Cytostatic effect of novel mTOR inhibitor, PRP-1 (galarmin) in MDA 231 (ER-) breast carcinoma cell line. PRP-1 inhibits mesenchymal tumors

Activation of the PI3K-Akt-mTOR pathway is implicated both in the establishment of tumors and as well as a target for therapy in many types of solid malignancy, its blockade represents an opportunity to improve outcomes in patients with tumors that are associated with poor prognosis. Our experimental data indicates that proline-rich polypeptide-1 (PRP-1, galarmin) is immunomodulator cytokine, produced by hypothalamic neurosecretory cells and exerts its antiproliferative effect on the tumor cells of mesenchymal origin via inhibiting mTOR kinase activity and repressing cell cycle progression. The goal of these investigations was to elucidate the antiproliferative action of PRP-1 on the breast carcinoma cell line MDA 231 (ER-) and to compare PRP-1 action previously reported on other mesenchymal tumors. These experiments confirmed maximum inhibition of cell growth at 0.5 and 1 μg/ml PRP-1 (71% and 63%, respectively) and inhibition at 10 μg/ml of 44%. There was no inhibitory effect observed on luminal T47-D (ER+) cells. Videomicroscopy results demonstrated dividing cells in the cytokine-treated MDA 231 (ER-), suggesting that the cells were not in the state of dormancy. The flow cytometry experiments confirmed that PRP-1-treated cells were accumulated in S phase. No apoptosis, caspase activation, or senescence was detected after treatment with this cytokine. Experiments with mTOR with PRP-1 (10 μg/ml) indicated statistically significant 40% inhibition of mTOR kinase activity in immunoprecipitates of the MDA 231 (ER-) cell line. PRP-1 is a novel mTOR inhibitor with strong antiproliferative action in mesenchymal tumors mostly resistant to radiation and chemotherapy

Utility of quantitative computerized pain drawings in a sample of spinal stenosis patients

To evaluate the utility of quantitative computerized pain drawings (CPDs) in a sample of spine patients before and after surgery. Analysis of changes in quantified CPDs, the Oswestry Disability Index (ODI), the Short Form-36 Health Survey Questionnaire (SF-36), and numerical ratings of pain intensity before and after surgery. Private clinic in large metropolitan area. Patients. Forty-six patients with spinal stenosis. Interventions. Surgery for the relief of pain due to spinal stenosis. A total points (TP) score was calculated from the CPD that reflected the total number of pixels filled by the patient, and the percentage of total pain area indicated as aching, stabbing, numbness, pins and needles, burning, and other, were each calculated separately. CPD scores, ODI score, Physical Components Summary (PCS) and Mental Components Summary scores of the SF-36, and pain intensity ratings (0-10 scale) were all recorded before and after surgical intervention. Results. After surgery, patients showed significant improvements in the extent of shaded pain area of the CPD, pain intensity ratings, ODI, and SF-36 PCS scores (paired t-test, P < or = 0.01). Changes in TP scores calculated from the CPDs were significantly correlated (P < or = 0.05) with changes in ODI scores (r = 0.34) and pain intensity ratings (r = 0.37). Changes in the percentage of total pain area covered by specific qualities of pain were not significant. Results from the present study provide initial support for the use of automated quantified data collected from CPDs to evaluate treatment interventions and to serve the clinician as a record of changes in spatial location, radiation or extent of pain, and the sensory quality of pain when evaluating individual patient needs

Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide‑1

Chondrosarcoma is a malignant bone neoplasm that is refractory to chemotherapy and radiation. With no current biological treatments, mutilating surgical resection is the only effective treatment. Proline rich polypeptide 1 (PRP‑1), which is a 15‑amino acid inhibitor of mammalian target of rapamycin complex‑1 (mTORC1), has been indicated to exert cytostatic and immunomodulatory properties in human chondrosarcoma cells in a monolayer. The aim of the present study was to evaluate the effects of PRP‑1 on an in vitro 3D chondrosarcoma tumor model, known as spheroids, and on the cancer stem cells (CSCs) which form spheroids. JJ012 cells were cultured and treated with PRP‑1. An ALDEFLUOR™ assay was conducted (with N,N‑diethylaminobenzaldehyde as the negative control) to assess aldehyde dehydrogenase (ALDH) activity (a recognized CSC marker), and bulk JJ012, ALDHhigh and PRP‑1 treated ALDHlow cells were sorted using flow cytometry. Colony formation and spheroid formation assays of cell fractions, including CSCs, were used to compare the PRP‑1‑treated groups with the control. CSCs were assessed for early apoptosis and cell death with a modified Annexin V/propidium iodide assay. Western blotting was used to identify mesenchymal stem cell markers (STRO1, CD44 and STAT3), and spheroid self‑renewal assays were also conducted. A clonogenic dose‑response assay demonstrated that 20 µg/ml PRP‑1 was the most effective dose for reducing colony formation capacity. Furthermore, CSC spheroid growth was significantly reduced with increasing doses of PRP‑1. Annexin V analysis demonstrated that PRP‑1 induced CSC cell death, and that this was not attributed to apoptosis or necrosis. Western blot analysis confirmed the expression of mesenchymal markers, and the spheroid self‑renewal assay confirmed the presence of self‑renewing CSCs. The results of the present study demonstrate that PRP‑1 eliminates anchorage independent CSC growth and spheroid formation, indicating that PRP‑1 likely inhibits tumor formation in a murine model. Additionally, a decrease in non‑CSC bulk tumor cells indicates an advantageous decline in tumor stromal cells. These findings confirm that PRP‑1 inhibits CSC proliferation in a 3D tumor model which mimics the behavior of chondrosarcoma in vivo

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas

Mutant IDH1 Depletion Downregulates Integrins and Impairs Chondrosarcoma Growth

Chondrosarcomas are a heterogeneous group of malignant bone tumors that produce hyaline cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including conventional and dedifferentiated chondrosarcomas. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into D-2-hydroxyglutarate (D-2HG), an oncometabolite. IDH mutations and D-2HG are thought to contribute to tumorigenesis due to the role of D-2HG as a competitive inhibitor of α-KG-dependent dioxygenases. However, the function of IDH mutations in chondrosarcomas has not been clearly defined. In this study, we knocked out mutant IDH1 (IDH1 ) in two chondrosarcoma cell lines using the CRISPR/Cas9 system. We observed that D-2HG production, anchorage-independent growth, and cell migration were significantly suppressed in the IDH1 knockout cells. Loss of IDH1 also led to a marked attenuation of chondrosarcoma formation and D-2HG production in a xenograft model. In addition, RNA-Seq analysis of IDH1 knockout cells revealed downregulation of several integrin genes, including those of integrin alpha 5 (ITGA5) and integrin beta 5 (ITGB5). We further demonstrated that deregulation of integrin-mediated processes contributed to the tumorigenicity of IDH1-mutant chondrosarcoma cells. Our findings showed that IDH1 knockout abrogates chondrosarcoma genesis through modulation of integrins. This suggests that integrin molecules are appealing candidates for combinatorial regimens with IDH1 inhibitors for chondrosarcomas that harbor this mutation

Treatment with AGI-5198 Impedes Colony Formation in IDH1-mutant JJ012 and HT1080 Human Chondrosarcoma Cells, While Sparing C28 Cells.

<p>A: Cell viability of JJ012, HT1080 and C28 cells. Cells were treated with increasing concentrations of AGI-5198 or DMSO and analyzed using MTS assay after 72 h of treatment. Relative cell viability (%) was calculated as the mean absorbance of AGI-5198-treated cells relative to DMSO-treated cells. B: Colony forming number of JJ012, HT1080 and C28 cells. Cells were treated with AGI-5198 or DMSO for 7 days, then the colonies were stained and counted. Error bars depict SEM from 3 independent experiments. *** <i>p</i><0.001.</p

Human Chondrosarcoma Cell Lines JJ012 and HT1080 Harbor IDH1 Mutations.

<p>A: PCR-based sequencing confirmed that JJ012 encoded an R132G (<u>C</u>GT><u>G</u>GT) missense mutation. B: HT1080 encoded an R132C (<u>C</u>GT><u>T</u>GT) missense mutation. Both cell lines harbored an <i>IDH1</i> mutation in a single allele whereas the other allele was wild-type. C: The chondrocyte cell line C28 had no mutation in either allele.</p

D-2HG Levels in AGI-5198 or DMSO-treated JJ012 and HT1080 Cell Pellets and Cell Culture Media.

<p>AGI-5198 reduced levels of D-2HG in both cell pellets and cell culture media of JJ012 (A) and HT1080 (B) in a dose-dependent fashion. Data was normalized by cell number and analyzed by one-tailed student’s unpaired t-Test between non-treated and drug-treated cells. Error bars depict SEM from 3 independent experiments. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

A Patient with IDH2 Mutant Dedifferentiated Chondrosarcoma Demonstrates a Drop in D-2HG Plasma and Urine Levels after Surgical Resection.

<p>A: Histologic image of this patient’s dedifferentiated chondrosarcoma showing neoplastic cartilage (Arrow a) juxtaposed to higher grade, and more cellular regions resembling fibrosarcomatous elements (dedifferentiated component, Arrow b.) B: plain radiograph pre- (left) and post-operation (right). Bracket delineates the tumor. C: PCR-based DNA sequencing showed that this patient’s tumor exhibits an IDH2 R172S (AG<u>G</u>>AG<u>T</u>) missense mutation. D: D-2HG levels measured in patient plasma samples showed a decrease after surgical resection. E: D-2HG levels measured in patient urine samples showed a decrease after surgical resection. Error bars depict one standard error of the mean (SEM) from three measurements.</p