Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells

Abstract

Complementary treatment possibilities for the therapy of cancer are increasing in demand due to the severe side effects of the standard cytostatics used in the first-line therapy. A common approach as a complementary treatment is the use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic activity of these extracts is attributed to Mistletoe lectins which are Ribosome-inactivating proteins type II. Besides these main constituents the extract of Viscum album L. comprises also a mixture of lipophilic ingredients like triterpene acids of the oleanane, lupane and ursane type. However, these constituents are not contained in commercially available aqueous extracts due to their high lipophilicity and insolubility in aqueous extraction media. To understand the impact of the extract ingredients in cancer therapy, the intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells was investigated in relation to the mistletoe triterpene acids, mainly oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized using cyclodextrins ("TT" extract). Afterwards, the uptake of either single compounds (isolated ML and the aqueous "viscum" extract) or in combination with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30, 60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies the A-chain of the hololectin. It could be shown that the intracellular uptake after 120 minutes amounted to 20% in all cell lines after incubation with viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and Ewing TC-71-cells was independent of the addition of TT extract. Interestingly, the uptake of ML by 143B-cells could only be measured after addition of triterpenes pointing to resistance to mistletoe lectin.

Fig 1. Cell viability of HL-60-cells.

The cells were treated with different ML concentrations (see Tables 2 and 3) for 30, 60 and 120 minutes. The isolated ML, three viscum extract batches and the viscum extract batch 161 in combination with TT 161 extract batch (25 μg/mL and 35 μg/mL OA) were used. The viability was determined with Annexin V-APC and propidium iodide by flow cytometry. The values are expressed as percentages of the untreated control cells. Error bars represent the standard deviation of n = 2 experiments.

Fig 2. Uptake of ML out of viscum extract without combination with TT extract.

The values were calculated using three viscum extract batches in two different ML concentrations (see Table 2) and are expressed as percentage of respective used concentration. Error bars represent the standard deviation of n ≥ 4 experiments. * Significant difference to the other cell lines, α ≤ 0.05.

Fig 3. ML uptake by 143B cells.

The averages of the uptake were calculated using isolated ML and viscum 155 extract batch in two different ML concentrations alone and in combination with TT 155 extract batch (20 μg/mL OA) (see Tables 2 and 3). The results are expressed as percentage of the respectively used concentration ± standard deviation of n = 5 experiments. * Significant difference to the isolated ML, α ≤ 0.05. ** Significant difference to the viscum 155 extract, α ≤ 0.05.

Fig 4. The TT concentration influence on the uptake by the 143B cells.

The averages were determined using isolated ML and viscum 155 extract batch in combination with TT 155 extract batch (see Tables 2 and 3) and are expressed as percentage of the respectively used concentration. Error bars represent the standard deviation of n = 5 experiments.

Fig 5. ML uptake out of viscum extract by the THP-1 cell line.

The values were calculated using viscum 161 extract batch in two different ML concentrations alone and in combination with TT 161 extract batch (25 μg/mL and 35 μg/mL OA) (see Table 2). The results are expressed as percentage of respective used concentration. Error bars represent the standard deviation of n = 4 experiments.