Characterization of Pt-, Pd-spermine complexes for their effect on polyamine pathway and cisplatin resistance in A2780 ovarian carcinoma cells

Abstract

We have previously showed that platinum drugs up-regulate SSAT and SMO and down-regulate ODC and SAMDC in the polyamine pathway. Several studies including our own established that platinum drugs combined with polyamine analog DENSPM produces synergistic increase in SSAT activity with polyamine depletion. Since polyamine pathway is an important therapeutic target, we investigated whether agents containing both platinum and polyamines have similar effects on the polyamine pathway. Two complexes i) Pt-spermine with two cisplatin molecules linked to a spermine in the center and ii) Pd-spermine with similar structure i, but Pd (II) substituted for Pt (II) were analyzed with respect to their effect on the expression of genes in polyamine pathway, SSAT and SMO protein expression, SSAT activity and polyamine pools. Pt-, Pd-spermine complexes induced significant down-regulation of SMO, arginase 2 and NRF-2, with no change in SSAT, while cisplatin as a single agent or in combination with DENSPM induced significant up-regulation of SSAT and SMO. The SSAT activity was not induced by either Pt- or Pd-spermine in A2780 cells; SMO protein levels were significantly elevated compared to the no-drug control and to a similar extent as cisplatin/DENSPM. The Pd-spm treatment induced a fall in putrescine levels to 33%, spermidine to 62% and spermine to 72% while Pt-spm did not induce such a decline. Comparative cytotoxicity studies in A2780 cells indicated the potency to be cisplatin> Pd-Spm>Pt-Spm. Although both complexes exhibit a lower potency, the degree of resistance itself is much lower for Pt-spermine and Pd-spermine in that order (2.5 and 7.5, respectively) compared to cisplatin ( approximately 12) as tested in cisplatin resistant A2780/CP cells. These studies suggest that Pd (II)-polyamine complexes may constitute a promising group of inorganic compounds for further studies in the development of novel chemotherapy/adjuvant chemotherapy strategies.

Figure 1

Structures of cisplatin (A), Pt, Pd-spermine complexes (B) and spermine (C).

Figure 2

Expression of genes represented on TLDA in A2780 human ovarian carcinoma cells treated with Pt-spermine and Pd-spermine complexes. Bars are log₁₀ RQ. RQ is the quantity of expression of a given gene relative to the drug untreated control. Bars going upward represent up-regulation and those going downwards down-regulation. Error bars are the confidence intervals compared to the no-drug controls in the first position of each cluster.

Figure 3

Expression of genes represented on TLDA in A2780 ovarian carcinoma cells treated with cisplatin, DENSPM or cisplatin/DENSPM. Bars are log₁₀ RQ. RQ is the quantity of expression of a given gene relative to the drug untreated control. Bars going upward represent up-regulation and those going downwards down-regulation. Error bars are the confidence intervals compared to the no-drug controls in the third position of each cluster.

Figure 4

Western blot analysis of SSAT (A) and SMO (B). A2780 cells were treated with Pd-spermine, Pt-spermine or cisplatin/DENSPM, all at 10 μM concentration each for 20 h followed by 24 h incubation in drug free medium. The cells were harvested thereafter and 60-80 μg of whole cell extracts were used for Western blot and probed with human SSAT antibody (A) or SMO antibody (B). Untreated cells were used as control for both. NIH3T3 cells treated with DENSPM were used as a positive control for SSAT (A) and HCT-116 cells treated with oxaliplatin/DENSPM were used as a positive control for SMO (B). A2780 cells treated with oxaliplain/DENSPM were also used as a positive control for SSAT and SMO. Note that fold changes could not be presented in 4A, as controls had no measurable SSAT protein.

Figure 5

Relative cytotoxic potency of cisplatin, Pt-spermine and Pd-spermine in A2780 cells. Cells were plated in a 96 well plate (1×10³ cells/well) on day ‘0’, followed by treatment with cisplatin or Pd-Spermine or Pt-spermine on day 1 at concentrations ranging from 0.1 to 100 μM for 72 h. Cells were then fixed and subjected to sulforhodamine-B-microculture colorimetric assay (SRB). Percent survival was determined as (OD₅₇₀ treated cells/OD₅₇₀ untreated cells) x100. Data presented are an average of 2 separate experiments, each experiment consisting of 5 replicates of each agent tested.

Figure 6

Cross resistance patterns for Pt (II) spermine and Pd (II) spermine in A2780/CP cisplatin resistant cells. Comparison of cytotoxicity in A2780 vs. A2780/CP cells for (A) cisplatin, (B) Pd-spermine and (C) Pt-spermine complexes. Data presented are an average of 2-3 separate experiments, each experiment consisting of 5 replicates of each agent tested.