Aims Extending work with brain tumours, the hypothesis that micronutrients may usefully augment anticancer regimens, chokeberry (Aronia melanocarpa) extract was tested to establish whether it has pro-apoptotic effects in AsPC-1, an established human pancreatic cell line, and whether it potentiates cytotoxicity in combination with gemcitabine. Pancreatic cancer was chosen as a target, as its prognosis remains dismal despite advances in therapy.
Methods An MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to assess the growth of the single pancreatic cancer cell line AsPC-1, alone and in comparison or combination with gemcitabine. This was backed up by flow cytometric DRAQ7 cell viability analysis. TUNEL assays were also carried out to investigate pro-apoptotic properties as responsible for the effects of chokeberry extract.
Results Chokeberry extract alone and its IC75 value (1 µg/mL) in combination with gemcitabine were used to assess the growth of the AsPC-1 cell line. Gemcitabine in combination with chokeberry extract was more effective than gemcitabine alone. TUNEL assays showed apoptosis to be a mechanism occurring at 1 µg/mL concentration of chokeberry, with apoptotic bodies detected by both colourimetric and fluorometric methods.
Conclusions The implication of this study, using single cancer cell line, is that chemotherapy (at least with gemcitabine) might be usefully augmented with the use of micronutrients such as chokeberry extract.
- CANCER RESEARCH
- PANCREATIC CANCER
- TUMOUR BIOLOGY
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Pancreatic cancer has a mortality rate strikingly similar to its incidence, making it a major health concern. The most common and aggressive form is pancreatic ductal adenocarcinoma (PDAC). Lacking early symptoms, it is difficult to combat. The standard treatment offered is surgery, radiotherapy and chemotherapy. Resection represents the only curative option, but only 10–20% are amenable to surgery.1 The side effects of radiotherapy compromise its therapeutic benefits. Although the introduction of the antimetabolite gemcitabine (GEM) in the 1990s has improved quality of life for patients with PDAC,2 there remains a dismal 5-year survival rate of 1–4% and a median survival time of 4–6 months.3 5-fluorouracil (5-FU), has been used as a second-line chemotherapeutic or in combination with GEM.4
The presence of mutations that resist apoptosis, trigger proto-oncogene expression and inactivate tumour suppressor genes makes pancreatic cancer cells resistant both to chemotherapy and radiotherapy. To improve responses, many studies address combining cytotoxics with novel agents or targets.5 The synergistic inhibition of PDAC cell growth by GEM and trichostatin A—an apoptosis inducer—has been suggested.6 It has also been reported that a combination of an antiepidermal growth factor receptor agent (Erlotinib) with GEM may be more effective than GEM alone. Other novel strategies involve angiogenesis inhibitors, matrix metalloproteinases inhibitors and antivascular endothelial growth factor agents, particularly bevacizumab.7
Many studies have explored the use of dietary chemopreventative agents, particularly polyphenols, such as flavonoids from fruits and vegetables, for their ability to promote apoptosis in a variety of cancer cells. A vitamin-A derivative has shown synergistic tumour growth inhibition in human pancreatic cancer cell lines in combination with GEM.8 Nuclear factor-κB (NF-κB) has vital roles in apoptosis, differentiation and control of cell growth.9 Apigenin inhibits (NF-κB)-DNA binding. Its use with GEM against pancreatic cancer cell lines showed enhanced cytotoxicity.10 Other polyphenols studied as therapeutic agents for pancreatic cancer include genistein from soya beans,11 epigallo-catechin-3-gallate (green tea),12 resveratrol (grapes, mulberries, peanuts),13 kaempferol (Ginkgo biloba)14 and curcumin (turmeric).15
There are no reports of anthocyanin-rich extracts from Aronia melanocarpa (chokeberry) in a pancreatic cancer context. We have previously shown that a number of polyphenolics including those from chokeberry (figure 1) have potential anticancer properties with respect to malignant brain tumours.16 Both chokeberry and curcumin extracts induced apoptosis and exhibited anti-invasive potential in an established glioblastoma cell line.17 The aim of this study is to establish whether these polyphenols also have the ability to induce apoptosis and enhance the chemotherapeutic effect of GEM in an established human pancreatic cancer cell line (AsPC-1) in vitro.
Materials and methods
Chokeberry extract was supplied as a dark purple-coloured powder by Artemis International Inc., Fort Wayne, Indiana, USA. This extract contains a number of flavonoids, rutin (a flavone) and a range of anthocyanins. GEM was purchased from Eli Lilly, USA. Chokeberry extract and GEM were diluted in dimethyl sulfoxide (DMSO-Sigma) prior to use.
Cell culture and maintenance
The human pancreatic adenocarcinoma cell line AsPC-1, was purchased from the American Type Culture Collection. Monolayers of AsPC-1 cells were cultured in RPMI-1640 medium supplemented with antibiotics (penicillin 104 IU/mL, streptomycin 10 mg/mL, cocktail added at 1% v/v), 10% v/v foetal calf serum and 1% v/v 200 mM L-glutamine) in a humidified incubator at 37°C and 5% CO2 in air–gas phase. At ∼80% confluence, the cells were passaged by trypsinisation or were frozen as appropriate.
Additionally, human umbilical vein endothelial cells (HUVEC) at passage 6 were sourced from TCS Cellworks, UK. Flasks for culturing HUVEC were coated with gelatine (1% w/v in PBS) and incubated at 37°C for 1 h before use. HUVEC monolayers were grown in Large Vessel Endothelial Cell Basal medium (TCS Cell Works, UK), supplemented with 0.2% antibiotic, 0.2% bFGF heparin, 0.2% EGF, 2% FCS and 0.1% hydrocortisone.
MTT residual viable biomass assay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) is reduced to insoluble formazan by the mitochondrial succinate dehydrogenase of viable cells.18 The formazan generated is dissolved in DMSO and the colour quantified by spectrophotometry at 570 nm. For treatment, cells were seeded into 96-well plates in triplicate before prepared concentrations of chokeberry extract, GEM or a combination of both were added. The plates were incubated for 48 h. Fresh complete medium and sterile distilled water was used to control for normal growth and non-specific colour, respectively.
Detection of apoptosis with annexin-FITC using flow cytometry
The binding of the calcium-dependent phosphatidylserine-binding protein, Annexin V to externalised residues, is a hallmark of apoptosis in many cells. A fluorochrome-labelled Annexin V used in combination with a DNA-binding dye, such as propidium iodide (PI) differentiates between live cells (negative for both dyes), cells in early apoptosis (Annexin positive but PI negative) and dead cells (positive for both dyes). Cells were treated with a range of concentrations of chokeberry and the percentage of live, dead and apoptotic cells assessed. Cells were harvested and washed before resuspension in 500 µL Annexin-V binding buffer (Pharmigen) with 2.5 µL Annexin-V-FITC (Pharmigen) and incubated at room temperature for 15 min. Fifty microlitres of PI (50 µg/mL, Sigma) was added just before measurement. Samples were analysed on a FACS Calibur (Becton Dickinson) with FITC fluorescence measured using a 530/30 bandpass filter and PI fluorescence using a 670 nm longpass filter. At least 10 000 events were acquired and data were analysed using CellQuest software (Becton Dickinson).
Dead End TUNEL systems
The colorimetric and fluorimetric Dead End TUNEL Systems (Promega) were used to determine apoptotic activity in AsPC-1 cell line following chokeberry extract treatment. These systems provide precise detection of apoptosis at a cellular level in situ through detection of Nuclear DNA fragmentation. Recombinant Terminal Deoxynucleotidyl Transferase, enzyme (rTdT) measures the fragmented DNA of apoptotic cells by catalytically incorporating labelled dUPT at 3′-OH DNA fragment ends. Staurosporine (1 µmol/mL), an apoptotic inducing agent, was used as a positive control; negative controls were untreated. The assays were carried out as detailed by the manufacturer. AsPC-1 cells were seeded initially at 105 cells per cover-slip in 0.5 mL of complete indicator-free RPMI, and incubated at 37°C, 5% CO2 for 24 h prior to treatment. For the colorimetric system, the peroxidase reporter molecule was visualised using diaminobenzidine, giving a brown product, and the nuclear counterstain was haematoxylin. Fluorimetric detection was achieved using fluorescein-12-dUTP and counterstained with 4′,6-diamidino-2-phenylindole (DAPI), exciting in the blue and ultraviolet spectral regions to give green and blue fluorescence, respectively.
Data derived from MTT viability assay was analysed using Microsoft Excel and Statistic Direct programs. Data from all the wells (n=24) at each concentration was expressed as % of positive control; this normalised results between experiments, hence every culture well accrued over the three experiments are able to be treated separately allowing calculation of SDs. Means±SD were plotted. The x-axes are non-linear to avoid bunching of high-dilution data points.
HUVEC (control) were treated with chokeberry extract to evaluate its toxicity over a range of concentrations. Figure 2 demonstrates HUVEC to be unaffected when treated with chokeberry extract up to 50 µg/mL.
The IC50 value for chokeberry-treated AspC-1 cells was not in the range tested (0–280 µg/mL), however, the data demonstrates that cytotoxicity is induced in combination of chokeberry extract and GEM. 1 µg mL of GEM alone reduced the residual viable biomass by 50% and become more cytotoxic (<20% rvb between 220 and 280 µg/mL, the highest concentrations used). In combination with 1 µg/mL chokeberry extract (selected for giving as a single agent at most 25% toxicity, ie, 75% rvb or higher), the rvb fell below 30% at 10 µg/mL and absorbances were at background levels by 260 µg/mL, as seen in figure 3. Comparisons between the two responses were statistically significant (p<0.001) by all tests applied.
Flow cytometry analysis
To augment the findings seen by MTT assay, flow cytometry analysis was also carried out using DRAQ7, a fluorochrome that stains the nucleus. It also showed decreased cell viability as drug concentrations increased, but less marked than in the MTT assay. Figure 4 indicates GEM and its combination with 1 µg/mL chokeberry extract yielding two curves closely following each other with the combination line showing only slightly more toxicity across the plot. Although two pairwise comparisons proved significant on applying an upaired t-test, two did not, and the overall ANOVA interaction p value was 0.129. We do not consider the responses to be different.
Dead end colorimetric and fluorimetric TUNEL system
Colorimetric and fluorometric TUNEL assay (figures 5 and 6, respectively) revealed the induction of apoptosis of AsPC-1 cells post-treatment with chokeberry extract at 1 µg/mL, in a dose-dependent manner compared with the positive (Staurosporine treated) and negative (untreated) controls.
In the face of challenging cancers, every little arguably helps. Nutraceutical approaches may well have a role in management. Chokeberry extract may, from preliminary work in our laboratories, possess pro-apoptotic, anti-invasive and antiangiogenic effects on brain tumour cells. Indeed, a published study demonstrated such activity by chokeberry extract in U373 cells; treating these glioblastoma cells with chokeberry extract for 48 h downregulated the gene expression of MMP-2,-14,-16 and -17, but killed the cells by non-apoptotic pathways.17
To establish whether comparable toxicity occurs in a single pancreatic cancer cell line, AsPC-1, the residual viable biomass assay (MTT) was used and results compared with those for GEM and combinations of the two drugs, to evaluate any supra-additive effect. HUVEC were also tested, as a non-transformed cell type which are similar to host components in tumours that are important for progression. HUVEC were unaffected when treated with chokeberry extract up to the highest concentration used (50 µg/mL). These cells are not transformed, yet similar phenotypes would be represented in in vivo cancers, and tumour vasculature has proved a popular therapeutic target. There are, thus, positive (that neoplastic cells may be more sensitive than normal cells) and negative (no indication of antiangiogenic effect) possibilities arising from their insensitivity to the extract. No normal pancreatic cells were available; such cells are not indexed in most catalogues. Although no recommended daily dose for chokeberry extract has yet been made, consumption of 150–400 mg with food is standard.19 However, one study20 has demonstrated anthocyanin having a cytotoxic effect on CRL-2606 normal endothelial cells at 300 µg/mL and above, while showing protective effects at lower concentrations inhibiting PGE-2 production by lipopolysaccharide stressed cells.
A range of 10−6 µg/mL–300 µg/mL of chokeberry extract was selected for AsPC-1 cells in this study, relying on preliminary dose-ranging MTT experiments, although 300 µg/mL is probably physiologically unachievable at a cellular level. A range from 0.1 to 60 µg/mL was selected for HUVEC treatment with chokeberry extract in the present study.
A dose-dependent curve for AsPC-1 was demonstrated in MTT assay of GEM treatments. Combination with chokeberry extract at minimally cytotoxic doses of the micronutrient showed strong supra-additive effects at lower doses of GEM. Whether these amount to synergy, strictly defined, is unclear from this study. 5-FU was used for pancreatic cancer treatment until 1997 before GEM was established as standard treatment.21 Trials in mixed regimens including GEM, had variable success but frequently failed in overall survival benefit over GEM only,22 the drug of choice for its safety quality.23
The differences in dose response by MTT and flow cytometry in this study probably argue in favour of a cytostatic as well as cytotoxic effect, giving less marked supra-additive effects of the combination when a snapshot live versus dead assay was used, as opposed to the cumulative residual viable biomass endpoint with MTT.
Chromatin condensation, nuclear fragmentation, or formation of apoptotic bodies are the characteristics used to determine apoptosis in TUNEL assay. The results obtained from the Colorimetric TUNEL were consistent with those of the Fluorometric TUNEL and, indeed, of the MTT experiments. It was observed that chokeberry extract induces apoptosis of AsPC-1 cells. Negative controls showed the absence of green fluorescent staining, and clearly depicted intact nuclei (figure 6A). A good positive control was obtained with staurosporine when more fragmented nuclei (figure 6B) were seen when 1 µg/mL of chokeberry extract was administered to the cells cultured (figure 6C).
Previous research in our laboratories involving different cell types reported an increase in caspase activation, p53 and bax protein leading to apoptosis induction postanthocyanins administration. Apoptosis induction via obstruction of cell cycle at G1 or G2/M phase resulting in cell cycle arrest has also been achieved with the use of anthocyanins. The experimental results described here using three in vitro indicators, one examining overall growth of biomass and two operating at the molecular level, suggest the anthocyanins may have significant chemopreventive properties in combination with drug therapy. The application of anthocyanins/flavonoids, such as those contained in chokeberry extract, has been inadequately researched. Their potential chemopreventative effects in inflammatory diseases24 ,25 and in cancers, and bioavailability and bioefficacy issues,26 ,27 have been examined. Data from clinical trials is, however, still scarce.
In conclusion, this study suggested that polyphenolics from chokeberry extract induces apoptosis in the established pancreatic cell line used. Furthermore, when cells were treated in combination with GEM, the IC50 values decreased, indicating that the effects seen are at least supra-additive, if not synergistic.
Take home messages
Chokeberry extract has pro-apoptotic properties. The cytotoxic action of gemcitabine in vitro is augmented by chokeberry extract in a supra-additive fashion in a single pancreatic cancer cell line.
Micronutrient supplementation should be considered as part of cancer therapy strategies.
What the paper adds
This work, first, adds reinforcement to the concept that therapy for intractable cancers might usefully be augmented by the inclusion of micronutrient supplementation into regimens. More specifically, it suggests that elements in chokeberry extract, while not intrinsically toxic, can have supra-additive effects in combination with at least one conventional cytotoxic drug. It builds on comparable studies using brain tumour cells.
The authors are grateful for the financial support by the Ministry of Higher Education, Malaysia and Have a Chance Inc, USA.
Abstract in Arabic
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Contributors NAAT made a substantial contribution to the design, organisation and conduct of the study (including acquisition of study data). SK made a substantial contribution to the organisation and conduct of the study. BAL critiquing the output for important intellectual content. AJC made a substantial contribution to the conception, design, organisation and conduct of the study. HKP made a substantial contribution to the conception, design, organisation and conduct of the study.
Funding The Ministry of Higher Education, Malaysia and Have a Chance Inc, USA.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement There is a body of work, both published and unpublished, on the effects of chokeberry extract on cells (primary cultures and cell lines) from the central nervous system. These are available from HKR, who is best contacted through (email@example.com). NAA (firstname.lastname@example.org) is the best contact on technical issues and has related work on other micronutrients, such as curcumin.
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