Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. We described recently that tumor growth inhibiting cytostatic proline-rich polypeptide 1, (PRP-1) significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocytes culture. In this study we hypothesized the existence and regulation of a functional marker in cancer stem cells, correlated to peptides antiproliferative activity. Experimental results indicated that among significantly downregulated miRNA after PRP-1treatment was miRNAs 302c*. This miRNA is a part of the cluster miR302-367, which is stemness regulator in human embryonic stem cells and in certain tumors, but is not expressed in adult hMSCs and normal tissues. PRP-1 had strong inhibitory effect on viability of chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type). Unlike chondrosarcoma, in glioblastoma, PRP-1 does not have any inhibitory activity on cell proliferation, because in glioblastoma miR-302-367 cluster plays an opposite role, its expression is sufficient to suppress the stemness inducing properties. The observed correlation between the antiproliferative activity of PRP-1 and its action on downregulation of miR302c explains the peptides opposite effects on the upregulation of proliferation of adult mesenchymal stem cells, and the inhibition of the proliferation of human bone giant-cell tumor stromal cells, reported earlier. PRP-1 substantially downregulated the miR302c targets, the stemness markers Nanog, c-Myc and polycomb protein Bmi-1. miR302c expression is induced by JMJD2-mediated H3K9me2 demethylase activity in its promoter region. JMJD2 was reported to be a positive regulator for Nanog. Our experimental results proved that PRP-1 strongly inhibited H3K9 activity comprised of a pool of JMJD1 and JMJD2. We conclude that inhibition of H3K9 activity by PRP-1 leads to downregulation of miR302c and its targets, defining the PRP-1 antiproliferative role.
Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent (
We have described recently that tumor growth inhibiting cytostatic proline-rich polypeptide 1, PRP-1 (galarmin), statistically significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocyte culture (
The chondrosarcoma cells JJ012 were cultured in monolayer. The chondrosarcoma cell line JJ012 was provided by Dr Joel A. Block (Department of Rheumatology, Rush-Presbyterian St. Luke’s Medical Center, Chicago, IL, USA), then cultured and propagated in our laboratory. Media consisted of Dulbecco’s modified Eagle’s medium (DMEM/MEM), supplemented with F12, 10% fetal bovine serum (ATCC), 25 μg/ml ascorbic acid, 100 ng/ml insulin, 100 nM hydrocortisone, and 1% penicillin/streptomycin (Sigma-Aldrich). The cells were trypsinized and used either for the rapid cell proliferation assays or for the mRNA extraction and miRNA arrays. The control samples were not treated with the peptide, whereas 10 μg/ml PRP-1 was added to the experimental series. PRP-1 was synthesized in our laboratory.
Cell Proliferation kit, EMD Biosciences (QIA127), was used for this assay. Cells were seeded at 5×104 cells/100 μl culture in the multi-well plate and incubated overnight at 37°C in 5% CO2 incubator. The actual assay was performed the next day. The rapid cell proliferation assay is based on the activity of mitochondrial enzymes active in viable cells. PRP-1 was added to corresponding wells just after seeding, before overnight incubation. The colorimetric 96-well assay measures the colorful product formazan formed by WST-1 tetrazolium salt cleavage by the mitochondrial dehydrogenases. The formazan formation was then quantified by measuring the change in absorbance at 450 nm in a microplate reader. The activity of mitochondrial dehydrogenases is proportional to the cell number. No washing, harvesting, or solubilization steps are required. In this series of experiments, PRP-1 was added to corresponding wells immediately after seeding and cell attachment followed by overnight incubation according to the manufacturer’s instructions.
A-172, T98G, and U87MG glioblastoma cell lines (purchased from ATCC) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Cells were grown for 24 h at a density of 500 cells per well in 384-well plates. IBET-151, PRP peptide, or vehicle controls (DMSO for IBET-151, saline for PRP) were added directly into the wells. Each condition was tested in triplicate. After 72 h, a CellTiter-Glo Luminescent Cell Viability assay (Promega) was performed according to the manufacturer’s recommendations. Briefly, the CellTiter-Glo reagents were added to the wells and the plate was briefly centrifuged. The plate was incubated 15 min at room temperature, and luminescence was read using an EnVision Multilabel plate reader (Perkin-Elmer). Dose-response curves were analyzed in GraphPad prism and fitted using a non-linear regression analysis.
Upon confluency, the cells were trypsinized and seeded in 6-well cluster dishes at a concentration of 1×106 cells/ml. The experimental samples were treated with PRP-1 in corresponding concentrations, whereas control samples were not treated with the peptide. The cells were incubated for 24 h in a 5% CO2 incubator at 37°C. The following day, the cells were washed with ice-cold phosphate-buffered saline. A protease inhibitor was added to the cell lysis buffer (C2978; Sigma-Aldrich, St. Louis, MO, USA) in a 1:100 ratio. The cells were collected with a scraper and centrifuged at 15, 000 × g at 4°C. The supernatant was collected and the protein concentration was measured. The pellets were frozen at −80°C until loading on the gel (20 μg/lane). Polyacrylamide gel electrophoresis and western blotting reagents were supplied by Lonza, Inc. (Allendale, NJ, USA), and all the related procedures followed the company’s protocol. The catalog numbers for the reagents and the suppliers are listed below.
Pager Gold Precast Gels (59502; 10% Tris-Glycine; Lonza, Inc.); ECL reagent (RPN2109; GE Healthcare, Little Chalfont, UK); Western Blocker solution (W0138; Sigma-Aldrich); ProSieve Quad Color Protein marker (4.6-300 kDa, 00193837; Lonza, Inc.); 20× reducing agent for ProSieve ProTrack Dual Color Loading buffer (00193861; Lonza, Inc.); ProTrack Loading buffer (00193861; Lonza, Inc.); ProSieve ProTrack Dual Color Loading buffer EX running buffer (00200307; Lonza, Inc.); ProSieve EX Western Blot Transfer buffer (00200309; Lonza, Inc.); Immobilon®-P Polyvinylidene difluoride membranes (P4188; Sigma-Aldrich).
MIAMI cells were grown as previously described (REF: PMID: 15173316). Briefly, whole BM cells were plated at 1×105/cm2 in T75 flasks (Costar) in the presence of D-MEM low glucose, 3% FBS, 100 U/ml penicillin (Gibco), 1 mg/ml streptomycin (Gibco). The cells were incubated at 37°C in a 100% humidified atmosphere of 3% O2, 5% CO2, and 92% N2. Half of the medium was changed after a week; thereafter, half the medium was replaced twice a week. MIAMI cells were cultured to 40–50% confluence. For expansion, MIAMI cells were replated at a density of 100 cells/cm2 in fibronectin-coated vessels in 95% D-MEM-low glucose, 3% lot-selected FBS, and 100 U penicillin/1,000 U streptomycin (expansion medium) at 3% O2, with 50% of the medium changed twice a week.
MIAMI cells were plated in triplicate at 2,000 cells/cm2 in 6-well plates in expansion medium. The following day cells were supplemented with 1, 2, and 10 μg/ml PRP-1. At the end of each assay-day, 7, 10, and 14, 21 cells were rinsed with PBS and detached with trypsin-EDTA, and then counted with a Neubauer hemacytometer chamber.
Primary: rabbit polyclonal anti-CDK/2 (M2), cat no. sc-1632 Santa Cruz Biotechnologies; mouse monoclonal (9E10) anti-c-Myc, cat no. SC-40 Santa Cruz Biotechnologies; rabbit polyclonal anti-p-c-Myc (Thr58/Ser 62), cat no. Sc-8000R, Santa Cruz Biotechnologies; mouse monoclonal anti-SCML2 (SCMAD14a), cat no. ab51506 Abcam; rabbit polyclonal antip-Src (Tyr416), cat no. 2101S, Cell Signaling; rabbit polyclonal anti-Src antibody, cat no. 2108S, Cell Signaling, rabbit monoclonal anti-p27 Kip1 (D69C12) XP® cat no. 3686; Santa Cruz Biotechnology; mouse monoclonal p21 (F-5), cat no. sc-6246, Santa Cruz Biotechnologies; mouse monoclonal anti Nanog, clone 7F7-1, cat no. MABD24, EMD Millipore; rabbit polyclonal anti-Bmi-1 antibody, cat no. ab38295, Abcam. Mouse monoclonal anti-tubulin, cat no. T5168, Sigma. Secondary: anti-mouse IgG (A9044; Sigma-Aldrich); and goat anti-rabbit IgG peroxidase conjugate (A0545; Sigma-Aldrich).
All experiments were performed in triplicate, and P<0.05 was considered statistically significant. Data analysis was perform using one-way analysis of variance (ANOVA) unpaired t-test (GraphPad Prism; GraphPad Software, San Diego, CA, USA).
This study pursued the identification of functional marker in cancer stem cells, correlated to peptides antiproliferative activity by comparing different cell lines, where miR302-367 cluster either induces or inhibits stemness properties, as well as marrow-isolated adult multilineage inducible (MIAMI) cells that express embryonic stem cell markers. We have previously demonstrated antiproliferative effect of PRP-1 reaching 80–90% inhibition in human chondrosarcoma cells (
The BET bromodomain inhibitor IBET-151 reduced glioblastoma cellular ATP levels with potency similar to that we previously reported (IC50 = 4.8 μM in A-172 cells, 9.3 μM in T98G cells, and 0.764 μM in U87MG cells) (
MIAMI cells resemble primitive stem cells in their capacity to differentiate, at least
The embryonic stem cell marker Nanog is one of the targets for miR302-367 cluster and it is expressed in many cancers. Nanogs expression was substantially decreased in human JJ012 chondrosarcoma cell line after the treatment with PRP-1 (
Western blot analysis revealed that PRP-1 reduced c-Myc (oncogene target for miR302c) and phosphorylated p-c-Myc expression (
The peptide was tested for its effect on the other oncogene, Src (albeit, its not the target for miR302c) and its phosphorylated form. PRP-1 decreased Src protein levels, but not p-Src expression (
It was important to check the expression of cell cycle regulatory proteins with or without the peptide treatment. PPP-1 increased the expression levels of p27, and CDK2 (
In the present study, we have demonstrated that miR302c, part of miR302-367 cluster, downstream factor of the embryonic stem cell regulation network determined the antiproliferative activity of cytostatic PRP-1. The cluster is known as a potential stemness regulator in human embryonic stem cells and its expressed in certain tumors but not in adult hMSC and normal cells. Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. We described recently that tumor growth inhibiting proline-rich polypeptide 1 (PRP-1, galarmin) significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocyte culture. Among the miRNAs was miRNA302c* 6.46-fold downregulated after PRP-1 (10 μM) treatment (
We have reported that inhibition of human chondrosarcoma cells, including primary and JJ012 cells with PRP-1 reached >80% (
Somatic cells reprogram to an embryonic stem cell (ESC) comparable induced pluripotent stem (iPS) cell state upon forced expression of exogenously delivered transcription factors, however, expression of exogenous miR-302 cluster (without miR-367) is efficient in attaining a fully reprogrammed iPS state. Methyl-DNA binding domain protein 2, (MBD2), is an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to Nanog promoter elements preventing transcriptional activation. When miR-302 cluster (without miR367) was overexpressed, significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing Nanog expression was observed (
Glioblastoma cell lines A-172, T-98G and U-87. Cells were grown for 24 h at a density of 500 cells per well in 384-well plates. IBET-151, PRP peptide, or vehicle controls (DMSO for IBET-151, saline for PRP) were added directly into the wells. Each condition was tested in triplicate. After 72 h, a CellTiter-Glo Luminescent Cell Viability assay (Promega) was performed.
MIAMI cells. Whole bone marrow cells were plated at 1×105/cm2 in T75 flasks, MIAMI cells were replated at a density of 100 cells/cm2 in fibronectin-coated vessels in 95% D-MEM-low glucose, 5% lot-selected FBS, and 100 U penicillin/1,000 U streptomycin (expansion medium) at 3% O2, with 50–60% of the medium changed twice a week.
PRP-1 attenuated significantly the expression of Nanog antibody in comparison to untreated control. Mouse monoclonal anti Nanog antibody, clone 7F7-1 was used in 1:1,000 dilution with secondary anti-mouse IgG antibodies. Mouse monoclonal anti-tubulin antibody was used at 1:2,000 and secondary anti-mouse IgG at 1:5,000. Gel exposure time <1 min. Nanog band was detected at 40 kDa and tubulin band at 55 kDa.
PRP-1 effect of on the expression of Bmi-1 in human JJ012 chondrosarcoma cell line. Rabbit polyclonal anti-BMI antibody was used at 1:1,000 and secondary goat anti-rabbit IgG peroxidase conjugate- at 1:5,000 Bmi-1 bands were detected at 33 kDa. Exposure time, 2–5 min.
PRP-1 effect on the expression of SCML2 in human JJ012 chondrosarcoma cell line. Mouse monoclonal anti-SCML2 (SCMAD14a), was used in 1:1,000 dilution, and secondary anti-mouse IgG at 1:5,000. Band was detected ~100 kDa region. Film exposure time, 2–5 min. All the antibody dilutions were made with Western Blocker solutions. Mouse monoclonal anti-tubulin antibody was used at 1:2,000 and secondary anti-mouse IgG at 1:5,000. Tubulin band was detected at 55 kDa.
Effect of PRP-1 on c-Myc and p-c-Myc. Mouse monoclonal (9E10) anti-c-Myc and rabbit polyclonal anti-p-c-Myc were used at 1:1,000 dilution, and secondary anti-mouse IgG and goat anti-rabbit IgG peroxidase conjugate at 1:5,000. Band was detected ~67 kDa. Film exposure time, 2–5 min.
Effect of PRP-1 on Src and p-Src. Rabbit polyclonal anti-p-Src (Tyr416) and rabbit polyclonal anti-Src antibodies were applied to the membranes at 1:1,000, whereas goat anti-rabbit IgG peroxidase conjugate was used at 1:5,000. Mouse monoclonal anti-tubulin antibody was used at 1:2,000 and secondary anti-mouse IgG at 1:5,000. On both gels tubulin band was detected at 55 kDa.
Effect of PRP-1 on the expression of p27 and CDK2 in human JJ012 chondrosarcoma cell line CDK2. Rabbit monoclonal anti-p27 Kip1 (D69C12) and rabbit polyclonal anti-CDK/2 both were used at 1:1,000 dilution in western blocker solution, whereas secondary antibodies goat anti-rabbit IgG peroxidase conjugate was used at 1:5,000. P27 band was detected at 27 kDa and cdk2 at 37 kDa.
Effect of PRP-1 on the p21 (detected at 21 kDa) expression, in human JJ012 chondrosarcoma cell line. Mouse monoclonal p21 (F-5), was applied at 1:1,000 dilution, and secondary anti-mouse IgG at 1:5,000 dilution. Mouse monoclonal anti-tubulin antibody was used at 1:2,000 and secondary anti-mouse IgG at 1:5,000. Tubulin band was detected at 55 kDa. Film exposure time, 2 min.