Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of IkappaBalpha

Abstract

Proteasome inhibitors are known to suppress the proteasome-mediated degradation of IkappaBalpha in stimulated cells. This results in the cytoplasmic retention of NFkappaB and its reduced nuclear transcriptional activity. In this study, we show that in the metastatic prostate cancer cells, the proteasome inhibitors exhibit a novel, previously unrecognized effect: they increase the cellular levels of IkappaBalpha, which then translocates to the nucleus, associates with the nuclear p65 NFkappaB, thus inhibiting the constitutive NFkappaB DNA binding activity and inducing apoptosis. The proteasome inhibition-induced nuclear translocation of IkappaBalpha is dependent on de novo protein synthesis, occurs also in other cell types, and does not require IkappaBalpha phosphorylation on Ser-32. Since NFkappaB activity is constitutively increased in many human cancers as well as in inflammatory disorders, the proteasome inhibition-induced nuclear translocation of IkappaBalpha could thus provide a new therapeutic strategy aimed at the specific inhibition of NFkappaB activity by the nuclear IkappaBalpha.

Figure 1. Proteasome inhibitor MG132 induces nuclear translocation of IκBα in prostate cancer PC-3 cells

(A) Western blotting of cytoplasmic (CE) and nuclear extracts (NE) prepared from PC-3 cells treated with increasing concentrations of MG132 for 24 hours, and analyzed by using IκBα, p65 and p50 NFκB antibodies. To confirm equal protein loading, the membranes were stripped and re-probed with actin antibody. The presence of cytoplasmic proteins in nuclear fraction was evaluated by re-probing the membrane with LDH antibody. Nuclear contamination in the cytoplasmic fraction was assessed by using lamin B specific antibody. Each lane corresponds to approximately 5×10⁴ cells. (B) Western blotting of CE and NE performed as described above, prepared from PC-3 cells treated with 50 μM MG132 for 0–24 hours. (C) Western blotting of whole cell extracts (WCE) prepared from unstimulated cells and cells treated with 50 μM MG132 for 0–24 hours. Each lane corresponds to approximately 2×10⁴ cells. (D) Proteasome activity measured in WCE prepared from PC-3 cells treated with increasing concentrations of MG132 for 24 hours. The 20S proteasomal activity is expressed as relative fluorescence units (RFU) of MG132-treated cells compared to untreated cells. The values represent the mean +/−SE of three independent experiments measured in duplicates.

Figure 2. The MG132-induced nuclear translocation of IκBα is associated with the inhibition of NFκB activity

(A) EMSA of NFκB and CREB DNA binding activities analyzed in nuclear extracts from PC-3 cells treated for 24 hours with increasing concentrations of MG132. (B) Specificity analysis of the constitutive NFκB and CREB activities in PC-3 cells. Nuclear extracts from untreated PC-3 cells were incubated with NFκB or CREB specific DNA probes alone or in the presence (+) of 30 molar excess of unlabeled wild type (wt) or mutant (mut) oligonucleotides. Antibodies used in supershift NFκB assays included antibodies to p50 and p65. (C) EMSA of NFκB and CREB activities analyzed in nuclear extracts from PC-3 cells treated with 50 μM MG132 for 0–24 hours. (D) Co-immunoprecipitation experiment from nuclear extracts of untreated or MG132-treated (50 μM, 24h) cells by using pre-immune IgG or IκBα-specific antibody. The western blots were analyzed with IκBα and p65 NFκB antibodies.

Figure 3. The MG132-Induced Nuclear Translocation of IκBα Results in the Induction of Apoptosis

(A) Western blotting of WCE prepared from PC-3 cells treated with increasing concentrations of MG132 for 24 hours, and analyzed by using Bcl-2, Bcl-xL, and actin antibodies. Each lane corresponds approximately to 2×10⁴ cells. (B) Apoptosis measured by the quantitative nucleosome enrichment assay in PC-3 cells treated 24 hours with increasing concentrations of MG132. (C) Western blotting of CE and NE prepared from PC-3 cells transfected with control and IκBα si RNA, followed by treatment with increasing concentrations of MG132 for 24 hours. The membranes were analyzed by using IκBα and actin antibodies. Each lane corresponds approximately to 4×10⁴ cells. (D) Apoptosis measured by the nucleosome enrichment assay in PC-3 cells transfected with control (full columns) and IκBα (empty columns) si RNA, followed by treatment with increasing concentrations of MG132 for 24 hours. The values represent the mean +/−SE of four independent experiments. Asterisks denote a statistically significant (P<0.01) inhibition.

Figure 4. De novo protein synthesis is required for the MG132-induced nuclear translocation of IκBα in PC-3 cells

Western blotting of CE and NE prepared from PC-3 cells treated with increasing concentrations of MG132 for 24 hours, in the absence and presence of 1h pre-treatment with CHX (10 μg/ml). The membranes were analyzed by using IκBα and actin antibodies. Each lane corresponds approximately to 5×10⁴ cells.

Figure 5. The MG132-induced nuclear translocation of IκBα is not dependent on IκBα phosphorylation by IκB kinase

(A) Western blotting of CE and NE prepared from PC-3 cells treated for 0–24 hours with control DMSO or 50 μM MG132, and analyzed by using Ser-32-phosho-IκBα (pIκBα) and IκBα antibodies. Each lane corresponds approximately to 5×10⁴ cells. (B) Western analysis of CE and NE prepared from PC-3 cells pre-treated for 1 hour with 10 μM IKK inhibitor VII (Calbiochem, 401486) or control DMSO, followed by 24 hour incubation with increasing concentrations of MG132. The samples were analyzed by using IκBα and actin antibodies; each lane corresponds approximately to 5×10⁴ cells. (C) Western analysis of cytoplasmic extracts prepared from PC-3 cells treated for 1 hour with 10 μM IKK inhibitor VII or control DMSO, followed by 0 – 30 minutes stimulation with 10 ng/ml TNF. The samples were analyzed by using IκBα and actin antibodies, and each lane corresponds approximately to 5×10⁴ cells. (D) Western analysis of CE and NE prepared from HeLa cells treated for 24 hours with increasing concentrations of MG132, and analyzed by using IκBα, Ser-32-phosho-IκBα (pIκBα) and actin antibodies. Each lane corresponds approximately to 5×10⁴ cells.

Figure 6. Current model of the regulation of nuclear IκBα translocation by proteasome inhibition