Breast cancer is the second leading cause of cancer-related death in women in the United States. It is estimated that in 2020, 30% of all newly diagnosed cancers in females will be breast cancer [1
]. Triple negative human breast cancer (TNBC) is an aggressive subtype of breast cancer, negative for estrogen and progesterone receptors, as well as for epidermal growth factor receptor 2. TNBC is usually diagnosed in females under the age of 50 with an incidence of 10–20% of all breast cancers. TNBC is therapy-resistant and is, therefore, characterized by poor prognosis. Non-halogenated anthracyclines and taxanes belong to the standard chemotherapeutic treatment regimen in breast patients including those with TNBC [2
]. Unfortunately, at present, no approved targeted therapy is available for TNBC [3
]. Therefore, novel agents for the prevention and treatment of breast cancer, particularly TNBC, are urgently required.
Plants and plant-derived compounds have a long-standing tradition of medicinal use. Even today, many cancer therapeutics are of natural origin as they are able to modulate pathways often deregulated in human cancers [4
]. However, a vast number of diverse secondary metabolites of medicinal plants have not yet been comprehensively investigated [6
]. Hence, natural compounds may still harbor new leads for the treatment of malignant diseases.
L. is a medicinal plant used in traditional Chinese medicine for the treatment of fever. Currently, the sesquiterpene lactone artemisinin originally isolated from Artemisia annua
is part of standard combination therapies to treat uncomplicated malaria [7
]. Artemisinin and its derivatives contain an endoperoxide group, which in the presence of ferrous ion generates reactive oxygen species (ROS). Artemisinin derivatives exhibit antiparasitic, antimalarial, and anticancer activities that are augmented in the presence of iron complexes [8
]. However, artemisinin and its derivatives are unstable leading to poor bioavailability [8
]. On the other hand, Artemisia annua
contains a variety of additional bioactive components worth to be investigated. Thus, the plant contains more than 50 different phenolic compounds (flavones, flavonols, coumarins, phenolic acids, etc.) making it one of the four medicinal plants with the highest oxygen radical absorbance capacity [8
]. As the dietary consumption of flavonoids correlates inversely with cancer occurrence, it has been assumed that flavonoids might prevent, delay, or help to cure cancer by modulating oxidative stress associated with cancerogenesis [8
]. In addition, Artemisia annua
contains plenty of structurally diverse polymethoxylated flavonoids, which can increase bioavailability and enhance the therapeutic efficacy of artemisinin. Such methoxylated flavones are believed to be more stable and to possess better pharmacokinetic properties compared to hydroxylated flavonoids [8
In the course of our investigations on antitumor efficacies of a number of commercially available Artemisia annua
nutraceuticals, we have identified a commercial Artemisia annua
extract (MoMundo GmbH, Bad Emstal, Germany) that exhibits potent cytotoxic activity in vitro [9
]. Using fingerprint analysis and fractionation of the Momundo extract, we found that it does not contain any detectable artemisinin yet high amounts of the cytotoxic methoxylated flavonols, casticin and chrysosplenol d
. Whilst some studies reported tubulin-binding and antiproliferative efficacy of casticin against breast, lung, and colon cancer cell lines [10
], almost no information is available as to potential anticancer activities of chrysosplenol d
]. Analysis of the structure-activity relationship of flavones revealed that the C2-C3 double bond, the C-3 hydroxyl- and the ortho-catechol moiety of ring B are important for high antiproliferative activity [8
]. Since chrysosplenol d
and casticin harbor several of these functionalities, the aim of the work was to analyze more closely their antiproliferative and apoptosis-inducing capacity in cancer cells in vitro and in vivo.
The present study provides evidence that the two Artemisia annua flavonols, chrysosplenol d and casticin, might be promising antitumor compounds with different mechanisms of action. The differential cytotoxicity to cancer cells with much lower cytotoxicity to PBMC and normal breast epithelial cells and no overt systemic toxicity to chick embryos point to selectivity of the flavonols for cancer cells and implicate low systemic toxicity in vivo. These findings encouraged us to further analyze potential molecular mechanisms of action of chrysosplenol d and casticin.
Uncontrolled proliferation caused by deregulated cell cycle control mechanisms is a common feature of neoplasia and many cancers are reported to be selectively vulnerable to the inhibition of cell cycle regulatory proteins [22
]. Hence, targeting the cell cycle is an attractive feature for an anticancer drug. Both compounds induced cell cycle aberrations. However, chrysosplenol d
increased the number of cells in the S- and G2
/M-phases, whereas casticin strongly increased the number of cells in the G2
/M-phase only. Analysis of cell viability demonstrated, that even relatively high concentrations of casticin, similar to paclitaxel, are cytotoxic only to a proportion of cells, whereas chrysosplenol d
eventually eliminated all cancer cells. These findings are consistent with a study reporting casticin to bind to tubulin and to induce G2
/M-phase arrest in MCF7 breast cancer and H1299 lung carcinoma cell lines [10
]. Hence, the data indicate that despite structural similarities, chrysosplenol d
and casticin target different signaling pathways in cancer cells.
Induction of apoptosis in cancer cells by a chemotherapeutic drug is an advantageous feature, because apoptosis is a highly regulated process that does not induce inflammation and does not negatively affect function of neighboring non-transformed cells [15
]. During early apoptosis, phosphatidylserine becomes exposed on the outer leaflet of the plasma membrane representing an initial signal for engulfment by phagocytes [15
]. This observation, together with the occurring later DNA-fragmentation, reveals that the Artemisia annua
flavonols chrysosplenol d
and casticin induce apoptosis in treatment-resistant MDA-MB-231 TNBC cells.
Permeabilization of the outer mitochondrial membrane is a central step in induction of intrinsic apoptosis [16
]. In casticin- as well as in paclitaxel-treated cells, mitochondrial damage did not precede the induction of apoptosis, but occurred at the same time as phosphatidylserine exposure. These data are in agreement with the finding that casticin and paclitaxel as tubulin binding agents do not primarily target mitochondria in cancer cells. In contrast, in chrysosplenol d
-treated cells, dissemination of the mitochondrial membrane potential took place 24 h earlier than any sign of apoptosis could be detected. This finding might suggest an active role of mitochondria in the induction of apoptosis by chrysosplenol d
. However, an increase in mitochondrial superoxide occurred quite late, at the time when also apoptosis signs have been detected. Differently, total ROS levels were increased quite early and addition of ROS scavengers attenuated chrysosplenol d
Early ROS induction may promote sustained ERK activation [20
]. MDA-MB-231 cells as used in our study express mutated K-ras [24
] and exhibit already significantly activated basal ERK1/2 phosphorylation. Still, treatment with chrysosplenol d
increased the basal ERK1/2 phosphorylation. Also, the flavonol quercetin, which is structurally similar to chrysosplenol d
, was shown to induce activation of ERK1/2 [25
]. Different to MDA-MB-231, PC-3 cells are relatively resistant to chrysosplenol d
. PC-3 lack the PTEN phosphatase, which results in aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway and no basal ERK1/2 activation as demonstrated by us and others [26
]. The mechanisms of the antagonistic relationship between ERK1/2 and AKT might involve a negative ERK1/2 feedback signaling affecting upstream Ras/ERK1/2 activators including prosurvival signaling of the phosphoinositide 3-kinase/AKT/mTOR [29
]. Hence, high basal ERK1/2 and low basal AKT activity might be indicative for the sensitivity of cells to chrysosplenol d
ERK1/2 are part of the pro-oncogenic Ras/Raf/MEK signaling pathway, which is deregulated in numerous cancers due to frequent activating mutations in Ras and B-Raf genes [30
]. Hence, ERK1/2, kinases downstream of Ras/Raf/MEK, are often activated in cancer promoting cell proliferation and resistance to apoptosis. Thus, basal ERK1/2 activation is necessary for ΔΨm
maintenance probably through activation of prosurvival BCL2 proteins [31
]. Accordingly, treatment with the MEK-inhibitor U0126 induced ΔΨm
dissipation in MDA-MB-231 cells.
Alternatively and paradoxically, under certain conditions, upregulated ERK1/2 can also induce apoptosis, autophagy, or senescence [32
]. Thus, similar to chrysosplenol d
, ERK activation by BCI, a dual MKP1/6 phosphatase inhibitor, which targets exclusively ERK1/2 [20
], induced concentration-dependent toxicity in different cancer cell lines including MDA-MB-231. Hence, an enhanced and sustained ERK1/2 activation may trigger proapoptotic effects.
There are several hypothesis how sustained ERK1/2 activation might promote apoptosis. Thus, ERK1/2 might induce expression of proapoptotic genes or directly activate caspase 8, albeit also through de novo
protein synthesis [20
]. In our experiments, ERK1/2 inhibition preceded treatment with chrysosplenol d
by 1 h, which might not suffice for new protein synthesis [26
]. Likewise, ERK1/2 activity has been demonstrated to directly target mitochondrial respiration, to promote disruption of mitochondrial membrane potential (ΔΨm
), to induce membrane permeability, and to induce the release of cytochrome c and other proapoptotic proteins. Furthermore, phosphorylated ERK1/2 has been localized on mitochondrial membranes [20
]. Our data, however, demonstrate a rather late increase in mitochondrial peroxide levels indicating that mitochondria might not be the primary target of chrysosplenol d
ERK1/2 can also bind to and stabilize p53 in different ways, thus augmenting p53 expression, which is required for the ERK1/2-induced apoptosis under certain conditions [20
]. In line with that, p53-deficient PC-3 cells are also resistant to treatment with chrysosplenol d
], whereas NSCLC A549 expressing wild type p53 (p53wt
) are particularly sensitive to chrysosplenol d
. However, the p53 status alone does not suffice to explain the differential sensitivity of cancer cell types to chrysosplenol d
. Thus, MCF7 breast cells are also p53wt
and are relatively resistant to chrysosplenol d
, whilst MDA-MB-231, which express gain of function p53mut
that promotes tumor growth independent from classical downstream targets of p53 [33
], are relatively sensitive to treatment. Also, MIA PaCa-2 harbor a gain of function mutation of p53 [35
], but are relatively resistant to chrysosplenol d
treatment. Hence, although the p53 expression status might contribute to apoptosis induction by chrysosplenol d
and casticin in some cancer cell types and should be considered, activation of other pathways should not be neglected.
Sustained ERK activation can induce autophagy [20
]. Indeed, cells treated with chrysosplenol d
exhibited increased prolonged ERK1/2 activation and an increased number and size of lysosomes indicating early induction of autophagy. Autophagy, though generally considered as a prosurvival process, might as well promote apoptosis [36
]. Remarkably, the ability of ERK1/2 to activate autophagy and proapoptotic pathways depends on the strength of their activation. Prolonged aberrant signaling through ERK kinases leads to a proteasome-dependent degradation of multiple phosphoproteins required for cell growth and cell-cycle progression and it is characterized by mitochondrial dysfunction. It has been suggested that such aberrant ERK1/2 activities are recognized by intracellular tumor-suppressor pathways, which promote apoptosis [29
]. This scenario is in agreement with the effects induced by chrysosplenol d
in cancer cells, i.e., increased ERK activation, increased formation of acidic organelles, and cytotoxicity with signs of both, apoptotic and autophagic cell death. In addition, both, chrysosplenol d
-induced toxicity and lysosomal aberrations were strongly attenuated by MEK/ERK1/2 inhibition. Differently, casticin-induced toxicity was not antagonized by U0126.
In summary, the data presented suggest that the Artemisia annua flavonols chrysosplenol d and casticin are potential antitumor compounds with different mechanism of action. Unlike the toxicity of casticin, the one of chrysosplenol d on cancer cells depends on sustained increased ERK1/2 activation.
4. Materials and Methods
4.1. General Experimental Procedures
Momundo Artemisia annua extract was obtained from MoMundo GmbH (Bad Emstal, Germany). Arteannuin B and arteannuic acid were from Carbosynth (Berkshire, UK), casticin and 6,7-dimethoxycoumarin from Extrasynthese (Genay cedex, France), chrysosplenol d from ChemFaces (Wuhan, Hubei, China), and artemisinin from Sigma (St. Louis, MO, USA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and further diluted with appropriate medium supplemented with 1% heat-inactivated fetal bovine serum (FBS) directly before the experiments. The final DMSO concentration in the medium was 0.5%. Propidium iodide, dual specificity protein phosphatase 1/6 inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), DNase-free RNase A, and paclitaxel were from Sigma. The XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazolium-5-carboxanilide) cell proliferation assay was purchased from Roche Diagnostics (Filderstadt, Germany). The mitochondrial potential sensor JC-1, H2DCFDA, MitoSOX™ Red, LysoTracker Red were from Molecular Probes (San Diego, CA, USA). FITC-labeled annexin V and phenol-free matrigel were purchased from BD Biosciences (Heidelberg, Germany).
4.2. Analytical Fingerprint of Momundo Artemisia annua Dietary Supplement by HPLC-DAD-MS Analysis
HPLC-MS analysis was performed on an Agilent 1260 Infinity system (Agilent, Santa Clara, CA) coupled with an AB API 2000 (Applied Biosystems, Foster City, CA, USA) triple quadrupole mass spectrometer via an electrospray ionization source (ESI). The data were obtained and processed through Analyst 1.6.1 software (Ab Sciex, Framingham, MA, USA).
Chromatographic separation was achieved using an analytical HPLC column (ReproSil-Pur Basic C18-HD, 3 µm, 125 × 3 mm, Dr. Maisch, Ammerbuch-Entringen, Germany) with a precolumn (ReproSil-Pur Universal RP, 5 µm, 10 × 4 mm, Dr. Maisch). The flow rate was 600 µL/min and the injection volume was 5 µL. The mobile phase consisted of eluent A (deionized, ultrapure water + 0.05% formic acid) and eluent B (acetonitrile + 0.05% formic acid). Initial conditions were 70% eluent A and 30% eluent B followed by a linear gradient to 95% eluent B over 18 min, then 95% eluent B until 24 min. Thereafter, followed a linear gradient to initial conditions until 25 min and reequilibration for additional 5 min. To stabilize the chromatographic system, the column was kept at 28 °C. The eluent was scanned with a photodiode array detector at 210 nm, 254 nm, and 280 nm. MS detection was accomplished in positive and negative atmospheric pressure electrospray ionization (ESI) modes, and in single quadrupole scan mode. The substances were identified by comparison of retention times and mass spectra with reference compounds. Chrysosplenol d
was subjected additionally to 1
H and 13
C NMR spectroscopy on a Bruker DRX 500 NMR spectrometer (Figures S1 and S2
4.3. Quantification of Artemisinin by HPLC-MS/MS
The HPLC system used including columns and the mass spectrometer are described above. For sample preparation, 30 mg of Momundo capsule content were dissolved in 1.5 mL acetonitrile and extracted for 1 h at RT with continuous stirring. After centrifugation (16,000 g, 10 min), 1 mL supernatant was added to 1 mL water and filtered through regenerated cellulose (0.45 µm). The resulting solution with a sample concentration of 10 mg/mL was analyzed in triplicates. For chromatographic separation, the flow rate was set to 600 µL/min and the injection volume to 70 µL. The mobile phase consisted of eluent A (deionized, ultrapure water + 0.1% acetic acid and 10 mM ammonium acetate) and eluent B (acetonitrile + 0.1% acetic acid). Initial conditions were 60% eluent A and 40% eluent B followed by a linear gradient to 90% eluent B over 10 min, then 90% eluent B until 13 min. Thereafter, a linear gradient continued to initial conditions until 15 min and reequilibration until 20 min. The column was kept constantly at 28 °C.
MS/MS analysis was performed in the positive atmospheric pressure ESI and multiple-reaction monitoring (MRM) detection modes. For quantification, the precursor ion at m/z 300.2 ([M + NH4]+) and the product ion of highest intensity at m/z 151.2 were selected. To achieve linearity and to determine the limit of detection (LOD) and the limit of quantification (LOQ), standard solutions in the range from 7.5 ng/mL to 100 ng/mL (6 levels) were analyzed, each in triplicates, yielding a LOD of 2 ng/mL and a LOQ of 8 ng/mL. To evaluate accuracy, the recovery was determined by using the standard addition method. Hence, a real sample was spiked at six levels, extracted as described above in sample preparation and analyzed, each in triplicates, yielding a recovery of 94.8% (± 9.6% SD). Precision was determined by analysis of a reference sample with six replicates at four different days yielding the intraday variation of 1.5% (RSD) and the interday variation of 1.8% (RSD). Sample analysis showed that the artemisinin concentration of the Momundo extract was below the LOD. The method was also used for analysis of three additional commercial Artemisia annua herbal preparations. The analysis revealed artemisinin contents below the LOQ in ‘Artemisinin′ (Euro Nutrador B.V., Landgraaf, Netherlands) and ‘Artemisia Extrakt 400 mg′ (Vita-World, Taunusstein, Germany), whereas ‘Artemisia annua intense® 600 mg′ (Novofrom Pharma GmbH, Gaggenau, Germany) contained 38 µg/mg artemisinin, which complies with the manufacturers specification of 4% artemisinin.
4.4. Cell Culture
Cell lines derived from advanced, therapy-resistant human tumors were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) or the DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). HeLa-Difluo™ hLC3 autophagy reporter cells were from Invivogen (San Diego, CA, USA). Cells were cultured according to the supplier′s recommendations. PBMC were isolated from whole blood of healthy donors by density gradient centrifugation using Biocoll separating solution (Biochrom GmbH, Berlin, Germany) as previously described [37
]. The collection and analysis of peripheral blood mononuclear cells used in this study was approved by the institutional Ethics Committee (# 177/18). The participating volunteers provided written informed consent to participate in this study.
4.5. Analysis of Cell Viability
Cell viability was analyzed by a XTT assay (Roche Diagnostics) according to the manufacturer′s instructions. Cells were seeded in 96-well plates and were treated with the Momundo Artemisia annua extract or compounds for 48 h. For some experiments, cells were pretreated with 5 µM of the MEK inhibitor U0126 (Biomol, Hamburg, Germany) or its inactive analogue U0124 (Bio-Techne, Minneapolis, MN, USA) for 1 h followed by incubation with chrysosplenol d or casticin (each 10 µM) for 48 h. Final DMSO concentrations did not exceed 0.5%. Absorbance of the orange formazan salt formed by mitochondrial reduction of the tetrazolium salt by viable cells was measured using an Tecan Infinite M1000 PRO plate reader (Männedorf, Switzerland) at 450 nm with a 630 nm reference filter.
4.6. Cell-Cycle Analysis
Cells were treated with different concentrations of the indicated compounds for 48 h. Then, cells were fixed with ice-cold 70% ethanol overnight. DNA was stained with propidium iodide in a buffer containing RNase for 1 h. Cells were analyzed by flow cytometry using a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Cell-cycle analysis was performed with FlowJo software (TreeStar Inc., Ashland, OR, USA).
4.7. Breast Cancer Xenografts
For analysis of tumor growth in vivo [17
], 0.75 × 106
MDA-MB-231 cells were xenografted onto the chick chorioallantoic membrane (CAM) in medium/matrigel (1:1, v
) 7 days after fertilization [40
]. Starting from day 1 after seeding, cells were treated topically for 3 consecutive days with either 20 µL of the compounds, doxorubicin, or 0.5% DMSO in 0.9% NaCl. Due to lower embryonic toxicity, doxorubicin instead of paclitaxel was chosen as positive control. On day 4 after initiation of the treatment, tumors were collected, fixed, and embedded in paraffin. Five µm-sections were stained with hematoxylin, eosin, or were further analyzed using antibodies against the nuclear proliferation marker Ki-67 (M7240, Dako, Glostrup, Denmark). Images were recorded with an Axio Lab.A1 microscope (Carl Zeiss, Göttingen, Germany) and a Zeiss 2/3” CMOS-camera using Progres Gryphax software (Jenoptik, Jena, Germany).
4.8. Analysis of Apoptosis
Early apoptotic cells were analyzed flow cytometrically by annexin V and propidium iodide double staining. Briefly, cells were treated with the compounds or paclitaxel for 24 and 48 h, harvested by trypsination, and incubated for 15 min in full growth medium for membrane regeneration at 37 °C. Cells were stained with fluorescein isothiocyante (FITC)-labeled annexin V in a buffer containing calcium for 30 min and propidium iodide was added 1 min before measurement. The percentage of cells with subdiploidal DNA content was analyzed by flow cytometry. After treatment with the compounds or paclitaxel for 48 h, DNA was stained with propidium iodide using the same protocol as described for cell cycle analysis [27
4.9. Analysis of ROS, Mitochondria, and Lysosomes
Cells were treated with the compounds for the indicated time, stained for 30 min with either 10 μg/mL JC-1 dye as mitochondrial potential indicator, LysoTracker Red (50 nM) as lysosomal stain, H2
DCFDA (10 µM) as a total ROS indicator, or MitoSOX™ Red (5 µM) as mitochondrial ROS indicator, and were further analyzed flow cytometrically or microscopically. Mitochondrial potential loss is presented as mean percentage of cells with a decreased red to green JC-1 fluorescence intensity ratio [27
]. For analysis of autophagy, HeLa cells expressing GFP-RFP-tagged LC3 were treated with chrysosplenol d
or rapamycin for 4 h and formation of fluorescent LC3-positive autophagic punctae was assessed by fluorescent microscopy at 400× magnification. Nuclei were counterstained with DAPI.
4.10. Human Phospho-Kinase Array and Western Immunoblotting
Analysis of kinase activation upon treatment with chrysosplenol d and casticin was performed according to manufacturer′s instructions (Proteome Profiler Human Phospho-Kinase Array ARY003B, R&D Systems, Minneapolis, MN, USA). Cells were serum starved for 12 h followed by treatment with chrysosplenol d, casticin (both 30 µM), U0126 (10 µM), or 0.5% DMSO vehicle control for 3 h. After lysis and quantification of protein levels using a BCA assay (Thermo Fisher Scientific, Waltham, MA, USA), equal amounts of protein (300 µg) were incubated with membranes spotted with antibodies against various phosphorylated kinases. Membranes were analyzed using an AmershamTM Imager 680 (GE Healthcare, Chicago, IL, USA). Alternatively, cells were treated with chrysosplenol d (10 µM), casticin (1 µM), or U0126 (10 µM) and whole cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride membranes. Antibodies used were as follows: anti-ERK1/2, anti-AKT-1, anti-phospho-ERK1/2 (T202/Y402), anti-phospho-AKT (S473) (all from Cell Signaling Technology, Danvers, MA, USA), and anti-actin (Merck Millipore, Darmstadt, Germany). Proteins were visualized with above antibodies, detected with corresponding horseradish peroxidase-coupled secondary antibodies and ECL™ Prime Substrate (GE Healthcare) using an AmershamTM imager 680. Autophagy was assessed by western immunoblot analysis of LC3II and p62 in whole cell lysates of MDA-MD-231 cells treated with vehicle or chrysosplenol d for 24 h. Antibodies were from Cell Signaling Technology. The anti-LC3 antibody used (#4108) recognizes preferentially the lipidated LC3II form.
4.11. Statistical Analysis
If not indicated otherwise, quantitative results are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. In case of two-group comparison and normally distributed data, analysis was done with the two-tailed Student´s t-test. Multigroup analysis was performed with the one-way analysis of variance or the Kruskal-Wallis test followed by the Newman-Keuls post hoc test. Correlations were investigated by the Pearson′s correlation test. For analysis, SigmaPlot software was used (Systat Software Inc., San Jose, CA, USA). Significance levels were set at * p < 0.05, ** p < 0.01, and *** p < 0.001.