Interaction of a standardized mistletoe (Viscum album) preparation with antitumor effects of Trastuzumab in vitro

U Weissenstein, M Kunz, K Urech, U Regueiro, S Baumgartner, U Weissenstein, M Kunz, K Urech, U Regueiro, S Baumgartner

Abstract

Background: Besides conventional anticancer therapy many breast cancer patients use complementary and alternative medicine (CAM) like the medicinal herb mistletoe (Viscum album L.). To gain more knowledge about possible herb-drug interactions between CAM and conventional anticancer medications, in the present in vitro study we investigated the effect of a standardized mistletoe preparation on the action of Trastuzumab, a drug used for the treatment of Her-2 positive breast cancer.

Methods: The Her-2 positive human breast carcinoma cell line SK-BR-3 was treated with Trastuzumab. Different doses of the drug were combined with Viscum album extract (VAE) in clinically relevant doses. Proliferation, apoptosis, cell cycle and the secretion of vascular endothelial growth factor (VEGF) were analyzed.

Results: No inhibition of antitumor efficacy of Trastuzumab by VAE was detected. VAE and Trastuzumab, either alone or in combination, inhibited proliferation of SK-BR-3 cells in vitro. At higher concentrations VAE induced apoptosis, which was not observed for Trastuzumab. Cells treated with Trastuzumab underwent a G0/G1 cell cycle arrest and cells treated with VAE a G2/M arrest. After application of the two drugs in combination both G0/G1 and G2/M arrest was observed. VEGF secretion of SK-BR-3 cells was significantly inhibited by sole treatment with Trastuzumab or VAE. Combined treatment of Trastuzumab and VAE at clinically relevant doses showed additive inhibitory effects on VEGF secretion.

Conclusions: VAE did not interfere with cytostatic effects of Trastuzumab on SK-BR-3 cells in vitro. Our in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to Trastuzumab and VAE simultaneously. In contrast, VAE and Trastuzumab seem to exhibit complementary anti-cancer effects in vitro.

Keywords: Apoptosis; Cell cycle; Cytostasis; Drug interactions; Her-2; Herceptin; Iscador; Mistletoe (Viscum album L.); Trastuzumab; VEGF.

Figures

Fig. 1
Fig. 1
Dose response effect of VAE, Trastuzumab and their combinations on the proliferation of SK-BR-3 cells. Proliferation inhibition by (a) VAE treatment after 3d and 7d, (b) Trastuzumab treatment after 3d and 7d and combined VAE and Trastuzumab treatment after (c) 3d and (d) 7d, are shown. Cell growth kinetic was assessed with the WST-1 assay. Results are presented as mean ± SE from three independent experiments
Fig. 2
Fig. 2
Apoptosis induction in SK-BR-3 cells by VAE. Flow cytometric analysis of apoptotic death in SK-BR-3 cells labelled with annexin-V FITC and 7-AAD after 3d (top) and after 7d (bottom) exposure to 10 μg/ml Trastuzumab in combination with 0 – 10 μg/ml VAE. This figure is representative of three independent experiments. The percentages in the graphs represent the percentage of cell numbers in each quadrant. Q1 and Q2: late apoptotic/necrotic cells, Q3: early apoptotic cells, Q4: living cells
Fig. 3
Fig. 3
Apoptosis induction in SK-BR-3 cells. Mean values (±SE) of (a) early apoptosis and (b) late apoptosis/necrosis after 3d and of (c) early apoptosis and (d) late apoptosis/necrosis after 7d treatment with different concentrations of Trastuzumab combined with different concentrations of VAE are presented
Fig. 4
Fig. 4
SK-BR-3 cell cycle analysis of cells treated with (a) VAE (b) Trastuzumab (c) 1 μg/ml Trastuzumab and VAE and (d) 10 μg/ml Trastuzumab and VAE. Cells were harvested after 3d, fixed, stained and analyzed for DNA content by flow cytometry. The distribution and percentage of cells in G0/G1, S and G2/M phase of the cell cycle are indicated. Results are presented as mean values from three independent experiments. (SE values are omitted for clarity). Significance values are given relative to the VAE and Trastuzumab untreated controls (**p < 0.01, ***p < 0.001). Discrepancies from 100 % can be attributed to slight gating differences and the display of mean values
Fig. 5
Fig. 5
Inhibitory effect of Trastuzumab, VAE and their combinations on VEGF production of SK-BR-3 human breast carcinoma cells in vitro. VEGF was measured by ELISA in cell culture supernatants after 3d of treatment. Results are presented as mean ± SE from three independent experiments

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