Enhancement of cisplatin sensitivity in Lewis Lung carcinoma by liposome-mediated delivery of a survivin mutant

Abstract

Background: A high concentration of cisplatin (CDDP) induces apoptosis in many tumor cell lines. CDDP has been administered by infusion to avoid severe toxicity. Recently, it has been reported that changes in survivin expression or function may lead to tumor sensitization to chemical and physical agents. The aim of this study was to determine whether a dominant-negative mouse survivin mutant could enhance the anti-tumor activity of CDDP.

Methods: A plasmid encoding the phosphorylation-defective dominant-negative mouse survivin threonine 34-->alanine mutant (survivin T34A) complexed to a DOTAP-chol liposome (Lip-mS) was administered with or without CDDP in Lewis Lung Carcinoma (LLC) cells and in mice bearing LLC tumors, and the effects on apoptosis, tumor growth and angiogenesis were assessed. Data were analyzed using one-way analysis of variance(ANOVA), and a value of P < 0.05 was considered to be statistically significant.

Results: LLC cells treated with a combination of Lip-mS and CDDP displayed increased apoptosis compared with those treated with Lip-mS or CDDP alone. In mice bearing LLC tumors and treated with intravenous injections of Lip-mS and/or CDDP, combination treatment significantly reduced the mean tumor volume compared with either treatment alone. Moreover, the antitumor effect of Lip-mS combined with CDDP was greater than their anticipated additive effects.

Conclusion: These data suggest that the dominant-negative survivin mutant, survivin T34A, sensitized LLC cells to chemotherapy of CDDP. The synergistic antitumor activity of the combination treatment may in part result from an increase in the apoptosis of tumor cells, inhibition of tumor angiogenesis and induction of a tumor-protective immune response.

Figure 1

Induction of apoptosis in LLC cells by treatment with Lip-mS and CDDP. LLC cells were treated with NS (a), CDDP (b), Lip-null(c), Lip-mS (d), or Lip-mS+CDDP (e). Flow cytometric analysis revealed the proportion of sub-G1 cells (apoptotic cells) to be 8.7% (a), 8.3% (b), 9.0%(c)44.6% (d), and 62.6% (e), respectively.

Figure 2

Lip-mS enhanced the antitumor effects of CDDP in vivo. Mice bearing LLC tumors were treated with NS, CDDP, Lip-mS or Lip-mS +CDDP. Combination treatment reduced the mean tumor volume on day 16 when compared with the Lip-mS or CDDP treatment group (P < 0.05).

Figure 3

Western blot analysis of caspase-9 expression in different groups. Expression of caspase-9 was found in all groups. While no significant difference in the expression of anti-actin was found among them, caspase-9 was found to be expressed to a higher extent in Lip-mS + CDDP treatment groups as compared to NS, CDDP alone, Lip-mS alone treatment groups.

Figure 4

Combination of Lip-mS and CDDP enhanced the induction of apoptosis in vivo. Tissue sections from tumor-bearing mice treated with NS (a), CDDP (b), Lip-mS (c), or Lip-mS + CDDP (d) were stained with FITC-DUTP. Percent apoptosis was determined by counting the number of apoptotic cells and dividing by the total number of cells in the field (5 high power fields/slide). (A) Representative Field from each group. (B) Percent apoptosis in each group. Values were expressed as means ± SE. An apparent increase in the number of apoptotic cells was observed within tumors treated with a combination of Lip-mS and CDDP compared with the other treatments (P < 0.05).

Figure 5

Inhibition of intra-tumoral angiogenesis assayed by CD31 staining of microvessels. Vascularization within tumors was detected by an antibody to CD31; representative images were taken under a light microscope (×400) in randomly-selected fields. Tumors of the NS (a) and CDDP (b) treatment groups demonstrated high microvessel density, while those of the Lip-mS (c) and Lip-mS + CDDP (d) treatment groups showed apparent inhibition of angiogenesis.