Омела белая Viscum album Mistletoe
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- Conclusions
- Mistletoe extracts and chemotherapeutic drugs
- Cell culture
- Proliferation assay
- Cell death analysis
- Drug concentrations in the assays
- Effects of a combined application of VAE and chemotherapeutic drugs on proliferation and apoptosis/necrosis in cancer cells
AbstractBackgroundGiven the importance of complementary and alternative medicine (CAM) to cancer patients, there is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Mistletoe (Viscum album L.) belongs to the medicinal herbs that are used as supportive care during chemotherapy. In the in vitro study presented here the effect of standardized mistletoe preparations on the cytostatic and cytotoxic activity of several common conventional chemotherapeutic drugs was investigated using different cancer cell lines. MethodsHuman breast carcinoma cell lines HCC1937 and HCC1143 were treated with doxorubicin hydrochloride, pancreas adenocarcinoma cell line PA-TU-8902 with gemcitabine hydrochloride, prostate carcinoma cell line DU145 with docetaxel and mitoxantrone hydrochloride and lung carcinoma cell line NCI-H460 was treated with docetaxel and cisplatin. Each dose of the respective chemotherapeutic drug was combined with Viscum album extract (VAE) in clinically relevant concentrations and proliferation and apoptosis were measured. ResultsVAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of our experimental settings. At higher concentrations VAE showed an additive inhibitory effect. ConclusionsOur in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE simultaneously. Keywords: Mistletoe (Viscum album L.), Iscador, Chemotherapy, Drug interactions, Cytostasis, Cytotoxicity BackgroundA primary cytotoxic mechanism of many conventional anticancer agents is based on the damage of DNA and the subsequent induction of apoptosis. Beside cytotoxic reactions cancer cells can also respond by cell cycle block or delay (cytostasis) [1]. Because chemotherapeutic agents preferably act on rapidly dividing normal cells, therapeutic treatments result in common side-effects like myelosuppression, hair loss, fatigue, infection etc. In an attempt to reduce the clinical toxicity of chemotherapeutic drugs, to consolidate the immune system and to improve the symptoms of their disease many cancer patients use mistletoe extracts as a complementary therapy in combination with standard regimens [2,3]. Mistletoe (Viscum album) preparations contain active components like mistletoe lectins and viscotoxins and are reported to show anti-tumoral properties by causing cell cycle delay or arrest and induction of apoptosis [4-7], affecting tumor angiogenesis [8,9] and exerting immune-potentiating activities that may enhance the host defense system against tumors [10-13]. Molecular compounds of mistletoe are reported to show in vitro inhibitory potential on P-glycoprotein (P-gp) also known as multidrug resistance protein 1 (MDR1) [14]. The analysis of clinical studies suggests that adjuvant treatment of cancer patients with mistletoe extracts is associated with a better survival, a reduction of side effects of conventional therapy and with an increase of quality of life [15-18]. In early stage breast cancer patients the frequency of relapse or metastasis within 5 years was not influenced by additional mistletoe therapy [19]. Oncologists, confronted with the decision of their patients to use complementary therapies, sometimes are concerned about possible interactions of herbal medicines with oncological drugs, which could influence the efficacy of the standard treatment. The aim of our study therefore was to investigate possible effects of clinically relevant doses of standardized VAEs on the cytostatic and cytotoxic efficacy of several standard chemotherapeutic agents on different cancer cell lines in vitro. MethodsMistletoe extracts and chemotherapeutic drugsThe aqueous, fermented mistletoe preparations Iscador M spec. 5 mg (VAE-M, host tree Malus domestica, Lot 1109/2103/2, total mistletoe lectin concentration 287 ng/ml) and Iscador Qu spec. 5 mg (VAE-Qu, host tree Quercus robur and Q. petraea, Lot 1204/2221/4, total mistletoe lectin concentration 399 ng/ml) were obtained from the Society for Cancer Research (Arlesheim, Switzerland). Doxorubicin hydrochloride, gemcitabine hydrochloride, docetaxel, and mitoxantrone hydrochloride were obtained from Sigma-Aldrich Logistik GmbH (Buchs, CH) and cisplatin from LuBio Science GmbH (Lucerne, CH). Cell cultureHuman breast carcinoma cell lines HCC1937 and HCC1143, pancreas adenocarcinoma cell line PA-TU-8902, prostate carcinoma cell line DU145 and lung carcinoma cell line NCI-H460 were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). HCC1937, HCC1143, DU145 and NCI-H460 cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% Penicillin – Streptomycin (Sigma-Aldrich). PA-TU-8902 cells were cultured in Dulbecco’s MEM High Glucose (Sigma-Aldrich) supplemented with 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 10% fetal calf serum and 1% Penicillin – Streptomycin in a humidified atmosphere with 5% CO2 at 37°C. Cell lines were maintained in exponential growth and cells from subconfluent monolayers were harvested by trypsin-EDTA (Sigma-Aldrich) to carry out the experiments. For measurement of the parameters, the cell cultures were used within 4–6 weeks after thawing. Proliferation assayProliferation was indirectly assessed using the cell proliferation reagent WST-1 (Roche, Mannheim, Germany). Cells (1.5 × 104 in 100 μl) were plated in triplicates in 96-well plates. After 4–6 hours to allow attachment, the drugs were added in various concentrations (see below). Proliferation rate was measured 4 h after incubation with the reagent in triplicate. The upper limit of absorbance was 2.0 - 2.1. Values are given in percent inhibition of proliferation relative to untreated control. Cell death analysisApoptosis/necrosis was measured using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences Pharmingen™, San Diego, CA, USA). Briefly: 2x105 cells were incubated with Annexin V-FITC and 7-AAD at room temperature in the dark. Thereafter, the samples were analysed in a flow cytometer (FACS Calibur, BD Biosciences, San Jose, CA). Early apoptotic cells: Annexin V-FITC positive and 7-AAD negative. Late apoptotic/necrotic cells: Annexin V-FITC positive and 7-AAD positive. Values are given in percent of total cell number. Cytotoxicity (%) was calculated as follows: early apoptotic cells (%)  + late apoptotic/necrotic cells (%).
Drug concentrations in the assaysPreceding the actual experiments the dose–response concentration range and the optimal incubation time was determined for each chemotherapeutic agent and each cell line individually using the WST-1 proliferation assay (data not shown). Cells were incubated for 48 h or 72 h respectively, depending on the maximal measurable anti-proliferative effect of cytostatic agents. Because of its own fluorescence, doxorubicin at higher doses interfered with the nucleic acid dye 7-AAD. Therefore the maximal doxorubicin concentration usable for the detection of apoptosis in the breast carcinoma cell lines HCC1143 and HCC1937 was 5 μg/ml. In the main experiments, the drugs were added in culture medium at the concentrations indicated in Table 1. Each dose of the respective chemotherapeutic drug was combined with VAE-M (HCC1937 and HCC1143) or VAE-Qu (PA-TU-8902, DU-145 and NCI-H460) at the concentrations of 0; 0.1; 1.0; 10; 100 μg/ml for the measurement of proliferation and of 0; 0.1; 1.0; 10 μg/ml for the measurement of apoptosis/necrosis. Typical clinical Iscador concentrations for subcutaneous application are 0.1 and 1 μg/ml, roughly corresponding to an injection of 5 mg Iscador when referring to the amount of circulating blood or body weight, respectively. Parameters were measured after the appropriate incubation time. As we intended to detect a minimal dose able to induce apoptosis in PA-TU-8902 cells we used considerably higher gemcitabine concentrations in apoptosis than in proliferation assay.
Effects of a combined application of VAE and chemotherapeutic drugs on proliferation and apoptosis/necrosis in cancer cellsFigure 2 presents the mean values of proliferation, early apoptosis and late apoptosis/necrosis of the breast carcinoma cell lines HCC1143 and HCC1937 treated with different concentrations of doxorubicin in combination with different concentrations of VAE-M. For HCC1143, the maximal cytostatic effect attained by the treatment with doxorubicin or VAE-M alone was about 75% or 65%, respectively. VAE-M generally enforced the antiproliferative effect of doxorubicin (Figure 2A). This enforcement was significant for 100 μg/ml VAE-M, compared to 0 μg/ml VAE-M, for the doxorubicin concentrations of 0.1–1 μg/ml (p For HCC1937, the maximal cytostatic effect attained by the treatment with doxorubicin or VAE-M alone was about 80% or 45%, respectively. VAE-M ≥10 μg/ml enforced the antiproliferative effect of doxorubicin (Figure 2B). This enforcement was significant for 100 μg/ml VAE-M, compared to 0 μg/ml VAE-M, for all doxorubicin concentrations applied (p A trend for an enhancement of the anti-proliferative effect of doxorubicin by VAE-M at the clinical relevant concentrations 0.1 and 1 μg/ml could be observed in the HCC1143 cell line, but not in HCC1937. This enforcement was not statistically significant. According to the apoptosis measurements, doxorubicin exerted a dose dependent cytotoxic effect on HCC1143 and HCC1937 cells (p In the pancreatic carcinoma cell line PA-TU-8902 the maximal inhibition of proliferation attained by the treatment with 10 μg/ml gemcitabine or 100 μg/ml VAE-Qu alone was about 60% or 35%, respectively. Proliferation inhibition through gemcitabine could not be augmented further by dose enhancement of gemcitabine (data not shown). Only VAE-Qu at a concentration of 100 μg/ml resulted in an additional increase of the antiproliferative effect (p The prostate carcinoma cell line DU145 was treated with the chemotherapeutic agents docetaxel or mitoxantrone, respectively, as well as VAE-Qu in various concentrations. The maximal cytostatic effect of all drugs applied alone was about 90%. An enforcement of chemotherapy induced cytostasis was detected at VAE-Qu concentrations of ≥10 μg/ml (p Docetaxel and mitoxantrone exerted a dose dependent cytotoxic effect on DU145 cells with a maximum of about 50% cytotoxicity each (p The treatment of the lung carcinoma cell line NCI-H460 with cisplatin at a concentration of 9 μg/ml resulted in a proliferation inhibition of 95% (Figure 5A), whilst VAE-Qu (100 μg/ml) inhibited proliferation by 50% (Figure 5A,B). The maximal cytostatic effect attained by the treatment with docetaxel (1 μg/ml) was about 40% and – as in PA-TU-8902 cells – could not further be augmented by dose enhancement (Figure 5B). Only VAE-Qu at a concentration of 100 μg/ml could additionally enhance the antiproliferative effect of docetaxel (Figure 5B), as it did for 0.3–3 μg/ml cisplatin (Figure 5A). The dose dependent cytotoxic effect of cisplatin and docetaxel on NCI-H460 revealed a maximal cytotoxicity for cisplatin of 85% and for docetaxel of 55%. In general, no significant influence of VAE-Qu at concentrations between 0.1 and 10 μg/ml was observed; only at 3 μg/ml cisplatin, VAE-Qu 1 and 10 μg/ml additionally enhanced early apoptosis (p |