Mechanistic added value of a trans-Sulfonamide-Platinum-Complex in human melanoma cell lines and synergism with cis-Platin

Abstract

Background: Cisplatin is a potent antitumor agent. However, toxicity and primary and secondary resistance are major limitations of cisplatin-based chemotherapy, leading to therapeutic failure. We have previously reported that mono-sulfonamide platinum complexes have good antitumor activity against different tumoral cell lines and with a different and better cytotoxic profile than cisplatin. Besides, N-sulfonamides have been used extensively in medicinal chemistry as bactericides, anticonvulsant, inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization, among others.

Methods: We aimed to compare the cytotoxic effects of cisplatin and a trans-sulfonamide-platinum-complex (TSPC), in two human melanoma cell lines that differ in their TP53 status: SK-MEL-5, TP53 wild type, and SK-MEL-28, TP53 mutated. We performed cytotoxicity assays with both drugs, alone and in combination, cell cycle analyses, western blotting and immunoprecipitation, and fluorescence immunocytochemistry.

Results: TSPC had similar antiproliferative activity than cisplatin against SK-MEL-5 (3.24 ± 1.08 vs 2.89 ± 1.12 μM) and higher against SK-MEL-28 cells (5.83 ± 1.06 vs 10.17 ± 1.29 μM). Combination of both drugs inhibited proliferation in both cell lines, being especially important in SK-MEL-28, and showing a synergistic effect. In contrast to cisplatin, TSPC caused G1 instead G2/M arrest in both cell lines. Our present findings indicate that the G1 arrest is associated with the induction of CDKN1A and CDKN1B proteins, and that this response is also present in melanoma cells containing TP53 mutated. Also, strong accumulation of CDKN1A and CDKN1B in cells nuclei was seen upon TSPC treatment in both cell lines.

Conclusions: Overall, these findings provide a new promising TSPC compound with in vitro antitumor activity against melanoma cell lines, and with a different mechanism of action from that of cisplatin. Besides, TSPC synergism with cisplatin facilitates its potential use for co-treatment to reduce toxicity and resistance against cisplatin. TSPC remains a promising lead compound for the generation of novel antineoplastic agent and to explore its synergism with other DNA damaging agents.

Keywords: Cell Cycle control; Cisplatin; Mechanisms of action; Melanoma; Mono-Sulfonamide; Transplatin.

Fig. 1

trans-Dichlorido [(rac)-2-(5-(dimethylamino)naphthalene-1-sulfonamido)cyclohexylamino] (dimethylsulfoxide)platinum(II) compound (TSPC). Chemical Formula: C₂₀H₃₁Cl₂N₃O₃PtS₂ Molecular Weight: 691.5924 g/mol

Fig. 2

Effect of CDDP and TSPC on melanoma cell lines. a Log-dose response curves for SK-MEL-5 and SK-MEL-28 cells following treatment with CDDP or TSPC at 72 h. b Distribution of cells between cell cycle phases after CDDP and TSPC exposure. (*, p < 0.05). c Apoptotic effect of CDDP and TSPC evaluated by APO-BrDUTunel assay. The experiments were carried out in triplicate. The results are shown as the mean ± standard deviation. d Effect of CDDP and TSPC in cell cycle regulatory proteins TP53, CDKN1A, CDKN1B, CDKN2B, CDK2, CDK4, CCND1 (cyclin D1), CCNE2 (cyclin E2), and P-CDK1 as determined by Western blot analysis. GAPDH is shown as loading control

Fig. 3

Quantification of effect of CDDP and TSPC in cell cycle regulatory proteins TP53, CDKN1A, CDKN1B, CDKN2B, CDK2, CDK4, CCND1, CCNE2, and P-CDK1 as determined by Western blot analysis of three independent experiments

Fig. 4

Co-immunoprecipitation assay of the interaction between CDK2 and CDKN1A, and CDK2 and CDKN1B in SK-MEL-5 and SK-MEL-28 cell lines after CDDP and TSPC exposure. Quantification of CDKN1A and CDKN1B binds to CDK2, detected in co-immunoprecipitated products. (*, p < 0.05)

Fig. 5

Cellular distribution of CDKN1A and CDKN1B in melanoma cell lines and its alteration after CDDP or TSPC treatment. a Subcellular distribution of CDKN1A and CDKN1B in SK-MEL-5 and SK-MEL-28 cells, after CDDP and TSPC treatment assessed by confocal microscopy. First and fourth columns: nuclei stained with DAPI; Second and fifth columns: CDKN1A (up) and CDKN1B (down) proteins stained with specific antibody and secondary antibody conjugated with Alexa Fluor 488; Third and sixth columns: colocalization. b Quantitative analysis of nuclear CDKN1A (left) and CDKN1B (right) staining in melanoma cell lines. Staining cells were counted at 200 × magnification from six to ten randomly selected fields. Total 100 cells were counted in each experiment. (*, p < 0.05)

Fig. 6

Effects of CDDP and TSPC combined treatment. a Cell recovery after treatment removal. Cell growth was assessed for 7 days in cells treated with either CDDP (triangles) or TSPC (circles) at their corresponding IC₅₀ for 72 h followed by drug removal. Untreated cells were used as control (squares). b Cell viability with drug treatment combination. Viability after treatment with different doses of CDDP ranging from 0.5 to 10 μM combined with TSPC 1 μM (right side-up triangles) or 5 μM (upside-down triangles). Cells just treated with CDDP were used as control (squares). c Fa-log CI plot. The line represents the additive effect and all the points under it show synergism. d Fa-log DRI plot. The line separates the favourable dose reduction (up) from the unfavourable (down). e Cell recovery after combination treatment removal. Clear shape symbol: SK-MEL-5; Dark shape symbol: SK-MEL-28

Fig. 7

TSPC induces cell cycle G1 arrest probably mediated by CDKN1A and CDKN1B CKIs in a TP53-independent manner, while CDDP induces S and G2/M arrest via TP53/CDKN1A pathway in SK-MEL-5, and preventing dephosphorylation of CDK1 in both cell lines