Paclitaxel Induce Apoptosis of Giant Cells Tumor of Bone via TP53INP1 Signaling.

OBJECTIVE: To evaluate the antitumor capability and to investigate the underlying molecular mechanism of paclitaxel. METHODS: First, cck-8 and apoptosis assays were used to determine survival and apoptotic effects of HS 737.T cells under treatment of paclitaxel. Next, RNA-seq and bioinformatics were used to determine the differentially expressed genes and to analyze the pathway involved. Quantitative real-time polymerase chain reaction was used to verify the accuracy of some differentially expressed genes (DEG). ClueGO was used to decode and visualize functionally grouped GO terms of differentially expressed genes, and to map the DEG protein-protein interactions (PPI) network. Western blotting was used to check the expression of target genes, the cleavage of Caspase-3 and PARP1, and the phosphorylation level of p53. Finally, transcriptomics, bioinformatics, and RNAi were used to estimate the antitumor capability and to identify the underlying mechanisms of paclitaxel in GCTB. RESULTS: Our data revealed that paclitaxel had significant time-dependent effects on the viability and induced apoptosis of HS 737.T cells. RNA-seq and bioinformatics analysis showed that apoptosis, death receptor signaling pathway, TNF signaling pathway, and TP53 regulated transcription of cell death genes pathway were closely associated with paclitaxel in the treatment of GCTB. Western bolt results revealed that paclitaxel induced cleavage of Caspase-3 and PARP1, and increased the phosphorylation level of p53 in HS 737.T cells. RNAi results showed that the expression level of TP53INP1 was significantly decreased in HS737.T cells (the decrease was more than 70%). In addition, we found that the inhibitory ratios of paclitaxel on HS737.T cells deficient in TP53INP1 were less than in HS737.T cells with empty vector (19.88 and 40.60%, respectively). Hence, our data revealed that TP53INP1 regulated paclitaxel-driven apoptosis in HS737.T cells. CONCLUSION: Paclitaxel can significantly repress cell proliferation and induce apoptosis of HS 737.T cells through activating Caspase-3, PARP1, p53, and TP53INP1. Paclitaxel may be an effective drug in the management of GCTB.

Therapeutic benefits of neoadjuvant and post-operative denosumab on sacral giant cell tumor: a retrospective cohort study of 30 cases.

PURPOSE: Denosumab, a new monoclonal antibody that inhibits receptor activator for nuclear factor Kß ligand (RANKL), has recently been approved by FDA for the treatment of aggressive giant cell tumor of bone (GCTB). So we initiated this study to evaluate the clinical benifits of denosumab used preoperatively or postoperatively. METHODS: Patients diagnosed with classic sacral GCT without metastasis were included in this study. Patients were assigned into 3 groups according to the use of denosumab: control group 1, post-operative group 2 and neoadjuvant group 3. The latter two groups were treated with 120 mg of subcutaneous denosumab every 4 weeks with loading doses on days 8 and 15 of the first cycle. The primary endpoints were event-free-survival (EFS) and objective response rate (OPR) based on RECIST criteria. A system (MUD system) proposed by our center was applied to score the sacral nerve deficit changes before surgery in group 3. RESULTS: A total 30 patients (13 men and 17 women, mean age 34.7 years, range 15-56) were enrolled from April 2014 to July 2016. Group 1 included 10 patients, group 2 9 and group 3 11. The study ended in March 01, 2017, and followup ranged from 3 to 36 months (mean 18.3). Two patients with PET-CT showed SUV max uptake down to muscle tissue level. In the neoadjuvant group 3 7 patients had partial responses and 4 stable disease (ORR 63.6%; 95% CI 35-92). Most (80%) patients achieved significant improvement in pain and great relief in the bladder and bowel functions. In 4 patients the urocatheter was removed after neoadjuvant denosumab. CONCLUSION: Neoadjuvant therapy with denosumab can significantly relieve the symptoms and neurologic deficits.

Osteonecrosis of the Jaw and Rebound Hypercalcemia in Young People Treated With Denosumab for Giant Cell Tumor of Bone.

Context: Denosumab, an inhibitor of receptor activator of nuclear factor κ-B ligand, is an approved treatment of giant cell tumor of bone (GCTB) in adults and "skeletally mature" adolescents. Safety concerns include oversuppression of bone remodelling, with risk of osteonecrosis of the jaw (ONJ) and atypical femur fractures during treatment in adults and rebound hypercalcemia after treatment cessation in children. To date, ONJ has never been reported in children or adolescents. Objectives: To describe serious adverse effects during and following high-dose denosumab therapy in GCTB patients. Patients: Two adolescents (14 and 15 years) and a young adult (40 years) received fixed-dose denosumab for GCTB for 1.3 to 4 years (cumulative dose, 47 to 98 mg/kg), which was stopped because of development of ONJ in one adolescent and bilateral femoral cortical stress reactions in the young adult. All three patients developed rebound hypercalcemia with acute kidney injury 5.5 to 7 months after denosumab cessation. Results: The ONJ necessitated surgical debridement. Rebound hypercalcemia (serum calcium, 3.1 to 4.3 mmol/L) was unresponsive to hyperhydration alone, requiring repeated doses of calcitonin or intravenous bisphosphonate treatment. Hypercalcemia recurred in two patients within 4 weeks, with normal serum calcium profiles thereafter. All patients were naive to chemotherapy, radiotherapy, bisphosphonates, and corticosteroids and were metastases free, confirming the causative role of denosumab in these complications. Conclusion: These suppression-release effects of high-dose denosumab on bone remodeling raise questions about safety of fixed dosing and treatment duration. In young people, weight-adjusted dosing and safety monitoring during and after antiresorptive therapy is required.

Use of warm Ringer's lactate solution in the management of locally advanced giant cell tumor of bone.

BACKGROUND: This study was conducted to discover the effectiveness and safety of using warm Ringer's lactate solution (RLS) as a local treatment in the management of locally advanced giant cell tumor of bone with marked soft tissue invasion, including nearby neurovascular bundles. PATIENTS AND METHODS: This was a longitudinal cohort study with an average follow-up period of 4.6 ± 0.3 years, ranging from 4.2 to 5.9 years. There were 21 patients (9 male and 12 female), with the ages of subjects ranging from 12 to 64 years. Eight patients (38 %) were tumor recurrence cases. Pathological fracture was found in 15 patients (71 %). After extended curettage, warm RLS (50 °C) was locally applied for 20 min. Bone stabilization and reconstruction were then performed. RESULTS: All patients survived the operation. No additional neurovascular injury resulting from the use of warm RLS was found. Patients who had neurological deficit before the operation experienced significant improvement in motor and sensory function during the follow-up period. Complication was found in one patient (5 %). Two patients (9.5 %), had tumor recurrence and 19 patients (90.5 %) were tumor-free with good to acceptable function. CONCLUSION: Use of warm Ringer's lactate solution as an adjunctive local treatment during intra-lesional curettage of giant cell tumor with locally soft tissue extension was found to be safe with relatively low recurrence rate. However, additional studies to identify the optimum thermoablation dose at each part of the body should be undertaken before this technique can be used as a standard treatment.

Autologous Platelet Gel Improves Bone Reconstruction of Large Defects in Patients with Bone Giant Cell Tumors.

BACKGROUND/AIM: Giant cell tumors are mostly benign but locally aggressive tumors. The excision of bone tumors can result in large defects, therefore bone reconstruction is still one the most demanding procedures in orthopedic surgery. Our study addresses the opportunity for improving surgical outcome by employing ß-tricalcium phosphate (ß-TCP) with platelet-rich plasma (PRP) at the surgical site. PATIENTS AND METHODS: We included 16 patients with giant cell tumors. After adjuvant therapy, the cavity was reconstructed with ß-TCP, bone graft material (ActifuserR Granules Baxter) and platelet gel application. RESULTS: Our explorative analysis suggests a positive effect of PRP on surgical outcome in patients with giant cell tumors treated with curettage. CONCLUSION: Use of platelet gel as an adjuvant significantly reduces the time required for bone healing following intralesional treatment of benign giant cell tumors, and achieves good functional results without promoting local recurrence.

Stimulation of osteogenic differentiation in stromal cells of giant cell tumour of bone by zoledronic acid.

Therapeutic effects of zoledronic acid (ZOL) on giant cell tumour of bone (GCT) have been proven. Apoptosis induction was considered to be one of the mechanisms of ZOL tumour inhibition. In this study, we presented the possibility of an osteogenic differentiation stimulation mechanism of ZOL and further investigated dosage and time effects. We treated stromal cells of GCT (GCTSC) with ZOL for 48 hours at different concentrations (0 µM, 0.01 µM, 0.1 µM, 1 µM, 5 µM, 30 µM) and assessed apoptotic and osteogenic differentiation markers with immunohistochemical techniques and real-time quantitative RT-PCR. Our results suggested that ZOL enhanced mRNA expression of Cbfa-1, osterix and osteocalcin genes with a maximum effect at 1 µM in GCTSC. Time course experiments indicated a time dependent osteogenic differentiation effect. In conclusion, ZOL may be considered as an adjuvant in the treatment of GCT not only by inducing apoptosis but also by stimulating osteogenic differentiation of remaining tumor stromal cells after surgery.

Pamidronate, farnesyl transferase, and geranylgeranyl transferase-I inhibitors affects cell proliferation, apoptosis, and OPG/RANKL mRNA expression in stromal cells of giant cell tumor of bone.

Giant cell tumor (GCT) is the most common nonmalignant primary bone tumor reported in Hong Kong. It usually affects young adults between the ages of 20 and 40. This tumor is well known for its potential to recur following treatment. To date no effective adjuvant therapy exists for GCT. Our project aimed to study the effects of pamidronate (PAM), farnesyl transferase inhibitor (FTI-277), geranylgeranyl transferase inhibitor (GGTI-298), and their combinations on GCT stromal cells (SC). Individual treatment with PAM, FTI-277, and GGTI-298, inhibited the cell viability and proliferation of GCT SC in a dose-dependent way. Combination of FTI-277 with GGTI-298 caused synergistic effects in reducing cell viability, and its combination index was 0.49, indicating a strong synergism. Moreover, the combination of FTI-277 with GGTI-298 synergistically enhanced cell apoptosis and activated caspase-3/7, -8, and -9 activities. PAM induced cell-cycle arrest at the S-phase. The combination of PAM with GGTI-298 significantly increased OPG/RANKL mRNA ratio and activated caspase-3/7 activity. Our findings support that the combination of bisphosphonates with GGTIs or FTIs with GGTIs may be used as potential adjuvants in the treatment of GCT of bone.

The efficacy of chemical adjuvants on giant-cell tumour of bone. An in vitro study.

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H(2)O(2)) and 50% zinc chloride (ZnCI(2)) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions. Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H(2)O(2) and 50% ZnCI(2). The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures. These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone.

Cytokine synthesis by chondroblastoma: relation to local inflammation.

PURPOSE: To investigate cytokine production by chondroblastoma in inducing local inflammation and adjacent-joint arthritis. METHODS: Immunohistochemical analyses of curetted tissues using anti-human interleukin (IL)-1 beta, IL- 6, IL-8, and tumour necrosis factor (TNF)-alpha were performed for 6 patients with chondroblastoma and 3 patients with giant cell tumour (GCT) of bone. In addition, prostaglandin E2, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, and TNF-alpha in the cyst fluid of one of the patients with chondroblastoma and 2 with GCT of bone were measured using immunoassay kits. RESULTS: More positive staining for IL-1 beta, IL-8, IL- 6, and TNF-alpha was shown in chondroblastoma than GCT of bone samples. Osteoclast-like giant cells in chondroblastomas showed positive staining for IL- 6 only. In addition, concentrations of IL-4, IL-6, and IL-8 in the cyst fluid were higher in the one patient with chondroblastoma than the 2 patients with GCT of bone. CONCLUSION: Cytokines such as IL-1 beta, IL-8, TNF-alpha, and particularly IL-6 play an important role in local inflammation in patients with chondroblastoma.

Adjuvant antiangiogenic therapy for giant cell tumors of the jaws.

PURPOSE: To further evaluate a novel treatment protocol for the management of aggressive giant cell lesions (GCLs) consisting of enucleation followed by adjuvant subcutaneous interferon alpha therapy. PATIENTS AND METHODS: Using a retrospective case series study design, a sample of patients with aggressive GCLs was enrolled between April 1995 and June 2006. Lesions were enucleated with preservation of vital structures. Postoperatively, the patients received daily subcutaneous interferon alpha (3 million units/m2 of body surface area). Interferon treatment continued with regular clinical and radiographic follow-up until the surgical defects filled in with bone, as demonstrated by panoramic radiographs and confirmed by computed tomography. Side effects, such as fever, fatigue, weight loss, decreased white blood cell count, decreased platelet count and elevated liver enzymes, were monitored. After completion of interferon therapy, patients followed for 2 years without evidence of recurrence were considered cured of disease. RESULTS: The study sample was comprised of 26 subjects (65% female) with a mean age of 18.5 years. At the time of this writing, 16 of the subjects have completed the protocol and are cured of disease, 6 are in remission, and 4 are in active treatment. Four subjects experienced significant side effects from the interferon, requiring modification of treatment. CONCLUSIONS: Enucleation of aggressive GCLs with preservation of vital structures and adjuvant interferon is an excellent strategy for managing aggressive GCLs. Approximately 15% of subjects developed significant side effects limiting interferon administration and necessitating alternative therapies.

Central giant cell granuloma of the jaw: a review of the literature with emphasis on therapy options.

Central giant cell granuloma (CGCG) is a benign lesion of the jaws with an unknown etiology. Clinically and radiologically, a differentiation between aggressive and non-aggressive lesions can be made. The incidence in the general population is very low and patients are generally younger than 30 years. Histologically identical lesions occur in patients with known genetic defects such as cherubism, Noonan syndrome, or neurofibromatosis type 1. Surgical curettage or, in aggressive lesions, resection, is the most common therapy. However, when using surgical curettage, undesirable damage to the jaw or teeth and tooth germs is often unavoidable and recurrences are frequent. Therefore, alternative therapies such as injection of corticosteroids in the lesion or subcutaneous administration of calcitonin or interferon alpha are described in several case reports with variable success. Unfortunately, randomized clinical trials are very rare or nonexistent. In the future, new and theoretically promising therapy options, such as imatinib and OPG/AMG 162, will be available for these patients.

[Case analysis on treatment and recurrence of giant cell tumor of bone].

OBJECTIVE: To analyze the clinical features, treatment methods, and recurrence factors of giant cell tumor of the bone and to investigate the surgical therapy choice for the tumor around the knees. METHODS: Thirty-eight patients (13 males and 25 females; average age 31.1 years, range 14-59 years) with giant cell tumor of the bone were treated and followed up from January 1993 to January 2005. The patients' diagnoses were established by biopsies of the specimens from the preoperative punctures or operations. The clinical features and the radiological and laboratory data from the 38 patients were reviewed. By the Campanacci's radiological grading system, 5 patients were in Grade I, 22 in Grade II, and 11 in Grade III . By the Enneking classification, 9 patients were in Grade I, 21 in Grade II, and 8 in Grade III . By the Jaffe's classification, 7 patients were in Grade I, 24 in Grade II, and 7 in Grade III. The intralesional excision (curettage) with the bone grafting was performed on 4 patients; the curettage with some adjuvant treatments (high-speed burring, phenol, alcohol, cement, hydrogen peroxide, 50% ZnCl2, 3% iodine tincture, or bone cement) was used in 26 patients; and resection of the whole tumor was performed on 8 patients. RESULTS: The follow-up of the 38 patients for 12-144 months (average, 67 months) revealed that giant cell tumor of the bone was found around the knees in 29 of the 38 patients (13 at the distal femur, 16 at the proximal tibia), at the proximal femur in 2, at the proximal ulna in 2, at the distal radius in 2, at the sacroiliac area in 2, and at lumbar spine in 1. Of the 38 patients, 4 had a recurrence after simple curettage, 8 had no recurrence after resection of the whole tumor, and 8 of the remaining 26 patients had a recurrence after curettage with some adjuvant treatments. Five patients in Grade I (Campanacci's radiological grading) had no recurrence, 6 of the 11 patients in Grade II had a recurrence, and 6 of the 11 patients in Grade III had a recurrence. Two of the 9 patients in Grade I (Enneking grading) had a recurrence, 6 of the 21 patients in Grade II had a recurrence, and 4 of the patients in Grade III had a recurrence; all the recurrent lesions were around the knee, with a duration of the recurrence ranging from 2 months to 36 months (average, 14.3 months). Of the patients with the recurrence, 12 underwent reoperations (8 by the total resection of the recurrent tumor, 4 by the curettage with adjuvant treatments), and there was no recurrence after the reoperation. CONCLUSION: Giant cell tumor of the bone usually recurs around the knee joint, especially at the proximal tibia, usually graded as Grade II or III by the Campanacci's radiological grading system. Simple curettage has a higher recurrence rate; therefore, extensive curettage and resection of the lesions combined with some adjuvant treatments after the correct diagnosis can be used to reduce the high recurrence rate of giant cell tumor of the bone.

Bisphosphonates may reduce recurrence in giant cell tumor by inducing apoptosis.

Giant cell tumor of bone is an aggressive tumor characterized by extensive bone destruction and high recurrence rates. This tumor consists of stromal cells and hematopoietic cells that interact in an autocrine manner to produce tumoral osteoclastogenesis and bone resorption. This autocrine regulation may be disrupted by novel therapeutic agents. Nonspecific local adjuvant therapies such as phenol or liquid nitrogen have been used in the treatment of giant cell tumor, but specific adjuvant therapies have not been described. The bisphosphonates pamidronate and Zoledronate can induce apoptosis in giant cell tumor culture in a dose-dependent manner. We established giant cell tumor cultures from patients with extensive destruction of bone. One of the four cultures formed osteoclastlike giant cells in vitro after more than six passages without exogenous receptor activator of NF-kappaB ligand or macrophage colony stimulating factor. Annexin V staining, presence of active cleaved form of caspase-3, and disappearance of poly (ADP-ribose) polymerase on Western blotting indicated activation of apoptosis by bisphosphonates in giant cell tumor. These results indicate that topical or systemic use of pamidronate or zoledronate can be a novel adjuvant therapy for giant cell tumor by targeting osteoclastlike giant cells, mononuclear giant cell precursor cells, and the autocrine loop of tumor osteoclastogenesis.

Thermal effects of acrylic cementation at bone tumour sites.

The use of acrylic bone cement as an adjunct to surgical excision of giant cell tumour of bone appears to reduce the incidence of tumour recurrence. Possible mechanisms for this apparent tumour inhibition include cytotoxic effects from the methylmethacrylate monomer and tissue hyperthermia from the heat of polymerization of the cement. This work presents a method for the prediction of temperature fields and resulting tissue necrosis arising from the implantation of polymethylmethacrylate (PMMA) at the site of a curretted giant cell tumour of bone. This is accomplished using a two-dimensional model based on geometry obtained from digitized MRI images of the distal femur. A general-coordinate, non-orthogonal grid generation technique is used and solutions are obtained with an alternating-direction implicit (ADI) finite-difference scheme. The nodal temperature histories are then used to evaluate the effect of variable defect size on the zone of thermally induced cell necrosis. The results suggest the depth of the necrotic region is quite sensitive to the size of the implant. In at least some cases, the heating effect is sufficient to cause significant necrosis of tumorigenic cells. Implanting a large mass of acrylic may risk overkill, damaging substantial amounts of healthy tissue.

Characterization of the osteoclast vacuolar H(+)-ATPase B-subunit.

During bone resorption, osteoclasts acidify the extracellular bone resorbing compartment via a vacuolar H(+)-ATPase (V-ATPase), which resides in the ruffled-border membrane. In an effort to characterize the composition of the osteoclast V-ATPase catalytic domain, we have isolated a cDNA clone that encodes the V-ATPase B-subunit from a cDNA library constructed from highly purified chicken osteoclasts. Comparison of the predicted amino-acid sequence with the published sequences of isoforms of V-ATPase B-subunits from other sources revealed that the chicken osteoclast B-subunit is brain type and not kidney type. Furthermore, only clones encoding the brain type isoform of subunit B could be generated by PCR from a cDNA library prepared from human osteoclastoma osteoclast-like cells. Northern blot analysis revealed that two B-subunit mRNAs, approx. 1.7 and 3.5 kb in length, are expressed in chicken bone marrow mono-nuclear cells, brain and kidney, although the relative amounts of these two transcripts were different in each tissue. In brain, the 3.5-kb mRNA was predominantly expressed. In bone marrow cells, the levels of the 1.7-kb mRNA were higher than in other tissues and expression of this message was increased by 1,25-dihydroxyvitamin D-3, suggesting that this mRNA is specifically upregulated during osteoclast differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Culturing of cells from giant cell tumour of bone on natural and synthetic calcified substrata: the effect of leukaemia inhibitory factor and vitamin D3 on the resorbing activity of osteoclast-like cells.

Osteoclastic cells from giant cell tumour of bone (GCT) of bone provide a rich source for investigation of cellular mechanisms leading to formation of multinucleated cells, the resorption process and involvement of hormones and cytokines in these events. In the present study we investigated the effect of 1,25-dihydroxyvitamin D3 (VD3) and leukaemia inhibitory factor (LIF) on the resorbing potential of osteoclast of GCT origin using quantitative image-analysis of resorption lacunae in an in vitro dentine model. While VD3 unsignificantly increased the number of resorption pits and implicated surface after 7 days of GCT cell culturing, the stimulative effect of LIF was statistically significant. In cultures supplemented with LIF (5000 U/ml) the number of lacunae and resorption surface increased by 38% and 55%, respectively, when compared with control cultures. We suggest that both osteotropic agents increased osteoclastic activity, as the number of multinucleated cells was similar in control and experimental cultures. Seeding of GCT cells on biphasic calcium phosphate substratum revealed the relative inability of osteoclastic cells to resorb this synthetic material.