Screening for Synergistic Reagents With Pazopanib Against Osteosarcoma Using a Compound Library.

BACKGROUND/AIM: Osteosarcoma (OS) is the most common malignant bone tumor. As the same agents have been in use since the mid-1970s, new therapeutic approaches are needed to improve prognosis. Pazopanib (PZP) has already demonstrated marked antitumor activity clinically and can be effective in patients with metastatic OS. We investigated the combination treatment of candidate agents with PZP and examined effects on tumor growth using an in vivo model. MATERIALS AND METHODS: A library of 324 compounds was used. MG63 OS cells were treated with PZP and each compound. Cell viability was measured. The antiproliferative effects of compound combination on four OS cell lines was tested. Cell signaling was evaluated by western blot analysis. In vivo antitumor testing was performed using 143B-bearing mice. RESULTS: The screening process identified crizotinib (CRZ) as the most effective drug for combination with PZP. The combination of PZP and CRZ demonstrated effects compared to control or single therapy. Cell signal investigation showed that dual therapy down-regulated c-MYC, p-AKT, p-STAT3, p-cyclin D1 and survivin and up-regulated cleaved caspase-3 and cleaved PARP compared to control or single therapy. In vivo analysis showed dual therapy achieved synergic effects for tumor growth compared to control or single-treatment groups. No significant difference in the change in body weight was observed among groups. CONCLUSION: Combined use of PZP and CRZ offers synergic anti-tumor effects against OS, inducing apoptosis in vitro and in vivo by down-regulating AKT and STAT3. Our data suggest that these agents can be used for patients clinically.

Prognostic Significance of Thrombomodulin mRNA in High-Grade Soft Tissue Sarcomas after 10 years.

OBJECTIVE: To elucidate the correlation between expression of thrombomodulin (TM) mRNA from 83 benign soft tissue tumors or soft tissue sarcomas (STS) and clinicopathological parameters and to analyze the outcome of high-grade STS patients after 10 years. METHODS: Total RNA was extracted from 83 primary soft tissue tumors (15 benign tumors, 68 STS). TM mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was measured with real-time quantitative polymerase chain reaction and compared to various clinicopathological parameters. The log-rank test and Cox proportional hazard analysis were used to evaluate recurrence-free survival, metastasis-free survival, and overall survival. RESULTS: Thrombomodulin mRNA levels were not significantly different between benign tumors and STS. In STS, TM mRNA levels were not significantly different between histologically high-grade (n = 57) and low-grade (n = 11) tumors. Following analysis of high-grade STS at the 10-year follow-up, 21 patients had experienced a recurrence, 22 patients had experienced metastasis, and 23 patients had died of disease (DOD). TM levels were significantly higher in patients with metastasis or DOD patients. Receiver operating characteristic analysis for identifying 5-year and 10-year DOD determined the threshold for best sensitivity and specificity as 0.283. We divided patients into those with high (<0.283) and low (≤0.283) TM mRNA levels. Based on Kaplan-Meier analysis, a significant difference between the two groups was seen for recurrence-free survival (5 years: low = 76.6%, high = 53.1%, 10 years: low: 67.0%, high 39.8%, P = 0.0122) and metastasis-free survival (5 years: low = 86.3%, high = 40.2%, 10 years: low: 73.3%, high: 35.2%, P = 0.00023). Furthermore, the high TM group showed significantly worse prognosis than the low TM group (5 years: low = 90.1%, high = 42.3%, 10 years: low: 76.4%, high 31.3%, P = 0.00031). Thus, high levels of TM mRNA are associated with highly recurrent and metastatic potential and lead to poor prognosis. In multivariate Cox proportional hazard analysis, only high TM showed a significant difference in metastasis-free survival (hazard ratio: 4.33, 95% confidence interval 1.61-11.6, P = 0.00359) and overall survival (hazard ratio: 3.69, 95% confidence interval 1.49-10.5, P = 0.00569). CONCLUSION: High levels of TM mRNA may be a significant predictor of recurrence, metastasis, and a poor outcome in STS patients after 10 years. TM is a candidate molecular marker and may be clinically useful for devising a therapeutic treatment strategy by prediction of prognosis.

Serum thrombomodulin as a metastatic and prognostic marker in soft tissue sarcomas.

BACKGROUND: Thrombomodulin (TM) has multiple biological functions and modulates not only anti-coagulation, but also cell proliferation, adhesion, and anti-inflammation activities. The main function of TM is to activate the anticoagulant pathway of protein C. Soluble TM is related to metastasis by its inactivation of thrombin. OBJECTIVES: To clarify the correlation between serum TM levels and clinicopathological parameters. METHODS: The plasma TM levels (FU/ml) of 135 primary soft tissue tumors (benign, 67; soft tissue sarcoma (STS), 68) were measured before biopsy or treatment. TM levels were analyzed and compared to various clinicopathological parameters. Log-rank test and Cox proportional analysis were used to evaluate recurrence-free survival, metastasis-free survival, and overall survival. RESULTS: STS tumors had significantly higher TM values (15.9) than benign tumors (13.7) (p= 0.0138). 5-year MFS was 81.1% in low TM and 40.0% in high TM (p= 0.00671), and 5-year OS was 85.5% in low and 52.5% in high TM in grades 1-3 (p= 0.0673). In multivariate COX proportional analysis, high-TM showed a significant difference (MFS: HR 4.37, p= 0.0147; OS: HR 3.60, p= 0.0557) in grades 1-3. CONCLUSIONS: We demonstrated that a high level of soluble TM has the potential to be a significant predictor of metastasis and poor prognosis in STS patients. TM is a candidate molecular marker for high metastatic potential and can be clinically useful for guiding therapeutic strategy.

Infiltrative tumor growth patterns on magnetic resonance imaging associated with systemic inflammation and oncological outcome in patients with high-grade soft-tissue sarcoma.

OBJECTIVE: The aim of this study was to determine whether the tumor infiltrative growth pattern on magnetic resonance imaging (MRI) was associated with blood inflammatory markers (C-reactive protein; CRP and Neutrophil-lymphocyte ratio; NLR) and survival in patients with high-grade soft-tissue sarcoma (STS). METHODS: The cohort for this retrospective study included 81 patients with a mean age of 63 years. The tumor depth was superficial or deep in 15 and 66 patients, respectively. The mean CRP and NLR were 1.31 mg/dL and 2.81, respectively. The assessment of a peripheral growth pattern which divided into three patterns on MRI was based on the largest midsection of the tumor. RESULTS: On MRI scans, diffuse-type, focal-type, and pushing-type growth patterns were observed in 18, 33, and 30 patients, respectively. Superficial high-grade STS were prone to show a focal-type pattern on MRI. There were no correlations between growth pattern type and clinicopathological factors such as age, sex, tumor size, and histological grade. However, the incidence of infiltrative growth was significantly higher in patients with elevated CRP (p = 0.0002). In multivariate analysis, growth pattern and CRP were independent prognostic factors for disease-specific survival, metastasis-free survival. Growth pattern was also related to local tumor control. CONCLUSIONS: There were significant associations between the tumor growth pattern and CRP levels in patients with high-grade soft-tissue sarcoma. An infiltrative growth pattern and elevated CRP may be associated with inferior disease-specific and metastasis-free survival rates in these patients. Therefore, careful post-treatment follow-up should be conducted in such patients.

STAT3 inhibitor, cucurbitacin I, is a novel therapeutic agent for osteosarcoma.

The development of clinical agents remains a costly and time-consuming process. Although identification of new uses of existing drugs has been recognized as a more efficient approach for drug discovery than development of novel drugs, little screening of drugs that might be used for a rare malignant tumor such as osteosarcoma (OS) has been performed. In this study, we attempted to identify new molecular targeted agents for OS by employing Screening Committee of Anticancer Drugs (SCADS) kits. To screen compounds for OS treatment, their effect on cell viability of the OS cell lines 143B, MG63, HOS, SAOS-2, and HUO9 were evaluated. Candidate drugs were narrowed down based on a global anti-proliferative effect against these five OS cell lines. After excluding cytotoxic compounds and compounds unsuitable for in vivo administration, cucurbitacin I was extracted. Cucurbitacin I has been found to have cytotoxic and anti-proliferative properties against several tumors through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Cucurbitacin I dose- and time-dependently inhibited the proliferation of all five OS cell lines. Following cucurbitacin I treatment, STAT3 was inactivated and analysis of Mcl-1, cleaved PARP and caspase-3 indicated apoptosis induction. Expression of cell cycle regulator proteins, such as phospho-cyclin D1, c-Myc and survivin, were suppressed. Finally, cucurbitacin I potently inhibited the tumor growth of human OS 143B cells in nude mice. Our in vitro and in vivo results suggest that STAT3 inhibition by cucurbitacin I will be an effective and new approach for the treatment of OS.

Minimally invasive surgery using intraoperative electron-beam radiotherapy for the treatment of soft tissue sarcoma of the extremities with tendon involvement.

BACKGROUND: When a soft tissue sarcoma (STS) is located at the distal part of an extremity and involves the tendon, a wide excision usually causes severe functional disability. We therefore developed a minimally invasive surgical technique using intraoperative electron-beam radiotherapy (IOERT) to reduce the incidence of post-operative functional disability in patients with peri-/intra-tendinous STS. We assessed the clinical outcomes of the novel minimally invasive surgery. METHODS: The study population included five patients who received treatment for distal extremity STSs. After elevating the tumor mass, including the tendon and nerve from the tumor bed with a wide margin, a lead board was inserted beneath the tumor mass to shield the normal tissue. IOERT (25-50 Gy) was then applied, and the tumor excised with care taken to maintain the continuity of the tendon. RESULTS: In a desmoid patient, local recurrence was observed outside the irradiated field. No cases of neuropathy or bone necrosis were observed. The mean limb function score was excellent in all patients. None of the high-grade sarcoma patients had local recurrence or distant metastasis. CONCLUSIONS: Although the current study is only a pilot study with a small number of patients, it shows that this minimally invasive procedure has the potential to become a standard treatment option for selected patients. TRIAL REGISTRATION: H17-250 (registered 2 November 2005) and H25-250 (modified from H17-250, registered 5 December 2013).

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease.

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N-acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using (32)P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H(2)O(2) with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.