Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 1. Adipose leptin mediates cell proliferation in androgen-independent TRAMP-C1 prostate cancer cells that express leptin receptor. (A) mRNA expression of the two signaling- competent leptin receptor isoforms muOB-Ra and muOB-Rb was profiled in TRAMP-C1 cells. RT-PCR analysis identified expression of these isoforms with primers that detect each of them specifically (muOB-Ra and muOB-Rb). PCR also amplified the common extracellular portion of the two isoforms (muOB-R). GAPDH served as a control. Presented are the reproducible results from one of the four repeated experiments. The experiment was performed using three independently prepared samples (samples 1, 2 and 3): three PCR were carried out simultaneously with each primer set, and PCR products were analyzed on the same gel. Three PCR showed consistent results with all primer sets. (B) TRAMP-C1 cells were serum-starved for 48 h and stimulated with DHT or leptin at indicated concentrations for 20 h. [3H] thymidine incorporation was measured during the last 5 h. Ethanol (Et) was used as a vehicle control for DHT. Values represent the mean±SD of quadruplicate samples of a representative experiment. ⁎Pb 0.0005 versus the controls (open bars). (C) Fat extracts were prepared from low-fat (Low) and high-fat (High) diet-fed male C57BL/6J mice, as well as regular diet-fed ob/ob mice (ob/ob), as described in Materials and methods. TRAMP-C1 cells were deprived of serum for 24 h and treated for 5 days with these fat extracts using serum-free medium as a control. Cell viability was measured by the enzymatic reduction of MTT (OD 550-670 nm) during the last 3 h. Values represent the mean±SD of quadruplicate samples of a representative experiment. ⁎Pb 0.01, ⁎⁎Pb 0.0005. (D) Serum- starved TRAMP-C1 cells were incubated for 5 days with the control serum-free medium (open bars) or fat extracts from high-fat diet-fed male C57BL/6J mice (solid bars) in the absence and presence of indicated concentrations of anti-leptin neutralizing antibody or non-specific mouse IgG; and cell viability was evaluated by the MTT assay. Values represent the mean±SD of quadruplicate samples of a representative experiment. ⁎P= 0.002. (E) Conditioned medium was prepared from in vitro differentiated human primary adipocytes as described before [9]. Serum-deprived DU145 cells were treated for 20 h with the control serum-free medium (open bars) or adipocyte condition medium (solid bars) in the absence and presence of anti-leptin neutralizing antibody or non-specific mouse IgG at indicated concentrations. [3H] thymidine incorporation was measured during the last 5 h. Values represent the mean±SD of quadruplicate samples of a representative experiment. ⁎Pb 0.02.

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Incubation, MTT Assay, In Vitro

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 2. Leptin activates the JNK-c-Jun pathway biphasically in androgen-independent DU145 prostate cancer cells. DU145 cells were serum-deprived for 24 h and incubated in serum-free medium (lanes 1, 8, and 13) or the serum-free medium containing 12.5 μg/ml leptin (lanes 2–7, 9–12, and 14–18) for indicated periods. (A) Cell lysates (250 μg protein) were subjected to the in vitro JNK assay with N-terminal c-Jun fusion protein as a substrate. Phosphorylation of the substrate protein on Ser-63 was detected by Western blot analysis using the specific antibody (Substrate (Ser-63)). To assess total JNK protein levels, cell lysates (50 μg protein) were applied to Western blot analysis using anti-JNK antibody that detects both active and inactive forms of JNK (p54 JNK and p46 JNK). Membranes probed with this antibody were stripped and re-probed with anti- GAPDH antibody to normalize variations in sample loading (GAPDH). (B) Cell lysates (50 μg protein) were analyzed by Western blotting. c-Jun phosphorylation at Ser-63 and-73 was assessed using anti-phospho-c-Jun (Ser-63) and (Ser-73) antibodies (p-c- Jun (Ser-63) and p-c-Jun (Ser-73)). To determine total c-Jun protein levels, membranes probes with these antibodies were stripped and re-probed with anti-c-Jun antibody that recognizes both phosphorylated and non-phosphorylated forms of c-Jun (c-Jun). The same membranes were stripped again and re-hybridized with anti-GAPDH antibody to normalize variations in sample loading (GAPDH). In some gels, a lane is removed and the remaining lanes are spliced together. Such alterations are indicated by leaving a white line between the gel pieces.

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Incubation, In Vitro, Phospho-proteomics, Western Blot

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 3. Leptin biphasically stimulates the DNA binding activity and Tyr-705 and Ser-727 phosphorylation of STAT3 in androgen-independent DU145 prostate cancer cells. Following 24-h serum starvation, DU145 cells were incubated in serum-free medium in the absence (lanes 1, 8, 13, and 19) and presence (lanes 2–7, 9–12,14–18, and 20–22) of 12.5 μg/ml leptin for indicated periods. (A) To assay STAT3 DNA binding activity, cell lysates (10 μg protein) were subjected to EMSA using 32P-end-labeled M67-SIE as a probe. The STAT3–DNA complex was confirmed by the supershift assay using anti-STAT3 antibody (lanes 19–22). A lane is removed from one of the gels, and the remaining lanes are spliced together. This alteration is indicated by putting a white line between the gel pieces. STAT3, STAT3–DNA complex; STAT3 Ab, anti-STAT3 antibody; SS, STAT3–DNA complex supershifted by the anti-STAT3 antibody; NS, non-specific. (B) Cell lysates (50 μg protein) were applied to Western blot analysis. STAT3 phosphorylation at Tyr-705 and Ser-727 was assessed with anti-phospho- STAT3 (Tyr-705) and (Ser-727) antibodies (p-STAT3 (Tyr-705) and p-STAT3 (Ser-727)). To determine total amounts of STAT3 protein, membranes hybridized with these antibodies were stripped and re-hybridized with anti-STAT3 antibody that detects both phosphorylated and non-phosphorylated forms of STAT3 (STAT3). The same membranes were stripped again and re-hybridized with anti-GAPDH antibody to normalize variations in sample loading (GAPDH).

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Binding Assay, Activity Assay, Phospho-proteomics, Incubation, Labeling, Western Blot

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 4. Leptin stimulates Akt phosphorylation at Ser-473 in a biphasic manner in androgen-independent DU145 prostate cancer cells. DU145 cells were serum-deprived for 24 h and incubated in serum-free medium in the absence (lanes 1, 8, and 13) and presence (lanes 2–7, 9–12, and 14–18) of 12.5 μg/ml leptin for indicated periods. Akt phosphorylated at Ser-473 was immunoprecipitated in cell lysates (1 mg protein) with immobilized anti-Akt (1G1) antibody and detected by Western blot analysis using anti- phospho-Akt (Ser-473) antibody (p-Akt (Ser-473)). To determine total levels of Akt protein, cell lysates (50 μg protein) were applied to Western blot analysis using anti-Akt antibody that recognizes both phosphorylated and non-phosphorylated forms of Akt (Akt). Membranes probed with this antibody were stripped and re-probed with anti- GAPDH antibody to normalize variations in sample loading (GAPDH). In some gels, a lane is removed and the remaining lanes are spliced together. Such alterations are indicated by putting a white line between the gel pieces.

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 5. JNK mediates the leptin-stimulated DNA binding activity and phosphorylation of STAT3 in androgen-independent DU145 prostate cancer cells. Following 24-h serum deprivation, DU145 cells were treated with 12.5 μg/ml leptin for 15 or 120 min or 100 ng/ml IL-6 for 15 min with and without pretreatment with Me2SO (vehicle control) or 10 μM SP600125 (pharmacological JNK inhibitor) for 30 min. Cells without any treatment (lanes 1, 7 and 13) and treated with Me2SO alone (lanes 2, 8 and 14) were included as controls. (A) To assess STAT3 DNA binding activity, cell lysates (10 μg protein) were subjected to EMSA with 32P-end-labeled M67-SIE as a probe. STAT3, STAT3–DNA complex; NS, non-specific. (B) Cell lysates (50 μg protein) were analyzed by Western blotting. STAT3 phosphorylation at Tyr-705 and Ser-727 was determined using anti-phospho-STAT3 (Tyr-705) and (Ser-727) antibodies (p-STAT3 (Tyr-705) and p-STAT3 (Ser-727)). To measure total STAT3 protein levels, membranes hybridized with these antibodies were stripped and re-hybridized with anti-STAT3 antibody that recognizes both phosphorylated and non-phosphorylated forms of STAT3 (STAT3). The same membranes were stripped again and re-hybridized with anti-GAPDH antibody to normalize variations in sample loading (GAPDH).

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Binding Assay, Activity Assay, Phospho-proteomics, Control, Labeling, Western Blot

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 6. The PI3K-Akt pathway mediates leptin-stimulated androgen-independent cell proliferation in DU145 cells. (A) Androgen-independent DU145 prostate cancer cells were serum-starved for 24 h and treated with 12.5 μg/ml leptin for 5 min with and without pretreatment with Me2SO (vehicle control) or 5 μM LY294002 (pharmacolo- gical PI3K inhibitor) for 30 min. Cells without any treatment (lanes 1 and 2) and treated with Me2SO alone (lanes 3 and 4) served as negative controls. Ser-473-phosphorylated Akt was immunoprecipitated in cell lysates (1 mg protein) with immobilized anti-Akt (1G1) antibody and assessed by Western blot analysis using anti-phospho-Akt (Ser-473) antibody (p-Akt (Ser-473)). To determine total Akt protein levels, cell lysates (50 μg protein) were subjected to Western blot analysis using anti-Akt antibody that detects both phosphorylated and non-phosphorylated forms of Akt (Akt). Membranes probed with this antibody were stripped and re-probed with anti-GAPDH antibody to normalize variations in sample loading (GAPDH). (B) After 48-h serum deprivation, DU145 cells were incubated for 20 h in serum-free medium in the absence (open bars) and presence (solid bars) of 12.5 μg/ml leptin with and without pretreatment with Me2SO or 5 μM LY294002 for 30 min. Cell proliferation was measured by [3H] thymidine incorporation during the last 5 h. Values represent the mean±SD of quadruplicate samples of a representative experiment. ⁎Pb 0.005 versus the controls (open bars).

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Control, Immunoprecipitation, Western Blot, Incubation

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 7. JNK is indispensable for leptin-stimulated Akt phosphorylation at Ser-473 in androgen-independent DU145 prostate cancer cells. DU145 cells were deprived of serum for 24 h and treated with 12.5 μg/ml leptin for 5 min or 4 h or 100 ng/ml IGF-I for 15 min with and without pretreatment with Me2SO (vehicle control) or 10 μM SP600125 (pharmacological JNK inhibitor) for 30 min. Cells without any treatment (lanes 1, 7 and 13) and treated with Me2SO alone (lanes 2, 8 and 14) were included as controls. Ser-473- phosphorylated Akt was immunoprecipitated in cell lysates (1 mg protein) with immobilized anti-Akt (1G1) antibody and detected by Western blot analysis using anti- phospho-Akt (Ser-473) antibody (p-Akt (Ser-473)). To determine total levels of Akt protein, cell lysates (50 μg protein) were subjected to Western blot analysis using anti- Akt antibody that recognizes both phosphorylated and non-phosphorylated forms of Akt (Akt). Membranes probed with this antibody were stripped and re-probed with anti-GAPDH antibody to normalize variations in sample loading (GAPDH).

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Phospho-proteomics, Control, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 8. Leptin activates JNK, STAT3 and Akt pathways in androgen-independent prostate cancer TRAMP-C1 cells. TRAMP-C1 cells were starved of serum for 24 h and stimulated with 12.5 μg/ml leptin for indicated periods. (A) Cell lysates were subjected to the in vitro JNK assay with N-terminal c-Jun fusion protein as a substrate (Substrate (Ser-63)) and Western blot analysis using anti-phospho-c-Jun (Ser-63) and (Ser-73) antibodies (p-c-Jun (Ser-63) and p-c-Jun (Ser-73)). The membrane probed with anti-phospho-c-Jun (Ser-63) antibody was stripped and re-probed with anti-c-Jun antibody to normalize c-Jun phosphorylation to total c-Jun protein levels (c-Jun). (B) Left panel, Cell lysates were applied to EMSA to assess STAT3 DNA binding activity using 32P-end-labeled M67-SIE as a probe. STAT3, STAT3–DNA complex. Right panel, STAT3 phosphorylation on Tyr-705 and Ser-727 was determined in cell lysates by Western blot analysis using anti-phospho-STAT3 (Tyr-705) and (Ser-727) antibodies (p-STAT3 (Tyr-705) and p-STAT3 (Ser-727)). To normalize STAT3 phosphorylation to total STAT3 protein levels, the membrane hybridized with anti-phospho-STAT3 (Tyr-705) antibody was stripped and re-hybridized with anti-STAT3 antibody (STAT3). (C) Akt phosphorylated at Ser-473 was immunoprecipitated with immobilized anti-Akt (1G1) antibody in cell lysates and analyzed by Western blotting using anti-phospho-Akt (Ser-473) antibody (p-Akt (Ser-473)). To normalize Akt phosphorylation to total amounts of Akt protein, cell lysates were subjected to Western blot analysis with anti-Akt antibody (Akt).

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: In Vitro, Western Blot, Membrane, Phospho-proteomics, Binding Assay, Activity Assay, Labeling, Immunoprecipitation

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig. 9. JNK mediates leptin-stimulated STAT3 DNA binding activity and phosphorylation, as well as Akt phosphorylation, in androgen-independent TRAMP-C1 prostate cancer cells. Following 24-h serum deprivation, TRAMP-C1 cells were treated with 12.5 μg/ml leptin for 15 min with and without pretreatment with Me2SO (vehicle control) or 10 μM SP600125 (pharmacological JNK inhibitor) for 30 min. Cells without any treatment (lane 1) and treated with Me2SO alone (lane 2) were included as controls. (A) To assess STAT3 DNA binding activity, cell lysates (10 μg protein) were subjected to EMSA with 32P-end- labeled M67-SIE as a probe. STAT3, STAT3–DNA complex; NS, non-specific. (B) Cell lysates (50 μg protein) were analyzed by Western blotting. STAT3 phosphorylation at Tyr-705 and Ser-727 was determined using anti-phospho-STAT3 (Tyr-705) and (Ser- 727) antibodies (p-STAT3 (Tyr-705) and p-STAT3 (Ser-727)). To measure total STAT3 protein levels, membranes hybridized with these antibodies were stripped and re- hybridized with anti-STAT3 antibody that recognizes both phosphorylated and non- phosphorylated forms of STAT3 (STAT3). The same membranes were stripped again and re-hybridized with anti-GAPDH antibody to normalize variations in sample loading (GAPDH). (C) TRAMP-C1 cells were deprived of serum for 24 h and treated with 12.5 μg/ ml leptin for 10 min with and without pretreatment with Me2SO or 10 μM SP600125 for 30 min. Cells without any treatment (lane 1) and treated with Me2SO alone (lane 2) were included as controls. Ser-473-phosphorylated Akt was immunoprecipitated in cell lysates (1 mg protein) with immobilized anti-Akt (1G1) antibody and detected by Western blot analysis using anti-phospho-Akt (Ser-473) antibody (p-Akt (Ser-473)). To determine total levels of Akt protein, cell lysates (50 μg protein) were subjected to Western blot analysis using anti-Akt antibody that recognizes both phosphorylated and non-phosphorylated forms of Akt (Akt). Membranes probed with this antibody were stripped and re-probed with anti-GAPDH antibody to normalize variations in sample loading (GAPDH).

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Binding Assay, Activity Assay, Phospho-proteomics, Control, Labeling, Western Blot, Immunoprecipitation

Journal: Biochimica et biophysica acta

Article Title: c-Jun NH(2)-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt.

doi: 10.1016/j.bbadis.2008.07.005

Figure Lengend Snippet: Fig.10. Model of leptin signaling network that mediates AIPC cell proliferation. Leptin activates JNK and JAK upon binding to the leptin receptor. After STAT3 activation is triggered by JAK-mediated Tyr-705 phosphorylation, JNK further stimulates and stabilizes STAT3 activation via Ser-727 phosphorylation. JNK also activates c-Jun through Ser-63 and -73 phosphorylation. Moreover, JNK mediates Akt activation through PI3K. Activation of these signaling pathways leads to expression of cell growth-relevant genes, resulting in androgen-independent cell proliferation. Transcription factor(s) that form a complex with c-Jun are unknown in leptin-stimulated AIPC cells. Signaling molecules downstream of Akt also need to be identified.

Article Snippet: The Mouse and Rat Leptin ELISA kit was bought from BioVendor Laboratory Medicine, Inc. (Modrice, Czech Republic).

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Protein-Protein interactions, Expressing