P4HA2-induced prolyl hydroxylation suppresses YAP1-mediated prostate cancer cell migration, invasion, and metastasis.

Yes-associated protein 1 (YAP1), a key player in the Hippo pathway, has been shown to play a critical role in tumor progression. However, the role of YAP1 in prostate cancer cell invasion, migration, and metastasis is not well defined. Through functional, transcriptomic, epigenomic, and proteomic analyses, we showed that prolyl hydroxylation of YAP1 plays a critical role in the suppression of cell migration, invasion, and metastasis in prostate cancer. Knockdown (KD) or knockout (KO) of YAP1 led to an increase in cell migration, invasion, and metastasis in prostate cancer cells. Microarray analysis showed that the EMT pathway was activated in Yap1-KD cells. ChIP-seq analysis showed that YAP1 target genes are enriched in pathways regulating cell migration. Mass spectrometry analysis identified P4H prolyl hydroxylase in the YAP1 complex and YAP1 was hydroxylated at multiple proline residues. Proline-to-alanine mutations of YAP1 isoform 3 identified proline 174 as a critical residue, and its hydroxylation suppressed cell migration, invasion, and metastasis. KO of P4ha2 led to an increase in cell migration and invasion, which was reversed upon Yap1 KD. Our study identified a novel regulatory mechanism of YAP1 by which P4HA2-dependent prolyl hydroxylation of YAP1 determines its transcriptional activities and its function in prostate cancer metastasis.

The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells.

Doxorubicin (Adriamycin, ADM) is an antimitotic drug used in the treatment of a wide range of malignant tumors, including acute leukemia, lymphoma, osteosarcoma, breast cancer, and lung cancer. Multidrug resistance-associated proteins (MRPs) are members of a superfamily of ATP-binding cassette (ABC) transporters, which can transport various molecules across extra- and intra-cellular membranes. The aim of this study was to investigate whether there was a correlation between MRP4 and primary ADM resistance in osteosarcoma cells. In this paper, we chose the human osteosarcoma cell line MG63, ADM resistant cell line MG63/DOX, and the patient's primary cell GSF-0686. We checked the ADM sensitivity and cytotoxicity of all the three cells by cell proliferation assay. The intracellular drug concentrations were measured by using LC-MS/MS. We also examined MRP4 gene expression by RT-PCR and Western Blot. We found that the intracellular ADM concentration of the parent osteosarcoma cell line MG63 was higher than the ADM resistant osteosarcoma MG63/DOX cell line or the GSF-0686 cell after ADM treatment (P < 0.05). In addition, MRP4 mRNA and protein levels in ADM resistant osteosarcoma cells were higher than in MG63 cell (P < 0.05). Taking together, this work suggests that overexpression of MRP4 may confer ADM resistance in osteosarcoma cells.

KLF8 knockdown suppresses proliferation and invasion in human osteosarcoma cells.

Krüppel-like factor 8 (KLF8) is a transcription factor that is important in the regulation of the cell cycle and has a critical role in oncogenic transformation and epithelial to mesenchymal transition (EMT). EMT is a key process in tumor metastasis. Although overexpression of KLF8 has been observed in a variety of human tumor types, the role of KLF8 in human osteosarcoma is yet to be elucidated. The present study aimed to investigate the biological impact of KLF8 on Saos-2 osteosarcoma cells. KLF8 gene expression was knocked down in vitro using a lentivirus-mediated small interfering (si)RNA method. Cell proliferation and cell cycle distribution were evaluated using 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide and colony formation assays, and flow cytometry, respectively. Cell invasion was analyzed using a Transwell® invasion assay. Knockdown of KLF8 was found to significantly inhibit proliferation and invasion in osteosarcoma cells. These data suggest that KLF8 may exhibit an important role in osteosarcoma tumorigenesis and that KLF8 may be a potential therapeutic target for the treatment of osteosarcoma.

Cyclophosphamide-hydroxycamptothecin as second-line chemotherapy for advanced Ewing's sarcoma: experience of a single institution.

AIM: To investigate the feasibility and efficacy of cyclophosphamide (CTX)-hydroxycamptothecin (HCPT) as second-line chemotherapy on advanced Ewing's sarcoma. METHODS: From April 2009 to November 2010, 27 patients with advanced Ewing's sarcoma who had progressive disease after the first-line chemotherapy regimen of vincristine, dactinomycin and cyclophosphamide and ifosfamide and etoposide were retrospectively reviewed in this analysis. CTX was given (0.6 g/m(2), i.v. push day 1) and HCPT (6 mg/m(2), i.v. drip days 1-5) as second-line chemotherapy every 3 weeks. The primary end-point was overall response rate, the secondary end-point included progression-free, overall survival, disease control rate and toxicities. RESULTS: A total of 134 cycles were given, median four cycles per patient (range 2-6). Overall response rate was 30% and disease control rate was 82%, with two complete response (8%), six partial remission (22%) and 14 stable disease (52%). The median time to progression and overall survival time were 7 months (95% CI 3-10) and 11 months (95% CI 5-18), respectively. Major severe toxicities (grade 3 and 4) were: nausea/vomiting (17%), alopecia (17%); leukopenia (27%) in total cycles. Mild toxicities (grade 1 or 2) were leukopenia (73%), nausea/vomiting (83%), hepatic lesion (14%) and anemia (44%). CONCLUSION: A CTX-HCPT regimen can control disease progression effectively and the side effects can be tolerable for Chinese advanced Ewing's sarcoma patients. Further assessment is necessary to confirm the safety and efficacy of this treatment.

Pirarubicin inhibits multidrug-resistant osteosarcoma cell proliferation through induction of G2/M phase cell cycle arrest.

AIM: Pirarubicin (THP) is recently found to be effective in treating patients with advanced, relapsed or recurrent high-grade osteosarcoma. In this study, the effects of THP on the multidrug-resistant (MDR) osteosarcoma cells were assessed, and the underlying mechanisms for the disruption of cell cycle kinetics by THP were explored. METHODS: Human osteosarcoma cell line MG63 and human MDR osteosarcoma cell line MG63/DOX were tested. The cytotoxicity of drugs was examined using a cell proliferation assay with the Cell Counting Kit-8 (CCK-8). The distribution of cells across the cell cycle was determined with flow cytometry. The expression of cell cycle-regulated genes cyclin B1 and Cdc2 (CDK1), and the phosphorylated Cdc2 and Cdc25C was examined using Western blot analyses. RESULTS: MG63/DOX cells were highly resistant to doxorubicin (ADM) and gemcitabine (GEM), but were sensitive or lowly resistant to THP, methotrexate (MTX) and cisplatin (DDP). Treatment of MG63/DOX cells with THP (200-1000 ng/mL) inhibited the cell proliferation in time- and concentration-dependent manners. THP (50-500 ng/mL) induced MG63/DOX cell cycle arrest at the G(2)/M phase in time- and concentration-dependent manners. Furthermore, the treatment of MG63/DOX cells with THP (200-1000 ng/mL) downregulated cyclin B1 expression, and decreased the phosphorylated Cdc2 at Thr(161). Conversely, the treatment increased the phosphorylated Cdc2 at Thr(14)/Tyr(15) and Cdc25C at Ser(216), which led to a decrease in Cdc2-cyclin B1 activity. CONCLUSION: The cytotoxicity of THP to MG63/DOX cells may be in part due to its ability to arrest cell cycle progression at the G(2)/M phase, which supports the use of THP for managing patients with MDR osteosarcoma.

Relationships between levels of CXCR4 and VEGF and blood-borne metastasis and survival in patients with osteosarcoma.

The present study was designed to determine whether the expression of chemokine receptor CXCR4 and vascular endothelial growth factor (VEGF) is correlated with the extent of metastasis and the survival of patients with osteosarcoma. Using tissue microarrays, we analyzed the expression of CXCR4 and VEGF in tumor tissues collected from 56 patients with osteosarcoma. A two-year follow-up was performed to evaluate tumor metastatic behavior and the overall survival of the patients. There was a significant correlation between the expression of CXCR4 and the expression of VEGF in tumor tissues of these patients (P = 0.002). Univariate analysis revealed that expression of these proteins was correlated with clinical stage, but not age, gender, or serum alkaline phosphatase levels. The patients with tumors expressing CXCR4 and VEGF had worse overall survival rates compared with the patients with tumors that did not express CXCR4 (P = 0.03) or VEGF (P = 0.04). These data indicate that CXCR4 and VEGF expression is highly correlated with metastatic progression in patients with osteosarcoma and had predictive value for the metastasis and survival of osteosarcoma patients.

Down-regulation of ribosomal protein L7A in human osteosarcoma.

PURPOSE: To explore the expression of ribosomal protein L7A (RPL7A) in osteosarcoma and its correlation with clinical features. METHODS: Ribosomal protein L7A mRNA expression was quantified by real-time reverse transcription (RT)-polymerase chain reaction (PCR) in 47 specimens from osteosarcoma, 8 from normal bone tissues and 12 from benign bone lesion tissues. Expression of RPL7A mRNA was also detected in human osteosarcoma cell line MG-63. The relationship between RPL7A mRNA expression and clinicopathological factors was statistically analyzed. The immunoblotting pattern of RPL7A was also analyzed in 20 osteosarcomas, 8 normal bone and 8 benign bone tissues. RESULTS: Ribosomal protein L7A mRNA expression in osteosarcoma samples was significantly down-regulated compared with that in samples from normal bone (P < 0.001) and benign bone lesion tissues (P < 0.001). Low expression of RPL7A mRNA was also found in osteosarcoma cell line MG-63. Low expression of RPL7A mRNA was significantly associated with increased serum level of alkaline phosphatase (ALP, P = 0.008), but was not correlated with other clinicopathological parameters including sex, age, serum lactate dehydrogenase (LDH), tumor location, histological type, histological grading, lung metastasis and overall survival. Interestingly, survival analysis suggested low RPL7A mRNA expression was a significant poor prognostic indicator for overall survival in patients with high grade lesion developed lung metastasis at the time of diagnosis of the primary osteosarcoma (P < 0.05). On western blot, reduced expression of RPL7A protein was observed in osteosarcoma samples (n = 20) compared with normal bone (n = 8) (P < 0.001) and benign bone tissues (n = 8) (P < 0.001). CONCLUSION: Under-expression of RPL7A may be involved in the carcinogenesis of osteosarcoma. Loss of RPL7A expression may be associated with poor survival of osteosarcoma patients with lung metastasis.

The expression of CRM1 is associated with prognosis in human osteosarcoma.

The nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) is involved in the nuclear export of proteins and messenger RNAs and, thus, mediates the subcellular distribution of important molecules. Osteosarcoma is a ubiquitous and highly aggressive malignant bone tumor. The expression of CRM1 protein in human osteosarcoma has not been reported to date. We investigated the expression of CRM1 in 57 human osteosarcoma and 5 normal cartilage tissues. Western blot investigation revealed expression of CRM1 was significantly increased in osteosarcoma compared with normal tissues. High expression of CRM1 was significantly associated with increased serum level of alkaline phosphatase (ALP, P=0.001) but did not associate with that of lactate dehydrogenase (LDH, P=0.06). In univariate analysis, a significant association between CRM1 expression and tumor size (P=0.014) as well as histological grade (P=0.003) was observed, while high CRM1 expression was not correlated with the other clinicopathological parameters. In Kaplan-Meier survival analysis, high CRM1 expression was a significant prognostic indicator for progression-free survival (P=0.016) as well as overall survival (P=0.008). Multivariate analysis demonstrated that expression of CRM1 was an independent prognostic parameter for longer overall survival (95% CI, 1.27-5.39). Additional prospective studies are required to investigate the prognostic role of high expression of CRM1.

Relationship of serum methotrexate concentration in high-dose methotrexate chemotherapy to prognosis and tolerability: A prospective cohort study in chinese adults with osteosarcoma.

BACKGROUND: Cancer that originates in the bone, termed primary bone cancer, is rare. Osteosarcoma (OS) occurs primarily in growing bone tissue and is more prevalent in children and adolescents. OS in adults is rare, with 3 to 5 cases per million population per year worldwide. There are limited data on treatment-related prognosis and adverse reactions in adults reported in the literature. OBJECTIVES: The aims of this study were to investigate factors that influence serum methotrexate (MTX) concentrations used in chemotherapy in Chinese adult patients with OS, and to determine the correlations (based on age, sex, and dosage), if any, between MTX and prognosis, in terms of disease-free survival (DFS) and overall survival (OAS), and tolerability. METHODS: Adult patients aged ≥30 years with OS received ≥3 courses (2 courses before surgery and 3-4 courses postsurgery) of high-dose MTX (6 or 8 g/m(2)) combined chemotherapy. The regimen consisted of day 1: MTX + folinic acid (herein referred to as citrovorum factor rescue); day 8: cisplatin; days 21 to 25: ifosfamide + mesna; and day 21: doxorubicin. Serum MTX concentrations were assessed immediately after the end of infusion (baseline) and at 24 and 48 hours using high-performance liquid chromatography. Changes in serum MTX concentrations, factors that influence serum MTX concentrations, and the relationship between serum MTX concentrations and prognosis and tolerability (determined by adverse reactions) were analyzed. Patients received a second course of treatment after a 3-week period. RESULTS: Ninety patients (58 men, 32 women; age range, 30-67 years) with OS were included in the study. A total of 532 courses of combined chemotherapy were administered. The serum MTX concentrations ranged widely at baseline (244.31-929.68 mol/L, Cmin and Cmax, respectively) and at 24 hours (0.73-28.24 mol/L, respectively), suggesting that the serum MTX concentrations varied significantly between different individuals and within the same individual at different time points. The serum MTX concentrations in ~23% of cases (122/532) determined at 24 and/or 48 hours were numerically higher than the safety values (according to Nirenberg's reference: irreversible damage if MTX concentration was >10 umol/L and > 1 umol/L at 24 and 48 hours, respectively). No correlation was found between high serum MTX concentration at baseline and high serum MTX concentration at 24 hours (r = 0.401). The prevalences of the 3 most common adverse reactions in these patients were depressed white blood cell count (44.03%), dental ulcer (23.0%), and rash (18.0%). However, in the remaining 410 courses in which serum MTX concentrations were lower than the safety values, these prevalences were 14.6%, 3-9%, and 2.4%, respectively. Neither age nor sex was significantly associated with MTX Cmax, but dosage was (P < 0.05). Patients with a serum MTX Cmax concentration >500 µmol/L at baseline had a significantly longer DFS rate than those with ≤500 umol/L (P = 0.040). There were no significant between-group differences in the OAS rates. conclusions: In these Chinese patients with OS, serum MTX concentrations measured at different time points were varied. The findings suggest that adverse reactions occurred in patients whose serum MTX concentrations at 24 and/or 48 hours were higher than the safety values. The dosage appeared to have influenced MTX Cmax, while sex and age did not, and the Cmax was significantly related to DFS but not OAS.

Triptolide inhibits cyclooxygenase-2 and inducible nitric oxide synthase expression in human colon cancer and leukemia cells.

Triptolide (TP), a traditional Chinese medicine, has been reported to be effective in the treatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines. This study investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia (K562), and elucidates the possible molecular mechanism involved. SW114 and K562 cells were treated with different doses of TP (0, 5, 10, 20, or 50 ng/ml). The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation of both tumor cell lines in a dose-dependent manner. To further investigate its mechanisms, the products prostaglandin E(2) (PGE(2)) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed that TP strongly inhibited the production of NO and PGE(2). Consistent with these results, the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulated both at the mRNA level and the protein expression level, as shown by real-time RT-PCR and Western blotting. These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activity could be involved in the antitumor mechanisms of TP.

[Effects of Imatinib mesylate on the development of dendritic cells derived from bone marrow mononuclear cells of patients with chronic myeloid leukemia].

OBJECTIVE: To investigate the effects of Imatinib mesylate (STI571) on the development of dendritic cells (DC) derived from the bone marrow mononuclear cells of patients with chronic myeloid leukemia (CML). METHODS: Bone marrow mononuclear cells (BMMNC) from CML patients were cultured initially using multiple cytokine combinations as follows: recombined human granulocyte/macrophage colony-stimulating-factor (rhGM-CSF) plus recombined human interleukin-4 (rhIL-4) as control groups, rhGM-CSF plus rhIL-4 and STI571 as experimental groups, and from day 8 added recombined human tumor necrosis factor-alpha (rhTNF-alpha) for stimulating maturation. The morphologic features of cells were observed by Wright's staining, Cytogenetic analysis was performed by Fluorescence in-situ hybridization (FISH), phenotypes were assessed by flow cytometry, and the functions of antigen-presenting were assayed by mixed lymphocyte reaction (MLR). The concentration of VEGF was detected by enzyme-linked immunosorbent assay (ELISA). NF-kappaB activation was evaluated by TransAM(TM) ELISA kit. RESULTS: CML experimental groups treated with STI571 displayed features in morphology which were similar to those of control groups with delicate membrane projections. FISH confirmed the DC of both CML groups were leukemic origin. In comparison with the CML control groups, the CML experimental groups showed an increased expression of CD80, CD86, CD83 and HLA-DR and showed more intense abilities of allogeneic antigen presentation. The concentration of VEGF was dramatically reduced, and yet NF-kappaB activation was increased in experimental groups. CONCLUSION: STI571 could promote the activation/maturation of DC derived from BMMNCs of patients with CML in vitro, which might be partially responsible for the fact that the inhibitory effect of VEGF on DC NF-kappaB activation was relieved through STI571 inhibiting the overproduction of VEGF in CML.

[Effects of ST1571 on the development of dendritic cells derived from bone marrow mononuclear cells in patients with chronic myeloid leukemia].

OBJECTIVE: To investigate the effects of ST1571 on the development of dendritic cells (DC) derived from bone marrow mononuclear cells of patients with chronic myeloid leukemia (CML). METHODS: Bone marrow mononuclear cells (BMMNC) from CML patients and healthy volunteers were cultured initially using multiple cytokine combinations as follows: recombinant human granulocyte/ macrophage colony-stimulating-factor (rhGM-CSF) plus recombinant human interleukin-4 (rhIL-4) as CML and normal control groups, rhGM-CSF plus rhIL-4 and ST1571 as CML experimental groups, and from day 8 recombinant human tumor necrosis factor-alpha ( rhTNF-alpha) was added to stimulate DC maturation. The morphologic features of cells were observed by Wright's staining and phenotypes were assessed by flow cytometry. Cytogenetic analysis was performed by fluorescence in-situ hybridization (FISH), and the antigen-presenting function was assayed by mixed lymphocyte reaction (MLR). The concentration of VEGF was detected by ELISA. RESULTS: CML experimental groups treated with STI571 displayed morphological features similar to those of control groups with delicate membrane projections. However, in comparison with the CML control groups, the CML experimental groups showed an increased expression of CD80, CD86, CD83 and HLA-DR and showed more intense abilities of allogeneic antigen presentation, which were similar to those of normal control groups. FISH confirmed that DCs of both CML, groups were of leukemic origin. The concentration of VEGF was dramatically reduced in CML experimental groups. CONCLUSION: In vitro, STI571 promotes the activation/maturation of DCs derived from BMMNCs of patients with CMI, and decreases VEGF production by the leukemic cells. The promotion of DC maturation may be partially due to decreased inhibitory effect of VEGF.