Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Gut Microbiota-Mediated Pharmacokinetic Drug-Drug Interactions between Mycophenolic Acid and Trimethoprim-Sulfamethoxazole in Humans.

Mycophenolic acid (MPA) and trimethoprim-sulfamethoxazole (TMP-SMX) are commonly prescribed together in certain groups of patients, including solid organ transplant recipients. However, little is known about the pharmacokinetic drug-drug interactions (DDIs) between these two medications. Therefore, the present study aimed to determine the effects of TMP-SMX on MPA pharmacokinetics in humans and to find out the relationship between MPA pharmacokinetics and gut microbiota alteration. This study enrolled 16 healthy volunteers to take a single oral dose of 1000 mg mycophenolate mofetil (MMF), a prodrug of MPA, administered without and with concurrent use of TMP-SMX (320/1600 mg/day) for five days. The pharmacokinetic parameters of MPA and its glucuronide (MPAG) were measured using high-performance liquid chromatography. The composition of gut microbiota in stool samples was profiled using a 16S rRNA metagenomic sequencing technique during pre- and post-TMP-SMX treatment. Relative abundance, bacterial co-occurrence networks, and correlations between bacterial abundance and pharmacokinetic parameters were investigated. The results showed a significant decrease in systemic MPA exposure when TMP-SMX was coadministered with MMF. Analysis of the gut microbiome revealed altered relative abundance of two enriched genera, namely the genus Bacteroides and Faecalibacterium, following TMP-SMX treatment. The relative abundance of the genera Bacteroides, [Eubacterium] coprostanoligenes group, [Eubacterium] eligens group, and Ruminococcus appeared to be significantly correlated with systemic MPA exposure. Coadministration of TMP-SMX with MMF resulted in a reduction in systemic MPA exposure. The pharmacokinetic DDIs between these two drugs were attributed to the effect of TMP-SMX, a broad-spectrum antibiotic, on gut microbiota-mediated MPA metabolism.

First study on the effect of transforming growth factor beta 1 and insulin-like growth factor 1 on the chondrogenesis of elephant articular chondrocytes in a scaffold-based 3D culture model.

Background and Aim: Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-ß1 (TGF-ß1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes. Materials and Methods: Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-ß1 or IGF-1 alone or 5-10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively. Results: TGF-ß1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses. Conclusion: Together, our results suggest that the single treatment of TGF-ß1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.

Mass Spectrometric-Based Proteomics for Biomarker Discovery in Osteosarcoma: Current Status and Future Direction.

Due to a lack of novel therapies and biomarkers, the clinical outcomes of osteosarcoma patients have not significantly improved for decades. The advancement of mass spectrometry (MS), peptide quantification, and downstream pathway analysis enables the investigation of protein profiles across a wide range of input materials, from cell culture to long-term archived clinical specimens. This can provide insight into osteosarcoma biology and identify candidate biomarkers for diagnosis, prognosis, and stratification of chemotherapy response. In this review, we provide an overview of proteomics studies of osteosarcoma, indicate potential biomarkers that might be promising therapeutic targets, and discuss the challenges and opportunities of mass spectrometric-based proteomics in future osteosarcoma research.

IMPDH2 and HPRT expression and a prognostic significance in preoperative and postoperative patients with osteosarcoma.

Osteosarcoma is one of the most aggressive bone tumors in children and adolescents. Development of effective therapeutic options is still lacking due to the complexity of the genomic background. In previous work, we applied a proteomics-guided drug repurposing to explore potential treatments for osteosarcoma. Our follow-up study revealed an FDA-approved immunosuppressant drug, mycophenolate mofetil (MMF) targeting inosine-5'-phosphate dehydrogenase (IMPDH) enzymes, has an anti-tumor effect that appeared promising for further investigation and clinical trials. Profiling of IMPDH2 and hypoxanthine-guanine phosphoribosyltransferase (HPRT), key purine-metabolizing enzymes, could deepen understanding of the importance of purine metabolism in osteosarcoma and provide evidence for expanded use of MMF in the clinic. In the present study, we investigated levels of IMPDH2, and HPRT in biopsy of 127 cases and post-chemotherapy tissues in 20 cases of high-grade osteosarcoma patients using immunohistochemical (IHC) analysis. Cox regression analyses were performed to determine prognostic significance of all enzymes. The results indicated that low levels of HPRT were significantly associated with a high Enneking stage (P = 0.023) and metastatic status (P = 0.024). Univariate and multivariate analyses revealed that patients with low HPRT expression have shorter overall survival times [HR 1.70 (1.01-2.84), P = 0.044]. Furthermore, high IMPDH2/HPRT ratios were similarly associated with shorter overall survival times [HR 1.67 (1.02-2.72), P = 0.039]. Levels of the enzymes were also examined in post-chemotherapy tissues. The results showed that high IMPDH2 expression was associated with shorter metastasis-free survival [HR 7.42 (1.22-45.06), P = 0.030]. These results suggest a prognostic value of expression patterns of purine-metabolizing enzymes for the pre- and post-chemotherapy period of osteosarcoma treatment.

Expression profiling of DNA methyl transferase I (DNMT1) and efficacy of a DNA-hypomethylating agent (decitabine) in combination with chemotherapy in osteosarcoma.

BACKGROUND: Abnormality in the DNA methylation process is one of the hallmarks of cancer. Emerging evidence strongly supports the idea that defects in DNA methyl transferases (DNMTs) are involved in tumor development and progression. This alteration has major effects at the transcription level of various cancer-associated genes. METHODS: Expression profiles of DNMT1 were investigated in fresh frozen tissues, patient-derived cells, and formalin-fixed paraffin-embedded tissues using immunoblotting and immunohistochemistry analysis. We also examined an anti-tumor effect of single DNA-hypomethylating agent (decitabine) and a combination of decitabine and chemotherapy in osteosarcoma cell lines. RESULTS: The results showed an overexpression of DNMT1 in most cases compared to normal cells and tissue samples. DNMT1 was also expressed at the same levels in paired primary cells derived from biopsy and post-chemotherapy tissues. Expression patterns of DNMT1 were examined in 77 osteosarcoma patients of whom 82% had positive DNMT1 with an IRS score > 0. Most of the cases expressed low to moderate levels of DNMT1 (IRS range 1-8, median = 2.0). Furthermore, we found that a combination of decitabine and chemotherapy had a synergistic effect in most of the tested osteosarcoma cells at a low dose therapeutic range of decitabine. CONCLUSIONS: Our study revealed DNMT1 expression patterns that indicated potential roles of DNMT1 in osteosarcoma transformation and progression. This finding also suggests the efficacy of a combination therapy of decitabine with chemotherapy for osteosarcoma treatment.

Mycophenolic acid is a drug with the potential to be repurposed for suppressing tumor growth and metastasis in osteosarcoma treatment.

Our previous review of proteomics data showed that in osteosarcoma, some overexpressed proteins were targets of FDA-approved immunosuppressive and anti-arrhythmic drugs, including mycophenolate mofetil (MMF), ribavirin, leflunomide, azathioprine and digoxin. Here, these drugs were screened for growth inhibitory effects in human osteosarcoma cell lines, including MNNG/HOS, U2OS, SaOS-2, MG-63 and 143B cells. Only mycophenolic acid (MPA), an active metabolite of MMF, efficiently inhibited osteosarcoma cell growth with IC50 values of 0.46-7.3 µM; these values are in the therapeutic range for organ transplant patients. At a therapeutic dose (10 µM), MPA significantly inhibited colony formation, caused cell cycle arrest in the S phase, and induced apoptosis. Moreover, the in vitro invasion of osteosarcoma cells was reduced by MPA by inhibiting cell migration capability. The in vivo antitumor effect of MMF was determined in nude mice harboring 143B cell xenografts. Daily oral administration of 200 mg/kg/day MMF for 2 weeks significantly suppressed tumor growth in treated mice, achieving 57.4 ± 11.1% tumor growth inhibition. Compared with the vehicle group, the MMF group treated with 50-200 mg/kg/day for 3 weeks had a significant reduction in the number of lung metastatic nodules in a tail vein-lung metastasis model of 143B cells. MMF doses of 50, 100 and 200 mg/kg/day are approximately equivalent to the non-toxic doses of 0.25, 0.5 and 1 g/day in humans, respectively. These findings indicate that MPA/MMF can effectively control osteosarcoma tumor growth and metastasis. Thus, the potential to repurpose MPA/MMF for use in osteosarcoma chemotherapy is of great interest.

Proinflammatory cytokines and lipopolysaccharides up regulate MMP-3 and MMP-13 production in Asian elephant (Elephas maximus) chondrocytes: attenuation by anti-arthritic agents.

BACKGROUND: Osteoarthritis (OA), the most common form of arthritic disease, results from destruction of joint cartilage and underlying bone. It affects animals, including Asian elephants (Elephas maximus) in captivity, leading to joint pain and lameness. However, publications regarding OA pathogenesis in this animal are still limited. Therefore, this study aimed to investigate the effect of proinflammatory cytokines, including interleukin-1 beta (IL-1ß), IL-17A, tumor necrosis factor-alpha (TNF-α), and oncostatin M (OSM), known mediators of OA pathogenesis, and lipopolysaccharides on the expression of cartilaginous degrading enzymes, matrix metalloproteinase (MMP)-3 and MMP-13, in elephant articular chondrocytes (ELACs) cultures. Anti-arthritic drugs and the active compounds of herbal plants were tested for their potential attenuation against overproduction of these enzymes. RESULTS: Among the used cytokines, OSM showed the highest activation of MMP3 and MMP13 expression, especially when combined with IL-1ß. The combination of IL-1ß and OSM was found to activate phosphorylation of the mitogen-activated protein kinase (MAPK) pathway in ELACs. Lipopolysaccharides or cytokine-induced expressions were suppressed by pharmacologic agents used to treat OA, including dexamethasone, indomethacin, etoricoxib, and diacerein, and by three natural compounds, sesamin, andrographolide, and vanillylacetone. CONCLUSIONS: Our results revealed the cellular mechanisms underlying OA in elephant chondrocytes, which is triggered by proinflammatory cytokines or lipopolysaccharides and suppressed by common pharmacological or natural medications used to treat human OA. These results provide a more basic understanding of the pathogenesis of elephant OA, which could be useful for adequate medical treatment of OA in this animal.

LL-37 alone and in combination with IL17A enhances proinflammatory cytokine expression in parallel with hyaluronan metabolism in human synovial sarcoma cell line SW982-A step toward understanding the development of inflammatory arthritis.

LL-37 is the only human cathelicidin-family host defense peptide and has been reported to interact with invading pathogens causing inflammation at various body sites. Recent studies showed high levels of LL-37 in the synovial-lining membrane of patients with rheumatoid arthritis, a common type of inflammatory arthritis. The present study aims to investigate the role of LL-37 on mechanisms associated with pathogenesis of inflammatory arthritis. The effects of LL-37 on the expression of proinflammatory cytokines, hyaluronan (HA) metabolism-related genes, cell death-related pathways, and cell invasion were investigated in SW982, a human synovial sarcoma cell line. Time-course measurements of proinflammatory cytokines and mediators showed that LL-37 significantly induced IL6 and IL17A mRNA levels at early time points (3-6 hr). HA-metabolism-related genes (i.e., HA synthase 2 (HAS2), HAS3, hyaluronidase 1 (HYAL1), HYAL2, and CD44) were co-expressed in parallel. In combination, LL-37 and IL17A significantly enhanced PTGS2, TNF, and HAS3 gene expression concomitantly with the elevation of their respective products, PGE2, TNF, and HA. Cell invasion rates and FN1 gene expression were also significantly enhanced. However, LL-37 alone or combined with IL17A did not affect cell mortality or cell cycle. Treatment of SW982 cells with both LL-37 and IL17A significantly enhanced IKK and p65 phosphorylation. These findings suggest that the chronic production of a high level of LL-37 may synchronize with its downstream proinflammatory cytokines, especially IL17A, contributing to the co-operative enhancement of pathogenesis mechanisms of inflammatory arthritis, such as high production of proinflammatory cytokines and mediators together with the activation of HA-metabolism-associated genes and cell invasion.

Protein profiling of osteosarcoma tissue and soft callus unveils activation of the unfolded protein response pathway.

Oncogenic drivers of osteosarcoma remain controversial due to the complexity of the genomic background of the disease. There are limited novel therapeutic options, and the survival rate of patients with osteosarcoma has not improved in decades. Genomic instability leads to complexity in various pathways, which is potentially revealed at the protein level. Therefore, the present study aimed to identify the mechanisms involved in the oncogenesis of osteosarcoma using proteomics and bioinformatics tools. As clinical specimens from patients are the most relevant disease­related source, expression patterns of proteins in osteosarcoma tissues were compared with soft tissue callus from donors containing high numbers of osteoblastic cells. Two­dimensional electrophoresis and liquid chromatography­tandem mass spectrometry (LC­MS/MS) successfully identified 33 differentially expressed proteins in the osteosarcoma tissues compared with the soft tissue callus. Among these proteins, 29 proteins were significantly upregulated in osteosarcoma. A functionally grouped network of the overexpressed proteins, that was created using the ClueGo and CluePedia applications, demonstrated that the unfolded protein response (UPR) pathway was activated mainly through the activating transcription factor 6 arm in osteosarcoma. The results of proteomics analysis were confirmed by elevated expression of UPR­related chaperone proteins, including 78 kDa glucose­related protein (GRP78), endoplasmin, calreticulin and prelamin­A/C, in the patient­derived primary cells and osteosarcoma cell lines. Furthermore, the expression of GRP78, a master regulator of the UPR, was enhanced in the osteosarcoma tissues of patients that were resistant to double regimen of doxorubicin and a platinum­based drug. The findings of the present study suggest that targeting the UPR pathway may be promising for the treatment of osteosarcoma.

Suppression of Cartilage Degradation by Zingerone Involving the p38 and JNK MAPK Signaling Pathway.

Zingerone, an active compound that is present in cooked ginger, has been claimed to be a bioactive ingredient that holds the potential of preventing and/or treating diseases involving inflammation. In this study, zingerone was used to discover its properties against joint inflammation using interleukin-1ß-induced osteoarthritis in cartilage explant and cell culture models. Zingerone was supplemented into the cartilage explant and cell culture media at different concentrations along with the presence of interleukin-1ß, an inducer of osteoarthritis. Markers indicating cartilage degradation, inflammation, and the signaling molecules involved in the inflammatory induction were investigated. Diacerien, an anti-osteoarthritic drug, was used as a positive control. Zingerone at a concentration of 40 µM reduced the level of matrix metalloproteinase-13 to about 31.95 ± 4.33 % compared with the interleukin-1ß-treated group and halted cartilage explant degradation as indicated by reducing the accumulative release of sulfated glycosaminoglycans by falling to the control concomitantly with an elevation of the remaining contents of uronic acid and collagen in the explant tissues when zingerone was added. In the SW1353 cell line model, zingerone efficiently suppressed the expression of TNF-α, interleukin-6, and interleukin-8 mRNA levels and tended to reduce the levels of both p38 and c-Jun N-terminal kinase phosphorylation. From the results of this study, it can be concluded that zingerone potentially reduced cartilage degradation, which is partially involved in p38 and c-Jun N-terminal kinases of the mitogen activator protein kinase signaling pathway leading to the reduction of proinflammatory cytokine amplification effects and cartilage-degrading enzyme syntheses. This finding supports the contention that ginger holds positive pharmaceutical effects against osteoarthritis.