Fig 1.
Structures of bendamustine (1) and the bendamustine esters 2–7.
Table 1.
Cytotoxicity of compounds 1–7 against selected human cancer cell lines of different origin.
Subgroups, representing hematological malignancies, sarcoma, carcinoma and malignant melanoma, are separated by horizontal lines. IC50 values (μM) after 96 hours of incubation.
Fig 2.
Antiproliferative activity of compounds 1, 4 and 5 against selected malignant tumor cells and spontaneously immortalized human keratinocytes upon long-term incubation.
Jurkat (A; acute T cell leukemia), SK-ES-1 (B; Ewing’s sarcoma), NCI-H460 (C; large cell lung cancer) and HT-29 (D; colorectal cancer) cells were treated with compounds 1, 4 and 5 at concentrations between 1 μM and 50 μM, HaCaT cells (E; spontaneously immortalized human keratinocytes) were incubated with 1, 4 and 5 at 0.1, 1 and 10 μM. Incubation period: 5 days. Antiproliferative and cytotoxic effects correspond to the left y-axes. The growth curves of untreated control cells (open circles) correspond to the right y-axes. Data are mean values ± SEM of at least 2 independent assays with 8 replicates per compound concentration. For additional data on long-term cytotoxity of compounds 1–7 against various tumor cell types and cytotoxicity of compound 2 against HaCaT cells cf. S3–S13 Figs.
Fig 3.
Induction of apoptosis in Jurkat cells.
Results of annexin V/propidium iodide staining, performed after incubating Jurkat cells with compounds 1, 2, 4 and 5 at a concentration of 10 μM for different periods. Annexin V+/PI- cells were defined as early apoptotic, whereas secondary apoptotic cells were annexin V+/PI+. Significances illustrated on top of the columns refer to the overall effect (apoptotic + secondary necrotic cells), those depicted in the columns refer to apoptotic cells. After 48 hours, apoptotic and secondary necrotic cells were significantly different (p < 0.001) (mean ± SEM, N = 3). One-way ANOVA and Bonferroni’s post-test were applied to calculate the significance of the apoptotic fraction and the sum of apoptotic and secondary necrotic cells; n.s.: not significant; *: p < 0.05; **: p < 0.01, ***: p < 0.001.
Fig 4.
Effect of 1, 2, 4 and 5 on the expression of p53 in NCI-H460 and HT-29 cells.
Western blot of p53 and histone H2B (loading control) of NCI-H460 (A) and HT-29 (B) cells. A: Nuclear extracts of NCI-H460 cells, incubated with different concentrations of 1 (10, 30 and 100 μM) or 10 μM of 2, 4, or 5 for 24 hours. Nuclear extracts (NE) and whole cell lysates (WCE) of untreated cells were used as control (Ctrl). B: Nuclear extracts of HT-29 cells, incubated with 10 μM of 1, 2, 4, 5 for 24 hours. Untreated cells served as control (Ctrl).
Fig 5.
Accumulation of 1 and derivatives by HT-29 and NCI-H460 cells.
Amount (mean ± SEM, N = 3–4) of cell-associated 1, 2, 4 and 5, expressed as nmol/106 cells (left y-axis) and cellular enrichment (right y-axis) in HT-29 (A) and NCI-H460 (B) cells. A mean cell volume of 3 pL was used to calculate accumulation factors. Significance was calculated using one-way Anova: n.s.: not significant; *: p < 0.05; **: p < 0.01, ***: p < 0.001.
Fig 6.
Confocal laser scanning microscopy of cellular ASP+ uptake via OCT1 and OCT3.
Uptake of fluorescent ASP+ [1 μM] (green) in the absence and presence of compound 5 or TPA after 5 minutes of pre-incubation. Nuclei were stained with Draq5 [5 μM] (red). A, B: HEK-Co (control) cells, treated with Draq5 (A) or ASP+ plus Draq5 (B). C-E: HEK-OCT1 cells, treated with ASP+ plus Draq5 (C), ASP plus Draq5 plus compound 5 [15 μM] (D), or ASP+ plus Draq5 plus TPA [200 μM] (E). F-H: HEK-OCT3 cells, treated with ASP+ plus Draq5 (F), ASP+ plus Draq5 plus compound 5 [15 μM] (G), or ASP+ plus Draq5 plus TPA [200 μM] (H).
Fig 7.
Specific uptake of ASP+ and Michaelis-Menten kinetics determined by flow cytometry.
A: Time dependency of the specific mean fluorescence intensity (MFI) after incubating HEK-OCT1 and HEK-OCT3 cells with 1 μM ASP+ (means ± SEM, N = 3). B: Concentration dependency of the initial velocity of the specific increase in the mean fluorescence intensity (MFI) caused by ASP+-uptake into HEK-OCT1 and HEK-OCT3 cells. HEK cells transfected with an empty vector served as control for unspecific uptake (means ± SEM, N = 3).
Table 2.
Inhibition of ASP+-uptake into HEK-OCT1 and HEK-OCT3 cells.
IC50 (μM) values ± SEM (N = 3).
Fig 8.
Concentration-dependent inhibition of ASP+-uptake by OCT expressing HEK-cells.
Inhibition of ASP+-uptake (1 μM) into HEK-OCT1 (A) and HEK-OCT3 (B) cells by TPA, 2, 4 or 5. The mean fluorescence intensities were normalized to uninhibited ASP+-uptake (N = 3).
Fig 9.
Expression of OCT1 and OCT3 by various cancer cells.
Gel electrophoretic analysis of SLC22A1 (encoding human OCT1) and SLC22A3 (encoding human OCT3) mRNA expression by different human cancer cell types after RT-PCR. Human SLC22A1 mRNA was detected using the primer pair oOCT1-RT.for–oOCT1-RT.rev, resulting in a specific band of 319 bp, human SLC22A3 mRNA by the primer pair oOCT3-RT.for–oOCT3-RT.rev, resulting in a band of 439 bp. As a positive control, the plasmids pOCT1.31 and pOCT3.31 (cDNA) were used as templates of SLC22A1 and SLC22A3, respectively. NTC = non-template control.