PAMAM-camptothecin conjugate inhibits proliferation and induces nuclear fragmentation in colorectal carcinoma cells

Abstract

Purpose: To synthesize and characterize a poly (amido amine) dendrimer-camptothecin (PAMAM-CPT) conjugate and evaluate its activity on human colorectal carcinoma cells (HCT-116).

Methods: The attachment of CPT to amine-terminated PAMAM was facilitated through a succinic acid-glycine linker. The conjugate was characterized for absence of small molecular weight impurities, size and drug content. Stability of the conjugate in PBS and growth media and its in vitro activity on HCT-116 were studied. Cell cycle arrest and nuclear fragmentation upon PAMAM-CPT treatment were investigated.

Results: The conjugate was stable under physiological pH (7.4) in PBS and in growth media (with 10% FBS) with minimal release of 4% and 6% drug, respectively, at 48 h. PAMAM-CPT inhibited proliferation of HCT-116 cells with an IC50 value of 1.6 ± 0.3 µM. The conjugate induced signs of cell cycle arrest with up to 68% of cells blocked in the G(2) phase. Confocal images of cells treated with PAMAM-CPT suggest nuclear fragmentation and formation of apoptotic bodies.

Conclusions: Results show that the PAMAM-CPT conjugate was active against colorectal cancer cells in vitro, inhibiting their growth and inducing nuclear fragmentation. Coupled with the ability to target macromolecular therapeutics to tumors, this conjugate shows promise for cancer chemotherapy.

Fig. 1

CPT released from PAMAM-CPT conjugate in PBS () and growth media (). 115 μM of the conjugate was incubated at 37°C, and aliquots were drawn at various time points over 48 h. Samples were later analyzed by HPLC. Control measurement shows bar with 100% equivalent of free CPT. Mean values±S.D. from three independent experiments are presented.

Fig. 2

Toxicity of different forms of CPT on HCT-116 cells. ▲ PAMAM G4.0; PAMAM-CPT; S-G-CPT; CPT. Cells were incubated with various concentrations of drugs for 48 h. Relative cell number was determined by a WST-8 assay. Mean values±S.D. from three independent experiments are presented. * indicates PAMAM G4.0 polymer alone treatment at corresponding conjugate equivalent concentrations.

Fig. 3

Changes in DNA content of HCT-116 cells due to incubation with different forms of CPT. Cells were incubated with 3×IC₅₀ concentrations of each drug for 24 h, fixed with ethanol and stained with propidium iodide. DNA content was measured by FACScan and data analyzed by ModFit software. Representative histograms from one experiment are shown.

Fig. 4

Alteration of cell cycle distribution in HCT-116 cells after treatment with 3×IC₅₀ concentrations of each analog. ■G₁; S; G₂. Cells were prepared, data acquired and analyzed as described in the legend of Fig. 3. Mean values±S.D. from three independent experiments are presented. Statistical comparisons between untreated and treated cells were performed. * indicates statistically significant data (p<0.05).

Fig. 5

Nuclear fragmentation and apoptotic body formation in HCT-116 cells due to CPT and PAMAM-CPT treatment. Cells were incubated with 3× IC₅₀ concentrations of CPT and PAMAM-CPT for 24 h, fixed and stained with DAPI. Cells were imaged using a confocal laser-scanning microscope Olympus FV1000. * indicates a cell undergoing mitosis in the untreated population. Arrows indicate apoptotic bodies. Bar in picture is 20 μm.

Scheme 1

Schematics of the three-step synthesis of PAMAM-CPT conjugate. CPT; Spacer (succinic acid-glycine); PAMAM dendrimer. For abbreviations, see abbreviations list.