Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). MLO-Y4 cells were treated with C4-2B conditioned-media (CM) (Left), VCaP CM (Right) or MLO-Y4 CM as control for 24 hours. CM and whole cell lysates were collected and subjected to cytokine array. (B). Spots were subjected to densitometry using Image J. Results are shown as change in optical density of the cells subjected to prostate cancer CM relative to Control CM. (C). Ovals indicate the differentially regulated proteins. The overlap indicates the cytokines regulated similarly in both VCaP and C4-2B cells. (D). MLO-Y4 cells treated with MLO-Y4 CM, VCaP CM, C4-2B CM or PC3 CM. After 24 hours, total RNA was collected and subjected to RT-PCR for endoglin mRNA. (E). MLO-Y4 cells treated with MLO-Y4 CM, VCaP CM, C4-2B CM or PC3 CM. After 24 hours, total RNA and proteins was collected and subjected to immunoblot for GDF15 protein (upper left figure) and RT-PCR for GDF15 mRNA (lower left graph). After 24 hours of cell culture, supernatants were collected and subjected to immunoblot (upper right figure) and ELISA (lower right graph) for GDF15 protein. Data are shown as the mean±SD of 3 independent experiments. ** P < 0.01.

Article Snippet: Cells were then transduced with GDF15 shRNA lentiviral particles (Cat. sc-39799-V, Santa Cruz Biotechnology, Santa Cruz, CA) or shRNA control lentiviral particles (Cat. sc-108080, Santa Cruz Biotechnology).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). Migration (left figure) and invasion (right figure) were assessed using a transwell assay. C4-2B cells (1.5×105 per cells) were treated with the indicated concentrations of rGDF15 for 24 hours. The membrane was stained using differential Quick staining kit and photographed under light microscopy (20x). The numbers of migrating (no Matrigel on membrane) and invading (Matrigel present on membrane) cells were counted in five random fields for each insert. (B). C4-2B cells (1.5×104 per well) were treated with the indicated concentrations of rGDF15 for 48 hours. Cell numbers were quantified using a hemocytometer. (C). Validation of GDF15 knockdown in MLO-Y4 cells: Total cell protein and RNA were collected and subject to immunoblot (left figure) and real-time PCR (middle graph), respectively. Supernatant was collected from the indicated cells after 24 hours of incubation and subjected to ELISA for GDF15 (right graph). (D). Upper Figure: C4-2B cells (1.5×104 per well) were treated with the indicated CM from the genetically-modified MLO-Y4 cells. In some cases, rGDF15 (500 pg/ml) was added as indicated. After 48 hours, cell numbers were quantified using a hemocytometer. Lower Figure: C4-2B cells (1.5×104 per well) were treated with the indicated CM with the addition of either anti-GDF15 antibody or isotype antibody from the genetically-modified MLO-Y4 cells. After 48 hours, cell numbers were quantified using a hemocytometer. (E). Migration (upper figures) and invasion (lower figures) were assessed using a transwell assay. C4-2B cells (1.5×105 per cells) were treated with CM from untreated or C4-2B CM pre-treated MLO-Y4 cells with knockdown (or control) of GDF15. In some cases, rGDF15 was added to the CM as indicated. After 24 hours, the membrane was stained using differential Quick staining kit and photographed under light microscopy (20x). The numbers of migrating (no Matrigel on membrane) and invading (Matrigel present on membrane) cells were counted in five random fields for each insert. Data are shown as the mean±SD of 3 independent experiments. * P < 0.05; ** P < 0.01. (F). Total protein and RNA from the indicated cell lines were subjected to immunoblot (left figure) and real time PCR (right graph) for GFRAL. The PCR data are shown as the mean±SD of 3 independent experiments.

Article Snippet: Cells were then transduced with GDF15 shRNA lentiviral particles (Cat. sc-39799-V, Santa Cruz Biotechnology, Santa Cruz, CA) or shRNA control lentiviral particles (Cat. sc-108080, Santa Cruz Biotechnology).

Techniques: Migration, Transwell Assay, Membrane, Staining, Light Microscopy, Biomarker Discovery, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Genetically Modified, Control

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). SCID mice injected with mixture of prostate cancer PC3-luc cells and siRNA (siGDF15 or siControl) into right tibia followed by an additional intratibial injection of siRNA at day 7. Tumor burden was evaluated using bioluminescent imaging (left graph and middle figure) and radiography (right figure). The tumor-laden tissue area is delineated by all 4 arrows within each radiograph. Results are shown as mean±SEM. * P < 0.05. (B). SCID mice injected with mixture of prostate cancer C4-2B-luc cells and siRNA (siGDF15 or siControl) into right tibia followed by an additional intratibial injection of siRNA at day 7. Tumor burden was evaluated using bioluminescent imaging (left and middle) and radiography (right). Results are shown as mean±SEM. * P < 0.05. (C). One set of mice for each tumor type and treatmen (n=5 per tumor and treatment) were sacrificed at day 14-post initial injection and the intratibial tumors were subjected to immunohistochemistry for Ki67, Caspase 3 and EGR1. Images (left) and quantification (right) of Ki67, Caspase 3 and EGR1 staining. * P < 0.05, ** P < 0.01. (D). One set of mice (n=5) were sacrificed at day 14 -post initial injection, the tibiae flash frozen, total RNA collected and subjected to qPCR for murine GDF15 and human EGR1 mRNA expression. ** P < 0.01. (E). A proposed model system for secreted GDF15 promoting prostate cancer progression. Prostate cancer cells disseminate to bone, interact with osteocytes to induce production and release of GDF15 into the bone microenvironment which enhances prostate cancer proliferation, migration and invasion resulting in progression of bone metastasis.. Furthermore, our finding of the GDF15 receptor, GFRAL, on the prostate cancer cells, suggests that GDF15 mediates its activity through GFRAL in the prostate cancer cells. Finally, GDF15 induced EGR1 signaling in the prostate cancer cells, suggesting that EGR1 may contribute to the pro-metastatic effects of GDF15. *The data for EGR1 activation was supported by our in vitro findings and consistent with our in vivo studies; however, it has not been evaluated critically in vivo as of yet.

Article Snippet: Cells were then transduced with GDF15 shRNA lentiviral particles (Cat. sc-39799-V, Santa Cruz Biotechnology, Santa Cruz, CA) or shRNA control lentiviral particles (Cat. sc-108080, Santa Cruz Biotechnology).

Techniques: Injection, Imaging, Immunohistochemistry, Staining, Expressing, Migration, Activity Assay, Activation Assay, In Vitro, In Vivo

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). Confirmation of siRNA-mediated GDF15 protein expression knockdown in mouse primary osteocytes cells by immunoblot (left figure) and ELISA (right graph). (B). SCID mice were subcutaneously injected with a C4-2B cells alone or a mixture of C4-2B cells and mouse primary osteocytes that had been pre-treated with siGDF15 or siControl. The ratio of cells injected was 10 C4-2B cells to 1 osteocyte. Subcutaneous tumor volume was measured using caliper. Results are reported as mean±SEM. (C). Subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl were stained using Ki67, Caspase 3 and EGR1. (D). Quantification of Ki67, Caspase 3 and EGR1 in subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl. ** P < 0.01. (E). EGR1 protein expression in subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl.

Article Snippet: Cells were then transduced with GDF15 shRNA lentiviral particles (Cat. sc-39799-V, Santa Cruz Biotechnology, Santa Cruz, CA) or shRNA control lentiviral particles (Cat. sc-108080, Santa Cruz Biotechnology).

Techniques: Expressing, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Staining

Journal: Biomedical Journal

Article Title: Caffeic acid phenethyl ester inhibits the growth of bladder carcinoma cells by upregulating growth differentiation factor 15

doi: 10.1016/j.bj.2021.10.006

Figure Lengend Snippet: Gene expression of GDF 15 in human bladder cells . The mRNA levels of GDF15 (A) and α-SMA (B) in bladder smooth muscle cells (HBdSMC), fibroblast cells (HBdSF), normal epithelial cells (HBdEC), and carcinoma cell lines (RT-4, HT1376, T24, and TSGH-8301) were determined by RT-qPCR assays. Data are expressed as the mean ratio of mRNA in relation to HJBdEC cells (n = 3). (C) The protein levels of α-SMA, UPK2, and β-actin in bladder cells as indicated were determined by immunoblot assays. (D) GDF15 secretions from bladder cells as determined by ELISA (n = 4).

Article Snippet: Cells were seeded into 6-well plates 1 day before the culture medium was replaced with RPMI-1640 medium plus 10% FCS and 5 μg/ml polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then transduced with GDF15 (sc-39798-V) or AMP-activated kinase (AMPK) α1/2 (sc-45312-V) shRNA transduction particles (Santa Cruz Biotechnology).

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Biomedical Journal

Article Title: Caffeic acid phenethyl ester inhibits the growth of bladder carcinoma cells by upregulating growth differentiation factor 15

doi: 10.1016/j.bj.2021.10.006

Figure Lengend Snippet: Effects of CAPE on GDF15 expression and proliferation of bladder carcinoma cells . (A) HT1376 and T24 cells were treated with various concentrations of CAPE as indicated for 24 h, then lysed, and the mRNA levels of GDF15 and β-actin were determined by RT-qPCR assays. Data are expressed as the mean ratio of mRNA relative to the control group (n = 3). (B) GDF15 protein levels in conditional media were determined by ELISA (n = 4). (C) T24 cells were treated with various concentrations of CAPE as indicated for 24 h and protein levels of GDF15 were determined by immunoblots. (D) The reporter activity of the GDF15 reporter vector in HT1376 cells treated with various dosages of CAPE for 24 h. Data are expressed as the mean percentage of luciferase activity relative to the mock-transfected group (n = 6). The mRNA (E) and protein (F) levels of target genes as indicated were determined by RT-qPCR and immunoblots. Data are expressed as the mean ratio of mRNA relative to the control group (n = 3). (G) Cell proliferation of T24 cells was determined by EdU assays after CAPE (30 μM) treatment for 24 h. Data are expressed as the mean percentage of Edu positive cells relative to the control group (DMSO-treated, n = 4). ∗∗ P < 0.01, ∗ P < 0.05.

Article Snippet: Cells were seeded into 6-well plates 1 day before the culture medium was replaced with RPMI-1640 medium plus 10% FCS and 5 μg/ml polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then transduced with GDF15 (sc-39798-V) or AMP-activated kinase (AMPK) α1/2 (sc-45312-V) shRNA transduction particles (Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Plasmid Preparation, Luciferase, Transfection

Journal: Biomedical Journal

Article Title: Caffeic acid phenethyl ester inhibits the growth of bladder carcinoma cells by upregulating growth differentiation factor 15

doi: 10.1016/j.bj.2021.10.006

Figure Lengend Snippet: Downregulation of cell proliferation and invasion by CAPE is GDF15-dependent in bladder carcinoma HT1376 cells . (A) HT_shCOL and HT_shGDF15 cells were treated with (+) or without (−) CAPE (30 μM) for 24 h. The protein levels of target genes, as indicated, were determined via immunoblotting. (B) HT_shCOL and HT_shGDF15 cells were treated with (+) or without (−) CAPE (30 μM) for 24 h and GDF15 secretions in the supernatants of culture media were determined by ELISA (n = 4). (C) HT_shCOL and HT_shGDF15 cells were treated with (+) or without (−) CAPE (30 μM) for 24 h, then the relative mRNA levels of GDF15, NDRG1, and maspin were determined by RT-qPCR assays (n = 3). (D) Cell proliferation of HT_shCOL and HT_shGDF15 cells after treated with CAPE (30 μM) for 48 h was determined by EdU assays (n = 4). (E) The quantitative result of EdU assays. (F) HT_shCOL and HT_shGDF15 cells were treated with 30 μM CAPE for 48 h and cell proliferation was determined by the CyQuant assay. The data were presented as the mean percentage compared with the control group (DMSO-treated, n = 8). (G) HT_shCOL and HT_shGDF15 cells were treated with CAPE (30 μM) for 24 h and the cell invasive ability was measured by the Matrigel invasion assay after 24 h of incubation (H) The quantitative result of invasion assays. The data were presented as the mean percentage compared with the control group (DMSO-treated, n = 3). ∗∗ P < 0.01, ∗ P < 0.05.

Article Snippet: Cells were seeded into 6-well plates 1 day before the culture medium was replaced with RPMI-1640 medium plus 10% FCS and 5 μg/ml polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then transduced with GDF15 (sc-39798-V) or AMP-activated kinase (AMPK) α1/2 (sc-45312-V) shRNA transduction particles (Santa Cruz Biotechnology).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, CyQUANT Assay, Control, Invasion Assay, Incubation

Journal: Biomedical Journal

Article Title: Caffeic acid phenethyl ester inhibits the growth of bladder carcinoma cells by upregulating growth differentiation factor 15

doi: 10.1016/j.bj.2021.10.006

Figure Lengend Snippet: CAPE induces the phosphorylation of ERK, JNK, and p38 to modulate the expressions of GDF15, NDRG1, and maspin in bladder carcinoma HT1376 cells . (A) HT1376 cells were treated with various concentrations of CAPE as indicated for 24 h and the mRNA levels of target genes as indicated were determined by RT-qPCR assays. (B) Protein levels of target genes, as indicated, were determined by immunoblot assays after treatment with various concentrations of CAPE as indicated for 16 h in HT1376 cells. The quantitative data were expressed as the intensity of protein bands of the target genes/β-actin relative to the control solvent-treated group (n = 3). The expressions of ERK, p-ERK (C, top), JNK, p-JNK (D, top), p38, and p-p38 (E, top) were determined by the immunoblots after 20 min of 30 μM CAPE treatment with (+) or without (−) pretreatment of the indicated MAPK inhibitors for 1 h in HT1376 cells. The protein level of GDF15, NDRG1, maspin, and β-actin of HT1376 cells after CAPE treatment with (+) or without (−) pretreatment with PD0325901 (C, bottom), SP600125 (D, bottom), or SB202190 (E, bottom). The quantitative data were expressed as the intensity of protein bands of the p-target gene/target gene or target genes/β-actin relative to the control solvent-treated group (n = 3). ∗∗ P < 0.01, ∗ P < 0.05.

Article Snippet: Cells were seeded into 6-well plates 1 day before the culture medium was replaced with RPMI-1640 medium plus 10% FCS and 5 μg/ml polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then transduced with GDF15 (sc-39798-V) or AMP-activated kinase (AMPK) α1/2 (sc-45312-V) shRNA transduction particles (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Quantitative RT-PCR, Western Blot, Control, Solvent

Journal: Biomedical Journal

Article Title: Caffeic acid phenethyl ester inhibits the growth of bladder carcinoma cells by upregulating growth differentiation factor 15

doi: 10.1016/j.bj.2021.10.006

Figure Lengend Snippet: CAPE upregulates the activities of the AMPKα1/2 signaling pathway in bladder carcinoma cells . (A) HT1376 cells were treated with various concentrations of CAPE for 24 h, and the protein levels of the AMPKα1/2 and p-AMPKα1/2 were determined using immunoblot assays. (B) The expression of AMPKα1 and AMPKα2 was determined by RT-qPCR in HT_shCOL and HT_shAMPKα1/2 cells (n = 3). (C) The expression of AMPKα1/2 and β-actin was determined by immunoblotting in HT_shCOL and HT_shAMPKα1/2 cells. The protein level of AMPKα1/2 and β-actin was determined by immunoblot assays (D) and the mRNA level of GDF15 was determined by RT-qPCR assays (E; n = 3) in HT_shCOL and HT_shAMPKα1/2 cells after (+) or without (−) 30 μM CAPE treatment. (F) The proliferation of HT_shCOL and HT_shAMPKα1/2 cells was determined by the EdU assays after (+) or without (−) 30 μM CAPE treatment for 24 h (n = 4). ∗∗ P < 0.01, ∗ P < 0.05.

Article Snippet: Cells were seeded into 6-well plates 1 day before the culture medium was replaced with RPMI-1640 medium plus 10% FCS and 5 μg/ml polybrene (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then transduced with GDF15 (sc-39798-V) or AMP-activated kinase (AMPK) α1/2 (sc-45312-V) shRNA transduction particles (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Quantitative RT-PCR