Peptide targeting of platinum anti-cancer drugs

Abstract

Besides various side effects caused by platinum anticancer drugs, they are not efficiently absorbed by the tumor cells. Two Pt-peptide conjugates; cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8) bearing the Asn-Gly-Arg (NGR) targeting sequence, a malonoyl linker, and low molecular weight miniPEG groups have been synthesized. The platinum ligand was attached to the peptide via the carboxylic end of the malonate group at the end of the peptide. The pegylated peptide is nontoxic and highly soluble in water. Platinum conjugates synthesized using the pegylated peptides are also water-soluble with reduced or eliminated peptide immunogenicity. The choice of carboplatin as our untargeted platinum complex was due to the fact that the malonate linker chelates platinum in a manner similar to that of carboplatin. Cell toxicity assay and competition assay on the PC-3 cells (CD13 positive receptors) revealed selective delivery and destruction of PC-3 cells using targeted Pt-peptide conjugates 7 and 8 significantly more than untargeted carboplatin. Platinum uptake on PC-3 cells was 12-fold more for conjugate 7 and 3-fold more for conjugate 8 compared to that of the untargeted carboplatin, indicating selective activation of the CD13 receptors and delivery of the conjugates to CD13 positive cells. Further analysis on effects of conjugates 7 and 8 on PC-3 cells using caspase-3/7, fluorescence microscopy, and DNA fragmentation confirmed that the cells were dying by apoptosis.

Figure 1

CNGRC model conjugate with platinum pro-drug

Figure 2. Cytotoxic effects of Pt complexes on PC-3 cells

Cyclic mPeg-CNGRC-mal (6), carboplatin, mixture of carboplatin and free peptide 9 (carboplatin + compound 9), Pt-peptide conjugates; cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8) on the proliferation of prostate cancer PC-3 (CD13 positive) cells. PC-3 cells were exposed to the compounds listed above and incubated for 48 h at different concentration. All points are a representative of mean +− SD of triplicate readings of the three independent experiment. *P = 0.0190 (carboplatin and conjugate 7); P = 0.0126 (carboplatin and conjugate 8); *P = 0.0407 (conjugate 7 and conjugate 8). The difference is statistically significant as P < 0.05 at 150 μM concentrations. Statistical difference was determined by student’s t test; one tailed by using Graph Pad Prism software version 5.0.

Figure 3. Role of the NRG peptide in cytoxicity

The effect of various concentrations of the free peptide 9 on 200 μM of carboplatin and conjugate 8. Two thousand PC-3 cells were incubated with various concentrations of the free peptide mPeg-CNGRC (9) and 200 μM of carboplatin and conjugate 8 for 48 h. All points are a representative of mean +− SD of triplicate readings of the three independent experiment.

Figure 4. Platinum uptake of PC-3 (CD13 positive) cancer cells

One million cells were incubated with 200 μM carboplatin or Pt-peptide conjugate 7 or 8 for 24 h. Pt levels in the washed cells were measured by atomic absorption spectroscopy as described in the Experimental section. All points are a representative of mean +− SD of triplicate readings of the three independent experiment.

Figure 5. JC-1 Dye staining of cells treated with Pt-compounds

Fluorescence microscopy of cells that have been treated with various compounds, incubated for 48 h at 37 °C and then imaged by JC-1 staining after a further 30 min of incubation. Image A shows PC-3 cells, treated with RPMI media only. Image B shows PC-3 cells, treated with 50% DMSO. Image C shows PC-3 cells, treated with 200 μM of Pt-peptide conjugate 8 in RPMI media. Images D and E shows PC-3 cells incubated with a mixture of carboplatin/free peptide 9 (1:50) and carboplatin only (200 μM), respectively. Scale bar: 10 μm.

Figure 6. DNA fragmentation assay

PC-3 cells were treated with 5 μl of 1Kb DNA marker (lane 1), medium only (Lane 2), and 100 μM each of compound 9 alone (lane 3), carboplatin alone (lane 4), compound 9 + carboplatin (lane 5), conjugate 7 (lane 6) and conjugate 8 (lane 7) for 48 h. DNA was extracted and internucleosomal cleavage visualized on a 1.5% agarose gel. Image is representative of three independent experiment.

Figure 7. Caspase activation as a result of Pt-drug treatment

The apo-ONE homogenous caspase-3/7 assay where PC-3 cells (10,000 cells/well) in triplicate were treated with medium only and 100 μM concentration of the compound 9, carboplatin alone, mixture of compound 9 and carboplatin, conjugate 7 and conjugate 8 for 24 h. After 24 h, the caspase activity was measured in the wells by adding 100 μl of the apo-ONE caspase-3/7 assay reagent. The cells were incubated for an additional hour at ambient temperature prior to recording fluorescence (485 Ex/527Em). The P values are statistically significant and calculated by student’s t-test; one tailed.

Figure 8

Predicted pathways of apoptosis by conjugate 7 or 8. The cell-extrinsic apoptosis is engaged through death receptors (DR4 and DR5) and cell-intrinsic apoptosis is triggered through mitochondrial.

Scheme 1

Coupling of CNGRC with di-tert-butyl 2-(3-aminopropyl)malonate.

Scheme 2

Platination reaction using Mal-Glut-CNGRC

Scheme 3

Synthesis of di-tert-butyl 2-(3-glutaricaminopropyl)malonate

Scheme 4

Formation of cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8).