The gallium complex KP46 exerts strong activity against primary explanted melanoma cells and induces apoptosis in melanoma cell lines

Abstract

The antineoplastic properties of gallium are well documented. Owing to their robust accumulation of gallium, melanoma cells should be amenable to gallium-based anticancer drugs. With the aim of improving the disappointingly low activity of inorganic gallium salts, we have developed the orally bioavailable gallium complex KP46 [tris(8-quinolinolato)gallium(III)] that had already been successfully studied in a phase I clinical trial. To assess its therapeutic potential in malignant melanoma, its antiproliferative effects were investigated in series of human cell lines and primary explanted melanoma samples by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and the Human Tumor Cloning Assay, respectively. When compared with other cell lines, the majority of melanoma cells rank among the KP46-sensitive cell lines (50% inhibitory concentration values: 0.8-3.7 micromol/l). Clinically achievable concentrations of KP46 proved to be highly effective in melanoma cells from primary explants of cutaneous and lymph node metastases. Colony growth was inhibited in 10 of 10 specimens by 5 micromol/l KP46 (corresponding to the steady-state plasma concentration measured earlier in a study patient) and in four of 10 specimens by 0.5 micromol/l KP46. In-vitro potency of KP46 is higher than that of dacarbazine or fotemustine and comparable with that of cisplatin. The effects induced by KP46 in melanoma cell lines involve cell-cycle perturbations (S-phase arrest) and apoptosis (activation of caspase-9, PARP [poly(ADP-ribose) polymerase] cleavage, formation of apoptotic bodies). No effects on DNA secondary structure could be observed in an electrophoretic mobility shift assay using double-stranded plasmid DNA. Thus, further studies on the therapeutic applicability of KP46 in malignant melanoma are warranted.

Fig. 1

Cytotoxicity of KP46 [tris(8-quinolinolato)gallium(III)] in 10 melanoma cell lines (black bars) and 15 other human tumor cell lines (hatched bars). The 50% inhibitory concentration (IC₅₀) mean graph representing the deviations from the mean log IC₅₀. Bars oriented to the left indicate a higher sensitivity, bars oriented to the right a lower sensitivity than the average. NSCLC, non-small-cell lung cancer; SCLC, small-cell lung cancer.

Fig. 2

Concentration–effect curves of KP46 [tris(8-quinolinolato)gallium(III)], gallium nitrate, dacarbazine and cisplatin (MTT assay, 96 h exposure) in five human melanoma cell lines: 518A2 (a), 607B (b), A375 (c), MEL-JUSO (d), and SK-MEL-28 (e).

Fig. 3

Single cells isolated from primary explanted melanoma and their colony growth after incubation for 3 weeks in the Human Tumor Cloning Assay using a double-layer soft-agar medium. (a) Untreated single cells (white arrows) after 5 days, accompanied by background growth of fibroblasts at an agar content of 0.25% in the upper layer; (b) colony formed by untreated cells after 3 weeks at an agar content of 0.3% in the upper layer, preventing fibroblast growth; (c) KP46 [tris(8-quinolinolato)gallium(III)]-treated cells (5 μmol/l) showing massive inhibition of colony formation; (d) Fotemustine-treated cells (5 μmol/l).

Fig. 4

Inhibition of colony growth of primary explanted melanoma cells by KP46, cisplatin, and fotemustine, determined at various concentrations by the Human Tumor Cloning Assay in soft-agar medium. (a) Comparison of all ten KP46 [tris(8-quinolinolato)gallium(III)]-treated specimens; (b) comparison of KP46 and fotemustine, indicating thestronger cytostatic effect of the gallium complex; and (c) comparison of KP46 and cisplatin, indicating comparable activity. T/C, treated/control.

Fig. 5

Impact of KP46 [tris(8-quinolinolato)gallium(III)] (exposure time 24 h) on the cell cycle of the melanoma cell lines 518A2 and SK-MEL-28 and the colon carcinoma cell line SW480, as visualized by histograms obtained from fluorescence-assisted cell sorting upon propidium iodide staining of treated and control cells. Treatment with KP46 results in an increase of the cell fractions in the S phase and a concomitant decrease of cells in the G₀/G₁ phase. The two melanoma cell lines show a more massive S-phase arrest than the colon carcinoma cell line SW480.

Fig. 6

Impact of KP46 [tris(8-quinolinolato)gallium(III)] (exposure time 48 h) on the cell cycle of the melanoma cell lines 518A and SK-MEL-28, measured by the same technique as in Fig. 5.

Fig. 7

Electropherograms of double strand DNA (plasmid pTZ18u) after incubation with 60 μmol/l cisplatin or KP46 [tris(8-quinolinolato)gallium(III)] for the indicated times as compared with untreated controls (c). In contrast to cisplatin, KP46 does not affect the mobility of the supercoiled (SC) form of the plasmid. C, control; OC, open circular.

Fig. 8

Morphological features of apoptosis in SK-MEL-28 cells treated with KP46 [tris(8-quinolinolato)gallium(III)] (48 h), fixed in 4% paraformaldehyde and stained with DAPI (4′,6-diamidine-2′-phenylindole). Control cells show normal nuclear morphology, whereas an increasing percentage of KP46-treated cells show either bigger nuclei (5 μmol/l) or advanced nuclear segmentation (10 and 50 μmol/l).

Fig. 9

Western blot analysis of 518A2 cells treated with KP46 [tris(8-quinolinolato)gallium(III)] (0, 1.25, 2.5, 5, and 10 μmol/l) for 2 days as compared with untreated control, showing the activation of caspase-9 and cleavage of PARP [poly(ADP-ribose) polymerase] as indicators for apoptosis.