Esters of bendamustine are by far more potent cytotoxic agents than the parent compound against human sarcoma and carcinoma cells

The alkylating agent bendamustine is approved for the treatment of hematopoietic malignancies such as non-Hodgkin lymphoma, chronic lymphocytic leukemia and multiple myeloma. As preliminary data on recently disclosed bendamustine esters suggested increased cytotoxicity, we investigated representative derivatives in more detail. Especially basic esters, which are positively charged under physiological conditions, were in the crystal violet and the MTT assay up to approximately 100 times more effective than bendamustine, paralleled by a higher fraction of early apoptotic cancer cells and increased expression of p53. Analytical studies performed with bendamustine and representative esters revealed pronounced cellular accumulation of the derivatives compared to the parent compound. In particular, the pyrrolidinoethyl ester showed a high enrichment in tumor cells and inhibition of OCT1- and OCT3-mediated transport processes, suggesting organic cation transporters to be involved. However, this hypothesis was not supported by the differential expression of OCT1 (SLC22A1) and OCT3 (SLC22A3), comparing a panel of human cancer cells. Bendamustine esters proved to be considerably more potent cytotoxic agents than the parent compound against a broad panel of human cancer cell types, including hematologic and solid malignancies (e.g. malignant melanoma, colorectal carcinoma and lung cancer), which are resistant to bendamustine. Interestingly, spontaneously immortalized human keratinocytes, as a model of “normal” cells, were by far less sensitive than tumor cells against the most potent bendamustine esters

Confocal laser scanning microscopy of cellular ASP⁺ uptake via OCT1 and OCT3.

<p>Uptake of fluorescent ASP⁺ [1 μM] (green) in the absence and presence of compound <b>5</b> 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 <b>5</b> [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 <b>5</b> [15 μM] (G), or ASP⁺ plus Draq5 plus TPA [200 μM] (H).</p

Effect of 1, 2, 4 and 5 on the expression of p53 in NCI-H460 and HT-29 cells.

<p>Western blot of p53 and histone H2B (loading control) of NCI-H460 (A) and HT-29 (B) cells. <b>A</b>: Nuclear extracts of NCI-H460 cells, incubated with different concentrations of <b>1</b> (10, 30 and 100 μM) or 10 μM of <b>2</b>, <b>4</b>, or <b>5</b> for 24 hours. Nuclear extracts (NE) and whole cell lysates (WCE) of untreated cells were used as control (Ctrl). <b>B</b>: Nuclear extracts of HT-29 cells, incubated with 10 μM of <b>1</b>, <b>2</b>, <b>4</b>, <b>5</b> for 24 hours. Untreated cells served as control (Ctrl).</p

Antiproliferative activity of compounds 1, 4 and 5 against selected malignant tumor cells and spontaneously immortalized human keratinocytes upon long-term incubation.

<p>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 <b>1, 4</b> and <b>5</b> at concentrations between 1 μM and 50 μM, HaCaT cells (E; spontaneously immortalized human keratinocytes) were incubated with <b>1</b>, <b>4</b> and <b>5</b> 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 <b>1–7</b> against various tumor cell types and cytotoxicity of compound <b>2</b> against HaCaT cells cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133743#pone.0133743.s003" target="_blank">S3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133743#pone.0133743.s013" target="_blank">S13</a> Figs.</p

Concentration-dependent inhibition of ASP⁺-uptake by OCT expressing HEK-cells.

<p>Inhibition of ASP⁺-uptake (1 μM) into HEK-OCT1 (A) and HEK-OCT3 (B) cells by TPA, <b>2</b>, <b>4</b> or <b>5</b>. The mean fluorescence intensities were normalized to uninhibited ASP⁺-uptake (N = 3).</p

Accumulation of 1 and derivatives by HT-29 and NCI-H460 cells.

<p>Amount (mean ± SEM, N = 3–4) of cell-associated <b>1</b>, <b>2</b>, <b>4</b> and <b>5</b>, 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; <b>*</b>: p < 0.05; <b>**</b>: p < 0.01, <b>***</b>: p < 0.001.</p

Structures of bendamustine (1) and the bendamustine esters 2–7.

<p>Structures of bendamustine (1) and the bendamustine esters 2–7.</p

Expression of OCT1 and OCT3 by various cancer cells.

<p>Gel electrophoretic analysis of <i>SLC22A1</i> (encoding human OCT1) and <i>SLC22A3</i> (encoding human OCT3) mRNA expression by different human cancer cell types after RT-PCR. Human <i>SLC22A1</i> mRNA was detected using the primer pair oOCT1-RT.for–oOCT1-RT.rev, resulting in a specific band of 319 bp, human <i>SLC22A3</i> 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 <i>SLC22A1</i> and <i>SLC22A3</i>, respectively. NTC = non-template control.</p

Specific uptake of ASP⁺ and Michaelis-Menten kinetics determined by flow cytometry.

<p>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).</p