Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression.

Abstract

p53 mutations occur in almost half of all soft tissue sarcomas (STS) and may contribute to multidrug resistance (MDR) in patients with STS. Doxorubicin (Dox) is one of the most active single agents in STS but is less effective in STS with p53 mutations. The effect of reintroducing wild type (wt) p53 into STS cells harboring p53 mutations on the cytotoxicity of DOX in vitro and in vivo was studied. The following cell lines were used in this study: SKLMS-1 STS cells, which do not express wt p53; two wt p53 stable transfectant cells derived from SKLMS-1 cells; and SKLMS-1 transfectant cells from a p53 temperature-sensitive mutant that expresses wt p53 at 32 degrees C and mutant p53 at 38 degrees C. The cytotoxicity of Dox was examined by [3-(4,5-dimethylthiazzol-2-yl)-2,5-diphenltetrazolium] (MTT) and clonogenetic assay, and the effect of reintroducing wt p53 on tumor suppression by Dox was evaluated with a tumorigenicity assay. DNA fragmentation was used to detect apoptosis. MDR-1 P-glycoprotein (P-gp) expression was detected by Western blot and immunohistochemical analyses of protein levels and by Northern blot analysis of mRNA levels, respectively. The intracellular accumulation of Dox was detected by flow cytometric analysis. The 50% inhibitory concentration (IC(50)) of Dox for the SKLMS-1 wt p53 transfectants decreased 16-fold compared with SKLMS-1 parental cells expressing mutant p53. Colony formation of SKLMS-1 cells after Dox treatment also was inhibited by wt p53 reintroduction. The tumorigenicity of SKLMS-1 cells was inhibited by wt p53 reintroduction alone or by Dox treatment alone and was inhibited further when p53 introduction was combined with Dox treatment in severe combined immunodeficient mice. Although no difference in DNA fragmentation, Bax expression, or Bcl-2 expression was detected among wt p53 transfectants and parental SKLMS-1 cells after Dox treatment, MDR-1 P-gp expression was decreased in wt p53 transfectants compared with parental SKLMS-1 cells. Furthermore, higher intracellular accumulations of Dox were found in wt p53 transfectants than that in SKLMS-1 cells. Reintroduction of wt p53 into STS cells harboring p53 mutations can enhance their chemosensitivity to Dox through the inhibition of MDR-1 P-gp expression. Thus, the combination of p53 gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients with STS.