Chemical modification of melphalan as a key to improving treatment of haematological malignancies

Abstract

Chemical modification of known, effective drugs is one method to improve chemotherapy. Thus, the object of this study was to generate melphalan derivatives with improved cytotoxic activity in human cancer cells (RPMI8226, HL60 and THP1). Several melphalan derivatives were synthesised, modified in their two important functional groups. Nine analogues were tested, including melphalan compounds modified: only at the amino group, by replacing the amine with an amidine group containing a morpholine ring (MOR-MEL) or with an amidino group and dipropyl chain (DIPR-MEL); only at the carboxyl group to form methyl and ethyl esters of melphalan (EM-MEL, EE-MEL); and in a similar manner at both functional groups (EM-MOR-MEL, EE-MOR-MEL, EM-DIPR-MEL, EE-DIPR-MEL). Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (comet assay) and the ability to induce apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling, TUNEL, phosphatidylserine externalisation, chromatin condensation, activity of caspases 3/7, 8 and 9 and intracellular concentration of calcium ions) in comparison with the parent drug. Almost all derivatives, with the exception of MOR-MEL and DIPR-MEL, were found to be more toxic than melphalan in all cell lines evaluated. Treatment of cultures with the derivatives generated a significant higher level of DNA breaks compared to those treated with melphalan, especially after longer incubation times. In addition, all the melphalan derivatives demonstrated a high apoptosis-inducing ability in acute monocytic and promyelocytic leukemia cells. This study showed that the mechanism of action of the tested compounds differed depending on the cell line, and allowed the selection of the most active compounds for further, more detailed investigations.

Figure 1

IC₅₀ ± SD values [µM] of compounds in RPMI8226, HL60 and THP1 human cancer cell lines.

Figure 2

Influence of melphalan and its derivatives on the induction of apoptosis in RPMI8226, HL60 and THP1 cancer cell lines, as estimated by annexin V/propidium iodide (A) and Vybrant® DyeCycle™ Violet/SYTOX^® AADvanced^™ (B) assays. Quantitative results of the compounds effect on the level of necrotic, and early and late apoptotic cells are represent by the mean ± standard deviation (SD) for three independent experiments.

Figure 3

Fluorescence images of RPMI8226, HL60 and THP1 cells at 48 h after treatment with melphalan and its derivatives. The cells were stained with an annexin V/propidium iodide mixture and visualised by fluorescence microscopy, magnification ×200. After drug treatment, high green (derived from annexin V- fluorescein isothiocyanate, FITC) and red fluorescence (derived from propidium iodide) in cells with exposed phosphatidylserine (PS) indicated damage to the integrity of cellular membranes, characteristic symptoms of the late stages of programmed cell death. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this article).

Figure 4

(A) Tail DNA (%) of cells treated with investigated compounds for 4, 24 and 48 h in RPMI8226, HL60 and THP1 cell lines. The number of analysed cells in each treatment was 100, and the analysis was repeated three times. (B) Influence of melphalan and its derivatives on the induction of DNA damage correlated with apoptosis in RPMI8226, HL60 and THP1 cancer cell lines, as estimated by TUNEL assay. Error bars denote SD.

Figure 5

Changes in activity of caspase-9, caspase-8 and caspase-3 after exposure of RPMI8226, HL60, THP1 cell lines to melphalan and analogues for 4, 24, 48 h. The measurements were carried out in the presence or absence of inhibitors. The cells were treated with an IC₅₀ dose of MEL and its derivatives. The final result obtained was a percentage activity of a particular caspase, where the fluorescence value (caspase-3) or luminescence (caspase-8 and -9) of the control, not treated with the compound, was taken as 100%. Each point represents the average ± SD of three independent experiments. Values shaded in grey indicate differences between samples incubated with new derivatives and those incubated with MEL.

Figure 6

The effect of melphalan and its derivatives on Ca²⁺ concentration in THP1, HL60 and RPMI8226 cell lines. The cells were treated with an IC₅₀ dose of the test compounds and then incubated for 2, 4, 24 or 48 h. The intensity of Fluo-4-NW probe fluorescence measured in control cells after 2, 24 or 48 h of incubation was taken as 100%. Each point represents the average ± SD of three independent experiments.

Figure 7

Proposed model of the molecular and cellular responses to the new MEL derivatives.

Figure 8

Chemical structures of melphalan derivatives.