Anti-Neoplastic Activity of Two Flavone Isomers Derived from Gnaphalium elegans and Achyrocline bogotensis

Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5–80 µM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones.


Introduction
There is a group of medicinal plants commonly known in the Andean regions of South America as vira-viras. These plants belong to the family Asteraceae, tribe Inuleae, and to the genera Gnaphalium, Achyrocline, and Gamochaeta [1,2,3]. They are annuals or perennials that grow between 2000 and 3200 meters above sea level, to an average height of 1.5 meters. Because of their morphological characteristics they can easily be confused with species belonging to different genera. The medicinal use of these plants is not limited to South America, since similar species belonging to these genera grow in various parts of the world, and are commonly used for diverse medicinal purposes. Thus, some Gnaphalium species are used in poultices to tend wounds, as a hemostatic, to fight infections, or ease inflammation. For the cure of cancer, it is recommended in the Andean regions of South America, the hot beverage obtained by decoction of Gnaphalium purpureum L., and Gnaphalium elegans H.B.K. [4]. Ethnobotanical data provide valuable information to further the research and identification of compounds derived from plants that have been traditionally used in medicinal preparations by various cultures. The search for compounds responsible for the therapeutic effects reported by ethnobotanical studies begins with the sequential fractionation of extracts derived from specific parts of the plant, followed by the isolation of molecules present in fractions which display biological activity. Among the molecules with highest therapeutic potential, flavonoids stand out for their extensive range of pharmacological and biological activities. In vitro and in vivo studies have shown that flavonoids possess not only cardioprotective [5,6] anti-inflammatory [7,8], antimicrobial [9,10], antioxidant, anti mutagenic, and anti-tumorigenic activities [11,12], but are among the most widespread secondary plant metabolites. Flavonoids potential as antitumor agents is based on mechanisms that include induction of apoptosis, cell cycle arrest, and modulation of protein kinase activities [13,14,15] on cancer cells. Several of these compounds and their derivatives have been studied in clinical trials, as is the case of genistein, flavopiridol, catechin, and quercetin [16].

Cell Culture
Well-differentiated and poorly-differentiated cell lines that originated in multiple tissue sites were obtained from the American Tissue Type Culture Collection (ATCC; Manassas, VA) including: colon (Caco-2, HCT-116), pancreatic (MiaPaca), breast (MCF-7, SK-BR3), and prostate (LNCaP, PC-3) cell lines. Human colon normal CCD-112 coN fibroblasts were also obtained from the ATCC. Tumor cell lines, and normal fibroblasts were grown in tissue culture according to ATCC instructions.
The Panc-28 cell line was a gift from Dr. Paul Chiao (University of Texas M. D. Anderson Cancer Center, Houston, TX) [19], and was grown in tissue culture in the same manner as pancreatic cell line Mia PaCa-2, in DMEM with hi-glucose media (GIBCO/ Invitrogen, Carlsbad, CA) supplemented with 10% serum and penicillin/streptomycin. All cells were seeded and allowed to reach 75% confluency before treatment with flavone A, B or vehicle (DMSO at a final maximum concentration of 0.01%).
Procedure to obtain 5, 7-dihydroxy-3, 6, 8-trimethoxy flavone (flavone A) 1.5 kg of G. elegans dried flowers were extracted with CHCl 3 . The extract was concentrated by dry vacuum, disolved in methanol and filtered to eliminate fats and hydrocarbons. The extract was concentrated and disolved in C 6 H 6 , followed by silica gel chromatography using C 6 H 6 :Me 2 CO (19:1) as eluent. 50 mg of the flavonoid was purified from fractions 12 through 18 by crystallizations in hexane. The compound was identified by its physical and spectroscopic properties as 5,7 dihydroxy-3,6,8 trimethoxy flavone, mp 170-171uC [3], 1H NMR (300 MHz MTT assay. Cells were seeded at a density of 4000/well in 48 well plates, grown overnight and treated with either vehicle, flavone A or flavone B in concentrations of 5, 10, 20, 40, 60, 80 mM; dissolution vehicle was dimethyl sulfoxide to yield a maximum final concentration of 0.01% in the treated well (Sigma-Aldrich, St. Louis, MO). After 24 hours of incubation 3-(4, 5-methyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added at 100 mg/well for 3 hours (Invitrogen). Formazan products were solubilized with acidified 2-propanol and optical density was measured at 570 nm using a Cary 50 (Varian, Palo Alto, CA). All experiments were done in triplicate. Data from assays displaying decrease of cell viability $50%, were evaluated by nonlinear regression analysis (GraphPad Prism, La Jolla, CA), and represented as the effective concentration required to decrease 50% of cell viability (EC 50 ). Phase contrast images of the treated cells were obtained using a Zeiss Axio Observer inverted microscope, equipped with a Zeiss AxioCam CCD camera.
TUNEL assay. Cells at 75% confluency were treated with 40mM flavone A, flavone B, or vehicle (DMSO at a maximum final concentration of 0.01%) for 90 minutes. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% sodium citrate and 0.1% Triton X. DNA fragmentation was determined by TdT-mediated dUTP nick end labeling (TUNEL) as described by the manufacturer (Roche Applied Science, Mannheim, Germany). Fluorescent images were obtained using an EVOS fluorescent microscope (AMG, Bothell, WA).

Data Treatment and Statistical Analysis
Data were analyzed for significant difference using two-way ANOVAs with compound and concentration as factors. Significant main effects were followed with Bonferroni-Holm post hoc tests (SAS 9.2; SAS Institute, Cary, NC). Statistical significance was set at p,0.05.      survival. Figure 4B shows flavone B effectively decreases cell viability on receptor negative (ER /PR ), poorly differentiated SK-BR3 human breast cancer cells, with a half maximal effective concentration of 11.06 mM (figure 4C).

Neither Flavone A nor flavone B is cytotoxic to human prostate carcinoma LNCaP and PC3 cells
To determine whether the flavones where cytotoxic to prostate cancer cells, we treated the cells with either one of the compounds. Flavone A and flavone B failed to decrease cell viability of the more differentiated [20][21] androgen-responsive LNCaP cells (figure 5A), as well as the poorly differentiated [22] PC3 androgen receptor negative cells (figure 5B) as indicated by MTT assay.

Neither flavone A nor flavone B is cytotoxic to normal fibroblasts
To determine whether the flavones were cytotoxic to normal cells, we treated normal fibroblasts with either one of the compounds. Cell viability of normal colon fibroblasts CCD-112 coN, is unaffected by either flavone as shown by MTT assay after a 24 hour treatment with increasing concentrations of the compounds between 5-80 mM (figure 5C).
The apoptotic effects of flavone A and flavone B, were confirmed via TUNEL assay as shown on figure 6 on Caco-2, Panc 28, and MIA PaCa cells.

Discussion
Gnaphalium elegans and Achyrocline bogotensis were selected for their medicinal properties reported in ethnobotanical studies [4]. These plants are indigenous to the Andean region of South America, but have successfully undergone preliminary domestication studies.
The flavones presented in this study are not exclusive to plants native to this geographic location. Flavone A has been isolated from Ainsliaea henryi, native to the southern and western portions of China [23]. Tomas-Lorente et al., has reported that flavone A was extracted from Helichrysum decumbens along with two other methylated flavonoids [24] Flavone B has been found in Helichrysum graveolens [25], Helichrysum odoratissimum [26] and Helichrysum compactum [27]. The plant extracts of the latter demonstrated to have antibacterial and antioxidant activities. Medicinal plants belonging to the genus Helichrysum, traditionally have been used in Rwanda and other African countries for their antibacterial, antitussive, and sedative properties. Research on the therapeutic use of flavonoids has advanced greatly in the last ten years, and some compounds are being tested in clinical trials.
Our findings identify flavone A and its isomer flavone B, as potential anti-tumor agents. Indeed, these compounds display cytotoxic activity against cell lines that have been categorized as being highly tumorigenic. High-expression levels of Aldehyde dehydrogenase (ALDH) is regarded as a very specific marker used in the detection of cancer-initiating cells as subpopulations in tumors, [28,29,30,31,32,33] as well as in the determination of tumorigenic status in established cancer cell lines. Flavone A and flavone B cytotoxic activity is directed towards highly tumorigenic cells that express high levels of ALDH: Caco-2 and HCT-116 [34,35], as well as pancreatic cancer Panc28 and MIA PaCa, [36], and breast cancer SK-BR3 [37] cells. On the contrary, Flavone A and B activities were observed to be significantly diminished in less tumorigenic cells, characterized by low expression levels of ALDH: breast cancer MCF7 cells [34,37], and prostate cancer PC3and LNCaP cells [38].
Cancer cell lines display variability in regards to differentiation status. Thus, more differentiated cells can be distinguished from poorly differentiated cells by the presence of specific polarity and differentiation markers, as well as doubling time. Among these highly tumorigenic cells upon which the flavones activity is targeted, we find that flavone A has a preferential effect on the more differentiated colon cancer Caco-2 [39] cells, and pancreatic cancer Panc28 cells. It is important to note that the latter has been classified as poorly differentiated when polarity markers are compared with better differentiated pancreatic cancer cells such as Capan-1. However, it is the general consensus that Panc28 cells possess a higher differentiation status than MIA PaCa cells, specifically when doubling time is considered [40]. Our results suggest that flavone A has a preference for the more-differentiated colon carcinoma Caco-2 cells and pancreatic cancer Panc28 cells as compared to greatly diminished activity on poorly differentiated cells. On the contrary, flavone B displays preferential activity towards poorly differentiated cancer cells of the colon (HCT-116), pancreas (MIA PaCa) and cadherin negative breast cancer SK-BR3 cells [41].
In line with previous reports [13], neither flavone A nor flavone B is cytotoxic to normal cells. However, it has been shown that this lack of cytotoxic activity on normal cells while targeting malignant cells, has several exceptions as is the case of the hydroxyflavones luteolin and apigenin. Recent studies conducted on methoxylated hydroxyflavones suggest that the presence of sequential methoxy-lated groups, as observed in flavone B, may dictate lack of cytotoxic activity on normal cells [42]. Extensive studies indicate that certain substitution patterns in flavones may increase their anticancer activity. These observations may benefit from additional information in regards to characteristics that may serve to categorize cancer cell lines prone to anticancer activity. In these flavone isomers, the positions of -OH and -OCH 3 groups not only alter the conformation but also the charge delocalization of the compounds, which may be relevant to cellular uptake. In flavone A, both 5-and 7-OH can resonate with the carbonyl, but the 5-OH next to the carbonyl has one more resonance structure. In flavone B, the 5-OH can resonate with the carbonyl but not the 3-OH. While optimization of these structures is yet to be obtained and analyzed, charge delocalization and resonance as a result of the position of substituents are likely to influence anti cancer activities.
At the cellular level, our results suggest an appreciable degree of neoplastic specificity. The preferential effect of these two isomers on cancers with varying differentiation status, suggest a bifurcation in the mechanisms of action of these compounds that may afford new insights on the study of these malignancies. Specific and effective growth inhibition of cancer cells by these compounds may provide alternative therapies urgently needed in highly aggressive  Recent reports have shown the use of flavonoids to augment the effectiveness of existing oncology medications [28,39,43]. The process by which these compounds are able to effectively induce the activation apoptosis in cancer cells is also of great interest [30,31]. The differential effects described in this study, suggest activation of distinct apoptotic pathways. Initial studies on the mechanism of action of these compounds, however, indicate that both induce apoptosis via the activation of the intrinsic caspase cascade. TUNEL assay which specifically detects apoptosis was carried out 1.5-4 hours after treatment with the flavones or vehicle, due to parallel occurrence of apoptosis activation and cellular detachment ( figure 6). These observations suggest that these compounds may interfere shortly after treatment with signaling pathways associated with cell-matrix and cell-cell adhesion molecules, as apoptosis takes place. Investigation in the mechanisms of activation upstream of the activation of caspases is necessary to better understand the potential use of these compounds in novel antineoplastic therapies.
This study presents two flavone isomers that may be responsible for the anticancer activities associated to Gnaphalium elegans and Achyrocline bogotensis. We also present evidence that suggest that these compounds display preferential targeting which may depend upon cellular tumorigenic and differentiation status. Further studies are necessary to elucidate the mechanisms responsible for the observed differential effects.