therapy

Abstract

Cancer is the major public health problem worldwide; consequently, the search for new chemotherapeutic drugs is constant. Most of these agents are derived from natural sources, which are the major consistent basis for the search for modern anticancer medicines. In this context, numerous studies indicate flavonoids as a potential new class of secondary metabolites for anticancer therapy. In this review, special attention was addressed to flavonoids present in Achyrocline satureioides, a widely used medicinal plant with several and a well established range of biological properties. Two of these flavonoids are extensively studied for anticancer therapy, quercetin and luteolin, followed by 3-O-methylquercetin. Achyrobichalcone, recently isolated from A. satureioides by our group, can also represent a promising chemotherapeutic biomolecule due to its similarity with other cytotoxic bichalcones to cancer cell lines. The anticancer properties of these flavonoids, specially quercetin and luteolin, type of cell death, mechanisms and molecular targets involved were described. In general, these effects were observed due to the inhibition of cell proliferation, cell cycle arrest, apoptosis, inhibition of angiogenesis, prevention of migration/metastasis and overcoming multidrug resistance, alone or in combination with commonly used chemotherapeutic drugs. All these successful findings in preclinical studies suggest that these flavonoids are promising biomolecules for the development of new anticancer drugs in the future.

---

Juliana Poglia Carini

Juliana Poglia Carini received her Doctor degree in Pharmaceutical Sciences, with emphasis in Pharmaceutical Technology, from the Federal University of Rio Grande do Sul (UFRGS), Brazil, in 2013. Her doctoral thesis was developed in the research group of Professor Valquiria Linck Bassani, which focused on isolation of new phytopharmaceuticals from Achyrocline satureioides and their biological evaluation, with emphasis on anticancer activity.

Fábio Klamt

Fábio Klamt is a Professor and Researcher at the Department of Biochemistry at the Federal University of Rio Grande do Sul (UFRGS), Brazil. He was a Postdoctoral fellow at the Division of Therapeutic Proteins/Food and Drug Administration (FDA), USA, studying molecular mechanisms of the elimination of tumor cells by oxidants. His experience is especially in the area of biochemistry and cell biology: development of in vitro models, study of the pathophysiology of oxidants in biological systems, cellular and molecular mechanisms involved in mitochondrial dysfunction and death by apoptosis in tumors and in neurons, and establishment of prognostic and predictive biomarkers in cancer. He is an Affiliate Member of the Brazilian Academy of Sciences.

Valquiria Linck Bassani

Valquiria Linck Bassani is a Full Professor in Pharmaceutical Technology at the Federal University of Rio Grande do Sul (UFRGS), Brazil. She was the Dean of Pharmacy School and Vice-rector for Graduate Courses of the UFRGS. Her multidisciplinary research group is focused in phytopharmaceuticals with emphasis on phytopharmaceutical technology and biological activities of several species from South America, specially Achyrocline satureioides. The influence of the extraction or drying methods, cyclodextrin association and other technological tools on the biological activity of the herbal products have been studied as a strategy for exploring the pharmacological potential of the Brazilian plants.

---

1. Introduction

Cancer is a major public health problem in many countries of the world.¹ Because of this, the disease receives the special attention of the World Health Organization from the International Agency for Research on Cancer. The GLOBOCAN 2008 project was one of the tools created by these international agencies that allowed estimation of the cancer incidence and mortality. The program indicated that about 14.9 million cancer cases and 8.9 million cancer deaths are estimated to occur in 2015 worldwide, with the majority of deaths occurring in the economically developing world.² In recent decades, the disease has become more common in developing countries, such as Brazil, where it is estimated there has been more than 500 000 new cases of cancer in 2013.³

Natural products are privileged structures created by strong biological and ecological pressure that are able to interact with a wide variety of biological targets, consequently originating effective drugs in a large variety of therapeutic indications.^4,5 Throughout human evolution, the importance of natural products for medicine and health has been enormous, from the earliest civilizations until today.⁶ The accumulated experience, knowledge and research over the years makes the secondary metabolites from natural sources, like plants, the most consistently successful approach for obtaining modern medicines.^6,7

According to an extensive work published by Newman and Cragg, in 2012, 175 small molecules were approved worldwide over the time frame from around the 1940s to 2010 for cancer treatment. Of this total, the majority (74.9%) were inspired by compounds found in nature.⁸ Since the discovery of successful natural anticancer drugs such as paclitaxel (Taxol®) (from Taxus brevifolia), camptothecins (from Camptotheca acuminata), vinca alkaloids, such as vincristine and vinblastine (from Catharanthus roseus) and many others,⁴ the clinical interest for secondary metabolites for cancer therapy increased over the years and currently receives major attention from researchers.

Flavonoids are an emerging potential class of secondary metabolites from plants for anticancer chemotherapy.⁹ The existence of a large number of recently published papers focused on the chemotherapeutic and/or chemopreventive properties of flavonoids^9–22 reinforces the great interest and importance of this issue today. Thus, this work aims to review the potential application of flavonoids for anticancer therapy focusing on the compounds present in Achyrocline satureioides (Lam.) DC. Asteraceae.

2. Flavonoids and anticancer therapy

Flavonoids have probably existed in the plant kingdom for over one billion years and they are present in practically all dietary plants, vegetables and in several medicinal plants used in folk medicine around the world.¹¹ Interest in the anticancer effects of flavonoids has emerged from in vitro and in vivo experimental evidence indicating they interfere with cancer processes such as proliferation, inflammation, angiogenesis, invasion and metastasis.^18,22 In addition, a growing number of epidemiological studies suggests that high flavonoid intake may be correlated with a decreased risk of cancer.¹¹ Usually, the molecular targets of flavonoids comprise the mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K or AKT) and β-catenin pathways, whose activity has been associated with malignant transformation and tumor promotion. Flavonoids can also interfere with the activation of the transcription factors such as nuclear factor kappa B (NFkB) and activator protein-1 (AP-1), while inducing cell cycle arrest and apoptosis.²³

Two flavonoid derivatives reached close to approval and being launched in the market as new drugs for cancer treatment. Phenoxodiol, a synthetic analog of the plant isoflavone genistein, is one of these molecules, which is effective as anticancer agent and/or chemosensitizer for several traditional chemotherapeutic drugs in resistant tumors. The molecule is active against several human cancers, such as ovarian cancer,^24–27 prostate cancer,²⁸ melanoma,^29,30 and squamous carcinoma,³¹ both in vitro and in vivo tests, reaching the step of clinical trials for some cancers.³² Phenoxodiol presented several promising and encouraging results, as the ability to increase the effectiveness of docetaxel in drug resistant ovarian carcinoma cells by at least 100 times,³³ besides having an inhibitory effect against Topoisomerase 2 in the same range of etoposide, but stronger than genistein.³⁴ Despite this, phenoxodiol failed in clinical trials for recurrent ovarian carcinoma after the “fast track” status for clinical use provided by FDA,⁹ the molecule (or its derivatives) still arouses great scientific^35–38 and economical interest, especially from the pharmaceutical industry.³⁹

Flavopiridol is another semisynthetic flavonoid with potential anticancer properties. The compound is a flavone derived from rohitukine, a natural anticancer agent isolated from an Indian tree.¹¹ Due to its interesting biological properties both in vitro and in vivo, as cell cycle arrest in G1 and G2 phases, cytotoxicity against the majority of tumor cell lines in the nanomolar range, suppression of the tumor growth in animals and synergistic activity with most anticancer drugs and radiation, flavopiridol was the first cyclin-dependent kinase (CDK) inhibitor to be tested in human clinical trials.^11,40,41 However, the molecule in Phase II clinical trials was ineffective against cancer and presented high toxicity to patients.^40–42 Nevertheless, currently 60 anticancer clinical trials can be found with flavopiridol, alone or combined with other drugs, whereby 6 of them have shown to be active in the recruiting phase.⁴³

2.1. Flavonoids from Achyrocline satureioides

Achyrocline satureioides (Fig. 1A), known as marcela, is a very popular medicinal plant in southern Brazil, being one of the most remembered and used by the local population.⁴⁴ The species is widely used in folk medicine as an anti-inflammatory, antispasmodic, digestive and carminative agent.^45–49 This popular use has motivated biological and ethnopharmacological studies of the species and revealed a wide range of pharmacological properties.^45–84

Fig. 1 Achyrocline satureioides inflorescences (A) and chemical structures of its major flavonoids: quercetin (QCT) (B); luteolin (LUT) (C); 3-O-methylquercetin (3OMQ) (D); and achyrobichalcone (ACB) (E). The main molecular mechanisms of anticancer activity for QCT and LUT are highlighted.

Some works have reported the ethnopharmacological use of Achyrocline genus (A. satureioides and A. lehmanii Heiron) for anticancer therapy.^50,85 A work of Santos and Elisabetsky, in 1999, investigated the ethnopharmacological use of plants to treat signs and sympoms related to cancer. From these analyses, A. satureioides was one of the species with the highest degree of importance.⁵⁸ Another study reported that the plant was used by cancer patients as a complementary therapy to radioterapy.⁸⁶

The anticancer properties of A. satureioides extracts could also be observed in biological assays. Methanolic extracts of its flowers inhibited growth of two human cancer cell lines: oropharyngeal epidermoid carcinoma cells (KB cells)⁵⁴ and hepatoblastoma cells (HepG2 cells).⁶³ The inhibition growth of KB cells was 66.8% after 72 hours of treatment with 50 μg mL⁻¹ of the methanolic extract.⁵⁴ The IC_50% of HepG2 cells after 48 hours of treatment was 237 μg mL⁻¹ (SD 56 μg mL⁻¹), with inhibition growth in a concentration-dependent manner.⁶³ The difference between these results can be related to the types of cell line, but also due to variations in methodology, as methods of evaluation of the cell death, treatment times or lack of standardization of the tested extracts. In another report, an aqueous extract of A. satureioides significantly inhibited cell division in the Allium cepa system, a vegetal test which measures the mitotic and replication indexes.⁸⁷ Since the main flavonoids are present in the plant as aglycone form, only low concentrations are present in aqueous extracts. Thus, the observed effect reported by Fachinetto et al., in 2007, can be related to other constituents of the plant rather than the flavonoid aglycones.

Polydoro et al., in 2004, noted that A. satureioides freeze dried extracts (prepared from hydroethanol extractive solutions using 80% or 40% v/v ethanol) were cytotoxic to Sertoli cells, at the concentrations of 0.25 and 0.125 mg mL⁻¹, increasing cell death over 30%. It was also observed that an enriched flavonoid fraction, which contained the highest total flavonoid aglycone, caused increase in the cell death and the extracts with higher quercetin contents (prepared with 80% hidroethanol solution) were more cytotoxic.⁶⁷ Nevertheless, another study showed that an aqueous extract of A. satureioides (2% infusion) had low acute toxicity when administered intraperitoneally and no toxicity upon oral administration,⁸⁸ that could be related to the very low flavonoid aglycone content, which presents reduced water solubility.

Most of the biological properties ascribed to A. satureioides extracts are related to the presence of flavonoids in its inflorescences.⁸⁹ Often, quercetin (QCT; Fig. 1B), luteolin (LUT; Fig. 1C) and 3-O-methylquercetin (3OMQ; Fig. 1D) are the main flavonoids found in extracts, mainly in hydroalcoholic preparations.^{45,49,90,91,92} More recently, our group identified a new flavonoid in A. satureioides, named achyrobichalcone (ACB; Fig. 1E).^91,93 Bearing in mind that a better understanding of the pharmacological activity of flavonoids is a key factor to improve the comprehension of the mode of action of many plant extracts,⁹⁴ the next topics will address a compilation of the main studies found in literature concerning the anticancer properties of flavonoids from A. satureioides. Noting that much information is found in scientific databases about this issue, the intention of this review is to describe the most relevant and recent findings.

2.2. Molecular targets and mechanisms of action

Effects of flavonoids from A. satureioides (mainly QCT and LUT) against cancer are multi-targeted and involve a wide range of biological activities, as antiproliferative action against several cancer cells of human origin, induction of apoptosis, cell cycle arrest, inhibition of cell migration/invasion and angiogenesis. Often, their anticancer molecular targets in the cell cycle are cyclins (cyclin A-cyclin T) and cyclin-dependent protein kinases (CDK1-CDK9).⁹⁵ Other related proteins are mitogen-activated protein kinases (MAPK), originally called extracellular signal-regulated kinase (ERK), phosphatidylinositide 3-kinases (PI3K), protein kinase C (PKC), among others. These proteins activate specific cell signaling pathways culminating in cell death.⁹⁶ The reviewed flavonoids induced apoptosis by activating the intrinsic or extrinsic pathways. In the intrinsic pathway or at mitochondrial level, the Bcl-2 family members were induced, as pro-apoptotic proteins (Bax, Bak, PUMA, NOXA, Bim, Bid) or suppressed, as anti-apoptotic proteins (Bcl-2, Mcl-1, Bcl-xL, Bfl-1/A1).⁹⁷ Other signaling proteins were inhibited, as survivin (member of inhibitor of apoptosis-IAP family) and nuclear factor kappa B (NFkB), or activated, as caspases. Flavonoids also act in p53 regulation.^98,99 The extrinsic apoptotic pathway is induced by extracellular ligands, such as CD95 and TRAIL, which bind to specific receptors on the membrane, for example, death receptors, and triggers a cascade of apoptotic signals culminating in activation of caspases. Caspases activation leads to the destruction and elimination of cancer cells.^100,101 Other particular mechanisms and pathways for cell death were observed and will be described opportunely.

In general, the direct anticancer effects of flavonoids from A. satureioides were involved with cell proliferation, cell cycle arrest, apoptosis, angiogenesis, migration/metastasis and overcome multidrug resistance. These effects were observed for flavonoids alone or in combination with commonly used chemotherapeutic drugs.

2.3. Quercetin (QCT)

In the literature, numerous works are described evaluating the anticancer properties of QCT. Table 1 displays the major findings related to in vitro studies, with some of them correlated with in vivo or/and ex vivo experiments. QCT was active against many types of cancers and human cell lines, usually in concentrations between 50 and 100 μM, in a time and dose-dependent manner, reaching various cellular targets.

Table 1 Overview of the major molecular targets and biological effects of quercetin in anticancer therapy

Cancer	Cell culture system	Concentration used	Molecular mechanism	Biological effect	Reference
Bladder	253J	50 μM	Activation of BKCa channels	Antiproliferative	102
	T24	50–100 μM	Inhibition of ecto-5′-NT/CD73 activity	Antiproliferative	103
	T24, EJ, J82	100 μM	Increase in G0/G1 phase, decrease in mutant p53 and survivin	Induction of apoptosis, cell cycle arrest	104
Neuro (glioma)	U87, T98G	25–50 μM	Blocker of IL-6/STAT3	Antiproliferative, inhibition of migration	105
	U87	50 μM	Inhibition of NFkB-induced by PLD1, decrease in MMP-2	Antiproliferative, anti-invasive	106
	A172	50 μM	Down-regulation of ERK, AKT and survivin	Induction of apoptosis	107
Colon	HT29	50–100 μM	Increase in G1 phase, activation of AMPK, up-regulation of p53 and p21	Induction of apoptosis, cell cycle arrest	108
	HT29/MCF-breast	25–200 μM	Increase in G1 phase, activation of AMPK-COX-2	Induction of apoptosis, cell cycle arrest	109
	HCT116	40 μM	Induction of NAG-1	Induction of apoptosis	110
Liver	HepG2	40–160 μM	Increase in G0/G1 phase and p53, decrease in survivin and Bcl-2, activation of caspase-9 and -3	Induction of apoptosis, cell cycle arrest	111
Stomach	BGC823	30–120 μM	Increase in S phase, decrease in Bcl-2/Bax ratio, activation of caspase-3	Induction of apoptosis, cell cycle arrest	112
Blood (leukemia)	CCRF-CEM, HL60, K562	12.5–100 μM	Suppression of telomerase activity	Induction of apoptosis	113
	HL60	25–100 μM	Induction of FasL, activation of caspase-8	Induction of apoptosis	114
Breast	MCF7	25–200 μM	Suppression of AMPK, decrease HIF-1α	Induction of apoptosis	115
	MCF7	10–175 μM	Increase in S phase, decrease in Bcl-2 and ΔΨm, activation of caspase-6, -8 and -9	Induction of apoptosis, cell cycle arrest	116
Skin (melanoma)	B16-BL6	330 μM	Decrease in PKC and MMP-9	Anti-invasive	117
Prostate	PC3	50–200 μM	Increase in G0/G1 phase, decrease in Bcl-2 and ΔΨm, increase in Bax, activation of caspase-3, -8 and -9	Induction of apoptosis, cell cycle arrest	118
	PC3 (and HUVECs)	20–40 μM	Down-regulation of AKT, mTOR and P70S6K	Induction of apoptosis, antiangiogenic	119
Lung	A549	—	Increase in G0/G1 phase, decrease in survivin and Bcl-2, activation of caspase-3	Induction of apoptosis, cell cycle arrest	120
	A549, H1299	20–80 μM	Increase in G2/M phase and p53, decrease in survivin	Induction of apoptosis, cell cycle arrest	121
	A549	14.5–58 μM	Activation of MEK–ERK	Induction of apoptosis	122

---

In the bladder cancer cell lines T24, EJ and J82, QCT inhibited the cell growth by cell cycle arrest in G0/G1 and induced apoptosis by decrease in the expression of mutant p53 and surviving.¹⁰⁴ Additionaly, Rockenbach et al., in 2013, observed the involvement of the ectonucleotidase pathway, by means of ecto-5′-NT/CD73, the enzyme responsible for AMP hydrolysis, in the antiproliferative effects of QCT on T24 cells.¹⁰³ The large conductance Ca²⁺-activated K⁺ (BK_Ca) channel influenced the proliferation of 253J bladder cancer cells only in the presence of QCT, suggesting that this channel may represent a valuable target for antiproliferative effects of QCT in these cells.¹⁰²

In U87 and T98G glioma cells, QCT exerted anticancer effects through abrogation of the IL-6/STAT3 signaling pathway, inhibiting IL-6 and consequently STAT3, a key player involved in cancer-related inflammation by activate cellular responses as cyclin D1, MMP2 and Bcl-2 responsible for cancer cell proliferation, invasion and survival, respectively.¹⁰⁵ Other work demonstrated that QCT inhibited U87 cell proliferation by abolishing the phospholipase D (PLD1) expression, a regulator of cell proliferation and tumorigenesis.¹⁰⁶ In A172 cells, QCT induced apoptosis through caspase-dependent mechanisms involving down-regulation of ERK, AKT and surviving.¹⁰⁷

QCT promoted cell cycle arrest through the increase in G1 phase in HT29 colon cancer cells and upregulated apoptosis-related proteins, such as AMPK, p53 and p21. Additionaly, in vivo experiments showed that QCT resulted in a significant reduction in tumor volume over 6 weeks of treatment and apoptosis-related protein induction was significantly higher in the treated group compared to the control group.¹⁰⁸ Other works observed that AMPK activation in HT29 colon cancer cells and MCF breast cancer cells seemed to be closely related to a decrease in COX-2 expression, suggesting that a possible mechanism involved in the anticancer activity of QCT is partly mediated through its anti-inflammatory action.^109,123 The expression of NAG-1 (non-steroidal anti-inflammatory drug activated gene-1) was induced during treatment of HCT116 colon carcinoma cells with the QCT and apoptosis process was activated. Early growth response-1 (EGR-1) and p53 were required for QCT mediated activation of the NAG-1 promoter.¹¹⁰

The apoptotic effect and cell cycle arrest of QCT in HepG2 hepatoma cancer cells was promoted at G0/G1 phases, with suppression of survivin and Bcl-2, elevation of p53 and activation of caspase-9 and caspase-3.^120,124 Similar results were found for the BGC-823 gastric cancer cell treated with QCT, where caspase-3 was activated and the Bcl-2/Bax ratio was decreased. Additionally, some morphological features of apoptosis were found, such as cell shrinkage or even the formation of an apoptotic body.¹¹²

Avci et al., in 2011, observed that QCT had antiproliferative and pro-apoptotic effects on acute lymphocytic leukemia (ALL) (CCRF-CEM), acute promyelocytic leukemia (HL60) and chronic myeloid leukemia (CML) (K562) cell lines by the suppression of the activity of telomerase, an enzyme that is one of the hallmarks of cancer because it is able to restore final sequences of DNA.¹¹³ QCT also activated the extrinsic apoptotic pathway in HL60 cells through interaction with death receptor Fasligand (FasL) in the plasma membrane, which is involved in ERK and JNK signaling pathways and consequent activation of caspase-8, induction of Bid cleavage and Bax conformation change.¹¹⁴

Despite its apoptotic effects, AMPK also can help the cancer cell in the adaptation and survival during hypoxic conditions. In MCF7 breast cancer cells under hypoxia, QCT was capable of inhibiting AMPK and decrease HIF-1α, which is a critical survival factor in hypoxia.¹¹⁵ Another study showed that QCT arrested the cell cycle in sub-G1 and S phases for MCF-7 cells. The arrest in S phase was mediated by decreasing the protein expression of CDK2, cyclins A and B while increasing the p53 and p57 proteins. Decreased levels of Bcl-2 protein and ΔΨ_m and increased activations of caspase-6, -8 and -9 also were observed.¹¹⁶ Similar results were found for metastatic breast cancer cells, as MDA-MB-231 (ref. 125 and 126) and MDA-MB-453 cells.¹²⁷

Antiangiogenic activity of QCT was evaluated in vitro in PC3 prostate cancer cells and HUVEC endothelial cells, besides in vivo and ex vivo. QCT suppressed vascular endothelial growth factor (VEGF) that induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR and P70S6K in HUVECs, that are responsible by promoting proliferation, migration and tube formation of endothelial cells. QCT (20 mg kg⁻¹) significantly reduced the volume and the weight of solid tumors in the prostate xenograft mouse model, inhibiting tumorigenesis by targeting angiogenesis. Apoptosis was induced in PC3 cells, which was correlated with the down-regulation of AKT, mTOR and P70S6K expressions.¹¹⁹ The actions of QCT suppressing tumor invasion, angiogenesis and metastasis in PC3 cells also were observed in other works^128,129 and in B16-BL6 melanoma cells.¹¹⁷ QCT promoted cell cycle arrest, increasing G0/G1 phase, decreasing Bcl-2 and ΔΨ_m, increasing Bax and activating of caspase-3, -8 and -9 in PC3 cells.¹¹⁸ These findings are in agreement with the results of Senthilkumar et al., in 2010, that demonstrated the induction of both intrinsic and extrinsic pathways to mediate apoptosis in PC3 cells after treatment with QCT.¹³⁰

QCT induced cytotoxicity and apoptosis in A549 and H1299 non-small lung carcinoma cells. Inhibition of cell growth was observed, with increase in G2/M phase and raised cyclin B1 and phospho-CDC2 proteins. Survivin proteins were decreased and levels of total p53, phospho-p53 and p21 proteins increased and translocated to the nuclei.¹²¹ Another work demonstrated that QCT reduced cell viability and DNA synthesis, with a rate of apoptosis equivalent, in A549 cells. QCT treatments led to increase in Bax and Bad, decrease in Bcl-2 and cleavage of caspase-3 and caspase-7. While Akt-1 was inhibited, ERK was phosphorylated following QCT treatment. The induction of MEK–ERK pathway in A549 cells led to activation of caspase-3 and apoptosis.¹²² However, QCT was not active against DMS114, a small lung carcinoma cell.¹³¹

Finally, some works pointed QCT as very potent inhibitor of topoisomerases (Top 1 and 2) having activity at concentrations similar to that of etoposide (50 μM).⁹

Co-treatments with QCT and traditional chemotherapeutics drugs. QCT effectively enhances the toxic effects of doxorubicin (DOX) in SMMC7721 and QGY7701 liver cancer cells and hepatoma xenografts in mices. Pro-apoptotic activity of QCT in DOX-treated liver cancer cells was mediated by p53, activation of the mitochondrial apoptotic pathway and cleavage of pro-caspases. In addition, QCT reduces hepatotoxicity of DOX in normal liver cells in vitro and in vivo.¹³² The synergistic action of QCT and DOX was also observed in the treatment of human breast cancer cells. QCT potentiated anticancer effects of DOX specifically in the highly invasive breast cancer cells and attenuated unwanted cytotoxicity by DNA damage in normal cells.¹³³ These findings were confirmed in vivo in a work of Du et al., in 2010, where QCT combined to an intratumoral DOX injection induced potent rejection of 4T1 breast cancer and led to long-term, tumor-free survival in mice bearing established breast tumor. QCT or DOX alone failed to cure tumor-bearing mice. The response against the tumor was achieved by the induction of immune responses at the same time that QCT and DOX induced cell death.¹³⁴

The resistance of death receptor to the fludarabine in leukemic cells isolated from chronic lymphocytic leukemia (CLL) patients was ameliorated by combined treatment with QCT.¹³⁵ In another work, QCT cooperated with arsenic trioxide to induce apoptosis in U937, THP-1 and HL-60 leukemia cells, increasing the clinical efficacy of the antileukemic drug.¹³⁶

QCT sensitized DB-1 melanoma (p53 wildtype) cells to temozolomide (TMZ) by a mechanism that involves the modulation of p53 family members. After treatment with TMZ, DB-1 cells demonstrated increased p53, however the cells were resistant to TMZ-induced apoptosis. QCT treatment in combination with TMZ abolished drug insensitivity and caused a more than additive induction of apoptosis compared to either treatment alone.¹³⁷

The antiproliferative effect of QCT and cisplatin (CIS) alone or in combination was evaluated in SPC212 and SPC111 malignant mesothelioma cell lines. QCT significantly reduced the proliferation of cell lines, altered the cell cycle distribution and increased the level of caspase-9 and -3. Additionally, the combination of QCT and CIS was found to be more effective when compared to single-drug treatment.¹³⁸ In another work, QCT exhibited synergistic effects with CIS against nasopharyngeal carcinoma cells. The dose reduction index found implied the possibility of reducing the CIS dosage required to treat with the addition of QCT, reducing the risk of CIS-associated toxicity.¹³⁹ In vincristine (VCR) resistant A549 lung adenocarcinoma cells, QCT exerted increasing chemosensitivity when combined with CIS and VCR.¹⁴⁰ It was also found that in lung cancer cells that QCT sensitizes, TRAIL-induced apoptosis and two independent pathways are involved: induction of DR5 through PKC and suppression of AKT mediated survivin expression.¹⁴¹

In the pancreatic cell line EPP85-181RDB, QCT seemed to sensitize resistant cells to daunorubicin (DAU). In parallel, the effect of both substances on the sensitive cell line was synergistic.¹⁴² QCT was an effective cytotoxic agent in gastric carcinoma cell lines. It had not only a synergistic effect with DAU on P-cells but also sensitized DOX-resistant cells. The analysis of p-glycoprotein (P-gp) activity showed that QCT may down-regulate this transporter.¹⁰⁰

The combination treatment of 5-fluorouracil (5-FU) and QCT increased apoptosis in CO115 colorectal cells. Mitochondrial pathway and p53 were involved in cell death.¹⁴³ In another work, QCT sensitized HCT116 colon cancer cells to CIS and etoposide (ETO) under hypoxic conditions by targeting AMPK.¹⁴⁴

2.4. Luteolin (LUT)

The anticancer properties of LUT were extensively studied as those of QCT. The findings described in Table 2 exhibited in vitro studies, with some of them correlated with in vivo or/and ex vivo experiments. Usually, at concentrations near 50 μM, LUT was active against many types of cancers and human cell lines, in a time and dose-dependent manner, reaching various cellular targets.

Table 2 Overview of the major molecular targets and biological effects of luteolin in anticancer therapy

Cancer	Cell culture system	Concentration used	Molecular mechanism/cellular targets	Biological effect	Reference
Bladder	RT112	10–50 μM	Increase in sub-G1 phase, poly(ADP-ribose) polymerase cleavage, DNA fragmentation	Induction of apoptosis, cell cycle arrest	145
Colon	COLO205, HCT116	40 μM	Inhibition of NFkB, activation of JNK	Induction of apoptosis	146
	HT29	40–60 μM	Increase in G1 and G2/M phases, poly(ADP-ribose) polymerase cleavage, decreased in p21^CIP1/WAF1, survivin, Mcl-1, Bcl-xL, and Mdm-2, activation caspase-3, -7 and -9	Induction of apoptosis, cell cycle arrest	147
Liver	HepG2	25–100 μM	Activation of AMPK, inhibition of NFkB	Induction of apoptosis	148
	HLF	50 μM	Blocker of STAT3	Antiproliferative, induction of apoptosis	149
Stomach	AGS	20–80 μM	Increase in G2/M phase, Bax and p53, decrease of Bcl-2, activation of caspase-3, -6, -9	Induction of apoptosis, cell cycle arrest	150
Blood (leukemia)	H60	10–50 μM	DNA fragmentation	Induction of apoptosis	151
Breast	MCF7, MDA-MB-231, SK-BR3	30 μM	Activation of ERK and p38	Induction of apoptosis	152
Skin (melanoma)	B16F10	5–50 μM	Inhibition of β3 integrin/FAK	Anti-invasive	153
Prostate	PC3 (and HUVECs)	20–40 μM	Suppression of VEGFR2, down-regulation of AKT, ERK, mTOR, P70S6K, MMP-2, MMP-9	Induction of apoptosis, antiangiogenic	154
	PC3	10–40 μM	Induction of E-cadherin	Anti-invasive	155
Lung	A549, H460	5–80 μM	Production of superoxide, degradation of MKP-1, activation of JNK	Induction of apoptosis	156
	A549	20–80 μM	Increase in G2/M phase, increase in Bax, activation of JNK, inhibition of NFkB, activation of caspase-3, -9	Induction of apoptosis, cell cycle arrest	157
	A549	25–50 μM	Increase in G1 phase, decrease in ΔΨm, disruption of actin	Induction of apoptosis, cell cycle arrest, inhibition of migration	158

---

The cytotoxic effects of LUT on two different cancer cell lines, including RT112 bladder carcinoma and K562 chronic myeloid leukemia, was described by Kilani-Jaziri et al., in 2012. The apoptosis was induced, with minor cell-cycle perturbations, but some increase in sub-G1 fraction was observed. The poly(ADP-ribose) polymerase was cleaved and a typical ladder of DNA fragments was observed in treated cells.¹⁴⁵

In COLO205 and HCT116 colorectal cancer cells and cervical cancer HeLa cells, LUT inhibited TNFα, a cell mediator that activates both cell death and cell survival pathways, which render most cancer cells resistant to its cytotoxicity. With the inhibition of TNFα, the activation of NFkB was decreased and consequently the induction of NFkB-targeted anti-apoptotic genes was inhibited. In addition, the inhibition of NFkB led to augmentation of JNK activation.¹⁴⁶ Another work showed that LUT induced G1 and G2/M cell cycle arrest, which was mediated by CDK2, CDK4 and CDC2 in HT29 colon cancer cells. In addition, LUT promoted apoptosis through down-regulation of several anti-apoptotic proteins.¹⁴⁷ The same group described that LUT down-regulates the activation of the PI3K/AKT and ERK1/2 pathways via reduction in insulin-like growth factor-I receptor (IGF-IR) signaling in HT29 cells.¹⁵⁹

LUT strongly induced cell death in HepG2 hepatoma cells and dramatically reduced the tumor volume in a tumor xenograft model. Both effects were accompanied by AMPK activation and a strong inhibitory effect on NFkB. LUT treatment causes reactive oxygen species (ROS) production and these intracellular ROS in turn mediate AMPK-NFkB signaling.¹⁴⁸ Selvendiran et al., in 2006, observed the pro-apoptotic effect of LUT on HLF hepatoma cells in vitro and in vivo. Apoptosis was found by caspase-8 activation and enhanced expression in Fas/CD95. Decrease of STAT3, a known negative regulator of Fas/CD95 transcription, lead to down-regulation on these target gene products, such as cyclin D1, survivin, Bcl-xL and vascular endothelial growth factor. An overexpression in STAT3 led to resistance to LUT, suggesting that STAT3 was a critical target of LUT. In nude mice with xenografted tumors using HAK-1B hepatoma cells, LUT significantly inhibited the growth of the tumors.¹⁴⁹

Antiproliferative and chemosensitizing effects of LUT on AGS gastric cancer cells treated with LUT and/or other chemotherapeutic agents were evaluated by Wu et al., 2008. LUT induced cell cycle arrest by an increase of the G2/M phase, besides reducing protein levels of CDC2, cyclin B1 and CDC25C and increasing the cyclin-dependent kinase inhibitor p21 (cip1/Waf1). The pro-apoptotic proteins, including caspase-3,- 6, -9, Bax and p53 were increased and the levels of anti-apoptotic protein Bcl-2 was reduced, thus shifting the Bax/Bcl ratio in favor of apoptosis. The combined treatment of CIS and LUT induced more effective cell growth inhibition, compared to CIS treatment alone.¹⁵⁰

In MCF7, MDA-MB-231 and SK-BR3 breast cancer cells, LUT induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. LUT induces caspase dependent and independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38.¹⁵² Another work with MDA-MB-231 cells showed that LUT arrested the cell cycle at the G2/M and S stages and activated the apoptosis by decreasing AKT, PLK1, cyclin B1, cyclin A, CDC2, CDK2 and Bcl-xL and increasing p21 and Bax expression. LUT-supplementation significantly reduced tumor burden in nude mice inoculated with MDA-MB-231 cells.¹⁶⁰

LUT suppressed the hypoxia-induced changes in B16F10 melanoma cells, as increased cellular adhesion and invasion. Hypoxia significantly decreased the expression of E-cadherin while increasing the expression of N-cadherin in the cells, which was reversed by LUT. LUT up-regulated the E-cadherin at least partly via inhibiting the β3 integrin/FAK signal pathway. In experimental metastasis model mice, treatment with LUT (10–20 mg kg⁻¹) reduced metastatic colonization in the lungs by 50%.¹⁵³

The antiangiogenic activity of LUT was evaluated by Pratheeshkumar et al., in 2012, in HUVECs cells and PC3 prostate cancer cells, using in vitro, ex vivo and in vivo models. In vitro, LUT at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, key events in the process of angiogenesis, which was also confirmed by ex vivo studies. LUT suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2 and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1b, IL-6, IL-8 and TNF-α were significantly reduced after treatment of PC3 cells with LUT. The apoptosis also was activated in PC3 cells. LUT (10 mg kg⁻¹) significantly reduced the volume and the weight of solid tumors in the prostate xenograft mouse model.¹⁵⁴ In another work, LUT inhibits invasion of PC3 prostate cancer cells through the induced expression of E-cadherin through MDM2. LUT inhibits MDM2 through down-regulation of AKT, suggesting that LUT regulates E-cadherin through the AKT/MDM2 pathway. In vivo experiments showed that LUT inhibited spontaneous lung metastasis of PC3 cells implanted onto the nude mice.¹⁵⁵ LUT also acted as an antiproliferative and anti-invasion agent in LNCaP prostate cancer cells.¹⁶¹

In A549 and H460 non-small cell lung carcinoma, LUT induced apoptotic and non-apoptotic death mediated by the induction of superoxide radicals. JNK was potently activated after superoxide accumulation and suppression of superoxide completely blocked LUT-induced JNK activation. The induction of JNK suppressed MKP-1 and promoted cell death.¹⁵⁶ Kim et al., in 2006, observed that caspase activation culminated in apoptosis in Lewis lung carcinoma cells treated with LUT.¹⁶² In another work, LUT blocked the NFkB pathway, sensitizing H23, H2009, H460 and A549 lung carcinoma cells to apoptosis.¹⁶³

The antiproliferative effects of LUT in multi-drug resistance (MDR) breast cancer cells (ADR/RES and MCF-7/Mito^R) that expressed high levels of P-glycoprotein and ABCG2 were evaluated by Rao et al., in 2012. LUT induces apoptosis in P-glycoprotein- and ABCG2-expressing MDR cancer cells without affecting the transport functions of these drug transporters. LUT induced apoptosis by ROS generation, DNA damage, activation of ATR → CHK2 → p53 signaling pathway, inhibition of NFkB signaling pathway, activation of p38 pathway and depletion of anti-apoptotic proteins.¹⁶⁴

Co-treatments with LUT and traditional chemotherapeutic drugs. LUT sensitized the 4T1 and MCF7 breast cancer cells to DOX in vitro and in vivo. In vitro, LUT had only a slight effect on cell growth and cytotoxicity under normoxia, but it could reverse cancer resistance to DOX and promote death of cells under hypoxia. In vivo, there is no reduction in tumor growth when LUT was administered alone, but it could offer superior efficacy and lesser toxicity when administred with DOX in 4T1 and MCF-7 bearing mice. LUT was able to suppress glycolytic flux (but did not affect glucose uptake), P-glycoprotein, antioxidative enzymes under hypoxia in vitro, consequently keeping the ideal intratumoral DOX level in vivo.¹⁶⁵

Tang et al., 2011, showed that LUT acted as a potent inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2). This transcription factor is redox-sensitive and regulates the expression of a battery of genes generally involved with cell defense, enhancing cancer cell survival and resistance to anticancer drugs. In A549 non-small lung cancer cells, which possess constitutively active Nrf2, LUT promoted a dramatic reduction in Nrf2, leading to decreased Nrf2 binding to antioxidant response elements (AREs), down-regulation of ARE-driven genes and depletion of reduced glutathione. Additionally, LUT significantly sensitized A549 cells to the anticancer drugs oxaliplatin (OXA), bleomycin (BLEO) and DOX.¹⁶⁶

2.5. 3-O-Methylquercetin (3OMQ)

Some works evaluating anticancer properties of 3OMQ are found in literature. 3OMQ was tested in vitro, demonstrating activity against human cancer cells at low concentration (often 5–10 μM and not greater than 50 μM) in a time and dose-dependent manner. Some cellular targets were also identified in two of these papers.

Antiproliferative effect of 3OMQ was observed in mouse skin epidermal JB6P1 cells, with induction of cell cycle arrest at the G2/M phase. It also suppressed neoplastic cell transformation, with inhibitory efficacy greater than QCT. The activation of AP-1 was suppressed after the cell treatment with 3OMQ. The inhibition of ERK kinase activity and attenuated phosphorylation of ERKs were also observed.¹⁶⁷

3OMQ caused significant growth inhibition of lapatinib (LAP) sensitive (SK–Br-3) and resistant (SK–Br-3–Lap R) breast cancer cells. 3OMQ had no effect on AKT or ERK signaling in resistant cells. However, it caused a pronounced G2/M arrest mainly through the CHK1–CDC25c–cyclin B1/CDK1 pathway in both cell types. Differently, LAP produced an accumulation of cells in G1 phase mediated through cyclin D1, but only in LAP sensitive cells. 3OMQ induced apoptosis by increasing levels of cleaved caspase-3, -7 and poly(ADP-ribose) polymerase (PARP) in both cell lines.¹⁶⁸

Rubio et al., in 2006, evaluated the cytotoxicity of 3OMQ against several human cancer cell lines (HL60-leukemia, A431-epidermoid carcinoma, HeLa-cervical cancer, SKOV3-ovarian carcinoma and HOS-hosteosarcoma). The study also compared the cytotoxicity of 3OMQ and QCT in these cells. 3OMQ acted at a lower range and values of IC_50% (15–50 μM) when compared to QCT (30–100 μM). Both were not active for HOS cells. The methylation of the hydroxyl group at position C3 of QCT yields 3OMQ, a molecule with a higher antiproliferative activity. The authors presuppose that methylated derivatives can pass through biological membranes more easily than QCT, consequently having higher intracellular concentration and lower IC_50% values.¹⁶⁹

3OMQ isolated from Inula viscosa showed antiproliferative activity against the MCF7 breast cancer cells and Hep2 hepatoma cells (IC_50% values of 11.23 μg mL⁻¹ and 26.12 μg mL⁻¹, respectively). The toxicity was limited against Vero cells, with IC_50% values above 150 μg mL⁻¹. 3OMQ induced apoptosis in MCF7 cells and DNA fragmentation. The molecule increased the number of apoptotic cells with fragmented DNA, compared with untreated MCF7 cells. Cytomorphological alterations corresponding to typical morphology of apoptosis were detected in MCF7 cells treated with 3OMQ, that included cell shrinkage, membrane blebbing and formation of apoptotic bodies.¹⁷⁰

2.6. Achyrobichalcone (ACB)

Achyrobichalcone (ACB) is a biflavonoid recently isolated from A. satureioides, found only in this plant so far.^91,93 ACB arouses scientific interest due to its structural similarity to other bichalcones with anticancer properties.¹⁷¹ Furthermore, ACB integrates the class of chalcones, which are extensively studied for anticancer therapy. Some studies are running in our group with the aim of assessing the anticancer properties of the molecule. This work is reaching positive and promising results that will be published opportunely.

2.7. Clinical trials and future trends

Despite promising anticancer properties evidenced in preclinical studies for QCT and LUT, clinical trials should be conducted to confirm the chemotherapeutic properties of these molecules and to propose the possible use as adjuvants in anticancer therapy. In the literature, one Phase I clinical trial was performed for QCT.¹⁷² Their data suggested a safe dose injection of 1.4 g m⁻² (about 35 mg kg⁻¹) administered via intravenous infusion at three-week or weekly intervals. At higher doses, renal toxicity was detected. In this study, QCT inhibited the lymphocyte tyrosine kinase activity associated with the progression of cancer in some patients with hepatoma and ovarian cancer refractory to cisplatin, evidencing the antitumor activity of QCT in human. Currently, two ongoing clinical trials are evaluating the chemopreventive properties of QCT,^173,174 but there is a lack of information about its chemotherapeutic properties. For LUT, no studies were found in clinical phase. Additionally, problems associated with the low bioavailability of QCT and LUT reported in pharmacokinetic studies^{172,175–178} in humans should also be investigated in future works. Although there is still a long run of research for the insertion of these flavonoids into the clinical routine, the overwhelming data obtained so far for QCT and LUT support the anticancer potential of these molecules.

3. Concluding remarks

Flavonoids from A. satureioides, especially QCT and LUT, are multi-target anticancer agents by modulation of multiple proteins, being advantageous by conducting to a higher clinical efficacy when compared with target-selective drugs. A wide range of cancers were affected after treatment with flavonoids, both when administered alone or combined to traditional chemotherapeutic drugs. In this sense, the successful anticancer preclinical studies with these flavonoids (especially QCT) encourage the clinical trials in human and, possibly, these molecules will emerge as strong candidates to anticancer drugs in the future.

Conflict of interest

The authors have declared no conflict of interest.

Abbreviations

MAPK	Mitogen-activated protein kinase
PKC	Protein kinase C
PI3K or AKT	Phosphatidylinositol 3-kinase
NFkB	Nuclear factor kappa B
CDKs(1–9)	(1–9)Cyclin-dependent kinases
ERK	Extracellular signal-regulated kinase
VEGF	Vascular endothelial growth factor
IGF-IR	Insulin-like growth factor-I receptor
P-gp	P-glycoprotein
Cip1/Waf1	Cyclin-dependent kinase inhibitor p21
AIF	Apoptosis-inducing factor
Nrf2	Nuclear factor erythroid 2-related factor 2
PARP	Poly(ADP-ribose) polymerase
Bax	Bcl-2-associated X protein
PUMA	p53 Upregulated modulator of apoptosis or BBC3, Bcl-2-binding component 3
NOXA	Phorbol-12-myristate-13-acetate-induced protein 1
Bak	Bcl-2 homologous antagonist/killer
Bid	BH3 interacting-domain death agonist
Bcl-2	Derived from B-cell lymphoma 2
Bcl-xL	Derived from B-cell lymphoma-extra large
Mcl-1	Induced myeloid leukemia cell differentiation protein
IAP	Inhibitor of apoptosis
p53	Protein 53 or tumor protein 53
TRAIL	TNF-related apoptosis-inducing ligand
survivin or BIRC5	baculoviral inhibitor of apoptosis repeat-containing 5
IL	Interleucine
STAT3	Signal transducer and activator of transcription 3
MMP	Matrix metalloproteinase
COX-2	Cyclooxygenase-2
PLD1	Phospholipase D1
p21	Cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1
NAG-1	Nonsteroidal anti-inflammatory drug-activated gene
EGR-1	Early growth response protein 1
HIF-1α	Hypoxia-inducible factor 1-α
FasL or CD95L	Fas ligand
p57	Cyclin-dependent kinase inhibitor 1C
ΔΨm	Mitochondrial transmembrane potential
mTOR	mammalian target of rapamycin
P70S6K	serine/threonine kinase
TNFα	Tumor necrosis factor
JNK	c-Jun NH2-terminal kinase
PLK1	Serine/threonine-protein kinase
ABCG2	ATP-binding cassette sub-family G member 2
MKP-1	Mitogen-activated protein kinase phosphatases
ARE	Antioxidant responsive element
ROS	Reactive oxygen species
QCT	Quercetin
LUT	Luteolin
3OMQ	3-O-methylquercetin
ACB	Achyrobichalcone
DOX	Doxorubicin
TMZ	Temozolomide
CIS	Cisplatin
VCR	Vincristine
DAU	Daunorubicin
5-FU	5-Fluorouracil
ETO	Etoposide
MDR	Multi-drug resistance
OXA	Oxaliplatin
BLEO	Bleomycin
LAP	Lapatinib.

Acknowledgements

The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support and scholarship.

References

1. R. Siegel, D. Naishadham and A. Jemal, Cancer Stat., 2013, 63, 11–30.
2. J. Ferlay, H. R. Shin, F. Bray, D. Forman, C. D. Mathers and D. Parkin, GLOBOCAN 2008, Cancer incidence and mortality worldwide: IARC Cancer Base n° 10, http://globocan.iarc.fr, International Agency for Research on Cancer, Lyon, France, 2010, last accessed 25/02/2013.
3. INCA, I. Nacional de Câncer José Alencar Gomes da Silva, Coordenação Geral de Ações Estratégicas, Coordenação de Prevenção e Vigilância, Estimativa 2012: incidência de câncer no Brasil, 2011, Rio de Janeiro.
4. G. M. Cragg, D. G. I. Kingston and D. J. Newman, Anticancer agents from natural products, Taylor & Francis, Boca Raton, 2005.
5. F. E. Koehn and G. T. Carter, Nat. Rev. Drug Discovery, 2005, 4, 206–220.
6. H. Ji, X. Li and H. Zhang, EMBO J., 2009, 10, 194–200.
7. A. Harvey, Drug Discovery Today, 2000, 5, 294–300.
8. D. J. Newman and G. M. Cragg, J. Nat. Prod., 2012, 75, 311–335.
9. P. Russo, A. Del Bufalo and A. Cesario, Curr. Med. Chem., 2012, 19, 5287–5293.
10. P. Knekt, R. Järvinen, R. Seppänen, M. Heliövaara, L. Teppo, E. Pukkala and A. Aromaa, Am. J. Epidemiol., 1997, 146, 223–230.
11. W. Ren, Z. Qiao, H. Wang, L. Zhu and L. Zhang, Med. Res. Rev., 2003, 23, 519–534.
12. A. Ghasemzadeh and N. Ghasemzadeh, J. Med. Plants Res., 2011, 5, 6697–6703.
13. S. Cimino, G. Sortino, V. Favilla, T. Castelli, M. Madonia, S. Sansalone, G. I. Russo and G. Morgia, Oxid. Med. Cell. Longevity, 2012, 1–8.
14. J. García-Tirado, C. Rieger-Reyes and P. Saz-Peiró, Medicina Clínica, 2012, 139, 358–363.
15. A. G. Mercader and A. B. Pomilio, Curr. Med. Chem., 2012, 19, 4324–4347.
16. K. Michalak and O. Wesołowska, Anti-Cancer Agents Med. Chem., 2012, 12, 880–890.
17. E. Park and J. M. Pezzuto, Anti-Cancer Agents Med. Chem., 2012, 12, 836–851.
18. D. F. Romagnolo and O. I. Selmin, J. Nutr. Gerontol. Geriatr., 2012, 31, 206–238.
19. C. Spatafora and C. Tringali, Anti-Cancer Agents Med. Chem., 2012, 12, 902–918.
20. D. Vira, S. K. Basak, M. S. Veena, M. B. Wang, R. K. Batra and E. S. Srivatsan, Cancer Metastasis Rev., 2012, 31, 733–751.
21. C. Weng and G. Yen, Cancer Metastasis Rev., 2012, 31, 323–351.
22. C. Weng and G. Yen, Cancer Treat. Rev., 2012, 38, 76–87.
23. Y. J. Surh, Nat. Rev. Cancer, 2003, 10, 768–780.
24. M. Kamsteeg, T. Rutherford, E. Sapi, B. Hanczaruk, S. Shahabi, M. Flick, D. Brown and G. Mor, Oncogene, 2003, 22, 2611–2620.
25. A. B. Alvero, D. O'Malley, D. Brown, G. Kelly, M. Garg, W. Chen, T. Rutherford and G. Mor, Cancer, 2006, 106, 599–608.
26. P. L. De Souza, W. Liauw, M. Links, S. Pirabhahar, G. Kelly and L. G. Howes, Cancer Chemother. Pharmacol., 2006, 58, 427–433.
27. J. R. Gamble, P. Xia, C. N. Hahn, J. J. Drew, C. J. Drogemuller, D. Brown and M. A. Vadas, Int. J. Cancer, 2006, 118, 2412–2420.
28. S. Mahoney, F. Arfuso, P. Rogers, S. Hisheh, D. Brown, M. Millward and A. Dharmarajan, J. Biosci., 2012, 37, 73–84.
29. F. Yu, R. N. Watts, X. D. Zhang, J. M. Borrow and P. Hersey, Anti-Cancer Drugs, 2006, 17, 1151–1161.
30. H. M. Kluger, M. M. McCarthy, A. B. Alvero, M. Sznol, S. Ariyan, R. L. Camp, D. L. Rimm and G. Mor, J. Transl. Med., 2007, 5, 1–15.
31. M. F. Aguero, M. M. Facchinetti, Z. Sheleg and A. M. Senderowicz, Cancer Res., 2005, 65, 3364–3373.
32. Clinical Trials, United State National Institutes of Health, 2013, http://clinicaltrials.gov/ct2/results?term=phenoxodiol%26Search=Search, last access: 28/02/2013.
33. E. Sapi, A. B. Alvero, W. Chen, D. O'Malley, X. Y. Hao, B. Dwipoyono, M. Garg, M. Kamsteeg, T. Rutherford and G. Mor, Oncol. Res., 2004, 14, 567–578.
34. A. I. Constantinou and A. Husband, Anticancer Res., 2002, 22, 2581–2585.
35. J. B. Howes, P. L. De Souza, L. West, L. J. Huang and L. G. Howes, BMC Clin. Pharmacol., 2011, 11, 3.
36. M. G. Kelly, G. Mor, A. Husband, D. M. O'Malley, L. Baker, M. Azodi, P. E. Schwartz and T. Rutherford, Int. J. Gynecol. Cancer, 2011, 21, 633–639.
37. L. Wu, T. De Luca, T. Watanabe, D. M. Morré and D. J. Morré, Biochim. Biophys. Acta, 2011, 1810, 784–789.
38. C. Yao, S. Wu, D. Li, H. Ding, Z. Wang, Y. Yang, S. Yan and Z. Gu, Mol. Oncol., 2012, 6, 392–404.
39. Marshall Edwards Pharma, 2013, available in: http://www.meipharma.com/, last acess: 28/02/2013.
40. M. V. Blagosklonny, Cell cycle, 2004, 3, 1537–1542.
41. C. McInnes, Drug Discovery Today, 2008, 13, 875–881.
42. M. Guha, Nat. Rev. Drug Discovery, 2012, 11, 892–894.
43. Clinical Trials, United State National Institutes of Health, 2013, http://www.clinicaltrials.gov/ct2/results?term=flavopiridol%26Search=Search, last access: 01/03/2013.
44. S. G. D. Oliveira, F. R. R. De Moura, F. F. Demarco, P. S. Nascente, F. A. B. Del Pino and R. G. Lund, J. Ethnopharmacol., 2012, 140, 428–437.
45. C. M. O. Simões, E. P. Schenkel, L. Bauer and A. A. Langeloh, J. Ethnopharmacol., 1988, 22, 281–293.
46. L. Silva and A. Langeloh, Lat. Am. J. Pharm., 1994, 13, 35–40.
47. C. M. O. Simões, L. A. Mentz, E. P. Schenkel, B. F. Irgang and J. R. Stehmann, Plantas da Medicina Popular no Rio Grande do Sul, Editora da Universidade Federal do Rio Grande do Sul, Porto Alegre, 1998.
48. C. Kadarian, A. M. Broussalis, J. Miño, P. López, S. Gorzalczany, G. Ferraro and C. Acevedo, Pharmacol. Res., 2002, 45, 57–61.
49. K. C. B. De Souza, V. L. Bassani and E. E. S. Schapoval, Phytomedicine, 2007, 14, 102–108.
50. J. F. Morton, Q. J. Crude Drug Res., 1975, 13, 97–121.
51. H. Wagner, A. Proksch, R. M. Vollmar, S. Odenthal, H. Stuppner, K. Jurcic, M. Le Turdu and J. N. Fang, Arzneimittelforschung, 1985, 35, 1069–1075.
52. J. Puhlmann, U. Knaus, L. Tubaro, W. Schaefer and H. Wagner, Phytochemistry, 1992, 31, 2617–2621.
53. C. Anesini and C. Perez, J. Ethnopharmacol., 1993, 39, 119–128.
54. M. Arisawa, Nat. Med., 1994, 48, 338–347.
55. A. R. De Arias, E. Ferro, A. Inchausti, M. Ascurra, N. Acosta, E. Rodriguez and A. Fournet, J. Ethnopharmacol., 1995, 45, 35–41.
56. C. Desmarchelier, J. Coussio and G. Ciccia, Braz. J. Med. Biol. Res., 1998, 31, 1163–1170.
57. A. L. G. Santos, D. Ripoll, N. Nardi and V. L. Bassani, Phytother. Res., 1999, 13, 65–66.
58. M. A. C. Santos and E. Elisabetsky, Rev. Bras. Plant. Med., 1999, 2, 7–17.
59. S. M. Zanon, F. S. Ceriatti, M. Rovera, L. J. Sabini and B. A. Ramos, Rev. Latinoam. Microbiol., 1999, 41, 59–62.
60. J. G. Graham, M. L. Quinn, D. S. Fabricant and N. R. Farnsworth, J. Ethnopharmacol., 2000, 73, 347–377.
61. J. R. Carney, J. M. Krenisky, R. T. Williamson and J. Luo, J. Nat. Prod., 2002, 65, 203–205.
62. A. Gugliucci and T. Menini, Life Sci., 2002, 71, 693–705.
63. M. J. Ruffa, G. Ferraro, M. L. Wagner, M. L. Calcagno, R. H. Campos and L. Cavallaro, J. Ethnopharmacol., 2002, 79, 335–339.
64. M. F. Arredondo, F. Blasina, C. Echeverry, A. Morquio, M. Ferreira, J. A. Abin-Carriquiry, L. Lafon and F. Dajas, J. Ethnopharmacol., 2004, 91, 13–20.
65. J. M. R. Bettega, H. F. Teixeira, V. L. Bassani, C. R. M. Barardi and C. M. O. Simões, Phytother. Res., 2004, 18, 819–823.
66. O. Hnatyzyn, V. Moscatelli, R. Rondina, M. Costa, C. Arranz, A. Balaszczuck, J. Coussio and G. Ferraro, Phytomedicine, 2004, 11, 366–369.
67. M. Polydoro, K. C. B. De Souza, M. E. Andrades, E. G. Da Silva, F. Bonatto, J. Heydrich, F. Dal-Pizzol, E. E. S. Schapoval, V. L. Bassani and J. C. F. Moreira, Life Sci., 2004, 74, 2815–2826.
68. M. C. T. Duarte, G. M. Figueira, A. Sartoratto, V. L. G. Rehder and C. Delarmelina, J. Ethnopharmacol., 2005, 97, 305–311.
69. A. Morquio, F. Rivera-Megret and F. Dajas, Phytother. Res., 2005, 19, 486–490.
70. D. Calvo, L. N. Cariddi, M. Grosso, M. S. Demo and A. M. Maldonado, Rev. Latinoam. Microbiol., 2006, 48, 247–255.
71. M. L. Dickel, S. M. K. Rates and R. Ritter, J. Ethnopharmacol., 2007, 109, 60–71.
72. M. Consentino, R. Bombelli, E. Carcano, A. Luini, M. Franca, F. Crema, F. Dajas and S. Lecchini, J. Ethnopharmacol., 2008, 116, 501–507.
73. G. Ferraro, C. Anesini, A. Ouvina, D. Retta, R. Filip, M. Gattuso, S. Gattuso, O. Hnatyszyn and A. Bandoni, Lat. Am. J. Pharm., 2008, 27, 626–628.
74. M. F. Blasina, L. Vaamonde, A. Morquio, C. Echeverry, F. Arredondo and F. Dajas, Phytother. Res., 2009, 23, 1263–1269.
75. C. L. C. Brandelli, R. B. Giordani, G. A. De Carli and T. Tasca, Parasitol. Res., 2009, 104, 1345–1349.
76. M. J. González and J. M. Marioli, J. Invertebr. Pathol., 2010, 104, 209–213.
77. J. R. Santin, M. Lemos, L. C. Klein Junior, R. Niero and S. F. De Andrade, J. Ethnopharmacol., 2010, 130, 334–339.
78. M. B. Joray, M. R. Rollán, G. M. Ruiz, S. M. Palacios and M. C. Carpinella, Planta Med., 2011, 77, 95–100.
79. D. C. Sabaté, M. J. Gonzaléz, M. P. Porrini, M. J. Eguaras, M. C. Audisio and J. M. Marioli, World J. Microbiol. Biotechnol., 2012, 28, 1415–1422.
80. D. C. Espiña, F. B. Carvalho, D. Zanini, J. B. Schlemmer, J. D. Coracini, M. A. Rubin, V. M. Morsch, M. R. C. Schetinger, D. B. R. Leal, C. R. Baiotto and J. A. S. Jaques, Cell Biochem. Funct., 2012, 30, 347–353.
81. P. Faral-Tello, S. Mirazo, C. Dutra, A. Pérez, L. Geis-Asteggiante, S. Frabasile, E. Koncke, D. Davyt, L. Cavallaro, H. Heinzen and J. Arbiza, Sci. World J., 2012, 1–5 , Article ID 174837.
82. M. C. Sabini, F. M. Escobar, C. E. Tonn, S. M. Zanon, M. S. Contigiani and L. I. Sabini, Nat. Prod. Res., 2012, 26, 405–415.
83. M. Trojan-Rodrigues, T. L. S. Alves, G. L. G. Soares and M. R. Ritter, J. Ethnopharmacol., 2012, 139, 155–163.
84. M. B. Joray, S. M. Palacios and M. C. Carpinella, Phytomedicine, 2013, 20, 258–261.
85. H. Garcia-Barriga, Flora medicinal de Colombia, Universidad Nacional, Bogota, vol. 2, pp. 3, 1975.
86. M. Vanini, R. L. Barbieri, R. M. Heck and E. Schwartz, Enfermería Global, 2011, 21, 1–6.
87. J. M. Fachinetto, M. D. Bagatini, J. Durigon, A. C. F. Da Silva and S. B. Tedesco, Rev. Bras. Farmacogn., 2007, 17, 49–54.
88. F. Rivera, E. Gervaz, C. Sere and F. Dajas, J. Ethnopharmacol., 2004, 95, 359–362.
89. D. Retta, E. Dellacassa, J. Villamil, S. A. Suárez and A. L. Bandoni, Ind. Crops Prod., 2012, 38, 27–38.
90. K. C. B. De Souza, E. E. S. Schapoval and V. L. Bassani, J. Pharm. Biomed. Anal., 2002, 28, 771–777.
91. M. H. Holzschuh, G. Gosmann, P. H. Schneider, E. E. S. Schapoval and V. L. Bassani, Pharmazie, 2010, 65, 650–656.
92. J. P. Carini, S. Kaiser, G. G. Ortega and V. L. Bassani, Phytochem. Anal., 2013, 24, 193–200.
93. J. P. Carini, G. G. Leitão, P. H. Schneider, C. C. Santos, F. N. Costa and V. L. Bassani, J. Med. Food, 2013 , manuscript submitted.
94. F. Dajas, J. Ethnopharmacol., 2012, 143, 383–396.
95. G. K. Schwartz and M. A. Shah, J. Clin. Oncol., 2005, 23, 9408–9421.
96. A. B. Granado-Serrano, M. A. Martin, L. Bravo, L. Goya and S. Ramos, J. Nutr., 2006, 136, 2715–2721.
97. D. H. Lee, M. Szczepanski and Y. L. Lee, Biochem. Pharmacol., 2008, 75, 2345–2355.
98. R. V. Priyadarsini, R. S. Murugan, S. Maitreyi, K. Ramalingam, D. Karunagaran and S. Nagini, Eur. J. Pharmacol., 2010, 649, 84–91.
99. G. Niu, S. Yin, S. Xie, Y. Li, D. Nie, L. Ma, X. Wang and Y. Wu, Acta Biochim. Biophys. Sin., 2011, 43, 30–37.
100. S. Borska, M. Chmielewska, T. Wysocka, M. Drag-Zalesinska, M. Zabel and P. Dziegiel, Food Chem. Toxicol., 2012, 50, 3375–3383.
101. C. Spagnuolo, M. Russo, S. Bilotto, I. Tedesco, B. Laratta and G. L. Russo, Ann. N. Y. Acad. Sci., 2012, 1259, 95–103.
102. Y. Kim, W. Kim and E. Cha, Korean J. Physiol. Pharmacol., 2011, 15, 279–283.
103. L. Rockenbach, L. Bavaresco, P. F. Farias, A. R. Cappellari, C. H. Barrios, F. B. Morrone and A. M. O. Battastini, Urol. Oncol., 2013, 31, 1204–1211.
104. L. Ma, J. M. Feugang, P. Konarski, J. Wang, J. Lu, S. Fu, B. Ma, B. Tian, C. Zou and Z. Wang, Front. Biosci., 2006, 11, 2275–2285.
105. J. Michaud-Levesque, N. Bousquet-Gagnon and R. Béliveau, Exp. Cell Res., 2012, 318, 925–935.
106. M. H. Park and D. S. Min, Biochem. Biophys. Res. Commun., 2011, 412, 710–715.
107. E. J. Kim, C. H. Choi, J. Y. Park, S. K. Kang and Y. K. Kim, Neurochem. Res., 2008, 33, 971–979.
108. H. Kim, S. Kim, B. Kim, S. Lee, Y. Park, B. Park, S. Kim, J. Kim, C. Choi, J. Kim, S. Cho, J. Jung, K. Roh, K. Kang and J. Jung, J. Agric. Food Chem., 2010, 58, 8643–8650.
109. Y. Lee, S. Y. Park, Y. Kim, W. S. Lee and O. J. Park, Exp. Mol. Med., 2009, 41, 201–207.
110. J. H. Lim, J.-W. Park, D. S. Min, J.-S. Chang, Y. H. Lee, Y. B. Park, K. S. Choi and T. K. Kwon, Apoptosis, 2007, 12, 411–421.
111. J. Tan, B. Wang and L. Zhu, Chem. Biodiversity, 2009, 6, 1101–1110.
112. P. Wang, K. Zhang, Q. Zhang, J. Mei, C. Chen, Z. Feng and D. Yu, Toxicol. in Vitro, 2012, 26, 221–228.
113. C. B. Avci, S. Yilmaz, Z. O. Dogan, G. Saydam, Y. Dodurga, H. A. Ekiz, M. Karta, F. Sahin, Y. Baran and C. Gunduz, Hematology, 2011, 16, 303–307.
114. W. Lee, Y. Chen and T. Tseng, Oncol. Rep., 2011, 25, 583–591.
115. Y. Lee and O. J. Park, Food Sci. Biotechnol., 2011, 20, 371–375.
116. C. Chou, J. Yang, H. Lu, S. Ip, C. Lo, C. Wu, J. Lin, N. Tang, J. Chung, M. Chou, Y. Teng and D. Chen, Arch. Pharmacal Res., 2010, 33, 1181–1191.
117. X. Zhang, S. Huang and Q. Xu, Cancer Chemother. Pharmacol., 2004, 53, 82–88.
118. K. Liu, C. Yen, R. S. Wu, J. Yang, H. Lu, K. Lu, C. Lo, H. Chen, N. Tang, C. Wu and J. Chung, Environ. Toxicol., 2012 DOI:10.1002/tox.21769.
119. P. Pratheeshkumar, A. Budhraja, Y. Son, X. Wang, Z. Zhang, S. Ding, L. Wang, A. Hitron, J. Lee, M. Xu, G. Chen, J. Luo and X. Shi, PLoS One, 2012, 7, 1–10.
120. J. Tan, L.-C. Zhu and B.-C. Wang, Chinese Pharmacol. Bull., 2008, 24, 1220–1224.
121. P. Kuo, H. Liu and J. Chao, J. Biol. Chem., 2004, 279, 55875–55885.
122. T. T. T. Nguyen, E. Tran, T. H. Nguyen, P. T. Do, T. H. Huynh and H. Huynh, Carcinogenesis, 2004, 25, 647–659.
123. R. Narayansingh and R. A. R. Hurta, J. Sci. Food Agric., 2009, 89, 542–547.
124. S. Tanigawa, M. Fujii and D. Hou, Biosci., Biotechnol., Biochem., 2008, 72, 797–804.
125. C. M. J. Conklin, J. F. Bechberger, D. MacFabe, N. Guthrie, E. M. Kurowska and C. C. Naus, Carcinogenesis, 2007, 28, 93–100.
126. S. Chien, Y. Wu, J. Chung, J. Yang, H. Lu, M. Tsou, W. G. Wood, S. Kuo and D. Chen, Hum. Exp. Toxicol., 2009, 28, 493–503.
127. E. J. Choi, S. M. Bae and W. S. Ahn, Arch. Pharmacal Res., 2008, 31, 1281–1285.
128. M. R. Noori-Daloii, M. Momeny, M. Yousefi, F. G. Shirazi, M. Yaseri, N. Motamed, N. Kazemialiakbar and S. Hashemi, Med. Pediatr. Oncol., 2011, 28, 1395–1404.
129. K. Senthilkumar, R. Arunkumar, P. Elumalai, G. Sharmila, D. N. Gunadharini, S. Banudevi, G. Krishnamoorthy, C. S. Benson and J. Arunakaran, Cell Biochem. Funct., 2011, 29, 87–95.
130. K. Senthilkumar, P. Elumalai, A. Arunkumar, S. Banudevi, N. D. Gunadharini, G. Sharmila, K. Selvakumar and J. Arunakaran, Mol. Cell. Biochem., 2010, 344, 173–184.
131. C. Tsimplouli, C. Demetzos, M. Hadzopoulou-Cladaras, P. Pantazis and K. Dimas, Eur. J. Nutr., 2012, 51, 181–190.
132. G. Wang, J. Zhang, L. Liu, S. Sharma and Q. Dong, PLoS One, 2012, 7, 1–12.
133. D. Staedler, E. Idrizi, B. H. Kenzaoui and L. Juillerat-Jeanneret, Cancer Chemother. Pharmacol., 2011, 68, 1161–1172.
134. G. Du, H. Lin, Y. Yang, S. Zhang, X. Wu, M. Wang, L. Ji, L. Lu, L. Yu and G. Han, Int. Immunopharmacol., 2010, 10, 819–826.
135. M. Russo, C. Spagnuolo, S. Volpe, A. Mupo, I. Tedesco and G.-L. Russo, Br. J. Cancer, 2010, 103, 642–648.
136. A. M. Ramos and P. Aller, Biochem. Pharmacol., 2008, 75, 1912–1923.
137. T. Thangasamy, S. Sittadjody, G. C. Mitchell, E. E. Mendoza, V. M. Radhakrishnan, K. H. Limesand and R. Burd, BMC Cancer, 2010, 10, 1–10.
138. A. Demiroglu-Zergeroglu, B. Basara-Cigerim, E. Kilic and G. Yanikkaya-Demirel, J. Biomed. Biotechnol., 2010, 1–7 , Article ID 851589.
139. M. Daker, M. Ahmad and A. S. B. Khoo, Cancer Cell Int., 2012, 12, 1–8.
140. X. Zhan, R. Zhang, Y. Xu, S. Yang, D. Xie and L. Tan, Chin.-Ger. J. Clin. Oncol., 2012, 11, 380–383.
141. W. Chen, X. Wang, J. Zhuang, L. Zhang and Y. Lin, Carcinogenesis, 2007, 28, 2114–2121.
142. S. Borska, M. Drag-Zalesinska, T. Wysocka, M. Sopel, M. Dumanska, M. Zabel and P. Dziegiel, Folia Histochem. Cytobiol., 2010, 48, 222–229.
143. C. P. R. Xavier, C. F. Lima, M. Rohde and C. Pereira-Wilson, Cancer Chemother. Pharmacol., 2011, 68, 1449–1457.
144. H. Kim, T. Wannatung, S. Lee, W. K. Yang, S. H. Chung, J. Lim, W. Choe, I. Kang, S. Kim and J. Há, Apoptosis, 2012, 17, 938–949.
145. S. Kilani-Jaziri, V. Frachet, W. Bhouri, K. Ghedira, L. Chekir-Ghedira and X. Ronot, Drug Chem. Toxicol., 2012, 35, 1–10.
146. R. Shi, C. Ong and H. Shen, Oncogene, 2004, 23, 7712–7721.
147. D. Y. Lim, Y. Jeong, A. L. Tyner and J. H. Y. Park, Am. J. Physiol.: Gastrointest. Liver Physiol., 2007, 292, G66–G75.
148. J. Hwang, O. J. Park, Y. K. Lee, M. J. Sung, H. J. Hur, M. S. Kim, J. H. Ha and D. Y. Kwon, Int. J. Mol. Med., 2011, 28, 25–31.
149. K. Selvendiran, H. Koga, T. Ueno, T. Yoshida, M. Maeyama, T. Torimura, H. Yano, M. Kojiro and M. Sata, Cancer Res., 2006, 66, 4826–4834.
150. B. Wu, Q. Zhang, W. Shen and J. Zhu, Mol. Cell. Biochem., 2008, 313, 125–132.
151. W. G. Ko, T. H. Kang, S. J. Lee, Y. C. Kim and B. H. Lee, Phytother. Res., 2002, 16, 295–298.
152. M. J. Kim, J. S. Woo, C. H. Kwon, J. H. Kim, Y. K. Kim and K. H. Kim, Cell Biol. Int., 2012, 36, 339–344.
153. J. Ruan, Y. Liu, L. Zhang, L. Yan, F. Fan, C. Shen, A. Wang, S. Zheng, S. Wang and Y. Lu, Acta Pharmacol. Sin., 2012, 33, 1325–1331.
154. P. Pratheeshkumar, Y. Son, A. Budhraja, X. Wang, S. Ding, L. Wang, A. Hitron, J. Lee, D. Kim, S. P. Divya, G. Chen, Z. Zhang, J. Luo and X. Shi, PLoS One, 2012, 7, 1–15.
155. Q. Zhou, B. Yan, X. Hu, X. Li, J. Zhang and J. Fang, Mol. Cancer Ther., 2009, 8, 1684–1691.
156. L. Bai, X. Xu, Q. Wang, S. Xu, W. Ju, X. Wang, W. Chen, W. He, H. Tang and Y. Lin, Mol. Pharmacol., 2012, 81, 549–555.
157. X. Cai, T. Ye, C. Liu, W. Lu, M. Lu, J. Zhang, M. Wang and P. Cao, Toxicol. In Vitro, 2011, 25, 1385–1391.
158. Y. Zhao, G. Yang, D. Ren, X. Zhang, Q. Yin and X. Sun, Mol. Biol. Rep., 2011, 38, 1115–1119.
159. D. Y. Lim, H. J. Cho, J. Kim, C. W. Nho, K. W. Lee and J. H. Y. Park, BMC Gastroenterol., 2012, 12, 1–10.
160. E. Lee, S. Oh and M. Sung, Food Chem. Toxicol., 2012, 50, 4136–4143.
161. K. Tsui, L. Chung, T. Feng, P. Chang and H. Juang, Int. J. Cancer, 2012, 130, 281–282.
162. J. Kim, E. Lee, H. Lee, J. Ku, M. Lee, D. Yang and S. Kim, Ann. N. Y. Acad. Sci., 2006, 1090, 147–160.
163. W. Ju, X. Wang, H. Shi, W. Chen, S. A. Belinsky and Y. Lin, Mol. Pharmacol., 2007, 71, 1381–1388.
164. P. S. Rao, A. Satelli, M. Moridani, M. Jenkins and U. S. Rao, Int. J. Cancer, 2012, 130, 2703–2714.
165. G. Du, Z. Song, H. Lin, X. Han, S. Zhang and Y. Yang, Biochem. Biophys. Res. Commun., 2008, 372, 497–502.
166. X. Tang, H. Wang, L. Fan, X. Wu, A. Xin, H. Ren and X. J. Wang, Free Radical Biol. Med., 2011, 50, 1599–1609.
167. J. Li, M. Mottama, H. Li, K. Liu, F. Zhu, Y. Cho, C. P. Sosa, K. Zhou, G. T. Bowden, A. M. Bode and Z. Dong, Carcinogenesis, 2012, 33, 459–465.
168. J. Li, F. Zhu, R. A. Lubet, A. De Luca, C. Grubbs, M. E. Ericson, A. D'Alessio, N. Normanno, Z. Dong and A. M. Bode1, Mol. Carcinog., 2013, 52, 134–143.
169. S. Rubio, J. Quintana, M. López, J. L. Eiroa, J. Triana and F. Estévez, Eur. J. Pharmacol., 2006, 548, 9–20.
170. W. H. Talib, M. H. A. Zarga and A. M. Mahasneh, Molecules, 2012, 17, 3291–3303.
171. L. K. Mdee, S. O. Yeboah and B. M. Abegaz, J. Nat. Prod., 2003, 66, 559–604.
172. D. R. Ferry, A. Smith, J. Malkhandi, D. W. Fyfe, P. G. deTakats, D. Anderson, J. Baker and D. J. Kerr, Clin. Cancer Res., 1996, 2, 659–668.
173. Clinical Trials, United State National Institutes of Health, 2013, http://clinicaltrials.gov/ct2/show/NCT01538316?term=quercetin%26rank=9, last access: 03/10/2013.
174. Clinical Trials, United State National Institutes of Health, 2013, http://clinicaltrials.gov/ct2/show/NCT00003365?term=quercetin%26rank=19, last acess: 03/10/2013.
175. P. C. H. Hollman, M. V. D. Gaag, M. J. B. Mengelers, J. M. P. Van Trijp, J. H. M. De Wries and M. B. Katan, Free Radical Biol. Med., 1996, 21, 703–707.
176. P. C. Hollman and M. B. Katan, Biomed. Pharmacother., 1997, 51, 305–310.
177. E. U. Graefe, H. Derendorf and M. Veit, Int. J. Clin. Pharmacol. Ther., 1999, 37, 219–233.
178. Y. J. Moon, L. Wang, R. DiCenzo and M. E. Morris, Biopharm. Drug Dispos., 2008, 29, 205–217.

---