Cancer cells frequently show high constitutive activity of the antiapoptotic
transcription factor nuclear factor kappaB (
NF-kappaB), which results in their enhanced survival. Activation of
NF-kappaB classically depends on degradation of its inhibitor
IkappaBalpha by the
26s proteasome. Specific
proteasome inhibitors induce apoptosis in
cancer cells and, at nonlethal concentrations, sensitize cells to the cytotoxic effects of ionizing radiation and chemotherapeutic drugs. Recently, the
protease coded by the HIV-I virus has been shown to share cleavage activities with the
proteasome. For this reason, we investigated whether the HIV-I
protease inhibitor saquinavir can inhibit
NF-kappaB activation, block
26s proteasome activity in
prostate cancer cells, and promote their apoptosis. The effect of
saquinavir on LPS/IFN-gamma-induced activation of
NF-kappaB was assessed by gel-shift assays and by Western analysis of corresponding
IkappaBalpha-levels. Its effect on 20s and
26s proteasome activity was analyzed with a fluorogenic
peptide assay using whole cell lysates from LnCaP, DU-145, and PC-3
prostate cancer cells pretreated with
saquinavir for 9 h.
Proteasome inhibition in living cells was assessed using ECV 304 cells stably transfected with an expression plasmid for an
ubiquitin/green fluorescence
protein fusion
protein (ECV 304/10). Apoptosis was monitored morphologically and by flow cytometry.
Saquinavir treatment prevented LPS/IFN-gamma-induced activation of
NF-kappaB in RAW cells and stabilized expression of
IkappaBalpha. It inhibited 20s and
26s proteasome activity in lysates from LnCaP, DU-145, and PC-3
prostate cancer cells with an IC(50) of 10 micro M and caused the accumulation of an
ubiquitin/green fluorescence
protein fusion
protein in living ECV 304/10 cells. Incubation of PC-3 and DU-145
prostate cancer, U373
glioblastoma, and K562 and Jurkat
leukemia cells with
saquinavir caused a concentration-dependent induction of apoptosis. In the case of PC-3 and DU-145,
saquinavir sensitized the surviving cells to ionizing radiation. We conclude that
saquinavir inhibits
proteasome activity in mammalian cells as well as acting on the HIV-I
protease. Because
saquinavir induced apoptosis in human
cancer cells, HIV-I
protease inhibitors might become a new class of cytotoxic drugs, alone or in combination with radiation or
chemotherapy.