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The use of HIV protease inhibitors (PIs) is associated with the “HIV protease-induced lipodystrophy syndrome”. PIs have been shown to suppress preadipocyte differentiation and promote adipocyte death by decreasing expression and altering nuclear localization of key adipogenic transcription factors including SREBP-1 and PPARγ. Because of the lipolytic effects of PIs, we investigated their impact on liposarcoma clonogenicity using the human SW872 liposarcoma cell line. SW872 cells were treated for 4 hours (h) with increasing concentrations, 2.5μM, 10 μM and 20 μM of the PIs: nelfinavir (NFV), ritonavir, indinavir and saquinavir; colonies were scored at 7-10 days. Clonogenic assays using only NFV were also performed with human HT1080 fibrosarcoma and 293 embryonic kidney cell lines to evaluate PI inhibition in non-lipid cell lines. Both NFV and ritonavir inhibited SW872 clonogenicity. NFV selectively inhibited SW872 clonogenicity at clinically achievable concentrations as low as 2.5 μM. To determine whether the inhibition of clonogenicity is mediated by apoptosis, fluorescence-activated cell sorting (FACS) analysis for Annexin V was performed. FACS analysis demonstrated dose-dependent SW872 apoptosis with NFV. To determine the mechanism(s) of inhibition of liposarcoma clonogenicity and induction of apoptosis, expression of SREBP-1 and PPARγ were evaluated in SW872 cells after incubation for 24 h with the NFV concentrations from the clonogenicity experiments. Similarly, these protein levels were examined temporally after exposure to 10μM of NFV for 48, 72 and 96 h. Both increasing the concentrations of NFV and prolonging the duration of treatment with NFV were associated with increased expression of both the precursor (125-kDa) and the active (68-kDa) forms of SREBP-1. Additionally, the differential expression of the precursor and active forms of SREBP-1 suggests inhibition of its proteolytic processing. Although increased expression was seen with SREBP-1, levels of PPARγ did not change with dose or duration of exposure to NFV despite the fact that PPARγ is a target gene of SREBP-1. Our results demonstrate that NFV selectively inhibits SW872 clonogenicity by inducing dose-dependent apoptosis at clinically relevant doses. Surprisingly, the mechanism underlying this observation does not appear to result from downregulation of the adipogenic transcription factors SREBP-1 and PPARγ as proposed for the HIV protease-induced lipodystrophy syndrome. Rather, our results demonstrate upregulation of SREBP-1 and suggest inhibition of its proteolytic cleavage. Interestingly, no downstream effect on PPARγ was observed. Further investigation is necessary to establish the signaling pathways linking NFV to liposarcoma-induced apoptosis, and thereby exploit its antitumor potential in the clinic.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]