Anti-tumor activity of N-trimethyl chitosan-encapsulated camptothecin in a mouse melanoma model

Abstract

Background: Camptothecin (CPT) has recently attracted increasing attention as a promising anticancer agent for a variety of tumors. But the clinical application is largely hampered by its extreme water insolubility and unpredictable side effect. It is essential to establish an efficient and safe protocol for the administration of CPT versus melanoma.

Methods: Camptothecin was encapsulated with N-trimethyl chitosan (CPT-TMC) through microprecipitation and sonication. Its inhibition effect on B16-F10 cell proliferation and induction of apoptosis was evaluated by MTT assay and flow cytometric analysis in vitro. The anti-tumor activity of CPT-TMC was evaluated in C57BL/6 mice bearing B16-F10 melanoma. Tumor volume, tumor weight and survival time were recorded. Assessment of apoptotic cells within tumor tissue was performed by TUNEL assay. Antiangiogenesis and antiproliferation effects of CPT-TMC in vivo were conducted via CD31 and PCNA immunohistochemistry, respectively.

Results: CPT-TMC efficiently inhibited B16-F10 cells proliferation and increased apoptosis in vitro. Experiment group showed significant inhibition compared with free CPT-treated group (81.3% vs. 56.9%) in the growth of B16-F10 melanoma xenografts and prolonged the survival time of the treated mice (P < 0.05). Decreased cell proliferation, increased tumor apoptosis as well as a reduction in angiogenesis were observed.

Conclusions: Our data suggest that N-trimethyl chitosan-encapsulated camptothecin is superior to free CPT by overcoming its insolubility and finally raises the potential of its application in melanoma therapy.

Figure 1

Inhibitory effect of CPT-TMC on B16-F10 cells proliferation in vitro. The proliferation of B16-F10 cells was assessed by the MTT assay. Data were assessed as percent cell viability in terms of media-only treated (non-treated) control cells at each drug concentration. It is clear that CPT-TMC caused a dose-dependent inhibition of proliferation in vitro. Means ± SD (n = 3). *P < 0.05

Figure 2

Induction of apoptosis on B16-F10 cells by CPT-TMC in vitro. Cellular apoptosis was verified by flow cytometric analysis. B16-F10 Cells were treated with (a) media-only, (b) TMC, (c) CPT, or (d) CPT-TMC, respectively. It is clear that the number of apoptotic cells in CPT-TMC and CPT treated group was significantly higher compared with other two groups. The apoptotic rate showed 62% in CPT-TMC-treated group versus 57.1% in CPT-treated group, 10% in TMC-treated group and 3.9% in media-only-treated group.

Figure 3

Anti-tumor efficacy of CPT-TMC in vivo. The tumor models were established in C57/BL6 mice (10/group) and then were treated with i.v. administration of 2.5 mg/kg CPT-TMC, 2.5 mg/kg free CPT, 25 mg/kg TMC, or NS twice per week, when tumors were palpable. (a) Tumor volume growth curve. Tumor sizes were measured every 3 days. CPT-TMC significantly inhibited tumor growth. There was a significant difference in tumor volume between CPT-TMC and control groups (P < 0.05). (b) Comparison of the tumor weight. At the third day after the last treatment, mice were sacrificed, and tumors were removed and weighed. Significant differences between CPT-TMC group and control groups are represented (*P < 0.05, **P < 0.01). Values are means ± SD. (c) Survival curve for tumor-bearing mice. A significant increase in survival in CPT-TMC-treated mice was also found when compared with the control groups (P < 0.05, by Log-rank test). And there was no statistical difference between TMC-treated mice and NS-treated mice (P > 0.05). (d) Lack of toxicity-dependent weight loss in tumor-bearing mice treated with CPT-TMC. There are no significant differences in weight among the four groups (P > 0.05). Values are means ± SD.

Figure 4

CD31, PCNA and TUNEL analyses for tumor tissue. (a) Tumor sections immunostained with an antibody against PCNA revealed that there were many strongly positive nuclei in control tumor tissues, whereas such nuclei were rare in tumor tissues of CPT-TMC-treated group. (b) Quantification of PCNA staining showed percentage of PCNA-positive nuclei in CPT-TMC-treated group was the lowest among the four groups (*P < 0.05, **P < 0.01). (c) Apoptosis of tumor tissues in different groups were calculated by TUNEL assays, which showed that CPT-TMC induced a significant enhancement of apoptotic cells in contrast to control therapies. (d) Quantification of TUNEL assay shows that apoptosis index of CPT-TMC-treated tumor was much higher than that of control groups (*P < 0.05, **P < 0.01). (e) Tumor sections immunostained with anti-CD31 antibody (brown) for angiogenesis assay. Representative sections were taken from tumor tissue of NS-treated, TMC-treated, CPT-treated and CPT-TMC-treated groups. (f) Histomorphometric assay for tumor microvessels revealed that MVD was significantly lower in CPT-TMC-treated group compared with the controls (*P < 0.05, **P < 0.01).