Shoji Shimose

Evaluation of systemic chemotherapy with magnetic liposomal doxorubicin and a dipole external electromagnet

The development of an active drug delivery system is an attractive approach to increase the targetability of anticancer agents. In the present study, we examined the efficiency of systemic chemotherapy with small magnetic liposomes containing doxorubicin (magnetic DOX liposomes) and an externally applied electromagnetic force in osteosarcoma‐bearing hamsters. Syrian male hamsters inoculated with osteosarcoma, OS515, in the limb were studied 7 days after inoculation. The efficiency of this system was evaluated by measuring the tissue distribution and tumor‐suppressing effects of DOX on primary tumor growth and lung metastases. A DC dipole electromagnet was used, and the hamsters tumor‐bearing limb was placed between 2 poles after the i.v. administration of liposomes. The dose of DOX and the magnetic field strength were fixed at 5 mg/kg and 0.4 T, respectively. Administration of magnetic DOX liposomes followed by 60 min application of magnetic field produced a 3‐ to 4‐fold higher maximum DOX concentration in the tumor. This newly designed systemic chemotherapy significantly suppressed primary tumor growth for at least 2 weeks, though other DOX treatments also suppressed compared to control. Histologic examination confirmed a greater antitumor effect of this systemic chemotherapy compared to standard methods. In addition, this approach significantly suppressed lung metastases measured at 3 weeks posttreatment. These results suggest that this systemic chemotherapy can effectively reduce primary tumor growth and suppress lung metastasis due to increased targeting of DOX. Such targeted drug delivery for anticancer agents would provide clinical advantages compared to current methods.

Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration

MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is limited by the lack of an effective delivery system to tumors. Exosomes are small vesicles that play important roles in intercellular communication. Here, we show that synthetic miR-143 introduced into cells is released enveloped in exosomes and that the secreted exosome-formed miR-143 is transferred to osteosarcoma cells. The delivery of exosome-formed miR-143 significantly reduced the migration of osteosarcoma cells. The delivery efficiency of exosome-formed miR-143 was less than that achieved with lipofection, but the migratory potential of osteosarcoma cells was similarly inhibited after both strategies. Our results suggest that exosomes can deliver synthetic miR-143 and are a potentially efficient and functional delivery system.

Antitumor effects of telomerase inhibitor TMPyP4 in osteosarcoma cell lines

Telomere studies in carcinomas have been extensively reported for prognostic utility and effective methods for targeting telomerase therapy has been described, but efficacy of telomerase inhibitor remained unknown in sarcoma cells. In this study, we investigated the effects of telomerase inhibitor cationic porphyrin TMPyP4 on telomerase activity, telomere length, cell growth, and apoptosis in osteosarcoma cell lines. TMPyP4 significantly inhibited telomerase activity in telomerase positive HOS and Saos‐2, but not in MG‐63. TMPyP4 significantly induced telomere shortening, and inhibition of the cell growth in HOS and Saos‐2 with over 17% apoptosis rates. In terms of MG‐63, TMPyP4 did not induce inhibition of both telomerase activity and cell growth, although it induced significant telomere shortening. Telomere length after treatment was 5.60 kb in HOS, 4.00 kb in Saos‐2, and 9.89 kb in MG‐63. These results may suggest that both telomerase activity loss and sufficient telomere shortening are necessary to inhibit cell growth in telomerase positive osteosarcoma cells. TMPyP4 did not induced telomere shortening but significantly inhibited the growth with 22.6% apoptosis rate in telomerase negative with extremely longer telomere‐U2OS, may indicating the antitumor effect of TMPyP4 may be related to DNA damage including telomere dysfunction through G‐quadruplex stabilization, independent on telomere length.

Efficacy of a nitrogen-containing bisphosphonate, minodronate, in conjunction with a p38 mitogen activated protein kinase inhibitor or doxorubicin against malignant bone tumor cells

PurposeWe recently reported the sarcoma-selective antitumor effects of a newly developed nitrogen-containing bisphosphonate, minodronate (MIN), on malignant bone tumors. The aim of this study was to develop efficient combination MIN therapy in malignant bone tumors.MethodsWe examined downstream molecular events of MIN in osteosarcoma and Ewing’s sarcoma cells to search for a partner to combine with MIN. Furthermore, we evaluated the combined effects of MIN and clinically available Doxorubicin (DOX).ResultsWe found that MIN inhibited Rap 1A prenylation, and extracellular signal-regulated kinase (ERK) or Akt phosphorylation in osteosarcoma (Saos-2) and Ewing’s sarcoma (SK-ES-1) cells. Interestingly, MIN activated p38 mitogen activated protein kinase (MAPK) only in SK-ES-1 cells and a p38 MAPK inhibitor augmented MIN-induced growth inhibition in SK-ES-1 cells. Doxorubicin (DOX) exerted synergistic effects on Saos-2 and SK-ES-1 cell lines. Daily injection of MIN enhanced the growth inhibition of SK-ES-1 xenograft sarcoma treated by DOX in nude mice.ConclusionsThese findings suggest that the inhibition of the p38 MAPK pathway may be attractive in overcoming cellular resistance against MIN. In the light of clinical settings, MIN may have a beneficial adjuvant role in the DOX treatment.

Telomere-Maintenance Mechanisms in Soft-Tissue Malignant Fibrous Histiocytomas

BACKGROUND Sarcomas are distinct from carcinomas in that a substantial portion of them use the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. The present study clarifies the prevalence of the ALT mechanism and examines the prognostic importance of telomere factors in soft-tissue malignant fibrous histiocytomas. METHODS We investigated a series of forty-three soft-tissue malignant fibrous histiocytoma samples from forty-three patients with regard to telomere length, telomerase activity, and human telomerase reverse transcriptase (hTERT) mRNA expression. Tumor samples were obtained from surgical specimens and were stored at -80 degrees C until use. Univariate analysis of the tumor samples from patients for whom data were available on age, sex, histological grade, tumor size, surgical margin, recurrence, and telomere factors was performed with use of the log-rank test. Multivariate analysis with only significant variables was then performed. RESULTS Telomerase activity was detectable in 79.1% of the tumor samples, hTERT expression was demonstrated in 90.7% of the tumor samples, and evidence of engagement of the ALT mechanism of telomere length maintenance was observed in 32.6% of the tumor samples. Among the variables tested, ALT-positive status emerged as the only independent prognostic factor for death of the patient (hazard ratio, 0.275; 95% confidence interval, 0.104 to 0.724; p=0.0089). There were no significant differences in survival rates between patients with ALT-positive, telomerase-positive tumors and those with ALT-positive, telomerase-negative tumors (p=0.301) or between patients with ALT-positive tumors that showed above-average telomerase activity and those with ALT-positive tumors that showed below-average telomerase activity (p=0.900). Therefore, telomerase activity does not affect the prognosis in patients with ALT-positive malignant fibrous histiocytoma. High telomerase expression is associated with a poor prognosis in patients with ALT-negative malignant fibrous histiocytoma (p=0.0027). CONCLUSIONS More detailed analysis will be needed to identify the most valuable prognostic factor in patients with malignant fibrous histiocytoma, and a more thorough understanding of telomere biology may give an indication of telomere-targeting therapy in the future.

Clinical Outcomes of 54 Pelvic Osteosarcomas Registered by Japanese Musculoskeletal Oncology Group

Background: To determine the clinical outcomes in patients with pelvic osteosarcoma, we reviewed the experience with 54 pelvic osteosarcoma patients. Methods: Thirty-five patients underwent surgical treatment with 30 undergoing limb salvage procedures and 5 requiring hemipelvectomies. Results: Oncological outcome was continuously disease free in 12 cases, no evidence of disease in 3, alive with disease in 3, died of disease in 34 and died of other causes in 2. The 5-year survival rate was 29.5%. For the patients who underwent surgery, statistically significant prognostic factors were chemotherapy, surgical margin, tumor location in sacrum, efficacy of chemotherapy, local recurrence, metastasis and treatment for metastases. With univariate analysis, local recurrence, surgical margin and tumors resected from the sacrum were significant prognostic factors for metastasis. Surgical margin, tumor resected from sacrum and metastasis were significant prognostic factors for local recurrence. With regard to postoperative functional mobility, patients with tumors resected from the sacrum exhibited poorer functional mobility when compared with other sites. Conclusions: Patients with tumors located in the sacrum had worse prognosis, and significant prognostic factor for local recurrence and metastasis. Treatment of pelvic osteosarcoma especially located in the sacrum requires new approaches and further improvements in order to ensure successful outcomes.

Reconstructed patellar tendon length after proximal tibia prosthetic replacement.

A common problem after proximal tibia prosthetic reconstruction is insufficient extensor mechanism or extensor lag. Maintaining the reconstructed patellar tendon length is an important way to minimize extensor lag. We measured the patellar tendon length and extensor lag serially. From 1994-2003, seven consecutive patients with malignant bone tumors of the proximal tibia had prosthetic reconstruction. The extensor mechanism was reconstructed by reattachment of the patellar tendon to the prosthesis with a synthetic material and augmented by a gastrocnemius flap. The patellar tendon length was measured according to the Insall-Salvati ratio. The ratios at 30° knee flexion decreased immediately postoperatively, and reverted to almost the same preoperative values within 18 months. The mean ratios at 60° knee flexion are significantly greater than 30° at 6 months postoperatively. These findings indicate that the patellar tendon stretched, whereas the extensor lag improved continuously for 12 months postoperatively. In the revision at 22 months postoperatively, although the patellar tendon was not reattached to the replaced prosthesis, the ratio and the extensor lag had not worsened. These results show that providing strong continuity of the patellar tendon, gastrocnemius flap, and leg extensors can decrease the extensor lag, although the patellar tendon stretches. Level of Evidence: Therapeutic study, Level IV (case series). See the Guidelines for Authors for a complete description of levels of evidence.

Oncolytic vesicular stomatitis virus administered by isolated limb perfusion suppresses osteosarcoma growth

A significant limitation to oncolytic virotherapy in vivo is the lack of a clinically relevant means of delivering the virus. We evaluated the oncolytic activity of vesicular stomatitis virus (VSV) in human osteosarcoma cells and explored isolated limb perfusion (ILP) as a novel oncolytic virus delivery system to extremity sarcoma in immune‐competent rats. Human and rat osteosarcoma cells transduced with rVSV‐lacZ uniformly expressed β‐gal. VSV was fully capable of replicating its RNA genome in all osteosarcoma cell lines, and efficiently killed them in time‐ and dose‐dependent manners, whereas normal bone marrow stromal cells were refractory to the virus. VSV delivered by ILP inhibited growth of osteosarcoma xenografts more potently than that injected intravenously and intratumorally in the hind limb of immune‐competent rats. Histopathological sections of tumor lesions treated by ILP‐delivered VSV showed positive for VSV‐G protein. There were no VSV‐G expressions in perfused leg muscle, nonperfused leg muscle, brain, lung, and liver in VSV‐treated rats. Our findings show efficient VSV gene expression and replication in osteosarcoma cells, suggesting that osteosarcoma may be a promising target for oncolytic virotherapy with VSV. Furthermore, we firstly showed that ILP of VSV against extremity sarcoma caused antitumor activity.

Major-nerve schwannomas versus intramuscular schwannomas:

Background: A schwannoma is a benign peripheral nerve tumor. Predicting the involvement of a nerve on symptoms or magnetic resonance (MR) findings is crucial to the diagnostic process. Purpose: To compare symptoms, MR findings, and histological findings between major-nerve schwannomas and intramuscular schwannomas. Material and Methods: Thirty-four patients with 36 palpable schwannomas (29 major-nerve schwannomas and seven intramuscular schwannomas) surgically excised and proven histologically were retrospectively reviewed. Results: Frequencies of the Tinel-like sign, split-fat sign, entering and exiting nerve, and low-signal margin indicate the presence of a nerve, and were significantly higher in major-nerve schwannomas than in intramuscular schwannomas. In tumor morphological patterns (target sign, inhomogeneous and homogeneous pattern), there were no significant differences between major-nerve schwannomas and intramuscular schwannomas. Schwannomas showing the target sign histologically tended to be less degenerative. All major-nerve schwannomas and five of the intramuscular schwannomas produced some characteristic symptoms and/or MR findings, but two intramuscular schwannomas did not have any characteristic symptoms and findings. Conclusion: In major-nerve schwannomas, the Tinel-like sign, split-fat sign, entering and exiting nerve, and low-signal margin are commonly observed and useful for diagnosis. In intramuscular schwannomas, these characteristic findings are less common, which makes diagnosis difficult.

Telomerase Activity in Giant Cell Tumors of Bone

BackgroundA giant cell tumor of bone (GCT) is a histologically benign neoplasma that has an unpredictable pattern of biological aggressiveness. In the present study, we investigated whether there was a correlation between telomere length or the levels of telomerase activity and other clinical features of GCTs, for the possible use of these factors as parameters of aggressiveness or prognosis.MethodsIn 16 surgically resected GCTs specimens, telomere length was assessed by terminal restriction fragments by Southern blot analysis. Telomerase activity was measured by a semiquantitative polymerase chain reaction–based telomeric repeat amplification protocol assay.ResultsTelomere length reduction was observed in 69% of the GCT samples. The telomere lengths of tumors were significantly shorter than those of normal tissue (P = .008). The mean telomere length of grade 3 tumors was significantly shorter than those of grade 1 and 2 tumors (P = .038). Telomerase activity was detected in 81% of tumor samples. The level of telomerase activity in tumors with local recurrence was significantly higher than in tumors without local recurrence (P = .011).ConclusionsThese results suggest that telomere length correlates with roentgenographic grade as a result of the frequency of cell division, and high telomerase activity indicates the aggressiveness of GCTs.