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. 2013;8(2):e55651.
doi: 10.1371/journal.pone.0055651. Epub 2013 Feb 13.

Norspermidine and novel Pd(II) and Pt(II) polynuclear complexes of norspermidine as potential antineoplastic agents against breast cancer

Tânia Magalhães Silva  1 , Sara Andersson, Sunil Kumar Sukumaran, Maria Paula Marques, Lo Persson, Stina Oredsson
Affiliations

Norspermidine and novel Pd(II) and Pt(II) polynuclear complexes of norspermidine as potential antineoplastic agents against breast cancer

Tânia Magalhães Silva et al. PLoS One. 2013.

Abstract

Background: New strategies are needed for breast cancer treatment and one initial step is to test new chemotherapeutic drugs in breast cancer cell lines, to choose candidates for further studies towards clinical use.

Methodology and findings: The cytotoxic effects of a biogenic polyamine analogue - norspermidine - and its trinuclear Pd(II) and Pt(II) complexes - Pd(3)NSpd(2) and Pt(3)NSpd(2), respectively - were investigated in one immortalized normal-like and three breast cancer cell lines. The normal-like MCF-10A cells were least sensitive to the compounds, while growth inhibition and cell death was observed in the cancer cell lines. Norspermidine and its Pd(II) complex were generally shown to have stronger antiproliferative effects than the corresponding Pt(II) complex. Moreover, both norspermidine and the Pd(II) complex reduced the cellular activity of the growth-related enzyme, ornithine decarboxylase (ODC) to a lower level than the Pt(II) complex in most of the cell lines examined. Treatment with norspermidine or the Pd(II) complex reduced the number of colonies formed in a soft agar assay performed with the breast cancer cell lines, indicating that these compounds reduced the malignancy of the breast cancer cells. The effect of norspermidine or the Pd(II) complex on colony formation was much stronger than that observed for the Pt(II) complex. The results from a new mammalian genotoxicity screen together with those of a single cell gel electrophoresis assay indicated that none of the drugs were genotoxic at a 25 µM concentration.

Main conclusions: Overall, norspermidine and its Pd(II) complex were shown to have strong antiproliferative effects. In comparison, the effects obtained with the Pd(II) complex were much stronger than that of the Pt(II) complex. The results obtained in the present study demonstrate that the trinuclear Pd(II) complex of norspermidine (Pd(3)NSpd(2)) may be regarded as a potential new metal-based drug against breast cancer, coupling a significant efficiency to a low toxicity.

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Conflict of interest statement

Competing Interests: The authors have the following interests. S.K. Sukumaran is employed by Anthem Biosciences Pvt. Ltd. Anthem Biosciences Genotox screen technology was used in this study and is patented as follows: Publication No. WO/2012/137186 was published on Oct. 11. Title of the invention: ″AN IN VITRO METHOD FOR HIGH THROUGHPUT SCREENING OF GENOTOXIC AGENTS IN EUKARYOTIC CELLS. Dr. Madhuri Subiah, Lakshmi Rajakrishna, and Salini Krishnan Unni from Anthem Bisociences assisted with designing and validating the Superscreen Assay and provided technical help. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Dose response effects of NSpd, Pd-NSpd or Pt-NSpd treatment.
The three breast cancer cell lines JIMT-1 (A–C), L56Br-C1 (D–F), MCF-7 (G–I) and the normal-like breast cell line MCF-10A (J–L) were used. Twenty-four h after seeding of cells in 96-well plates, the polyamine analogue and its complexes were added to the final concentrations shown in the figure and the cells were treated for 24, 48 and 72 h, before evaluation using an MTT assay. The results are expressed as % of control (n = 12 independent samples from two independent experiments) with bars representing ± SD.
Figure 2
Figure 2. Effects of NSpd, Pd-NSpd or Pt-NSpd treatment on the proliferation of JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells.
Twenty-four h after seeding of cells (0 h time of treatment in the figure), NSpd, Pd-NSpd or Pt-NSpd was added to give a final concentration of 25 µM (A–D) or 100 µM (E–H). Cells were harvested by trypsinization and counted in a hemocytometer. The results are presented as mean values (n = 3−6 independent samples from one or two independent experiments) and bars represent ± SD. When not visible, the bars are covered by the symbols. I–L: Cells were seeded and NSpd, Pd-NSpd or Pt-NSpd was added to the final concentration of 25 µM after 24 h of seeding. After 72 h of treatment, the drug-containing medium was aspirated and drug free culture medium was added. After an additional 72 h of incubation, cells were harvested by trypsinization and counted in a hemocytometer. These 7 days were defined as one treatment cycle. The total recovery time between each treatment was 96 h. The cells were reseeded at the same density as at the previous passage and treated with the same drug for the next treatment cycle. All together this was repeated for 5 treatment cycles. The data are presented as the total amount of cells (mean values (n = 3−6 samples from one or two independent experiments) and bars represent ± SD) that theoretically would have accumulated if all cells had been reseeded with a known cell density after each treatment cycle. When not visible, the bars are covered by the symbols. Please note that the y-axis has different scales for the different cell lines because of different rates of cell proliferation.
Figure 3
Figure 3. Effects of NSpd, Pd-NSpd or Pt-NSpd on the uptake of 3H-spermidine in JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells.
The cells were seeded in 12 well plates and incubated for 48 h, whereupon the polyamine analogue or its complexes were added to give the final concentrations shown in the figure. The concentration of 3H-spermidine used was 1 µM. The results are presented as mean values (n = 4 samples from two independent experiments) and bars represent ± SD.
Figure 4
Figure 4. ODC activity in JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells treated with NSpd, Pd-NSpd or Pt-NSpd.
Twenty-four h after seeding of cells (0 h time of treatment in the figure), NSpd, Pd-NSpd or Pt-NSpd was added to give a final concentration of 25 µM. The ODC activity was determined using a radiometric assay. The results are presented as mean values (n = 3 independent samples from one independent experiment) and bars represent ± SD.
Figure 5
Figure 5. Sub-G1 region and cell cycle phase distribution of JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells treated with NSpd, Pd-NSpd or Pt-NSpd.
Twenty-four h after seeding the cells, NSpd, Pd-NSpd or Pt-NSpd were added to give a final concentration of 100 µM. At 24, 48 and 72 h of treatment, both detached and attached cells were harvested, pooled and fixed in 70% ice-cold ethanol. The nuclei were stained with propidium iodide and the analysis was performed using flow cytometry. The results are presented as mean values (n = 3−6 independent samples from one or two independent experiments) and bars represent ± SD.
Figure 6
Figure 6. Intracellular accumulation of Pd-NSpd and Pt-NSpd in L56Br-C1 and MCF-10A cells.
At 72 h of treatment with 25 µM of Pd-NSpd and Pt-NSpd, cells were harvested, pooled and digested in HNO3. The supernatant was used for analysis of Pd and Pt by ICP-MS and the data used to calculate the intracellular Pd-NSpd and Pt-NSpd concentrations. The results are presented as mean values (n = 3 independent samples from one independent experiment) and bars represent ± SD.
Figure 7
Figure 7. Genotoxic effects of NSpd, Pd-NSpd or Pt-NSpd in genetically engineered reported-based HCT-p21-GADD-p53 cells.
The genetically engineered reporter-based HCT-p21-GADD-p53 cells carrying the DNA damage early sensors p21, GADD153 and p53 were seeded in 96-well plates and allowed to attach for 16 h. The p21 promotor was operatively linked to Renilla luciferase reporter gene, the GADD 153 promotor operatively linked to firefly luciferase reporter gene and the p53 response elements operatively linked to β-galactosidase reporter gene. Sixteen h after seeding, the polyamine analogue and its complexes were added to the final concentrations shown in the figure and incubated for 72 h. The samples were sampled and analyzed for Renilla luciferase, firefly luciferase and β-galactosidase. The results are presented as mean values (n = 3 independent samples from one independent experiment) and bars represent ± SD.
Figure 8
Figure 8. The single cell gel electrophoresis (SCGE) assay was used to evaluate DNA damage in L56Br-C1 cells.
Twenty-four h after seeding of L56Br-C1 cells, NSpd, Pd-NSpd or Pt-NSpd was added to give a final concentration of 25 µM. After 72 h of treatment, cells were harvested for SCGE analysis. The ethidium bromide-stained nucleoids were photographed and then examined using the Comet Score™ Freeware. A. Images of comets obtained by the SCGE assay. DNA damage results in comets with head and tail, whereas undamaged DNA results in a round head. B. Percentage DNA in tail on the x-axis versus tail length on the y-axis for individual cells. C. Tail moment TMOM (%DNA in tail multiplied by tail length) for individual cells. Data were collected from three independent experiments, n = 207 cells. D. Table showing the mean TMOM value of the 10% highest TMOM values i.e. 20 highest values ± SD. *p<0.05 compared to control; ***p<0.001 compared to control.
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Grants and funding

The authors also acknowledge financial support from the Portuguese Foundation for Science and Technology – SFRH/BD/46364/2008, projects PTDC/QUI/66701/2006 (co-financed by the European Community fund FEDER) and Pest-OE/Qui/UIOO700/2011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.