Inhibition of tumor angiogenesis and growth by nanoparticle-mediated p53 gene therapy in mice

Abstract

Mutation of the p53 tumor suppressor gene, the most common genetic alteration in human cancers, results in more aggressive disease and increased resistance to conventional therapies. Aggressiveness may be related to the increased angiogenic activity of cancer cells containing mutant p53. To restore wild-type p53 function in cancer cells, we developed polymeric nanoparticles (NPs) for p53 gene delivery. Previous in vitro and in vivo studies demonstrated the ability of these NPs to provide sustained intracellular release of DNA, thus sustained gene transfection and decreased tumor cell proliferation. We investigated in vivo mechanisms involved in NP-mediated p53 tumor inhibition, with focus on angiogenesis. We hypothesize that sustained p53 gene delivery will help decrease tumor angiogenic activity and thus reduce tumor growth and improve animal survival. Xenografts of p53 mutant tumors were treated with a single intratumoral injection of p53 gene-loaded NPs (p53NPs). We observed intratumoral p53 gene expression corresponding to tumor growth inhibition, over 5 weeks. Treated tumors showed upregulation of thrombospondin-1, a potent antiangiogenic factor, and a decrease in microvessel density vs controls (saline, p53 DNA alone, and control NPs). Greater levels of apoptosis were also observed in p53NP-treated tumors. Overall, this led to significantly improved survival in p53NP-treated animals. NP-mediated p53 gene delivery slowed cancer progression and improved survival in an in vivo cancer model. One mechanism by which this was accomplished was disruption of tumor angiogenesis. We conclude that the NP-mediated sustained tumor p53 gene therapy can effectively be used for tumor growth inhibition.

Figure 1. Tumor Growth Inhibition by p53NPs

(A) Tumor growth after a single intratumoral injection (day 0) of p53NP, p53DNA, p53(-)NP, or saline (n = 8 per group). (B) Area under the curve by day 45 (time of first animal death) demonstrates an overall reduction in tumor growth in the p53NP group compared to each control group. Statistical analysis was performed using Student’s t test. *p ≤ 0.05.

Figure 2. Improved Animal Survival with p53NP

Kaplan-Meier plot shows improvement in animal survival after a single intratumoral injection with p53NPs compared to controls. Log-rank test of p53NP and each control group yields p ≤ 0.05 (*).

Figure 3. Intratumoral p53 Gene Expression

Expression of p53 relative to saline controls for p53NP, p53DNA, and p53(-)NP groups at 1, 3, and 5 weeks post treatment as determined by real-time RT-PCR. Data represented as mean ± standard deviation (n = 1–4 tumors, triplicate samples). Tumors treated with p53NP demonstrated higher levels of p53 expression, which was sustained over 5 weeks.

Figure 4. Intratumoral thrombospondin (TSP-1) protein expression

Immunohistochemical analysis of TSP-1 in tumors 1, 3, and 5 weeks post treatment with with saline (A-C), p53(-)NP (D-F), p53DNA (G-I), and p53NP (J-L).

Figure 5. Intratumoral expression of angiogenesis marker CD31

Immunohistochemical analysis for CD31 (marker of angiogenesis) after 1, 3, and 5 weeks post treatment with saline (A-C), p53(-)NP (D-F), p53DNA (G-I), and p53NP (J-L). Arrows indicate blood vessels (positive stain for CD31).

Figure 6. Intratumoral microvessel density

Quantification of microvessels (represented as number of blood vessels per field) in tumors after 1, 3, and 5 weeks post treatment with p53NP, saline, p53DNA, p53(-)NP. Tumors treated with p53NPs were less vascular compared to controls at all time points (*p ≤ 0.05).

Figure 7. Apoptosis in tumors

TUNEL staining for apoptotic cells (arrows) 1, 3, and 5 weeks post treatment with saline (A-C), p53(-)NP (D-F), p53DNA (G-I), and p53NP (J-L).