Combination effects of platinum drugs and N1, N11 diethylnorspermine on spermidine/spermine N1-acetyltransferase, polyamines and growth inhibition in A2780 human ovarian carcinoma cells and their oxaliplatin and cisplatin-resistant variants

Abstract

Purpose: To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively.

Methods: Parental and Pt-resistant cells were treated with platinum agent alone, DENSPM alone or combination (10 μM each, 20 h). QRT-PCR, radioactive product measurement and HPLC were used for mRNA, activity and polyamine pools, respectively; drug interaction on cell growth was by SRB and isobologram analysis.

Results: Both platinum agents induced SSAT mRNA in parental A2780 cells, but not in resistant cells. Platinum drug/DENSPM combinations produced high levels of SSAT activity in parental cells with significant depletion of spermine and spermidine, but not in resistant cells. Co-treatment with platinum agents increased the levels of DENSPM in all cell lines. Oxaliplatin/DENSPM combination was superior to cisplatin/DENSPM in the inhibition of cell growth in parental cells. No synergy was observed in the resistant cells.

Conclusions: Increased DENSPM levels following co-treatment with Pt agents enhances the translation and stability of SSAT protein leading to polyamine pool depletion, facilitating more Pt-DNA adduct formation in parental cells. Oxaliplatin/DENSPM combination is superior to cisplatin/DENSPM in cell growth inhibition as DACH-Pt DNA adducts are cytotoxic even at relatively fewer numbers. Reduced platinum uptake in Pt-resistant cells contributes to reduced SSAT mRNA induction and absence of synergy when combined with DENSPM.

Fig. 1

a Comparative dose–response curves for oxaliplatin in A2780 ovarian carcinoma and the oxaliplain-resistant A2780/C10B cells following a 72-h continuous exposure to oxaliplatin. b Comparative dose–response curves for cisplatin in A2780 ovarian carcinoma cells and the cisplatin-resistant A2780/CP cells following a 72-h continuous exposure to cisplatin. Data represent mean ± SE where n = 3. The IC₅₀ for oxaliplatin in A2780 and A2780/C10B are 0.2 and 3.0 μM, respectively. The IC₅₀ for cisplatin in A2780 and A2780/CP are 0.54 and 6.58 μM, respectively

Fig. 2

a Effect of oxaliplatin alone, DENSPM alone or oxaliplatin/DENSPM combination on SSAT mRNA in A2780 and A2780/C10B cells. b Effect of cisplatin alone, DENSPM alone or cisplatin/DENSPM combination on SSAT mRNA in A2780 and A2780/CP cells. Cells were exposed to 10 μM platinum agent or 10 μM DENSPM singly, or in combination for 20 h followed by another 24-h incubation in drug-free medium prior to SSAT mRNA measurements. Data shown are fold induction relative to drug untreated controls. Data represent mean ± SE where n = 2–5 separate cell pellets in up to 3 separate experiments. In a, the fold-induction in A2780 after oxaliplatin, DENSPM and oxaliplatin/DENSPM are 60.6 ± 12.3, 15.8 ± 7.8 and 95.5 ± 15, respectively; the fold induction in A2780/C10B cells after the same treatments are 2.8 ± 0.1, 2.3 ± 0.4, and 7.4 ± 1.3, respectively. In b, the fold-induction in A2780 cells after cisplatin, DENSPM and cisplatin/DENSPM are 42.3 ± 1.1, 15.8 ± 7.8 and 69.0 ± 20.2, respectively; the fold induction in A2780/CP cells after the same treatments are 3.5 ± 1.3, 3.7 ± 1.9 and 5.9 ± 0.6, respectively

Fig. 3

a Effect of oxaliplatin alone, DENSPM alone or oxaliplatin/DENSPM combination on SSAT activity in A2780 and A2780/C10B cells. b Effect of cisplatin alone, DENSPM alone or cisplatin/DENSPM combination on SSAT activity in A2780 and A2780/C10B cells. Data shown are SSAT activity in pmol/min/mg protein. Data represent mean ± SE where n = 3–9 separate cell pellets in up to 3 separate experiments. In a, SSAT activity in A2780 for control (no-drug treatment) cells, and in cells treated with oxaliplatin, DENSPM and oxaliplatin/DENSPM were 15.0 ± 1.1, 57.3 ± 11.7, 53.9 ± 28.7 and 6130.2 ± 1182.0 pmol/min/mg protein, respectively; the same for A2780/C10B cells were 11.0 ± 3.0; 24.7 ± 3.8, 25.7 ± 1.8; 46.7 ± 0.9 pmol/min/mg protein, respectively. In b, SSAT activity in A2780 for control cells and in cells treated with cisplatin, DENSPM and cisplatin/DENSPM were 23.7 ± 1.7, 52.6 ± 12.5, 272.2 ± 30.1 and 5807 ± 1218 pmol/min/mg protein, respectively; the same for A2780/CP cells were 20.9 ± 5.5, 33.4 ± 8.1, 165.2 ± 18.7, 533 ± 76.9 pmol/min/mg protein, respectively

Fig. 4

Effect of oxaliplatin alone, DENSPM alone or oxaliplatin/DENSPM combination on polyamine pools in A2780 (a) and A2780/C10B (b) cells and cisplatin alone, DENSPM alone or cisplatin/DENSPM on polyamine pools in A2780 (c) and A2780/CP (d) cells. Data shown are pmoles/mg protein. Note that N ¹ acetyl spermidine was detected only in oxaliplatin/DENSPM combinations. DENSPM levels in cells when administered alone or in combination with the platinum agent is also presented for all the cell lines. Note that DENSPM levels are higher when administered with the platinum agent. Data represent mean ± SE where n = 3–9 separate cell pellets in up to 3 separate experiments

Fig. 5

a Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780 cells. b Isobolograms of cisplatin and DENSPM in combination treatment of A2780 cells. c Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780/C10B cells. d Isobolograms of cisplatin and DENSPM in combination treatment of A2780/CP cells. In the axis legend, ‘×’ refers to the IC concentrations for each drug. The diagonal dashed line represents additivity and data below this line implies synergism and data above represent antagonism

Fig. 5

a Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780 cells. b Isobolograms of cisplatin and DENSPM in combination treatment of A2780 cells. c Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780/C10B cells. d Isobolograms of cisplatin and DENSPM in combination treatment of A2780/CP cells. In the axis legend, ‘×’ refers to the IC concentrations for each drug. The diagonal dashed line represents additivity and data below this line implies synergism and data above represent antagonism

Fig. 5

a Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780 cells. b Isobolograms of cisplatin and DENSPM in combination treatment of A2780 cells. c Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780/C10B cells. d Isobolograms of cisplatin and DENSPM in combination treatment of A2780/CP cells. In the axis legend, ‘×’ refers to the IC concentrations for each drug. The diagonal dashed line represents additivity and data below this line implies synergism and data above represent antagonism

Fig. 5

a Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780 cells. b Isobolograms of cisplatin and DENSPM in combination treatment of A2780 cells. c Isobolograms of oxaliplatin and DENSPM in combination treatment of A2780/C10B cells. d Isobolograms of cisplatin and DENSPM in combination treatment of A2780/CP cells. In the axis legend, ‘×’ refers to the IC concentrations for each drug. The diagonal dashed line represents additivity and data below this line implies synergism and data above represent antagonism