Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Mass spectrometry-based prediction of tumor suppressors and the effect of enolase 1 and ubiquitin C. EO = EO771 mammary tumor cells, CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, BML = BML284, Eno1 = Enolase 1, and Ubc = ubiquitin C. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. (A) List of 25 top tumor suppressor candidates identified by mass spectrometry-based proteomics analysis. (B) Reduction in MTT-based proliferation of EO771 mammary tumor cells by 9 recombinant proteins (5 μg/mL) in 48 h. (C) Levels of enolase 1 and ubiquitin C in β-catenin-overexpressing and BML284-treated EO771 CMs by ELISA. (D) Inhibition in the scratch-based migration of EO771 mammary tumor cells by enolase 1 and ubiquitin C in 24 h. (E) Expression of β-catenin and enolase 1 in EO771 cells that were treated with AP-Ⅲ-a4, an inhibitor of enolase 1. (F&G) Repressive effects of AP-Ⅲ-a4 on the proliferation (in 48 h) and migration of EO771 cells (in 24 h) by β-catenin overexpressing iTS CM.

Article Snippet: Eno1 (500 ng/mL, MBS2009113), and Ubc (500 ng/mL, MBS2029484, MyBioSource, San Diego, California, USA) recombinant proteins were given to EO771 cells, and cells were incubated for 24 h. A pharmacological inhibitor of Eno1 (ENOblock - AP-III-a4, MBS385406, MyBioSource) and an inhibitor of E3 ubiquitin ligase (Pomalidomide, MBS8005710, MyBioSource) were applied to the cells for 24 h , .

Techniques: Mass Spectrometry, Ubiquitin Proteomics, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Inhibition, Migration, Expressing

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Effect of silencing enolase 1 and ubiquitin C. CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, siEno1 = Enolase 1 siRNA, siUbc = ubiquitin C siRNA, EO = EO771 mammary tumor cells, TR = TRAMP prostate cancer cells, and PA = PANC-1 pancreas cancer cells. The double asterisk indicates p < 0.01. (A) siRNA-mediated knockdown of enolase 1, and ubiquitin C in EO771 breast cancer cells. (B&C) Promotion of MTT-based proliferation, and scratch-based migration of EO771 breast cancer cells by enolase 1 and ubiquitin C siRNA-treated CMs in 2 days. (D-I) Effects of enolase 1 and ubiquitin C siRNAs. Silencing these two proteins significantly prevented the reduction in EdU-based proliferation and Transwell invasion of EO771, TRAMP, and PANC-1 cells by their own β-catenin-overexpressing iTS CMs in 2 days.

Article Snippet: Eno1 (500 ng/mL, MBS2009113), and Ubc (500 ng/mL, MBS2029484, MyBioSource, San Diego, California, USA) recombinant proteins were given to EO771 cells, and cells were incubated for 24 h. A pharmacological inhibitor of Eno1 (ENOblock - AP-III-a4, MBS385406, MyBioSource) and an inhibitor of E3 ubiquitin ligase (Pomalidomide, MBS8005710, MyBioSource) were applied to the cells for 24 h , .

Techniques: Ubiquitin Proteomics, Control, Knockdown, Migration

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Tumor selectivity and the involvement of CD44. A5 = MLO-A5 osteocytes, β-cat = β-catenin plasmids, BML = BML284, Eno1 = Enolase 1, Ubc = ubiquitin C. The signal and double asterisk indicates p < 0.01 and p < 0.05, respectively. (A) Comparison of MTT-based viability of four non-tumor cells (KTB34, KTB6, MC3T3, MSC) and four tumor cells (EO771, TRAMP, 4T1.2, PANC-1) in response to β-catenin-iTS CM and BML-iTS CM in 2 days. (B) Tumor selectivity of EO771 CM, 4T1.2 CM, TRAMP CM, and PANC-1 CM. MTT-based tumor selectivity is defined as the ratio of (reduction in tumor cells) to (reduction in non-tumor cells). The selectivity value above 1 indicates that MTT-based inhibition is more selective to tumor cells than non-tumor cells. Of note, N.D. = not defined since the viability of non-tumor cells is stimulated. (C&D) Tumor selectivity of Enolase 1 and ubiquitin C. (E) CD44 was co-immunoprecipitated with Eno1. The protein extracts of EO771 cells were incubated with anti-Eno1 antibody using the protein A/G beads. Immunoprecipitates and total cell lysates were analyzed by Western blotting with anti-CD44 and anti-Eno1 antibodies as indicated. (F&G) siRNA knockdown of CD44 suppressed Eno1-mediated inhibition of the proliferation of EO771 cells. (H) siRNA knockdown of CD44 suppressed Eno1-mediated downregulation of MMP9, Runx2, and Snail in EO771 cells.

Article Snippet: Eno1 (500 ng/mL, MBS2009113), and Ubc (500 ng/mL, MBS2029484, MyBioSource, San Diego, California, USA) recombinant proteins were given to EO771 cells, and cells were incubated for 24 h. A pharmacological inhibitor of Eno1 (ENOblock - AP-III-a4, MBS385406, MyBioSource) and an inhibitor of E3 ubiquitin ligase (Pomalidomide, MBS8005710, MyBioSource) were applied to the cells for 24 h , .

Techniques: Ubiquitin Proteomics, Comparison, Inhibition, Immunoprecipitation, Incubation, Western Blot, Knockdown

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Effects of enolase 1, ubiquitin C, and iTS CM on the expression of tumor-promoting and tumor-suppressing genes. CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, siEno1 = Enolase 1 siRNA, siUbc = ubiquitin C siRNA, EO = EO771 mammary tumor cells. (A&B) Expression of MMP9, Runx2, Snail, p53, and TRAIL in response to enolase 1 and ubiquitin C in EO771 breast cancer cells. (C&D) Expression of MMP9, Runx2, Snail, p53, and TRAIL in response to β-catenin-overexpressing iTS CM impaired by siRNAs specific to enolase 1 and ubiquitin C. (E) Expression of PDL1 in EO771 mammary tumor cells in response to β-catenin-overexpressing iTS CM, enolase 1, and ubiquitin C. (F&G) Expression of MMP9, Runx2, Snail, p53, TRAIL, and caspase 3 in EO771 mammary tumor cells in response to β-catenin-overexpressing pre-treatment tumor cell-derived CM. (H) Low survival for cancer patients with a high transcript level of MMP9, Runx2, or Snail. (I) Proposed regulatory mechanism to inhibit tumor progression by iTS-CM. According to the mechanism, β-catenin-overexpressing iTS cells secrete ubiquitin C (Ubc), enolase 1 (Eno1), p53, and Trail. They suppress the progression of tumor cells by downregulating MMP9, Runx2, Snail, and PDL1, while upregulating cleaved-caspase 3. It should be noted that Eno1 interacts with CD44 and inhibits MMP9, Runx2, and Snail.

Article Snippet: Eno1 (500 ng/mL, MBS2009113), and Ubc (500 ng/mL, MBS2029484, MyBioSource, San Diego, California, USA) recombinant proteins were given to EO771 cells, and cells were incubated for 24 h. A pharmacological inhibitor of Eno1 (ENOblock - AP-III-a4, MBS385406, MyBioSource) and an inhibitor of E3 ubiquitin ligase (Pomalidomide, MBS8005710, MyBioSource) were applied to the cells for 24 h , .

Techniques: Ubiquitin Proteomics, Expressing, Control, Derivative Assay